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MALDI-TOF Mass Spectrometry

Matrix-assisted laser desorption ionization (MALDI) is a mass spectrometry ion source ideal for the analysis of biomolecules. Instead of ionizing compounds in the gaseous state, samples are embedded in a matrix, which is struck by a laser. The matrix absorbs the majority of the energy; some of this energy is then transferred to the sample, which ionizes as a result. Sample ions can then be identified using a time-of-flight analyzer (TOF).

This video covers principles of MALDI-TOF, including matrix selection and how TOF is used to elucidate mass-to-charge ratios. This procedure shows the preparation of a MALDI plate, the loading of samples onto the plate, and the operation of the TOF-mass spectrometer. In the final section, applications and variations are shown, including whole-cell analysis, characterization of complex biological samples, and electron spray ionization.

 Biochemistry

Soft Lithography

Many BioMEM devices, such as microfluidic channels, are fabricated using the soft lithography technique. Here, a microscale pattern is replicated by curing an elastomeric polymer over the 3D structure. These polymeric structures are then used to create a wide range of devices, ranging from microfluidic channels for biosensing applications to microscale bioreactors for the visualization of micro-colonies.

This video introduces photolithography and demonstrates the technique in the laboratory. Then, some applications of the technique and how the structures are used in the bioengineering field are examined.

 Bioengineering

Multi-Step Reactions

Chemical reactions often occur in a stepwise fashion involving two or more distinct reactions taking place in a sequence. A balanced equation indicates the reacting species and the product species, but it reveals no details about how the reaction occurs at the molecular level. The reaction mechanism (or reaction path) provides details regarding the precise, step-by-step process by which a reaction occurs. Each of the steps in a reaction mechanism is called an elementary reaction. These elementary reactions occur in sequence, as represented in the step equations, and they sum to yield the balanced chemical equation describing the overall reaction. In a multistep reaction mechanism, one of the elementary steps progresses slower than the others — sometimes significantly slower. This slowest step is called the rate-limiting step (or rate-determining step). A reaction cannot proceed faster than its slowest step, and hence, the rate-determining step limits the overall reaction rate.

Unlike balanced equations representing an overall reaction, the equations for elementary reactions are explicit representations of the chemical change. An elementary reaction equation depicts the actual reactant(s) undergoing bond-breaking/making and the product(s) formed. Rate laws may be derived directly from the balanced chemical equations for elementary reactions. However, this is not the case for most chemical reactions, where balanced equations often represent the overall change in the chemical system resulting from multistep reaction mechanisms. Therefore, the rate law must be determined from experimental data, and the reaction mechanism must be subsequently deduced from the rate law.

For instance, consider the reaction of NO₂ and CO:

The experimental rate law for this reaction at temperatures above 225 °C is:

According to the rate law, the reaction is first-order with respect to NO₂ and first-order with respect to CO. However, at temperatures below 225 °C, the reaction is described by a different rate law that is second-order with respect to NO₂:

This rate law is not consistent with the single-step mechanism, but it is consistent with the following two-step mechanism:

The rate-determining (slower) step gives a rate law showing second-order dependence on the NO₂ concentration, and the sum of the two elementary equations gives the net overall reaction.

In general, when the rate-determining (slower) step is the first step in the reaction mechanism, the rate law for the overall reaction is the same as the rate law for this step. However, when the rate-determining step is preceded by an elementary step involving a rapidly reversible reaction, the rate law for the overall reaction may be more difficult to derive, often due to the presence of reaction intermediates.

In such instances, the concept that a reversible reaction is at equilibrium when the rates of the forward and reverse processes are equal can be utilized.

 Core: Organic Chemistry

Ethers from Alkenes: Alcohol Addition and Alkoxymercuration-Demercuration

Overview

Ethers can also be prepared from alkenes through acid-catalyzed addition of alcohols and alkoxymercuration–demercuration.

Preparation of Ethers by Acid-Catalyzed Addition of Alcohol to Alkenes

The acid-catalyzed addition of alcohol to an alkene involves treating the alkene with an excess of alcohol in the presence of an acid catalyst to form an ether under suitable conditions. The hydrogen will add to the less substituted carbon so that the nucleophile can attack the more substituted carbon across an alkene forming an ether.

Preparation of Ethers by Alkoxymercuration–Demercuration

Alkoxymercuration–demercuration is a reaction in which an alkene and alcohol react in the presence of a mercuric acetate reagent followed by demercuration or reduction with sodium borohydride to yield an ether.

The alkoxymercuration–demercuration mechanism follows Markovnikov's regioselectivity with the alkoxy group attached to the most substituted carbon and the H attached to the least substituted carbon. A variety of alcohols and alkenes can be used in the reaction. Ditertiary ethers cannot be prepared by this method due to steric hindrance.

 Core: Organic Chemistry

Regioselectivity and Stereochemistry of Acid-Catalyzed Hydration

The rate of acid-catalyzed hydration of alkenes depends on the alkene's structure, as the presence of alkyl substituents at the double bond can significantly influence the rate.

The reaction proceeds with the slow protonation of an alkene by a hydronium ion to form a carbocation, which is the rate-determining step.

The reaction involving a tertiary carbocation intermediate is faster than a reaction proceeding through a secondary or primary carbocation. This can be justified by comparing their relative stabilities and the delocalization of the positive charge. Tertiary carbocations are the most stable and thus formed faster.

Regiochemical Outcome

The formation of a stable carbocation intermediate determines the regiochemical outcome as it directs the nucleophilic addition of water to the more substituted carbon following Markovnikov's orientation.

Stereochemical Outcome of Achiral Alkenes

For an achiral alkene such as 1-butene, protonation results in a secondary carbocation.

The trivalent carbon is sp²-hybridized with a plane of symmetry. It can react with water either from the top or the bottom face with equal probability.

The reaction from the top face leads to (S)-2-butanol, while the reaction from the bottom face leads to (R)-2-butanol. Thus, the formation of a new chiral center leads to a racemic mixture of enantiomeric products.

Stereochemical Outcome of a Chiral Alkene

The protonation of a chiral alkene forms a chiral carbocation with no plane of symmetry. The carbocation does not react equally from the top and bottom faces because one of the faces is more accessible than the other due to different steric setups, leading to a mixture of R and S products. Thus, two diastereomeric products are produced in unequal amounts, and the mixture is optically active.

Rearrangement of a Carbocation

In some cases, the carbocation formed in the first step can rearrange to a more stable carbocation. For example, the protonation of 3-methyl-1-butene forms a 2° carbocation intermediate, which rearranges to a more stable 3° carbocation via a 1,2-hydride shift.

 Core: Organic Chemistry

Structure and Physical Properties of Alkynes

Introduction:

In nature, compounds containing both carbon and hydrogen are known as "hydrocarbons". Aliphatic hydrocarbons are compounds whose molecules contain saturated single bonds (i.e., alkanes) or unsaturated double or triple bonds. Alkenes contain carbon–carbon double bonds and have a structural formula C_nH_2n. Unsaturated hydrocarbons containing carbon–carbon triple bonds are called "alkynes" and are structurally represented by the formula C_nH_2n-2.

The simplest alkyne is ethyne, or acetylene, a colorless gas, which undergoes combustion at high temperatures and is used as a fuel for welding. Alkynes are found in a variety of substances of both natural and synthetic origin. For example, natural alkynes can be found in the poison obtained from South American tree frogs. On the other hand, synthetic alkyne-containing compounds play an important role in drugs like oral contraceptives such as ethynylestradiol. Other alkyne-based drugs, such as selegiline, are used in combination with synthetic dopamine or L-dopa to treat Parkinson's disease. 

Hybridization of Alkynes:

The triple bond in alkynes occurs due to the overlap of the carbon–carbon (C–C) bond's sp orbitals forming a sigma (σ) bond, and the lateral overlapping of the 2p_y and 2p_z orbitals forming the two pi (π) bonds, respectively. The overlap of the carbon sp orbital with the 1s orbital of the hydrogen atom results in the carbon–hydrogen (C–H) sp–1s sigma bond of the alkyne. Alkynes show an sp hybridization with a 50% s character. As electrons in the s orbital show lower energies than the p orbitals, an increase in the atom's s character increases its electronegativity.

Molecular Geometry: Bond length, Bond angles, and Bond Strengths

Owing to the sp hybridization involving the lateral overlap of the orbitals, alkynes have a linear geometry with the bond angle being 180°. The C–C triple bond's length in acetylene is measured to be 121 pm or 1.21 Å, which is lesser than that of alkenes (134 pm or 1.34 Å) and alkanes (153 pm or 1.53 Å) due to the increased number of bonds holding the two carbons together.

The C–H bond in alkynes (1.06 Å in acetylene) is also shorter compared to that in alkenes and alkanes due to the increased s-character of the sp hybridized carbon orbital forming the sp-1s σ bond with the hydrogen. In addition to having lower energies, electrons in the s-orbital are closer to the atomic nucleus and are bound more tightly than those in the p orbitals. As a result, a greater s-character increases the strength of the bond. Hence, C-C triple bonds and C-H bonds in alkynes are shorter and stronger than those of alkenes and alkanes, mainly due to their increased s character. The bond dissociation energy of alkynes is 966 kJ or 231 kcal/mol, which is higher than that of alkanes and alkenes due to the presence of these shorter and stronger bonds.

Physical Properties of Alkynes:

Alkynes show similar physical properties as their parent alkane or alkene. They are nonpolar compounds having a lower density than water and are insoluble in water and polar solvents. However, they show good solubility in nonpolar organic solvents. The lower-molecular weight alkynes such as ethyne and propyne exist as gases at room temperature, while higher molecular weight alkynes such as 1-octyne and 1-decyne are liquids.

 Core: Organic Chemistry

Microtubules in Cell Motility

Microtubules are thick hollow cylindrical proteins that help form the cytoskeleton. Microtubules have varied roles in the cell. These filaments help form cellular appendages like cilia and flagella, which are responsible for locomotion. The cilia arise from basal bodies, separated from the main body by a membrane-like structure forming the transition zone. This zone is the gate for the entry of lipids and proteins, creating a unique composition of lipids and proteins in the ciliary membrane and body. The central strand of the flagellum is called the axoneme and has a 9+2 arrangement of microtubules.

Microtubules help cells move using mechanisms like modulating actin polymerization by regulating Rho GTPase signaling pathways. During actin polymerization, with the help of +TIPs complex, microtubules sequester signaling molecules and actin assembly factors. These molecules are only released upon the disassembly of microtubules, thus regulating lamellipodia and filopodia formation.

Microtubules can also regulate directional migrations when they act as tracks for motor proteins to transport intracellular cargo and signaling molecules to the leading edge of the migrating cells. The cortical microtubules associated with the focal adhesion junctions help recycle focal adhesion proteins from the cell membrane during cell motility. They also facilitate the cross-talk between different cytoskeletal components. These microtubules undergo repeated cycles of rescue and catastrophe near the cell boundaries to regulate cell motility.

 Core: Cell Biology

Acid-Catalyzed Ring-Opening of Epoxides

Epoxides that are three-membered ring systems are more reactive than other cyclic and acyclic ethers. The high reactivity of epoxides originates from the strain present in the ring. This ring strain acts as a driving force for epoxides to undergo ring-opening reactions either with halogen acids or weak nucleophiles in the presence of mild acid. The acid catalyst converts the epoxide oxygen, a poor leaving group, into an oxonium ion, a better leaving group, making the reaction feasible. The reaction follows the S_N2 mechanism, and the protonated oxygen, unlike other leaving groups, does not detach from the molecule. The regiochemistry of the products formed is governed by either steric or electronic effects. In the case of an asymmetrical epoxide bearing a primary and a secondary carbon, the steric effect dominates and favors the nucleophilic attack at a less-hindered carbon. However, in epoxides where one of the carbons is tertiary, the electronic effect comes into play and favors the attack at a more-hindered carbon. The stereochemistry of the products is similar to an S_N2 reaction, where the nucleophile attacks anti to the leaving group. Notably, the anti-attack at a chiral carbon causes an inversion of configuration.

 Core: Organic Chemistry

Esters to β-Ketoesters: Claisen Condensation Overview

Regular Claisen condensation is a base-promoted reaction involving identical esters with two α hydrogens, condensing to produce β-ketoesters. It is a nucleophilic acyl substitution reaction wherein one of the ester molecules, upon deprotonation by the base, forms a nucleophilic enolate ion, while the other molecule serves as an electrophile.

The condensation reaction requires specific nonaqueous bases, such as alkoxide, which must be similar to the alkoxy group of the ester molecule. The use of different alkoxide ions often leads to the transesterification product. Also, the use of hydroxide bases is avoided as they result in irreversible hydrolysis of the esters to carboxylate ions.

 Core: Organic Chemistry

Intrinsically Disordered Proteins

 Core: Cell Biology

Regulation of Angiogenesis and Blood Supply

Rapidly dividing tumors, embryos, and wounded tissues require more oxygen than usual, lowering the oxygen concentration in the blood. At low oxygen or hypoxic conditions, an oxygen-sensitive transcription factor called the hypoxia-inducible factor 1 or HIF1 is activated. HIF1 is a dimeric protein of alpha (ɑ) and beta (β) subunits.  Under optimal oxygen conditions, HIF1β is present in the nucleus while HIF1ɑ remains in the cytosol. HIF1ɑ is hydroxylated by prolyl hydroxylase and factor inhibiting HIF-1. Hydroxylated HIF1 alpha binds von Hippel Lindau (VHL) E3 ubiquitin ligase and undergoes degradation. 

Hypoxia promotes HIF1ɑ accumulation. HIF1ɑ translocate to the nucleus and binds HIF1-beta, forming the HIF1 dimers. HIF dimer associates with CBP/P300 transcriptional regulator and binds HIF1 response elements of target genes, initiating their transcription. Some pro-angiogenic genes regulated by HIF1 include erythropoietin, angiopoietin, and vascular endothelial growth factor (VEGF). VEGF signaling is critical for regulating angiogenesis.

VEGF is a dimeric protein. They bind the transmembrane RTKs called  VEGF receptors (VEGFR). They have five isoforms, of which VEGF-A is most important in regulating angiogenesis and binds VEGFR1 with high affinity. VEGFA binding stimulates endothelial cells to differentiate into tip cells. Tip cells express high levels of delta-like notch ligand four or DLL4. DLL4 of tip cells interacts with the notch receptors of the neighboring cells promoting their differentiation to stalk cells.

Another essential ligand/ RTK signaling includes the angiopoietin/ tie-2 receptor that works closely with the VEGF signaling pathway in the latter stages of angiogenesis.  Angiogepoeitin 1/ tie-2 signaling promotes endothelial cell survival, initiates vascular branching, and helps stabilize newly formed vessels.

A third ephrin-B/ephrin-B RTK signaling pathway is also essential for angiogenesis and helps specify endothelial cells into arterial and venous cell types.

Apart from these ligand/receptor interactions, some molecules help mediate cell-cell interactions and cell-matrix interactions and regulate angiogenesis. For example, matrix metalloproteases allow basement membrane degradation and endothelial tip migration to the target tissue. Alternatively, protease inhibitors prevent matrix degradation and stabilize them once the vessel is formed. VE-Cadherins, N cadherins, and occludin are essential junctional proteins that stabilize the newly formed endothelial lining.

 Core: Cell Biology

Transcription Elongation Factors

 Core: Cell Biology

Alkylation of β-Ketoester Enolates: Acetoacetic Ester Synthesis

Acetoacetic ester synthesis is a method to obtain ketones from alkyl halides and β-keto esters. The reaction occurs in the presence of an alkoxide base that abstracts the acidic proton of the β-keto esters. The step results in an enolate ion which is doubly stabilized. The enolate then reacts with an alkyl halide via the S_N2 process to produce an alkylated ester intermediate with a new C–C bond. The hydrolysis of the intermediate, followed by acidification, results in an alkylated β-keto acid. Under high-temperature conditions, the β-keto acid undergoes decarboxylation to form a ketone. However, if the alkylation is repeated before hydrolysis and decarboxylation steps, a disubstituted ketone is obtained.

 Core: Organic Chemistry

Cooperative Binding of Transcription Regulators

 Core: Cell Biology

Chemistry of Carbohydrates

 Core: Cell Biology

The Replisome

 Core: Cell Biology

Canonical Wnt Signaling Pathway

 Core: Cell Biology

Subcellular Fractionation

The homogenate obtained after cell lysis contains various membrane-bound organelles that can be further separated into pure fractions by subcellular fractionation. These isolates are used to study specific cellular components, analyze localized protein activity, and are even employed in diagnostics. Fractionation is typically achieved using centrifugation methods, the most common being density-gradient and differential centrifugation.

Differential Centrifugation

Differential centrifugation is a relatively simple  method that separates the cellular components based on size and density. Sequential centrifugation with increasing speeds (ranging from 10,000 X g to 150,000 x g) sediments the differently sized components. However, since multiple organelles can be of similar size and density, this method usually produces crude fractions.

Density Gradient Centrifugation:

Highly purified fractions of cellular components can be obtained by separating the homogenate in a density gradient solution. A density gradient is prepared in a centrifuge tube by layering solutions with increasing densities, such as increasingly concentrated sucrose solutions, with the densest layer at the bottom of the tube. Such gradients are used in rate-zonal centrifugation to separate cellular organelles based on their size and shape. Upon centrifugation, the organelles sediment at different rates, based on their sedimentation coefficients, as they move through the different density layers.

Alternatively, a continuous density gradient can also be prepared by mixing solutions of different densities in gradual proportions along the length of the tube. During centrifugation, each component immobilizes at the position that matches their density — their equilibrium position. Hence this method is also known as equilibrium or buoyant sedimentation. This separation of cellular components and molecules is thus based on their density, not their size.

 Core: Cell Biology

Fixation and Sectioning

Two basic types of preparation are used to visualize specimens with a light microscope: wet mounts and fixed specimens.

The simplest type of preparation is the wet mount, in which the specimen is placed in a drop of liquid on the slide. A liquid specimen can be directly deposited on the slide using a dropper. Solid specimens, such as skin scraping, can be placed on the slide before adding a drop of liquid to prepare the wet mount. Sometimes the liquid is simply water, but stains are often added to enhance contrast. Once the liquid has been added to the slide, a coverslip is placed on top, and the specimen is ready for examination under the microscope.

The second method of preparing specimens for light microscopy is fixation. The “fixing” of a sample refers to the process of attaching cells to a slide. Fixation is often achieved by heating (heat fixing) or chemically treating the specimen. In addition to attaching the specimen to the slide, fixation also kills microorganisms in the specimen, stopping their movement and metabolism while preserving the integrity of their cellular components for observation.

To heat-fix a sample, a thin layer of the specimen is spread on the slide (called a smear), and the slide is then briefly heated over a heat source. Chemical fixatives are often preferable to heat for tissue specimens. Chemical agents such as acetic acid, ethanol, methanol, formaldehyde (formalin), and glutaraldehyde can denature proteins, stop biochemical reactions, and stabilize cell structures in tissue samples.

In addition to fixation, staining is almost always applied to color certain features of a specimen before examining it under a light microscope. Stains, or dyes, contain salts made up of a positive ion and a negative ion. Depending on the type of dye, the positive or the negative ion may be the chromophore (the colored ion); and the other uncolored ion is called the counterion. If the chromophore is the positively charged ion, the stain is classified as a basic dye; if the negative ion is the chromophore, the stain is considered an acidic dye.

Dyes are selected for staining based on the chemical properties of the dye and the specimen being observed, which determine how the dye will interact with the specimen. In most cases, it is preferable to use a positive stain, a dye that will be absorbed by the cells or organisms being observed, adding color to objects of interest to make them stand out against the background. However, there are scenarios where it is advantageous to use a negative stain absorbed by the background but not by the cells or organisms in the specimen. Negative staining produces an outline or silhouette of the organisms against a colorful background.

Because cells typically have negatively charged cell walls, the positive chromophores in basic dyes tend to stick to the cell walls, making them positive stains. Thus, basic dyes such as basic fuchsin, crystal violet, malachite green, methylene blue, and safranin typically serve as positive stains. On the other hand, the negatively charged chromophores in acidic dyes are repelled by negatively charged cell walls, making them negative stains. Commonly used acidic dyes include acid fuchsin, eosin, and rose bengal.

Some staining techniques involve the application of only one dye to the sample; others require more than one dye. In simple staining, a single dye is used to emphasize particular structures in the specimen. A simple stain will generally make all of the organisms in a sample appear the same color, even if the sample contains more than one type of organism. In contrast, differential staining distinguishes organisms based on their interactions with multiple stains. In other words, two organisms in a differentially stained sample may appear different colors. Differential staining techniques commonly used in clinical settings include Gram staining, acid-fast staining, endospore staining, flagella staining, and capsule staining.

This text is adapted from Openstax, Microbiology 2e, Section 2.4: Staining microscopic specimens

 Core: Cell Biology

Interphase

The cell cycle occurs over approximately 24 hours (in a typical human cell) and in two distinct stages: interphase, which includes three phases of the cell cycle (G1, S, and G2), and mitosis (M). During interphase, which takes up about 95 percent of the duration of the eukaryotic cell cycle, cells grow and replicate their DNA in preparation for mitosis.

Phases of Interphase

Following each period of mitosis and cytokinesis, eukaryotic cells enter interphase, during which they grow and replicate their DNA in preparation for the next mitotic division.

During the G1 (gap 1) phase, cells grow continuously and prepare for DNA replication. During this phase, cells are metabolically active and copy essential organelles and biochemical molecules, such as proteins.

In the subsequent S (synthesis) phase of interphase, cells duplicate their nuclear DNA, which remains packaged in semi-condensed chromatin. During the S phase, cells also duplicate the centrosome, a microtubule-organizing structure that forms the mitotic spindle apparatus. The mitotic spindle separates chromosomes during mitosis.

In the G2 (gap 2) phase, which follows DNA synthesis, cells continue to grow and synthesize proteins and organelles to prepare for mitosis.

In human cells, the G1 phase spans approximately 11 hours, the S phase takes about 8 hours, and the G2 phase lasts about 4 hours. During G1, cells are diploid (2n, a pair of each chromosome). Following replication in S phase, cells increase their DNA content to 4n. Cells remain 4n until cytokinesis, at which point their DNA content is reduced to 2n.

 Core: Cell Biology

Spindle Assembly

 Core: Cell Biology

Nondisjunction

Nondisjunction is the failure of homologous chromosomes or sister chromatids to separate correctly and move to the opposite poles of the cells. This produces daughter cells with abnormal chromosome numbers.  Nondisjunction is common during anaphase I or anaphase II of meiosis.  Mutations in synaptonemal complex proteins that attach homologous chromosomes increase the chances of nondisjunction in anaphase I of meiosis I. In contrast, mutations in topoisomerases and condensins that hold sister chromatids together promote nondisjunction during anaphase II of meiosis II.

Nondisjunction of chromosomes in germ cells results in gametes possessing additional or fewer chromosomes than normal. Nondisjunction is more frequent during oogenesis than during spermatogenesis. When a gamete with abnormal chromosomes fertilizes a gamete with a normal chromosome number, the resulting zygote has an abnormal number of chromosomes or aneuploidy. Such aneuploid zygotes can have fewer chromosomes than normal, leading to monosomy (45; 2n-1), or more chromosomes than normal, leading to trisomy (47; 2n+1). Some females lack one of the X chromosomes, a typical case of monosomy (45, X), and develop Turner Syndrome. In other instances, individuals who develop Down Syndrome have trisomy with three copies of chromosome 21.

 Core: Cell Biology

Mouse Models of Cancer Study

 Core: Cell Biology

Tissue Renewal without Stem Cells

After cellular or tissue damage, the resident stem cells present in the human body can locally repair and regenerate the damaged tissue or organ. However, even though some tissues do not have stem cells, they can repair and regenerate with the help of pre-existing cells. For example, beta cells of the pancreas and hepatocytes of the liver can divide to renew and regenerate the tissue. Here, both cell division and cell death are well regulated by homeostasis.

However, failure of such a system may result in life threatening diseases. Dysfunction in insulin-secreting cells, as well as the target cells’ responsiveness to insulin, can lead to a condition called diabetes mellitus. Interestingly, a recent study also found that as a backup, both the liver and pancreas have a few stem cells which are activated under extreme conditions to produce differentiated cell types. In this case, both the liver and pancreas revert back to their normal mechanisms of repair and renewal.

 Core: Cell Biology

Types Of Column Chromatography

The stability and compatibility of column material with samples are crucial for efficient purification in chromatographic techniques. Various operating parameters such as pH, temperature, or solvent affect the packing of the column material, thereby determining the purification efficiency. The choice of column material also plays an essential role in deciding the operating parameters and can be modified based on the proteins that need to be purified.

Gel Filtration Chromatography

When the protein's chemical nature is unknown, gel filtration chromatography is used for purification based on size. Matrices such as agarose, polyacrylamide, dextran derivatives, or silica beads are used for low-pressure systems, whereas polymeric resins are used for higher flow rates. The varying degree of cross-linking of these polymeric materials determines the pore size of the matrix, which decides the size range of proteins that can be separated.

Ion Exchange Chromatography

Ion-exchange chromatography can purify proteins based on their net charge at a particular pH determined by their isoelectric point or pI. The net charge on matrix resin is used to purify oppositely charged proteins by replacing the cations or anions from the matrix resin with the charged proteins. The resins are categorized based on the type of ion that is exchanged — positively charged proteins replace cations from cation-exchange resins, and vice versa. Strong ion exchangers with stability over a wide range of buffer pH include quaternary ammonium, sulfonate, and sulfopropyl resins. In contrast, weak ion exchangers that are effective only in a narrow pH range include diethyl aminoethyl (DEAE) or carboxymethyl (CM) resins.

Affinity Chromatography

Affinity chromatography involves immobilizing protein-specific antibodies or enzyme-specific substrates to the column material, usually agarose beads. For example, a column with the immobilized antibody of choice is prepared to separate specific antigens. When the mixed antigen sample is passed through the  column, the target antigens bind to the immobilized antibodies and separate from the mixture. To separate recombinant proteins tagged with enzymes such as beta-galactosidase, the corresponding substrate Isopropyl-β-D-thiogalactopyranoside (IPTG) is immobilized on the column to purify the enzyme.

High-pressure liquid chromatography (HPLC) and gas chromatography (GC) are the most commonly used column chromatography techniques. In HPLC, the solvent flows at high pressure through a tightly packed column that improves efficiency. Gas chromatography analyzes volatile compounds by converting them to their gaseous forms upon heating. The volatilized sample is pushed through the column by inert gasses such as nitrogen and helium.

 Core: Cell Biology

Protein Organization

 Core: Anatomy and Physiology

Microtubules In Cell Motility

 Core: Anatomy and Physiology

Transcription

 Core: Anatomy and Physiology

Statistical Analysis: Overview

When we take repeated measurements on the same or replicated samples, we will observe inconsistencies in the magnitude. These inconsistencies are called errors. To categorize and characterize these results and their errors, the researcher can use statistical analysis to determine the quality of the measurements and/or suitability of the methods.

One of the most commonly used statistical quantifiers is the mean, which is the ratio between the sum of the numerical values of all results and the total number of results. Another commonly used quantifier is the median, which is the middle value amongst all the results arranged in the increasing or decreasing order of their numerical values. In the presence of extremely large or small values in the data set, the median can be a more suitable measure for the data set overall. Both the mean and median can be useful measures of the central value of a set of measurements. In addition to the central value, the range is another way to characterize the distribution of a set of values. It is the numerical difference between the highest and lowest values in a set of results.

Precision and accuracy are two important measures of observed errors in the data set. Precision is the measure of closeness between the replicate measurements. In a precise data set, the values of different measurements cluster close together. The values are farther apart or more scattered in an imprecise data set. Often, the range of an imprecise data set will be high. On the other hand, accuracy is a measure of closeness between the measurements and the true or expected value. This means that the magnitude of errors in a more accurate data set is smaller than that of a less accurate data set.

 Core: Analytical Chemistry

Difference from Background: Limit of Detection

The limit of detection (LOD) is the smallest amount of analyte that can be distinguished from the background noise. The LOD value corresponds to the concentration at which the analyte signal is three times larger than the standard deviation of the blank signal. Below this value, the analyte signal cannot be differentiated from the background noise. It is calculated by dividing the calibration slope by 3 times the standard deviation of the blank signals.

The LOD indicates the presence or absence of an analyte but is usually too low to be reliably quantified. For quantification, we need another value called the limit of quantification, which is defined as the lowest quantity of analyte that the instrument can quantify. Its value corresponds to the concentration at which the signal is ten times larger than the standard deviation of the blank signal.

 Core: Analytical Chemistry

Complexation Equilibria: The Chelate Effect

In complexation reactions, metal atoms or cations interact with ligands to form donor-acceptor adducts called metal complexes. Ligands that bind through one donor site are monodentate, ligands with two donor sites are bidentate, and those with more than two donor sites are polydentate ligands. For example, ethylene diamine is a bidentate ligand that binds through two nitrogen donor atoms, forming a five-membered ring. EDTA is a polydentate ligand that binds through four oxygen and two nitrogen atoms.

Bidentate and polydentate ligands are also called chelating agents, and the corresponding complexes are called chelates. Chelate is a Greek word that means "claw-like." Metal complexes formed by the chelating agents are more stable than those formed by their monodentate counterparts, as the reaction for their formation is entropically favored. This property is known as the chelate effect or the entropy effect.

 Core: Analytical Chemistry

Complexometric EDTA Titration Curves

EDTA titration curves determine the free metal ion concentration. The titration curve represents the change in concentration of free metal ions (p function) as a function of the volume of EDTA added. This curve consists of three regions: before, at, and after equivalence points. Excess free metal ions are present before the equivalence point. Equal concentrations of metal ions and EDTA are present at the equivalence point. After the equivalence point, excess EDTA exists. This means slight dissociation can be observed at and after the equivalence point.

The complex's conditional formation constant (K_f′) calculates the free metal ion concentration at and after the equivalence point, and the shape of the titration curve is affected by K_f′ of the complex. For example, the Ca–EDTA complex has a larger K_f′ than the Sr–EDTA complex. As a result, the Ca–EDTA titration curve has a larger break at the equivalence point.

The K_f′ of the complex depends on the pH of the solution. For instance, Ca–EDTA exhibits various shapes at different pH. At higher pH, Ca–EDTA has a larger K_f′, and complex formation is more favorable. The curve has a large break at the equivalence point. At lower pH, the K_f′ of the complex is small, indicating less favorable complex formation. As a result, the curve has a small break at the equivalence point.

 Core: Analytical Chemistry

Mass Spectrometry: Complex Analysis

Mass spectrometry is an important technique for the identification of pure compounds. However, it has some limitations for the analysis of complex mixtures, often due to excessive fragmentation making the spectrum too complicated to decipher. Mass spectrometry can be combined with suitable separation methods in sequence, forming hyphenated methods, which are useful in the analysis of complex mixtures.

GC–MS is a powerful hyphenated method commonly used in forensics and environmental laboratories for precise analysis of mixtures. Gas chromatography uses narrow capillary columns to separate components of a thermally stable volatile mixture and passes them to the mass spectrometer for analysis.

LC–MS is another hyphenated method that couples liquid chromatography with a mass spectrometer to analyze nonvolatile mixtures. To make liquid chromatography compatible with a mass spectrometer, suitable pressure-maintaining ionization interfaces or atmospheric-pressure ionization techniques like electrospray ionization, which is applicable for polar and ionic compounds, or atmospheric-pressure chemical ionization, which applies to less polar molecules, are used.

Another hybrid method, called tandem mass spectroscopy, uses multiple mass analyzers in sequence. Compared to other hyphenated methods, tandem spectroscopy is faster, more sensitive, and more selective due to smaller chemical noise. Tandem spectroscopy can be further combined with separation techniques to form GC–MS/MS or LC–MS/MS for more complex mixture analysis.

Capillary electrophoresis–mass spectrometry is a very sensitive technique commonly used to analyze large biomolecules like DNA, proteins, and polypeptides. This method feeds quadrupole mass analyzers with capillary effluents after passing through an electrospray ionization interface.

 Core: Analytical Chemistry

Ion Exchange

Ion exchange chromatography separates charged molecules from a solution by reversibly exchanging them with mobile, or 'active', ions associated with the oppositely charged stationary phase. This method can be used to separate ions, soften and deionize water, and purify solutions. The polymers comprising the ion-exchange column are high-molecular-weight and chemically stable polymers, crosslinked to be porous and essentially insoluble. They are also functionalized with either acidic or basic groups. When functionalized with acidic groups, the columns or resin are known as cation exchangers and contain negatively charged polymers with positive counter ions. These allow cations to be exchanged from the mobile phase. When functionalized with basic groups, the columns or resin are known as anion exchangers, which contain positively charged polymers with negative counter ions and exchange anions from the solution.

During ion exchange, the counterions associated with the solid-phase polymers enter the solution while ions from the solution interact with the polymer in their place. Note that the ability to exchange ions increases as the extent of cross-linking increases and the number of ion-exchange groups increases in the polymer. This exchange is reversible and continues until equilibrium is established.

Acid cation exchangers can be functionalized with either strong acids, such as sulfonic acid (−SO₃H), seen in polystyrene sulfonic acid, or weak acids, such as carboxylic acid (−COOH), seen in poly(methyl methylacrylic) acid. Similarly, anion exchangers are classified as either strong or weak. Strong anion exchangers often contain quaternary ammonium groups (−CH₂N(CH₃)₃⁺), such as those in polystyrene quaternary ammonium chloride, and weak anion exchangers are often functionalized with substituted amines (−NH₃⁺), such as those in polystyrene tertiary amine hydroxide.

 Core: Analytical Chemistry

Specialized Characteristics of Cardiac Muscles

The primary role of cardiac muscles is to propel blood throughout the cardiovascular system. The cardiac muscle cells, or cardiomyocytes, exhibit specialized characteristics that allow them to perform this function.

Cardiac muscle cells are smaller than skeletal muscles, averaging 10–20 mm in diameter and 50–100 mm in length. However, they have large energy demands for continuous contraction and relaxation. This energy is almost exclusively derived from aerobic metabolism of energy reserves in the form of glycogen and lipid inclusions. Additionally, the sarcoplasm of these cells contains large numbers of mitochondria and abundant reserves of myoglobin, which store the oxygen needed to break down energy reserves during peak activity.

In contrast to skeletal muscles, cardiomyocytes can contract independently without relying solely on nerve stimulation. The pacemaker cells, a distinct type of cardiomyocyte, can generate action potentials and initiate contractions in cardiac muscles approximately 75 times per minute. This depolarization wave swiftly spreads through the muscle cells through gap junctions, prompting all the cells to contract and relax together as one unit, known as the functional syncytium.

Cardiomyocytes also differ from skeletal muscles in that the depolarization wave triggers the gradual release of calcium ions into the sarcoplasm of the contractile cells from both the sarcoplasmic reticulum and the interstitial fluid. This results in the contraction cycle of cardiac muscles that lasts for about 200 ms, which is longer than the skeletal muscle contractions that last only 40 to 120 ms. The extended cycle also increases the absolute refractory period of cardiac muscles, which coincides with the repolarization phase of the action potential. This feature enables the muscle to fully relax before another action potential is generated, preventing sustained or tetanic contractions.

 Core: Anatomy and Physiology

Muscles of the Shoulder

The muscles surrounding the shoulder girdle, including the clavicle and scapula, primarily stabilize the scapula. This stable base allows other muscles to move the humerus effectively. Scapular movements often mirror those of the humerus and extend its range of motion. For instance, raising the arm above the head would not be feasible without simultaneous upward rotation of the scapula.

Anterior Thoracic Muscles

The anterior thoracic muscles include the serratus anterior, subclavius, and pectoralis minor. The serratus anterior, often known as the boxer’s muscle, starts from the first to eighth or ninth ribs. It inserts along the superior and inferior angle of the scapula as well as its medial border. This muscle is crucial for abducting and rotating the scapula upward. When the scapula is fixed, it helps elevate the ribs, assisting in punching movements.

The pectoralis major joins the pectoral girdle to the thorax. It originates from the anterior surface of the superior margins of the second or third to fourth or fifth ribs and inserts on the coracoid process of the scapula. It tilts the scapula anteriorly and pulls it inferiorly. It also elevates the thorax, aiding in respiration.

The subclavius muscle originates on the first rib and inserts into the lower surface of the lateral clavicle. Its primary function is to stabilize the clavicle during shoulder and arm movements. It also depresses the clavicle and moves it anteriorly.

Posterior Thoracic Muscles

The posterior thoracic muscles include the trapezius, levator scapulae, and the rhomboid minor and major muscles. The trapezius muscle, one of the broadest and most distinct muscles, has its roots in the bony structure of the cervical and thoracic regions. The muscle originates from the external protuberance of the occipital bone, ligamentum nuchae, and the spinous process of the seventh cervical and all thoracic vertebra. It inserts at the clavicle along its lateral third in addition to the acromion and the spine of the scapula. It is categorized into three parts — the upper, middle, and lower fibers, each performing unique actions. The upper fibers elevate the scapula and extend the neck, the middle fibers adduct the scapula, and the lower fibers depress the scapula.

The levator scapulae originate from the transverse process of the cervical vertebrae, C1-C4, and insert into the scapulae at the superior part of its medial border. It elevates the scapula and causes its downward rotation. It also flexes the neck laterally.

Originating from the nuchal ligament and spinous processes of C7 and T1, the rhomboid minor inserts into the vertebral border of the scapula, superior to the spine. On the other hand, the rhomboid major originates from the spinous processes of the T2 to the vertebral border of the scapula, inferior to the spine. These muscles help retract the scapula and rotate it medially.

 Core: Anatomy and Physiology

Action Potential

 Core: Anatomy and Physiology

Association Areas of the Cortex

Association areas are regions of the cerebral cortex that do not have a specific sensory or motor function. Instead, they integrate and interpret information from various sources to enable higher cognitive processes such as memory, learning, and decision-making. Some key association areas include the following:

Prefrontal Association Area: This area is located in the frontal lobe and is involved in planning, decision-making, and moderating social behavior. It connects with primary motor areas, sensory areas, and the limbic system, which processes emotions.

Parietotemporal Association Area: Found at the junction of the parietal and temporal lobes, this area is crucial for understanding language and spatial awareness. It receives input from the primary auditory cortex, somatosensory cortex, and other sensory areas.

Limbic Association Area: This area is essential for memory formation and emotional processing. Located in the medial part of the temporal lobe, it connects with the hippocampus, amygdala, and other parts of the limbic system.

Broca's Area: Broca's area, located in the posterior part of the left inferior frontal gyrus, plays a crucial role in speech production. It connects with the primary motor cortex, responsible for controlling speech muscles, and Wernicke's area through a bundle of nerve fibers called the arcuate fasciculus.

Damage to Broca's area can result in Broca's aphasia, a condition characterized by non-fluent speech, difficulty forming complete sentences, and trouble finding the right words. However, comprehension usually remains relatively intact.

Wernicke's Area: Wernicke's area, situated in the superior temporal gyrus of the left temporal lobe, is responsible for language comprehension. It receives input from primary auditory and visual cortices, allowing it to process spoken and written language. Wernicke's area connects with Broca's through the arcuate fasciculus, enabling communication between speech production and comprehension.

Damage to Wernicke's area can lead to Wernicke's aphasia, a condition where individuals have fluent but nonsensical speech with poor comprehension. They may also need help understanding written language.

Association Areas of Special Senses: The association areas of special senses are responsible for processing and interpreting information from our primary sensory cortices. These include:

Visual Association Area: The visual association area, or the visual association cortex, is located in the brain's occipital lobe. This region plays a crucial role in processing and interpreting visual information, allowing us to make sense of what we see. It receives input from the primary visual cortex (V1), which detects basic features such as edges, colors, and motion. The visual association area then integrates this information to create a more comprehensive understanding of the visual scene, enabling us to recognize objects, faces, and other complex elements. Damage to the visual association area can result in visual agnosia, where individuals can see objects but cannot recognize or identify them.

Auditory Association Area: Found in the temporal lobe, this area interprets sounds and speech from the primary auditory cortex. Damage to the auditory association area can lead to auditory agnosia, where individuals can hear sounds but cannot understand or interpret them.

Somatosensory Association Area: This area is situated in the parietal lobe. It integrates and interprets tactile information from the primary somatosensory cortex. Damage to the somatosensory association area can result in difficulties recognizing objects by touch, astereognosis, or identifying body parts' position in space, and agnosia for body schema.

Orbitofrontal Cortex

The orbitofrontal cortex (OFC) is a region in the brain's frontal lobes, situated just above the orbits of the eyes. It involves various cognitive functions, including decision-making, emotional regulation, and reward processing. While not directly responsible for visual processing, the OFC is interconnected with other brain regions that handle visual information, such as the visual association cortex and the fusiform face area.

The OFC contributes to our ability to make decisions based on visual stimuli, such as choosing between different food items or evaluating the attractiveness of a potential mate. Additionally, it helps process the emotional content of visual information, allowing us to respond appropriately to facial expressions and other emotionally charged visual cues.

Facial Recognition Area

The facial recognition area, commonly known as the fusiform face area (FFA), is located in the fusiform gyrus of the temporal lobe. This region is dedicated explicitly to recognizing faces and distinguishing them from other objects. The FFA allows us to identify familiar faces quickly and accurately interpret facial expressions and emotions. The FFA works with other brain regions, such as the occipital face area (OFA) and the superior temporal sulcus (STS), to create a robust facial recognition system. While the FFA focuses on processing the invariant aspects of faces (e.g., identity), the OFA and STS deal with more changeable aspects, such as gaze direction and expression.

Frontal Eye Field Area

The frontal eye field (FEF) is a region within the frontal cortex that explicitly controls eye movements and visual attention. The FEF guides our eyes toward relevant visual stimuli and away from irrelevant or distracting information. It coordinates saccadic eye movements, which are rapid, voluntary shifts in gaze that allow us to explore our visual environment efficiently. Additionally, the FEF contributes to higher-order cognitive processes, such as working memory and attention. The FEF enables us to prioritize and process relevant information more effectively by directing our visual attention to specific locations or objects.

 Core: Anatomy and Physiology

Spinal Nerves: Anatomy

Spinal nerves are pivotal conduits in the nervous system, bridging the central nervous system (CNS) with the peripheral nervous system (PNS). These nerves enable a complex communication network between the brain, spinal cord, and the rest of the body, facilitating sensory input, motor output, and autonomic functions.

There are 31 bilateral pairs of spinal nerves, each emerging from the spinal cord through the intervertebral foramina—openings between adjacent vertebrae. These nerves are symmetrically organized and are categorized based on the region of the spine from which they emerge —  8 cervical (C1-C8), 12 thoracic (T1-T12), 5 lumbar (L1-L5), 5 sacral (S1-S5), and 1 coccygeal (Co1).

• • Cervical nerves (C1-C8): These eight nerve pairs transmit signals to and from the head, neck, and upper extremities. The cervical nerves are:
  • C1: Suboccipital nerve
  • C2: Greater occipital nerve
  • C3: Lesser occipital nerve
  • C4: Supraclavicular nerve
  • C5: Dorsal scapular nerve
  • C6: Superior plexus root
  • C7: Middle plexus root
  • C8: Inferior plexus root
• • Thoracic nerves (T1-T12): The 12 pairs of thoracic nerves correspond to the 12 thoracic vertebrae and control the sensation and motor function of the chest, upper back, and abdominal muscles. The T1 to T11 nerves are the intercostal nerves, while T12 is the subcostal nerve.
• • Lumbar nerves (L1-L5): Five pairs of lumbar nerves control the lower back, hips, and parts of the legs. The five lumbar nerves are:
  • L1: Iliohypogastric nerve
  • L2: Ilioinguinal nerve
  • L3: Genitofemoral nerve
  • L4: Lateral femoral cutaneous nerve
  • L5: Lumbar plexus root
• • Sacral nerves (S1-S5): The sacral nerves consist of five pairs, and they are responsible for the function of the lower extremities, pelvic organs, and some muscles in the hips and legs. The sacral nerves are:
  • S1: Superior gluteal nerve
  • S2: Inferior gluteal nerve
  • S3: Sciatic nerve
  • S4: Pudendal nerve
  • S5: Sacral plexus root
• • Coccygeal nerve (Co1): The single coccygeal nerve pair, called anococcygeal nerve, provides sensory innervation to the skin over the coccyx and surrounding area, as well as to some of the muscles in the pelvic floor. Its motor functions are minimal and not as well defined as those of the other spinal nerves.

Structure of Spinal Nerve

Each spinal nerve is formed by the union of two distinct roots — the dorsal (posterior) root and the ventral (anterior) root.

• • Dorsal Root: The dorsal root carries sensory information from the peripheral receptors to the spinal cord. It contains sensory neurons whose cell bodies are located in the dorsal root ganglion, a bulge in the root just before it merges with the ventral root.
• • Ventral Root: In contrast, the ventral root conveys motor information from the spinal cord to the muscles and glands. This root consists of axons from motor neurons whose cell bodies are housed within the gray matter of the spinal cord.

These two roots combine to form a mixed spinal nerve carrying both sensory and motor fibers. Shortly after emerging from the spinal column, each spinal nerve splits into two primary branches — the dorsal ramus and the ventral ramus.

• • Dorsal Ramus: This branch innervates the muscles and skin of the posterior trunk, providing sensory and motor functions to these areas.
• • Ventral Ramus: The larger of the two branches, the ventral ramus, supplies the anterolateral parts of the trunk and the limbs. It carries fibers that form the nerve plexuses — networks of interconnecting nerves that give rise to peripheral nerves innervating the limbs.

Additionally, each spinal nerve has a small meningeal branch that reenters the vertebral column to innervate the vertebrae, ligaments, blood vessels, and meninges of the spinal cord. Spinal nerves also feature communicating rami that connect with the sympathetic trunk of the autonomic nervous system (ANS). These rami consist of:

• • Gray Rami Communicantes: These carry postganglionic sympathetic fibers back to the spinal nerves for distribution to the target organs and tissues.
• • White Rami Communicantes: These are present only in the thoracic and upper lumbar regions. They carry preganglionic sympathetic fibers from the spinal nerve to the sympathetic trunk.

 Core: Anatomy and Physiology

Sleep-Wake Cycles

Sleep is an essential physiological process vital to maintaining overall well-being. The reticular activating system (RAS), a network of neurons in the brainstem, regulates wakefulness and sleep. While it may seem passive, sleep consists of distinct cycles, each with its unique characteristics and functions. Two key sleep phases are non-rapid eye movement (NREM) and  rapid eye movement (REM).

NREM Sleep

NREM sleep comprises four progressive stages that seamlessly merge:

1. Stage 1  is the transitional phase bridging wakefulness and sleep. This stage usually lasts between 1 and 7 minutes, during which the person feels relaxed and has closed eyes. Individuals awakened during this stage often express a sense of not having been asleep.
2. Stage 2, characterized as light sleep, marks the onset of the sleeping state. It is relatively easy to rouse someone during this stage. Fragmented dreams may occur, and the eyes may exhibit slow horizontal movements.
3. Stage 3 denotes a period of moderately deep sleep. The individual experiences decreased body temperature and blood pressure, making it slightly more challenging to awaken them. Typically, Stage 3 emerges about 20 minutes after falling asleep.
4. Stage 4 signifies the deepest level of sleep. Although brain metabolism considerably decreases and there is a slight drop in body temperature, most reflexes remain intact, and muscle tone only undergoes minimal reduction. Waking an individual during this stage becomes exceedingly difficult.

REM Sleep

REM sleep is an intriguing phase characterized by rapid eye movements and intense brain activity including vivid dreams. While the body appears still and relaxed, internally, the brain is highly active. The muscles are temporarily paralyzed to prevent acting out dreams.

Sleep Disorders

Sleep-related health issues are a serious concern and can manifest in various forms, including insomnia, sleep apnea, and narcolepsy. Insomnia is characterized by persistent difficulty in falling or maintaining sleep, often as a consequence of stress, overconsumption of caffeine, disrupted sleep-wake cycles, or depression. Sleep apnea, in contrast, is a condition where the sufferer's breathing involuntarily stops for periods exceeding 10 seconds during sleep, usually due to the throat's muscular tone diminishing, causing the airway to constrict. Narcolepsy is unique in that it is not associated with an inability to sleep but rather a failure to stay awake. Sufferers experience sudden bouts of sleep during their waking hours. Research has linked this to a lack of the neuropeptide orexin, also known as hypocretin, which aids in maintaining wakefulness. A malfunction in the hypothalamus, where orexin is produced, can be a critical factor in the development of narcolepsy.

 Core: Anatomy and Physiology

Anatomy of the Eyeball

The eye is a spherical, hollow structure composed of three tissue layers. The outer layer — the fibrous tunic, comprises the sclera — a white structure — and the cornea, which is transparent. The sclera encompasses some of the ocular surface, most of which is not visible. However, the 'white of the eye' is distinctively visible in humans compared to other species. The cornea, a clear covering at the front of the eye, enables light penetration. The eye's middle layer, the vascular tunic, primarily consists of the choroid, the ciliary body, and the iris. The choroid is a highly vascularized connective tissue supplying blood to the eyeball, situated behind the ciliary body. The ciliary body, a muscular entity, is linked to the lens by zonule fibers or suspensory ligaments. These aid in lens curvature, facilitating the focus of light onto the rear of the eye. The iris, the eye's colored portion, overlays the ciliary body and is visible at the front of the eye. Iris, a circular muscle, dilates or constricts the pupil, the central eye aperture that permits light entry. The iris contracts the pupil in bright light, which widens the pupil in dim light. The innermost layer, the neural tunic or retina, houses the nervous tissue in light perception.

The eye can be segmented into two distinct sections: the front cavity and the back cavity. The front cavity between the cornea and the lens — encapsulating the iris and ciliary body — is filled with a light liquid known as aqueous humor. On the other hand, the back cavity expands from the area behind the lens to the back of the inner eyeball, where the retina is positioned. This cavity is filled with a thicker fluid referred to as vitreous humor.

The retina is a complex structure composed of numerous layers with distinct cells dedicated to the preliminary processing of visual signals. Photoreceptors, namely rods and cones, respond to light energy by altering their membrane potential. This change influences the quantity of neurotransmitters that the photoreceptors dispatch onto the bipolar cells in the outer synaptic stratum. In the retina, it is the bipolar cell that links a photoreceptor to a retinal ganglion cell (RGC) situated in the inner synaptic layer. Amacrine cells aid in processing within the retina before the RGC generates an action potential. Positioned at the retina's deepest layer, the RGCs' axons aggregate at the optic disc and exit the eye, forming the optic nerve. Since these axons traverse the retina, there is an absence of photoreceptors at the eye's rear, where the onset of the optic nerve lies. This results in a "blind spot" in the retina and an equivalent blind spot in our field of vision.

The intricate structure of the retina comprises multiple layers populated with different cells, all of which play a critical role in the initial interpretation of visual cues. The photoreceptors, specifically rods and cones, are sensitive to light energy, and this sensitivity prompts a shift in their membrane potential. This shift subsequently determines the amount of neurotransmitter released onto the bipolar cells in the outer synaptic layer. The bipolar cell is the intermediary between a photoreceptor and a retinal ganglion cell (RGC) in the inner synaptic layer within the retina. The processing within the retina is assisted by Amacrine cells before the RGC generates an action potential. The axons of RGCs, nestled in the innermost layer of the retina, converge at the optic disc, exiting the eye as the optic nerve. Due to the course these axons take through the retina, the rear of the eye, where the optic nerve originates, is devoid of photoreceptors. This results in a "blind spot" in the retina, reflecting an identical blind spot in our visual field.

It is important to note that the photoreceptors (rods and cones) within the retina are behind the axons, RGCs, bipolar cells, and retinal blood vessels. These structures absorb a considerable amount of light before it reaches the photoreceptor cells. Yet, the fovea is at the retina's center — a small area devoid of supporting cells and blood vessels, only housing photoreceptors. As such, visual acuity — the clarity of vision — is optimal at the fovea due to minimal absorption of incoming light by other retinal structures. As one moves away from the foveal center in any direction, there is a noticeable drop in visual acuity. Each of the fovea's photoreceptor cells is connected to a single RGC. It follows that the RGC does not need to amalgamate inputs from multiple photoreceptors, enhancing the precision of visual transduction.

Conversely, at the peripheries of the retina, several photoreceptors converge on RGCs (via the bipolar cells) in ratios as high as 50 to 1. The disparity in visual acuity between the fovea and the peripheral retina is starkly evident — focus on a word positioned in the middle of this paragraph without moving your eyes, and words at the beginning or end appear blurry and out of focus. The peripheral retina is responsible for creating the images in your peripheral field of view; however, these images often have indistinct, fuzzy edges, and the words must be more clearly discernible. So, a significant portion of the neural function of the eyes is concentrated on moving the eyes and head to ensure important visual stimuli are centered on the fovea.

Photoreceptors, the cells responsible for capturing light in the eye, are composed of two distinct components: the internal and external segments. The former harbors the nucleus and various other cell organelles, while the latter is a niche area enabling photoreception. Two distinct photoreceptor types exist — rods and cones — characterized by the morphology of their external segments. The rods — named for their rod-like segments — house membranous disks filled with the light-sensitive pigment rhodopsin. The cone photoreceptors, on the other hand, hold their light-sensitive pigments within the cell membrane's invaginations, and their external segments take on a conical shape. Cone photoreceptors possess three photopigments, namely opsins, each responsive to a specific light wavelength. The color of visible light is determined by its wavelength, and the photopigments in the human eye are adept at discerning three fundamental colors: red, green, and blue.

 Core: Anatomy and Physiology

Regulation of Hormone Secretion

Regulation of hormone secretion is a finely tuned orchestration driven by various types of stimuli, encompassing neural, humoral, and hormonal signals. Environmental cues instigate neural stimuli, where action potentials traverse nerve fibers to reach their designated targets. An illustrative scenario is the body's response to stress, wherein the sympathetic nervous system releases epinephrine from the adrenal glands, inducing the well-known 'fight or flight' reaction.

Humoral stimuli, conversely, involve concentration fluctuations of specific ions or nutrients in the bloodstream. A case in point is the response to diminished blood calcium levels, triggering the secretion of parathyroid hormones to bolster circulating calcium concentrations.

Hormonal stimuli manifest when one hormone induces the secretion of another hormone from a distinct endocrine organ. For instance, the hypothalamus secretes the thyrotropin-releasing hormone, instigating the anterior pituitary lobe to release the thyroid-stimulating hormone (TSH). TSH, in turn, stimulates the thyroid glands to discharge thyroid hormones like T₃. As circulating T₃ levels rise, a sophisticated negative feedback loop comes into play. This loop inhibits the continuous production of T₃ by dispatching inhibitory signals to both the pituitary gland and hypothalamus. The endocrine system maintains a delicate balance in this intricate interplay of signals and responses, ensuring precise regulation and harmony in hormone secretion.

 Core: Anatomy and Physiology

Nervous Tissue: Glial Cells

Glia, or neuroglia, are vital support cells that assist neurons in their functions. The term "glia" originates from the Greek word for "glue," reflecting their role in holding the nervous system together. These cells can be categorized into six types: four in the central nervous system (CNS) and two in the peripheral nervous system (PNS).

The CNS glial cell includes the astrocytes, the oligodendrocytes, the microglia, and the ependymal cells.

Astrocytes are star-shaped glial cells that interact with neurons, blood vessels, and the pia mater, a connective tissue covering the CNS. Their primary role is maintaining chemical balance, removing excess signaling molecules, contributing to the blood-brain barrier, and guiding neuronal growth and connections during embryonic development. Oligodendrocytes, smaller than astrocytes, form and maintain the myelin sheath around CNS axons. This myelin sheath is a multilayered lipid and protein covering that insulates axons. Microglial cells or microglia function as phagocytes, removing cellular debris and damaged nervous tissue. Ependymal cells, arranged in a single layer, line the brain's ventricles and the spinal cord's central canal. These cells produce, monitor, and help circulate cerebrospinal fluid. They also form the blood-cerebrospinal fluid barrier.

In the PNS, Schwann cells encircle axons, similar to oligodendrocytes, forming the myelin sheath around them. However, while a single oligodendrocyte can myelinate several axons, each Schwann cell myelinates only one axon. Finally, satellite cells surround the cell bodies of neurons in the PNS ganglia. They provide structural support and regulate material exchange between neuronal cell bodies and interstitial fluid in the PNS.

 Core: Anatomy and Physiology

Charge and Current

Electric charge is the most fundamental quantity in an electric circuit. The effects of electric charge are encountered daily, such as when a wool sweater sticks to the human body or when a person receives a shock while walking on a carpet.

Charge is an inherent property of the atomic particles that make up matter and is measured in units called coulombs (C). Matter is composed of atoms, each consisting of electrons, protons, and neutrons. Electrons have a negative charge (-e), while protons carry a positive charge (+e), with both charges having the same magnitude. An atom is electrically neutral when it has an equal number of protons and electrons.

Key points about electric charge include:

• • The coulomb (C) is a relatively large unit for measuring charges, and practical charge values are often in picocoulombs (pC), nanocoulombs (nC), or microcoulombs (μC).
• • Observations reveal that natural charges exist only as integral multiples of the elementary charge (e), the charge carried by an electron or proton.
• • The law of conservation of charge asserts that electric charge cannot be created nor destroyed; it can only be transferred. As a result, the total electric charge in a system remains constant. Charge can be moved from one place to another and converted into different forms of energy.

The movement of positive charges in one direction and negative charges in the opposite direction gives rise to electric current. Typically, a conventional current flow is defined as the movement of positive charges, and it is measured in units known as amperes (A). When the current remains constant over time, it is called direct current (DC). In cases where the current varies with time, it is denoted as a time-varying current, often referred to as alternating current (AC). AC is commonly used in households to power appliances like air conditioners, refrigerators, and washing machines.

 Core: Electrical Engineering

Sum and Difference OpAmps

Operational amplifiers (op-amps) are versatile devices that extend beyond amplification. In this context, two specific op-amp configurations are explored: the summing and difference amplifiers.

A summing amplifier, or an adder, utilizes an op-amp to merge multiple input signals into a single output signal. When audio signals are introduced into its input channels, the input resistors initiate currents that traverse feedback resistors, resulting in an output voltage. Applying Kirchhoff's current law and Ohm's Law, the output voltage becomes a weighted sum of the input signals. This basic differential op-amp circuit plays a role in mitigating audio track noise in audio mixer applications.

Conversely, a difference amplifier, often called a subtractor, amplifies the disparity between two input signals. Two equations are established by applying Kirchhoff's current law at the inverting and non-inverting nodes. An output voltage expression is derived when equal voltages exist across both nodes. However, specific resistance ratio conditions must be satisfied for the difference amplifier to nullify common signals between the inputs. When the resistances in the corresponding branches are equal, the difference amplifier functions as a subtractor.

In summary, operational amplifiers offer amplification and the ability to perform addition and subtraction tasks. The summing amplifier combines inputs into a weighted sum. The difference amplifier amplifies the disparity between two inputs and rejects common signals. Understanding these configurations opens the door to many practical applications in signal processing and circuit design.

 Core: Electrical Engineering

RL Circuit with Source

When an RL (Resistor-Inductor) circuit is connected to a DC source, the complete response of the circuit can be divided into two parts: the transient response and the steady-state response.

The transient response of the circuit is its temporary reaction to the sudden application of the DC source. This response is characterized by a current that exponentially decays to zero as time approaches infinity. During this transitional period, the inductor behaves like a short circuit, causing the source voltage to drop across the resistor.

Once the transient response has decayed to zero, the circuit enters its steady-state phase. In this phase, the current in the circuit becomes stable, equaling the ratio of the source voltage to the resistance of the circuit. This is the steady-state response.

The constant term in the transient response can be determined by substituting the initial current through the inductor at the time the switch is closed (t=0). This gives us the initial condition for the transient response.

Plotting the complete step response of the circuit shows that the initial current decreases exponentially until it reaches a steady-state value. From this current response, the voltage response is derived, which also follows an exponential decay from a maximum value to zero.

If the initial current in the circuit is zero, the complete step response shows the current increasing exponentially until it reaches the steady-state value. Correspondingly, the voltage response starts at the source voltage and decreases exponentially to zero.

In conclusion, understanding the complete response of an RL circuit to a DC source provides valuable insights into how these circuits react to sudden changes in input voltage. This knowledge is essential for designing and analyzing electronic circuits, particularly in applications such as power supply filtering and signal processing, where inductors are used extensively.

 Core: Electrical Engineering

Thévenin Equivalent Circuits

The household power distribution system, encompassing distribution lines and transformers, serves as the primary network. Electrical appliances within a household can be represented as load impedance. To simplify this intricate distribution system, Thévenin's theorem can be applied to create a Thévenin equivalent circuit. If an AC circuit is partitioned into two parts (circuit A and circuit B), connected by a single pair of terminals as shown in Figure 1.

Figure 1: Circuit portioned into two parts

Figure 2:Circuit A replaced by its Thévenin equivalent circuit

Replacing circuit A with its Thévenin equivalent circuit (a voltage source in series with an impedance) does not alter the current or voltage of any element in circuit B (shown in Figure 2). The values of the currents and voltages of all the circuit elements in circuit B will be the same irrespective of whether circuit B is connected to circuit A or its Thévenin equivalent. Two parameters are required to find the Thévenin equivalent circuit: the Thévenin voltage and the Thévenin impedance. Figure 3 shows an open circuit connected across the terminals of circuit A to determine the open-circuit voltage V_oc , while Figure 4 indicates that the Thévenin impedance Z_t is the equivalent impedance of circuit A*.

Figure 3:Thévenin equivalent circuit with V_oc .

Figure 4:Thévenin equivalent circuit showing Z_t. .

Circuit A* is formed from circuit A by replacing all independent voltage sources with short circuits and all independent current sources with open circuits. Generally, the Thévenin impedance Z_t can be determined by replacing series or parallel impedances with equivalent impedances repeatedly.

 Core: Electrical Engineering

Rules of Significant Figures

 Core: Mechanical Engineering

Titration of a Weak Acid with a Strong Base

In titrating a weak acid with a strong base, different calculation methods are applied at various stages. Initially, the pH of a weak acid like acetic acid is calculated using its dissociation constant (K_a) and an ICE table. Upon addition of a strong base such as sodium hydroxide, a buffer forms, and its pH is determined using the Henderson-Hasselbalch equation. As more base is added and the titration reaches the halfway point, the pH becomes equal to the pK_a of the acid, indicating equal concentrations of the acid and its conjugate base. At the equivalence point, all the acid is converted to its conjugate base, and the pH is calculated using the base's dissociation constant (K_b) and an ICE table. Beyond the equivalence point, the pH is governed by the concentration of the excess strong base.

 Core: Analytical Chemistry

Yeast Maintenance

Research performed in the yeast Saccharomyces cerevisiae has significantly improved our understanding of important cellular phenomona such as regulation of the cell cycle, aging, and cell death. The many benefits of working with S. cerevisiae include the facts that they are inexpensive to grow in the lab and that many ready-to-use strains are now commercially available. Nevertheless, proper maintenance of this organism is critical for successful experiments.

This video will provide an overview of how to grow and maintain S. cerevisiae in the lab. Basic concepts required for monitoring the proliferation of a yeast population, such as how to generate a growth curve using a spectrophotometer, are explained. This video also demonstrates the hands-on techniques required to maintain S. cerevisiae in the lab, including preparation of media, how to start a new culture of yeast cells, and how to store those cultures. Finally, the video shows off some of the ways these handling and maintenance techniques are applied in scientific research.

 Biology I

Chick ex ovo Culture

One strength of the chicken (Gallus gallus domesticus) as a model organism for developmental biology is that the embryo develops outside the female and is easily accessible for experimental manipulation. Many techniques allow scientists to examine chicken embryos inside the eggshell (in ovo), but embryonic access can be limited at later stages of development. Fortunately, chicks can also be cultured ex ovo, or outside of the eggshell. The major advantage to ex ovo culture is greater access to tissues that might otherwise be obstructed by the shell or the orientation of the chick within the egg, especially for embryos in later stages of development.

There are two principle strategies to ex ovo culture: whole yolk culture and explant culture. During whole yolk culture, the eggshell is cracked and the contents are transferred to a simple housing vessel. However, in explant culture methods, the embryo is excised from the yolk and mounted in the housing vessel to maintain membrane tension, which is important for normal development.

Basic protocols for whole-yolk and explant techniques will be provided in this video, along with a discussion of the pros and cons of culturing chicks outside of the shell. Finally, experimental applications of ex ovo culture will be discussed, demonstrating how this approach is used to improve access to the embryo for microscopy and genetic manipulation of late stage embryos.

 Biology II

An Introduction to Behavioral Neuroscience

Behavioral neuroscience is the study of how the nervous system guides behavior, and how the various functional areas and networks within the brain correlate to specific behaviors and disease states. Researchers in this field utilize a wide variety of experimental methods ranging from complex animal training techniques to sophisticated imaging experiments in human subjects.

This video first offers a historical overview of some of the major milestones that lead to our current understanding of the brain’s control over behavior. Then, some of the fundamental questions asked by behavioral neuroscientists are presented, which all involve the study of neural correlates, or specific brain regions whose activation is responsible for a given function. Next, prominent methods used to answer those questions are reviewed for both human and animal subjects, such as operant conditioning and functional neuroimaging. Finally, experimental applications of these techniques are presented, including animal training using a Skinner box, and the use of electroencephalography to investigate human neurological disease.

 Neuroscience

Embryonic Stem Cell Culture and Differentiation

Culturing embryonic stem (ES) cells requires conditions that maintain these cells in an undifferentiated state to preserve their capacity for self-renewal and pluripotency. Stem cell biologists are continuously optimizing methods to improve the efficiency of ES cell culture, and are simultaneously trying to direct the differentiation of ES cells into specific cell types that could be used in regenerative medicine.

This video describes the basic principles of ES cell culture, and demonstrates a general protocol to grow and passage ES cells. We also take a closer look at the hanging drop method, which is used to differentiate ES cells. Finally, this video will describe a few applications of ES cell culture and differentiation techniques, including a method used to generate functional heart muscle cells in vitro.

 Developmental Biology

An Introduction to Motor Control

Motor control involves integration and processing of sensory information by our nervous system, followed by a response through our skeletal system to perform a voluntary or involuntary action. It is vital to understand how our neuroskeletal system controls motor behavior in order to evaluate injuries pertaining to general movement, reflexes, and coordination. An improved understanding of motor control will help behavioral neuroscientists in developing useful tools to treat motor disorders, such as Parkinson's or Huntington's disease.

This video briefly reviews the neuroanatomical structures and connections that play a major role in controlling motion. Fundamental questions currently being asked in the field of motor control are introduced, followed by some of the methods being employed to answer those questions. Lastly, the application sections reviews a few specific experiments conducted in neuroscience labs interested in studying this phenomenon.

 Behavioral Science

Genome Editing

A well-established technique for modifying specific sequences in the genome is gene targeting by homologous recombination, but this method can be laborious and only works in certain organisms. Recent advances have led to the development of “genome editing”, which works by inducing double-strand breaks in DNA using engineered nuclease enzymes guided to target genomic sites by either proteins or RNAs that recognize specific sequences. When a cell attempts to repair this damage, mutations can be introduced into the targeted DNA region.

In this video, JoVE explains the principles behind genome editing, emphasizing how this technique relates to DNA repair mechanisms. Then, three major genome editing methods—zinc finger nucleases, TALENs, and the CRISPR-Cas9 system—are reviewed, followed by a protocol for using CRISPR to create targeted genetic changes in mammalian cells. Finally, we discuss some current research that applies genome editing to alter the genetic material in model organisms or cultured cells.

 Genetics

An Introduction to Cell Death

Necrosis, apoptosis, and autophagic cell death are all manners in which cells can die, and these mechanisms can be induced by different stimuli, such as cell injury, low nutrient levels, or signaling proteins. Whereas necrosis is considered to be an “accidental” or unexpected form of cell death, evidence exists that apoptosis and autophagy are both programmed and “planned” by cells.

In this introductory video, JoVE highlights key discoveries pertaining to cell death, including recent work done in worms that helped identify genes involved in apoptosis. We then explore questions asked by scientists studying cell death, some of which look at different death pathways and their interactions. Finally, several methods to assess cell death are discussed, and we note how researchers are applying these techniques in their experiments today.

 Cell Biology

Enzyme Assays and Kinetics

Enzyme kinetics describes the catalytic effects of enzymes, which are biomolecules that facilitate chemical reactions necessary for living organisms. Enzymes act on molecules, referred to as substrates, to form products. Enzyme kinetic parameters are determined via assays that directly or indirectly measure changes in substrate or product concentration over time. 

This video will cover the basic principles of enzyme kinetics (including rate equations) and kinetic models. The concepts governing enzyme assays are also discussed, followed by a typical colorimetric assay. The applications section discusses an enzyme assay via Förster resonance energy transfer (FRET) analysis, characterizing extracellular enzyme activity in the environment, and investigating DNA repair kinetics using molecular probes.

 Biochemistry

Overview of Bioprocess Engineering

Bioprocessing is a method that uses living organisms to produce a desired target product. Often, bioprocessing refers to the use of bioreactors to produce protein products from genetically engineered organisms. This field is responsible for the large-scale manufacture of biotherapeutics; drugs that have become essential to improving the quality of life for many with complex diseases like cancer, autoimmune diseases and HIV/AIDS.

This video will introduce the engineering approach to designing a targeted protein-production system. The prominent methods in the field, as well as some key challenges, and applications of the technology are also considered.

 Bioengineering

Bond Dissociation Energy and Activation Energy

Bond energy is the energy required to break a bond homolytically. These values are usually expressed in units of kcal/mol or kJ/mol and are referred to as bond dissociation energies when given for specific bonds or average bond energies when indicated for a given type of bond over many compounds. Firstly, the bond dissociation energy for a single bond is weaker than that of a double bond, which in turn is weaker than that of a triple bond. Secondly, hydrogen forms relatively strong bonds with carbon, nitrogen, and oxygen. Finally, with the exception of carbon and hydrogen, single bonds between atoms of the same element are relatively weak. Reactions between organic compounds involve the making and breaking of bonds. Hence, the strengths of bonds and their resistance to breaking are essential concepts in organic chemistry.

Reactions in which bonds are broken pass through a high-energy transition state before transforming into products. In order to reach this transition state, the reactant molecules must be oriented in a suitable direction and must be supplied with certain threshold energy. The activation energy, ΔG^‡, is the energy provided to the reactants to raise them to the transition state. Overall, for a reaction to occur, the reacting molecules must collide or otherwise interact. The necessary activation energy for the reactant–transition-state jump is provided by the kinetic energy of the colliding particles. Lastly, the colliding molecules must collide in a specific orientation so as to maximize the impact of the collision.

 Core: Organic Chemistry

Ethers to Alkyl Halides: Acidic Cleavage

Ethers are generally unreactive and unsuitable for direct nucleophilic substitution reactions since the alkoxy groups are strong bases and, therefore, poor leaving groups. However, ethers readily undergo acidic-cleavage reactions. Ethers can be converted to alkyl halides when heated with strong acids such as HBr and HI in a sequence of two substitution reactions.

In the first step, the ether is converted into an alkyl halide and alcohol.

In the second step, the alcohol reacts with the excess HX acid to form another equivalent of an alkyl halide.

Acidic cleavage of ethers can occur either by the S_N1 mechanism or S_N2 mechanism, depending on the substrate. Ethers with primary and secondary alkyl groups as substrates react with the S_N2 mechanism in which the nucleophile attacks the protonated ether at the less hindered site. Ethers with a tertiary, benzylic, or allylic group undergo the S_N1 mechanism because the substrates can produce more stable intermediate carbocations. Reactivity of the halogen acids with ethers increases relative to the nucleophilicity of the halide ions. HI and HBr are more reactive, HCl is less efficient, and HF is unsuitable.

 Core: Organic Chemistry

Oxymercuration-Reduction of Alkenes

Oxymercuration–reduction of alkenes is one of the major reactions converting alkenes to alcohols. It involves the hydration of alkenes with mercuric acetate in a mixture of tetrahydrofuran and water, forming an organomercury adduct. This is followed by a demercuration step in which the adduct is reduced to an alcohol using sodium borohydride.

In the mixture of water and tetrahydrofuran, tetrahydrofuran acts as a solvent dissolving the alkene and the aqueous mercuric acetate solution, while water functions as a reactant and a solvent for mercuric acetate.

Oxymercuration–Demercuration Mechanism

Consider the conversion of 2-methyl-2-butene to yield 2-methyl-2-butanol.

The mechanism proceeds with the dissociation of mercuric acetate, forming an electrophilic mercuric cation and an acetate anion. The alkene π bond attacks the electrophilic mercuric cation, resulting in a bridged-mercurinium-ion intermediate.

Regiochemical and Stereochemical Outcome

The bridged-mercurinium-ion intermediate is a resonance hybrid of a carbocation and a bridged mercurinium ion. The partial positive charge is shared between the more substituted carbon atom and the mercury atom, minimizing the chance of a carbocation rearrangement. Furthermore, the carbon–mercury bond to the more substituted carbon is longer and can be easily broken.

The factors mentioned above lead to the nucleophilic attack by water exclusively at the more substituted carbon, opening the three-membered ring.

The oxymercuration step is stereospecific, as the attack by water on the bridged mercurinium ion leads to the anti addition of the hydroxyl group. A proton transfer completes the oxymercuration step, forming an organomercury compound.

Lastly, the oxymercuration adduct is treated with sodium borohydride through a process called demercuration to yield an alcohol with Markovnikov's orientation.

During the demercuration step, as the hydrogen can replace the mercury species in either a syn or an anti fashion with respect to the hydroxyl group, the overall reaction produces a racemic mixture of two enantiomeric alcohols.

 Core: Organic Chemistry

Electrophilic Addition to Alkynes: Halogenation

Introduction

Halogenation is another class of electrophilic addition reactions where a halogen molecule gets added across a π bond. In alkynes, the presence of two π bonds allows for the addition of two equivalents of halogens (bromine or chlorine). The addition of the first halogen molecule forms a trans-dihaloalkene as the major product and the cis isomer as the minor product. Subsequent addition of the second equivalent yields the tetrahalide.

Reaction Mechanism

In the first step, a π bond from the alkyne acts as a nucleophile and attacks the electrophilic center on the polarized halogen molecule, displacing the halide ion and forming a cyclic halonium ion intermediate. In the next step, a nucleophilic attack by the halide ion opens the ring and forms the trans-dihaloalkene. Since the nucleophile attacks the halonium ion from the backside, the net result is an anti addition where the two halogen atoms are trans to each other.

The addition of a second equivalent of halogen across the alkene π bond also proceeds via the formation of a bridged halonium ion to give the tetrahalide as the final product.

For example, the addition of bromine to 2-butyne in the presence of acetic acid and lithium bromide favors anti addition and preferentially forms the trans or (E)-2,3-dibromo-2-butene as the major product. The corresponding cis isomer, (Z)-2,3-dibromo-2-butene, is formed in lower yields. A second addition gives 2,2,3,3-tetrabromobutane.

Reactivity of alkynes and alkenes towards electrophilic addition

Alkynes are less reactive than alkenes towards electrophilic addition reactions. The reasons are twofold. First, the carbon atoms of a triple bond are sp hybridized in contrast to the double bonds that are sp² hybridized. Since the sp hybrid orbitals have a higher s-character and are more electronegative, the π electrons in C≡C are held more tightly than in C=C. As a result, in alkynes, the π electrons are not readily available for the nucleophilic attack, making them less reactive towards electrophilic addition than alkenes.     

Secondly, the cyclic halonium ion formed from alkynes is a three-membered ring with a double bond where the 120° bond angle of an sp² carbon is constrained into a triangle.

Alkyne halonium ion	Alkene halonium ion

In contrast, the cyclic intermediate in alkenes is a three-membered ring with an sp³ hybridized carbon where a bond angle of 109° is constrained into a triangle. Therefore, the larger ring strain associated with the alkyne halonium ions makes them more unstable and hinders their formation. 

 Core: Organic Chemistry

Drugs that Stabilize Microtubules

Microtubules are dynamic structures that undergo cycles of catastrophe and rescue. The microtubules play a central role in cell division by forming the spindle apparatus for segregating the chromosomes. This makes them ideal targets for regulating dividing cells in tumors and malignant cancer cells. Microtubule stabilizing drugs help stabilize the microtubule formation and promote its polymerization. Paclitaxel was the first microtubule stabilizing agent used as anticancer drug in chemotherapy after medical approval in 1993.

Some important microtubule-stabilizing drugs include taxanes, epothilones, laulimalide, and dictyostatin. These drugs can be used to treat tumors in breast, lung, prostate, and ovarian cancers. Taxanes are the widely used class of microtubule-stabilizing drugs that target microtubules in the spindle apparatus and cytoskeleton and disrupt cellular processes, including cell division. If given in low concentration, these drugs lead to the formation of multipolar spindle apparatus during the G1 phase, a process called mitotic slippage. This leads to cell cycle arrest and apoptosis. If the drugs are given in a higher dosage, it causes mitotic arrest of cells during the G2/M phase of mitosis. The mitotic arrest prevents chromosome segregation, resulting in tetraploid G1 cells, with eventual apoptosis of cancer or tumor cells. However, these drugs have certain limitations because ABC transporters overexpressed in tumor, which pump them out of the cell.

These drugs have specific binding sites on the tubulin dimers. Taxane sites are hydrophobic pockets between the lateral interface of two adjacent protofilaments in the microtubule lumen. Epothilones used in paclitaxel-resistant tumors also use hydrophobic taxane sites for binding with microtubules.

 Core: Cell Biology

Base-Catalyzed Ring-Opening of Epoxides

Due to their highly strained structures, epoxides can readily undergo ring-opening reactions through nucleophilic substitution, either in the presence of an acid or a base. The nucleophilic substitution reactions in the presence of acid are called acid-catalyzed ring-opening reactions, and nucleophilic substitution reactions in the presence of a base are called base-catalyzed ring-opening reactions. Epoxides undergo base-catalyzed ring-opening reactions in the presence of a strong nucleophile or a base. A variety of nucleophiles like sodium hydroxide, sodium alkoxide, sodium hydrosulfide, sodium cyanide, lithium aluminum hydride, and Grignard reagent can open the epoxide ring. The nucleophilic attack on the epoxide ring proceeds via an S_N2 mechanism and involves an alkoxide intermediate. The product formed in base-catalyzed reactions shows S_N2-like stereoselectivity and regioselectivity. The nucleophile attacks at the less-hindered carbon and anti to the leaving group, leading to the inversion of configuration at a chiral center. In contrast to epoxides, acyclic ethers do not undergo direct nucleophilic substitutions, as the reaction is thermodynamically unfavorable.

 Core: Organic Chemistry

[4+2] Cycloaddition of Conjugated Dienes: Diels–Alder Reaction

The Diels–Alder reaction is an example of a thermal pericyclic reaction between a conjugated diene and an alkene or alkyne, commonly referred to as a dienophile. The reaction involves a concerted movement of six π electrons, four from the diene and two from the dienophile, forming an unsaturated six-membered ring. As a result, these reactions are classified as [4+2] cycloadditions.

From a molecular orbital perspective, the interacting lobes of the two π systems must be in phase to permit the formation of new σ bonds in a synchronous manner. For molecules in the ground state, the interaction between HOMO (diene) and LUMO (dienophile) or HOMO (dienophile) and LUMO (diene) satisfies the orbital symmetry requirements. The two π components interact suprafacially in each case, making Diels–Alder a thermally allowed [4+2] cycloaddition reaction.

 Core: Organic Chemistry

Protein Complex Assembly

 Core: Cell Biology

Multipotency of Hematopoietic Stem Cells

The hematopoietic stem cells or HSCs are multipotent, meaning they can differentiate and give rise to all blood and immune cells. HSCs are maintained in the quiescent stage until an external stimulus initiates their differentiation. The multipotent HSCs exist as two heterogeneous populations, long-term repopulating cells (LTRC) and short-term repopulating cells (STRC). The two HSC populations have different surface markers or receptors and are classified based on quiescence and long-term renewal capacity.

The long-term HSCs rarely divide as they maintain long gaps between successive cell divisions while keeping their metabolic activity to the bare minimum. Thus, the LTRCs undergo a few rounds of symmetric cell divisions before entering the state of dormancy. This allows them to expand the number of HSCs and maintain a stem cell pool before exhausting their regenerative and self-renewal potential.

LTRCs produce the more active multipotent short-term repopulating cells (STRC) in response to external stimuli. STRCs undergo fewer self-renewal divisions than the LTRCs and differentiate into specific blood or immune cells. LTRCs and STRCs find application in engraftment and therapeutic studies. The LTRCs can sustain hematopoiesis for upto four months upon transplantation into the recipients, while transplantation of STRCs supports hematopoiesis for only a few weeks. A delicate balance between dormancy and differentiation stimuli maintains the HSC population for the long run. A slow or non-responsive quiescent HSCs leave the body devoid of differentiated blood cells such as erythrocytes, phagocytes, and lymphocytes which are necessary for transporting nutrients or providing immune surveillance. In contrast, highly active HSCs exhaust the stem cell population making them unavailable to renew, repair, or replace blood cells lost due to an injury or infection.

 Core: Cell Biology

Pre-mRNA Processing: RNA Splicing

 Core: Cell Biology

Alkylation of β-Diester Enolates: Malonic Ester Synthesis

Malonic ester synthesis is a method to obtain α substituted carboxylic acids from ꞵ-diesters such as diethyl malonate and alkyl halides.

The reaction proceeds via abstraction of the acidic α hydrogen from a ꞵ-diester to produce a doubly stabilized enolate ion. The nucleophilic enolate attacks the alkyl halide in an S_N2 manner to form an alkylated malonic ester intermediate with a new C–C bond. Further treating the intermediate with aqueous acid or base results in the hydrolysis of the two ester groups to give a 1,3-dicarboxylic acid. The resulting ꞵ-diacid is unstable at high temperatures and readily eliminates CO₂ through a cyclic six-membered transition state, forming an enol. The enol tautomerizes to its more stable keto form producing a monosubstituted carboxylic acid. However, a disubstituted carboxylic acid is achieved if the deprotonation and alkylation steps are repeated before hydrolysis and decarboxylation.

 Core: Organic Chemistry

Prokaryotic Transcriptional Activators and Repressors

 Core: Cell Biology

Nucleic Acids

 Core: Cell Biology

Telomeres and Telomerase

 Core: Cell Biology

Non-Canonical Wnt Signaling Pathways

 Core: Cell Biology

Principles Of Column Chromatography

The chromatography technique was first invented in 1901 by Michael S. Tswett, a Russian botanist, to separate plant pigments using organic solvents. Further, in 1941, Archer John Porter Martin and R. L. M. Synge modified the technique by packing silica gel into a column. A mixture of amino acids was then separated on the packed column using chloroform and water mixture as the mobile phase. This was the first report on column chromatography. At present, column chromatography is a widely used technique for separating various types of compounds from a sample mixture.

Factors Influencing Efficient Separation of Proteins

Various parameters like column material, packing, and operational conditions such as flow rates and temperature determine the efficiency of separation by column chromatography.

The choice of column material or matrix determines the extent of interaction with the sample. The matrix material must be tightly and uniformly packed in the column. Air bubbles, debris, large particles, and precipitates interfere with the uniform flow of the solvent through the column, affecting its separation efficiency. The column should also be free from particulate matter.

The sample injected into the column should be clear and free from aggregates that might clog the column, hindering solvent flow. The flow rate of solvent also affects the separation. Very high or very low flow rates of solvents result in inefficient separation of compounds and impure preparations. Very high rates may also disturb the column packing, affecting process efficiency. In addition, the composition of elution buffer is also an important factor. It should be non-corrosive and compatible with the sample and also the column material to prevent in situ precipitation or dissolution.

Another operational parameter, temperature, also plays an important role in the process. It decides the stability of the sample, column material, and solvent buffer. Also, a constant temperature throughout the column efficiently resolves compounds. After completing the separation process, the columns must be washed thoroughly by repeatedly passing a suitable solvent to avoid sample contamination in further runs. Occasionally, the solvent is passed in a column in the reverse direction to remove any clogged material.

Limitations

Though a very widely used technique, the method still has some limitations. It is a very time-consuming method as the flow rates need to be slower for better resolution of the compounds. Also, large quantities of highly pure solvents required in the mobile phase make the process expensive. This also raises the scaling-up cost when higher yields of pure compounds are needed.

 Core: Cell Biology

Immunofluorescence Microscopy

A fluorescence microscope uses fluorescent chromophores called fluorochromes, which can absorb energy from a light source and then emit this energy as visible light. Fluorochromes include naturally fluorescent substances (such as chlorophylls) and fluorescent stains that are added to the specimen to create contrast. Dyes such as Texas red and FITC are examples of fluorochromes. Other examples include the nucleic acid dyes 4’,6’-diamidino-2-phenylindole (DAPI), and acridine orange.

The microscope irradiates the sample with short wavelength excitation, such as ultraviolet or blue light. The chromophores absorb the excitation light and emit visible light of longer wavelengths. The excitation light is then filtered out (partly because ultraviolet light is harmful to the eyes) so that only visible light passes through the ocular lens, producing an image of the specimen in bright colors against a dark background.

Fluorescence microscopes can identify pathogens, find particular species within an environment, or find the locations of particular molecules and structures within a cell. Approaches have also been developed to distinguish living from dead cells based on whether they take up particular fluorochromes. Sometimes, multiple fluorochromes are used on the same specimen to show different structures or features.

One of the most important applications of fluorescence microscopy is immunofluorescence, which is used to identify certain microbes by observing whether antibodies bind to them. (Antibodies are protein molecules the immune system produces that attach to specific pathogens to kill or inhibit them.) This technique has two approaches: direct immunofluorescence assay (DFA) and indirect immunofluorescence assay (IFA). In DFA, specific antibodies (e.g., those that target the rabies virus) are stained with a fluorochrome. If the specimen contains the targeted pathogen, one can observe the antibodies binding to the pathogen under the fluorescent microscope. This is a primary antibody stain because the stained antibodies attach directly to the pathogen.

In IFA, secondary antibodies are stained with a fluorochrome rather than primary antibodies. Secondary antibodies do not attach directly to the organism but bind to primary antibodies. When the unstained primary antibodies bind to the pathogen, the fluorescent secondary antibodies can be observed binding to the primary antibodies. Thus, the secondary antibodies are attached indirectly to the pathogen. Since multiple secondary antibodies can often attach to a primary antibody, IFA increases the number of fluorescent antibodies attached to the specimen, making it easier to visualize its features.

This text is adapted from Openstax, Microbiology 2e, Section 2.4: Staining Microscopic Specimens.

 Core: Cell Biology

The Cell Cycle Control System

 Core: Cell Biology

Attachment of Sister Chromatids

 Core: Cell Biology

What is Cancer?

 Core: Cell Biology

Cancer Prevention

 Core: Cell Biology

Unrenewable Cells

In humans, the photoreceptor cells of the eye and sensory hair cells of the ear lack stem cells. These cells are thus unrenewable and cannot be replaced when they are damaged or destroyed.

Photoreceptors

The retina is composed of several layers and contains specialized cells called photoreceptors. The photoreceptors (rods and cones) change their membrane potential when stimulated by light energy. There are two types of photoreceptors—rods and cones—which differ in the shape of their outer segment. The rod-shaped outer segments of the rod photoreceptor contain a stack of membrane-bound discs that contain the photosensitive pigment rhodopsin. The cone-shaped outer segments of the cone photoreceptor contain their photosensitive pigments in infoldings of the cell membrane. Since the eye photoreceptors lack stem cells, the loss of rods and cones is permanent and cannot be regenerated. The loss of photoreceptors or damage can be caused by age-related problems or exposure to high-intensity light.

Sensory Hair Cells

Like photoreceptors, the ear's sensory hair cells also do not contain stem cells. The hair cells are present in the organs of Corti, which are named for the hair-like stereocilia extending from the cell’s apical surfaces. When the pressure waves from the scala move the basilar membrane, the tectorial membrane slides across the stereocilia. This bends the stereocilia either toward or away from the tallest member of each array. When the stereocilia bend toward the tallest member of their array, the tension in the protein tethers opens ion channels in the hair cell membrane. This will depolarize the hair cell membrane, triggering nerve impulses that travel down the afferent nerve fibers attached to the hair cells. When the stereocilia bend toward the shortest member of their array, the tension on the tethers slackens, and the ion channels close. When no sound is present and the stereocilia are standing straight, a small amount of tension still exists on the tethers, keeping the membrane potential of the hair cell slightly depolarized. The loss or damage of hair cells is also permanent and often leads to permanent deafness.

This text is adapted from Openstax Anatomy and Physiology 2e, Section 14.1: Sensory Perception.

 Core: Cell Biology

Handwashing III: During the Procedure and Post-Procedure Steps

To wash hands properly, follow these steps:

1. Wet the hands. Use enough soap to cover all surfaces of the hands.
2. Rub both hands palm to palm.
3. Rub the back of the hand: Use the right palm over the left dorsum or back of the left hand with interlaced fingers, then switch hands.
4. Rub palm to palm with fingers interlaced.
5. With fingers interlocked, rub the backs of the fingers of the opposite hands. 
6. Rub the left thumb clasped in the right palm in a circular motion and repeat with the other thumb.
7. Rub the tips of the fingers. Rotationally rub backward and forward with clasped fingers of the right hand in the left palm and vice versa.
8. Rub each wrist with the opposite hand.
9. Rinse hands with water, as soap residue can lead to irritation and damage to the skin. Damaged skin does not provide a barrier to infection for the healthcare worker and can become colonized with potentially pathogenic bacteria, leading to cross-infection in the patient.
10. Ensure all surfaces of the hands are cleaned. Areas that are missed can be a source of cross-infection as well.
11. Following hand washing, turn off the faucet with a paper towel or use the elbow to prevent contact with the faucet. Dispose of paper towels.
12. With another towel, dry the hands. Wet hands encourage the growth of bacteria. Dispose of used paper towels in a foot-operated waste bin to prevent contamination of the hands.

 Core: Nursing

Introduction To Enzymes

 Core: Anatomy and Physiology

Mechanism of Ciliary Motion

 Core: Anatomy and Physiology

Translation

 Core: Anatomy and Physiology

Types of Errors: Detection and Minimization

Error is the deviation of the obtained result from the true, expected value or the estimated central value. Errors are expressed in absolute or relative terms.

Absolute error in a measurement is the numerical difference from the true or central value. Relative error is the ratio between absolute error and the true or central value, expressed as a percentage.

Errors can be classified by source, magnitude, and sign. There are three types of errors: systematic, random, and gross.

Systematic or determinate errors emerge from known sources and are reproducible during replicate measurements. Defective equipment and experiment design flaws are familiar sources of these errors. These errors can be minimized by employing standard reference materials, independent analysis, or varying the sample size.

Random errors or indeterminate errors are difficult to reproduce by repeating measurements. These errors originate from uncontrolled variables like electronic noise in the circuit of an electrical instrument and irregular changes in the heat loss rate from a solar collector due to changes in the wind.

Gross errors are caused by human mistakes. The magnitude of these errors is often high. The origin of such errors is entirely based on the observer.

 Core: Analytical Chemistry

Quantifying and Rejecting Outliers: The Grubbs Test

Sometimes, a data set can have a recorded numerical observation that greatly  deviates from the rest of the data. Assuming that the data is normally distributed, a statistical method called the Grubbs test can be used to determine whether the observation is truly an outlier.  To perform a two-tailed Grubbs test, first, calculate the absolute difference between the outlier and the mean. Then, calculate the ratio between this difference and the standard deviation of the sample. This number is known as the Grubbs statistic, 'G.' When the calculated G value exceeds the G critical value for a given confidence level and the number of observations, the questionable observation is considered an outlier and removed from the data set. On the contrary, if the calculated G value is smaller than the critical G value, the questionable observation is not considered an outlier and therefore retained in the data set.

 Core: Analytical Chemistry

Redox Equilibria: Overview

A reduction-oxidation reaction is commonly called a redox reaction. In a redox reaction, electrons are transferred from one species to another rather than being shared between or among atoms. The reducing agent or reductant is the species that loses electrons and gets oxidized in the process. The species that gains electrons and gets reduced in the process is the oxidizing agent or oxidant. Redox reactions are represented as two separate equations called half-reactions, where one equation represents the species that gains electrons, and the other represents the species that loses electrons.

Generally, the thermodynamics of a reaction is expressed in terms of the change in Gibbs free energy (ΔG), which is a function of concentrations of reactants and products. However, the thermodynamics of a redox reaction is expressed in terms of electrochemical potential (E)  and the Nernst equation, as the reaction involves the movement of electrons. The Nernst Equation expresses the relationship between the electrochemical potential and the concentrations of the reactants and products.

 Core: Analytical Chemistry

Effects of EDTA on End-Point Detection Methods

Different methods, such as visual observance of metal-ion indicators, spectroscopic techniques, and potentiometric methods, can determine the endpoint of an EDTA titration.

In the visual method, metal-ion indicators (metallochromic dyes), which have distinct colors in their free and complex forms, are added to the mixture to signal the titration's end point. They form stable complexes with metal ions, but these complexes are weaker than the corresponding metal–EDTA complexes. As a result, EDTA will displace the indicator to form a more stable metal–EDTA complex as the endpoint is reached.

Since the color of the free indicators also depends on pH, titration must be performed in the appropriate pH range.

If the titration solution is already intensely colored or the endpoint color change is subtle, the visual method might not be precise enough. In such cases, the spectroscopic method is employed. Here, the endpoint is detected by monitoring the absorbance of the mixture at a particular wavelength.

Another method is potentiometric titration, which detects the endpoint by measuring the change in the potential of the metal ion during the titration using an appropriate electrode for the metal.

 Core: Analytical Chemistry

Tandem Mass Spectrometry

Tandem mass spectrometry is a technique that uses multiple mass analyzers in series to obtain a higher selectivity and signal-to-noise ratio for the analyte. Instruments with multiple analyzers separated by an interaction cell enable secondary fragmentation and selected study of the fragment ions.

Secondary fragmentations occur in the interaction cell and can be induced by various factors. Fragmentation induced by collision with inert gases, such as N₂, Ar, He, etc., is called collision-induced dissociation (CID). Other techniques involve interaction with the surface through surface-induced dissociation (SID); interaction with low-energy electrons, called electron-capture dissociation (ECD); or through an intense laser beam, called photo-induced dissociation (PID).

Tandem mass spectrometry can be used for various experiments by varying the mode of operation of the two mass analyzers. When the first mass analyzer selects a particular precursor ion and the second mass analyzer scans all the fragmented product ions, it is called a product-ion scan, and the spectra obtained by this experiment are called product-ion spectra. Alternately, when the first mass analyzer scans all the precursor ions and the second mass analyzer selects a particular product ion, it is called 'precursor-ion mode', and the spectra are termed precursor-ion spectra. Another experiment can be performed by scanning both the analyzers simultaneously with an offset of a mass equal to a neutral fragment lost by the ion to be monitored. Such a mode is called 'neutral-loss scanning mode', and the corresponding spectra are called neutral-loss spectra.

The instrumentation of tandem mass spectrometry can be varied. Tandem-in-space spectrometers use two independent mass analyzers in two different regions in space, for example, a triple quadrupole mass spectrometer and TOF–TOF spectrometer. On the other hand, a tandem-in-time spectrometer, such as a quadrupole ion-trap instrument, uses the same spatial region to fragment the ions and dissociate and analyze a selected ion of interest, but at different times by expelling the unwanted ions. Tandem-in-time spectrometers can be used to perform MSⁿ experiments.   

 Core: Analytical Chemistry

EDTA: Indirect and Alkalimetric Titration

Unlike direct titration, back-titration, and displacement titration, indirect titration is an EDTA titration method for quantifying anions. In the indirect titration method, anions are precipitated as their insoluble salts with excess metal ions. The filtrate containing the excess metal ions is directly titrated with standard EDTA until the endpoint is achieved. Another approach involves extracting the metal ion and back-titrating with standard EDTA to obtain the endpoint. In this way, the amount of anion is indirectly estimated.

Another EDTA titration method is alkalimetric titration. This titration method is similar to acid–base titration. During the titration, disodium EDTA is added to a metal ion solution, forming the metal–EDTA complex. This process involves the liberation of two equivalents of hydrogen ions in the solution, which is titrated using a base such as standard sodium hydroxide in the presence of appropriate acid–base indicator until the end-point concentration is obtained.

 Core: Analytical Chemistry

Structure and Organization of Smooth Muscles

Smooth muscle tissue is a type of muscle tissue that can be found lining various vital organs in the human body, including the lungs, blood vessels, digestive tract, and respiratory tract. This type of tissue is responsible for regulating the movements of these organs, playing crucial roles in the functioning of various systems, including the vascular, digestive, respiratory, and urinary systems.

Structure of smooth muscle cell

Smooth muscle cells are spindle-shaped with tapering ends and a single nucleus in the center. They typically range from 30 to 200 µm in length and have a diameter of about 5-10 µm. Unlike the striated appearance of skeletal and cardiac muscles, smooth muscle fibers have a uniform, non-striated appearance. This appearance is due to the lack of organized sarcomeres. The contractile proteins actin and myosin, which form the thin and thick filaments, respectively, are arranged obliquely and irregularly along the long axis of the cell. The thin filaments are anchored to the sarcolemma by dense bodies interconnected via the intermediate filament network. Instead of T-tubules, these muscle fibers have caveolae — small pockets of invaginated sarcolemma that help influx Ca²⁺ ions during contraction cycles.

Smooth muscles can be further categorized into two types: multi-unit and single-unit (visceral) smooth muscle.

Multi-unit smooth muscle

In contrast, multi-unit smooth muscle fibers consist of individual muscle fibers that operate more independently of each other. Each fiber has its own nerve supply, and there are fewer gap junctions between cells. This arrangement allows for more precise and localized control of contraction. Multiunit smooth muscle is found in areas where fine control is necessary, such as in the iris of the eye, where it controls pupil size, and in the walls of blood vessels, where it regulates blood flow and pressure.

Visceral smooth muscle

Visceral smooth muscle is the most common type of smooth muscle. It is found in the walls of hollow organs such as the stomach, intestines, uterus, and bladder. These cells are connected by gap junctions, which allow electrical and chemical signals to pass directly from one cell to another. As a result, when one cell becomes excited and contracts, the signal quickly spreads to neighboring cells, causing the muscle layer to contract as a synchronized unit. This property is essential for functions like peristalsis in the gastrointestinal tract, where coordinated waves of contraction move contents through the system.

 Core: Anatomy and Physiology

Muscles that Move the Arm

Nine muscles are involved in arm movements. Two of these, the pectoralis major and latissimus dorsi, originate from the axial skeleton and are called axial muscles. The other seven originate from the scapula and are called the scapular muscles.

The pectoralis major has two origins. Its clavicular head originates on the medial half of the clavicle. In contrast, the sternocostal head originates on the costal cartilages of ribs 1-6, the sternum, and the aponeurosis of the external oblique of the anterior abdominal wall. Both these heads merge and insert on the intertubercular sulcus of the humerus. Its clavicular head flexes the humerus, and the sternocostal head extends a flexed arm back to the anatomical position. When the head works together, they adduct and medially rotate the humerus.

The latissimus dorsi originates from the spinous processes of the thoracic vertebrae T7 to T12, crests of sacrum and ilium, and ribs 9 to 12. It inserts anteriorly on the intertubercular sulcus of the humerus. Acting with the scapular muscle, teres major, it extends, adducts, and medially rotates the humerus. The origin of the scapular muscle, teres major, is at the inferior angle of the scapula. It inserts into the humerus at the medial lip of its intertubercular sulcus. Its actions are the same as that of latissimus dorsi.

The scapular deltoid is the most superficial muscle of the shoulder joint and is responsible for its rounded shape. Its anterior fibers originate on the lateral of the clavicle — the lateral fibers originate from the acromion of the scapula, and the posterior fibers originate from the spine of the scapula. All the heads merge and insert on the deltoid tuberosity of the humerus. While the lateral fibers abduct the arm, the anterior fibers flex and medially rotate it, and the posterior fibers extend and laterally rotate it.

The scapular muscles — supraspinatus, infraspinatus, teres minor, subscapularis, and their corresponding tendons, combine to create the rotator cuff. The origin of the supraspinatus muscle lies on the supraspinous fossa of the scapula. Conversely, the infraspinatus muscle originates from its infraspinous fossa. Both these muscles are inserted at the greater tubercle of the humerus, aiding in arm abduction and lateral rotation, respectively.

Another muscle, the teres minor, also inserts at the greater tubercle of the humerus but originates from the inferior angle of the scapula. It acts along with the infraspinatus muscle to laterally rotate and extend the arm. The origin of the subscapularis muscle is at the subscapular fossa of the scapula. It inserts on the lesser tubercle of the humerus and aids in the medial rotation of the arm. Finally, the last scapular muscle involved in arm movements is the coracobrachialis. The origin of this muscle is at the coracoid process of the scapula, and the insertion is on the middle surface of the medial shaft of the humerus. It helps with arm flexion and adduction.

The rotator cuff muscles are prone to injuries, especially in people active in sports. Due to its positioning between the head of the humerus and the acromion of the scapula, the supraspinatus muscle is particularly prone to strain and degenerative changes.

 Core: Anatomy and Physiology

Action Potential: Phases of Stimulation

The action potential is a complex electrical event that occurs in excitable cells, such as neurons and muscle cells. It consists of several distinct phases, each with specific characteristics.

Resting Phase:

In this phase, the cell's membrane is at its resting potential, typically around -70 millivolts (mV) for neurons. Inside the cell, there is a higher concentration of potassium ions (K⁺) and a lower concentration of sodium ions (Na⁺). Voltage-gated sodium channels are closed, and voltage-gated potassium channels are closed but capable of opening.

Depolarization Phase:

A graded potential, often an excitatory postsynaptic potential (EPSP), reaches the threshold level (typically around -55 mV). This triggers the voltage-gated sodium channels to open rapidly, allowing an influx of sodium ions into the cell. This rapid sodium influx causes a sharp increase in membrane potential, turning it more positive. The influx of sodium ions further depolarizes the membrane, leading to a positive feedback loop that triggers more sodium channels to open.

Peak of the Action Potential:

At the peak of the action potential, the sodium channels begin to inactivate or close, reducing sodium influx. Voltage-gated potassium channels start to open slowly in response to the increasing membrane potential.

Repolarization Phase:

As voltage-gated potassium channels open fully, potassium ions exit the cell. This movement of positively charged ions out of the cell helps to restore the negative membrane potential. The membrane potential gradually returns to the resting potential of around -70 mV.

Hyperpolarization Phase (Undershoot):

The movement of potassium ions continues for a brief period, causing the membrane potential to dip below the resting potential, typically around -80 mV. The delayed closure of some potassium channels contributes to this temporary hyperpolarization.

Refractory Period:

During and immediately after an action potential, it is impossible to trigger another one. This prevents the action potential from moving backward. This is called the absolute refractory period.

Following the absolute refractory period, it is possible to initiate another action potential, but it requires a stronger stimulus than usual. This is known as the relative refractory period.

The phases of an action potential are essential for transmitting electrical signals in neurons. This rapid and coordinated sequence of events allows for the unidirectional propagation of signals along the length of the neuron, enabling communication within the nervous system and with other cells.

 Core: Anatomy and Physiology

Diencephalon: Anatomical Regions

The diencephalon, etymologically translated as 'through brain,' plays an integral role as the conduit between the cerebrum and the vast extent of the nervous system. However, the olfactory system is an exception, as it interfaces directly with the cerebrum. The diencephalon, deeply ensconced beneath the cerebrum, primarily consists of three paired structures — the thalamus, hypothalamus, and epithelamus. It also includes accessory structures such as the subthalamus, which houses the subthalamic nucleus as part of the basal nuclei.

Thalamus

The thalamus operates as a central processing unit, encompassing various nuclei that transmit information between the cerebral cortex, spinal cord, and brainstem. All sensory information, except olfaction, is routed through the thalamus for preprocessing before it reaches the cortex. Axons from the peripheral sensory organs, or intermediary nuclei, synapse in the thalamus, and thalamocortical neurons project directly to the cerebrum. It is essential to note that the thalamus is more than a passive courier; it actively processes incoming information. For instance, the segment of the thalamus receiving visual data will dictate which stimuli warrant attention.

The thalamus also receives information from the cerebellum, typically in the form of motor commands. This interaction involves crosstalk with the cerebellum and other brain stem nuclei. An intricate network of connections exists between the cortex and basal nuclei involving the thalamus. The neuronal information relayed by the basal nuclei is directed toward the thalamus, which subsequently relays this output to the cerebral cortex. The cortex, in turn, transmits data to the thalamus, which influences the activity of the basal nuclei.

Hypothalamus

The hypothalamus, another primary region of the diencephalon, is positioned anterior and slightly below the thalamus. The hypothalamus includes a series of nuclei primarily tasked with maintaining homeostasis. This region serves as the command center governing the autonomic nervous system and the endocrine system via regulation of the anterior pituitary gland. Additional sections of the hypothalamus participate in memory and emotional processes as constituents of the limbic system.

Epithalamus

The epithalamus, the smallest part of the diencephalon, is situated posterior to the thalamus. This area contains the habenular nuclei, which relay information from the limbic system to the midbrain. The habenula is also thought to influence reward and punishment processing behavior. Furthermore, the epithalamus houses the pineal gland, which helps regulate sleep-wake cycles through melatonin release. It is one of the few parts of the brain thought to maintain some degree of neuroplasticity throughout life. The epithalamus is an essential part of the circuitry connecting the limbic system to motor regions, and it plays a key role in integrating sensory input from other areas. Additionally, it helps control endocrine functions by regulating hormones produced by the pituitary gland. Research also suggests that the epithalamus plays a role in emotion, learning, and memory formation.

 Core: Anatomy and Physiology

Spinal Nerves: Plexus I

Nerve plexuses are networks of interlacing nerves that serve as communication hubs to distribute and organize nerve action across various body regions. The nerve plexuses are organized into the cervical plexus located in the neck region, brachial plexus in the shoulder area, lumbar plexus found in the lower back, sacral plexus situated in the pelvis, and coccygeal plexus located in the coccygeal region.

The Cervical Plexus

The cervical plexus, formed by the anterior rami of the first four cervical spinal nerves (C1-C4), and partially the fifth cervical spinal nerve (C5), is situated in the neck region. This plexus innervates the skin and muscles of the head, neck, and shoulders, facilitating both sensory and motor functions.

• • Motor Innervation: The phrenic nerve is a primary motor nerve emerging from the cervical plexus. It innervates the diaphragm and is essential in controlling the respiratory process. Without the proper function of this nerve, breathing would be significantly impaired.

Other motor components of the cervical plexus include the ansa cervicalis and segmental branches. The ansa cervicalis innervates some infrahyoid muscles, aiding in swallowing and speech. The segmental branches supply motor fibers to the deep muscles of the neck, contributing to the stability and movement of the head.

• • Sensory Innervation: The cervical plexus also comprises four primary sensory nerves: the lesser occipital, great auricular, transverse cervical, and supraclavicular nerves. These nerves provide sensation to the skin of the neck, ear area, and parts of the shoulder and chest, ensuring sensory input from these regions reaches the central nervous system.

The Brachial Plexus

The brachial plexus extends from the neck into the axilla and is formed by the anterior rami of the fifth to eighth cervical nerves (C5-C8) and the first thoracic nerve (T1). This plexus supplies the shoulder and upper limbs, orchestrating a wide range of movements and sensory functions. The brachial plexus gives rise to five significant nerves:

• • Axillary Nerve: This innervates the deltoid and teres minor muscles, enabling shoulder abduction and playing a role in the sensation of the shoulder area.
• • Musculocutaneous Nerve: This nerve supplies the flexor muscles in the front of the arm, facilitating elbow flexion, and provides sensory information from the lateral forearm.
• • Radial Nerve: By innervating the muscles on the posterior aspect of the upper limb, this nerve supports elbow extension, wrist and finger extension, and thumb abduction. It also conveys sensory input from the posterior arm and hand.
• • Median Nerve: This nerve is primarily responsible for flexing the wrist and fingers, along with thumb opposition. It also carries sensory information from the palmar aspect of the hand.
• • Ulnar Nerve: The intricate movements of the fingers and wrist are controlled by this nerve. It provides sensation to the little finger and part of the ring finger, highlighting its importance in grip and hand movements.

 Core: Anatomy and Physiology

Higher Mental Functions of the Brain: Learning and Memory

Memory is one of the most vital higher mental functions of the brain. Memory is closely related to learning because it enables us to retain information and experiences from our past to use them in our present life. It also helps us to remember facts, events, and skills, such as riding a bike or swimming. There are two types of memory — declarative memory, which involves memorizing facts or events, and procedural memory, which enables us to remember how to do something like writing or playing an instrument.

Memory can be further divided into short-term and long-term memory. Short-term memory temporarily stores information, while long-term memory stores the information for longer. The hypothalamus consolidates short-term memories in our brain, which means that the nervous system will transfer information from the hypothalamus to the cerebral cortex for permanent storage. This means that consolidated short-term memories get converted into long-term memories.

Amnesia, characterized by forgetfulness, refers to the absence or impairment of memory. It can manifest as a complete or partial inability to recall past experiences. Anterograde amnesia involves explicitly the loss of memory for events that transpire after the underlying trauma or disease, failing to form new memories. On the other hand, retrograde amnesia pertains to the loss of memory for events before the trauma or disease, leading to an inability to recollect past events.

 Core: Anatomy and Physiology

Focusing of Light in the Eye

Light rays enter the eye through the cornea, a transparent dome-shaped tissue that is the eye's outermost layer. The cornea bends or refracts, light rays traveling to the pupil. The shape of the cornea determines how much of the light is bent and whether the image will be focused correctly on the retina at the back of the eye. Once the light has passed through both refraction layers, it converges into a single focal point onto a small area. This is where photoreceptors start transforming visible photons into electrical signals sent along nerve fibers up to your brain for interpretation - ultimately resulting in what is known as sight.

The refractive power of the human eye is its ability to bend light rays as they enter the eye to focus them onto the retina at the back of the eye. This is necessary because light entering the eye is initially divergent, but the images we perceive must be clear and focused. The refractive power of the human eye is measured in diopters (D).

The cornea is the primary refractive surface of the eye, providing approximately two-thirds of the eye's refractive power. The lens of the eye, located behind the iris, provides the remaining one-third of the eye's refractive power.

The lens can change shape, known as accommodation, allowing the eye to focus on objects at different distances. The eye's crystalline lens is located directly behind the iris and pupil and further focuses light onto the retina. Unlike a rigid camera lens, our crystalline lenses are elastic and can change shape depending on one's vision needs for near or far objects.

When looking at an object closer than 20 feet away from you (near vision), your eyes must accommodate by becoming more curved to focus on near objects properly. When looking at an object farther than 20 feet away from you (distant vision), your eyes must become less curved to focus on distant objects properly.

The amount of bend or curve placed upon incoming light rays depends on one's refractive power index: The higher power index means more curvature required of incoming light, and vice versa. People with myopia (nearsightedness) have too much anatomical curvature in their eyes. This causes incoming light to focus too soon before reaching the retina, causing blurry vision when looking at distant objects. Inversely, people with hyperopia (farsightedness) have anatomical curvature that is too slight in their eyes. This causes incoming light rays to not bend enough before reaching their retinas and, as a result, causes blurry vision when looking at nearby objects.

Some other types of refractive errors can occur in the human eye, including:

1. Astigmatism occurs when the cornea, the eye's clear front surface, is shaped like a football instead of spherical, causing blurry and distorted vision at any distance.
2. Presbyopia is an age-related condition that typically occurs when the eye lens is less flexible, resulting in difficulty focusing on close-up objects.

These refractive errors can be corrected using appropriate glasses or contact lenses or surgery in some extreme cases.

 Core: Anatomy and Physiology

The Pituitary Gland

The pituitary is a small endocrine organ in the sphenoid bone under the hypothalamus. Primarily, the pituitary in adults has two distinct anatomical and functional regions— the anterior and posterior lobes. During human fetal development, a third pituitary gland region called the pars intermedia atrophies and disappears. However, some of its cells migrate and exist adjacent to the anterior pituitary in adults.

• The anterior lobe comprises the pars distalis and the pars tuberalis. These areas consist of glandular epithelial tissue that produces various hormones. The anterior lobe is linked to the hypothalamus through the hypophyseal portal system. The hormones secreted by the ventral hypothalamus cells travel through this portal system and regulate hormone-secreting cells of the anterior lobe.

The posterior pituitary lobe has two parts: the infundibulum, a funnel-shaped stalk, and the pars nervosa. The infundibulum allows the passage of the supra-optic and paraventricular nuclei axons of the hypothalamic-hypophyseal tract, maintaining a neural connection with the hypothalamus. The hypothalamic-hypophyseal tract delivers hormones secreted by hypothalamic neurosecretory cells into the posterior pituitary lobe for storage and release.

 Core: Anatomy and Physiology

Ligand-gated Ion Channels

 Core: Anatomy and Physiology

Voltage

The movement of electrons in a conductor requires some form of energy or work, usually provided by an external force, like a battery. This force is called the electromotive force or voltage. The voltage between two points, referred to as points "a" and "b," in an electric circuit is the energy (or work) needed to move a unit charge from point "a" to point "b," and this relationship is expressed mathematically as

In this equation, "w" represents the energy measured in joules (J), and "q" represents the charge measured in coulombs (C). The voltage, denoted as "v_ab" is measured in volts (V).

Voltage, often referred to as the potential difference, signifies the energy required to move a unit charge through an element within the circuit. It is important to note that the value of a voltage can be either positive or negative, with its direction determined by its polarities, indicated as (+) and (-). In electrical terminology, it is customary to state that a voltage exists across an element. The notation "v_ab" represents the voltage between points "a" and "b" and can be interpreted in two distinct ways:

• • Point "a" is at a potential of "v_ab" volts higher than point "b."
• • The potential at point "a" with respect to point "b" is "v_ab" volts.

A constant voltage is categorized as a direct current (DC) voltage, typically represented as "V." DC voltages are commonly generated by sources such as batteries. On the other hand, a voltage that varies sinusoidally with time is called an alternating current (AC) voltage, represented as "v." AC voltages are typically produced by electric generators.

 Core: Electrical Engineering

Characteristics of Practical Op Amps

A difference amplifier, a crucial component in numerous electronic devices, ideally amplifies only the difference-mode signal, which is the difference between two input signals. However, in practical circuits, the output voltage depends on both the differential gain and the common-mode gain.

The ratio of differential gain to the common-mode gain is defined as the common-mode rejection ratio (CMRR). This ratio quantifies the ability of operational amplifiers (op-amps) to reject common-mode signals, which are identical signals present at both inputs of the amplifier.

For instance, consider a sound system where an op-amp picks up both music and electrical noise. A high CMRR allows the op-amp to effectively ignore the noise and amplify only the music, enhancing the sound quality of the system.

Another crucial characteristic of op-amps is the gain-bandwidth product. This is the product of the op-amp's open-loop voltage gain and the frequency at which this gain is measured. It remains constant for any given op-amp, providing a useful parameter for comparing the performance of different op-amps.

A unity gain buffer, or voltage follower, is a specific configuration of an op-amp that provides a voltage gain of one. This means that the output voltage matches the input voltage. It has low output impedance and high input impedance, making it ideal for signal isolation and impedance matching.

In an audio system, a voltage follower plays a vital role in preventing a low-impedance speaker from effectively shorting the audio signal from a high-impedance source to the ground. By matching the impedance levels, it ensures that maximum power is transferred from the source to the speaker without distortion or loss.

In conclusion, understanding the characteristics and functionalities of op-amps, such as the CMRR, gain-bandwidth product, and unity gain buffer configuration, is essential in designing effective and efficient electronic systems. These principles form the backbone of many modern audio and communication systems, influencing their performance and quality.

 Core: Electrical Engineering

Second-Order Circuits

Integrating two fundamental energy storage elements in electrical circuits results in second-order circuits, encompassing RLC circuits and circuits with dual capacitors or inductors (RC and RL circuits). Second-order circuits are identified by second-order differential equations that link input and output signals.

Input signals typically originate from voltage or current sources, with the output often representing voltage across the capacitor and/or current through the inductor. For example, in an RLC circuit, initial energy stored in the capacitor and inductor initiates the circuit. Applying Kirchhoff's voltage law and performing a time derivative yields a second-order differential equation.

 Its coefficients, determined by resistance, capacitance, and inductance, manifest as the damping coefficient and resonant frequency.

The damping coefficient plays a critical role in these circuits, signifying the extent of damping caused primarily by the resistor. It directly influences the pace at which energy dissipates within the system, effectively controlling the rate of energy loss. On the other hand, the resonant frequency is a key characteristic that represents the circuit's innate oscillation frequency. It measures how quickly energy is exchanged between the inductor and capacitor in the circuit, illustrating the circuit's natural tendency to oscillate at a particular frequency.

The damping coefficient dictates how fast energy is lost in the system due to resistance. At the same time, the resonant frequency highlights the circuit's inherent oscillation speed as energy shifts between the inductor and capacitor. These two factors are crucial in understanding and analyzing the behavior of second-order circuits.

 Core: Electrical Engineering

Norton Equivalent Circuits

Norton's theorem is a fundamental concept in the field of electrical engineering that allows for the simplification of complex AC circuits. The theorem states that any two-terminal linear network can be replaced with an equivalent circuit that consists of an impedance, which is parallel with a constant current source. Figure 1 shows the AC circuit portioned into two parts: Circuit A and Circuit B, while Figure 2 depicts the circuit obtained by replacing Circuit A by its Norton equivalent circuit.

Figure 1: A circuit portioned into two parts 

Figure 2: Norton equivalent circuit

To calculate the value of the parallel impedance, one must replace the source with its internal impedance, resulting in a circuit with an equivalent impedance known as the Norton impedance. The Norton impedance is the same as the Thévenin impedance and is used to determine the Norton current, which is the current flowing through the circuit.

Determining the Norton current requires placing the sources back into the circuit and analyzing the open-circuit voltage, also known as the Thévenin voltage. The value of the Thévenin voltage is determined by multiplying the source current by the Thevenin impedance and is used to drop the same voltage across the load impedance when it is placed in a parallel configuration.

By using the relationship between the Norton current, the Thévenin voltage, and the Norton current values, one can determine the Norton current of the circuit. This relationship makes Norton's theorem beneficial for analyzing and designing systems containing complex AC circuits since it simplifies their analysis by breaking the circuit down into smaller, more manageable sections.

 Core: Electrical Engineering

Random and Systematic Errors

 Core: Mechanical Engineering

Mixtures of Acids

The pH of a solution containing an acid can be determined using its acid dissociation constant and initial concentration. If a solution contains two different acids, then its pH can be determined using one of several methods depending on the relative strength of the acids and their dissociation constants.

In a strong and weak acid mixture, the strong acid dissociates completely and becomes a source of almost all the hydronium ions present in the solution. In contrast, the weak acid shows partial dissociation and produces a negligible concentration of hydronium ions. The high concentration of hydronium ions produced by the strong acid further reduces the dissociation of the weak acid. According to Le Chatelier's principle, this happens: "When a chemical system at equilibrium is disturbed, the system shifts in a direction that minimizes the disturbance." The excess hydronium ions produced by the strong acid disturb the equilibrium, and thus, the reaction will move in the reverse direction until the equilibrium is established. This leads to a decrease in the dissociation of the weak acid. Because of this decrease, the pH of a strong and weak acid mixture can be calculated from the concentration of the strong acid only. For example, the pH of a mixture with an equal concentration of hydrochloric acid (HCl), a strong acid, and formic acid (HCHO₂), a weak acid, can be determined from the concentration of HCl only. If the concentration of the HCl in the mixture is 0.0020 M, its pH can be calculated as follows.

pH = −log(0.002) = 2.7

Here, the concentration of hydronium ions produced by HCHO₂ and the autoionization of water is negligible and thus can be ignored.

A Mixture of Two Weak Acids with Different Dissociation Constants

In a mixture of two weak acids, the pH of a mixture will be determined by the stronger acid if its dissociation constant is significantly higher than the weaker acid. For example, in a mixture with an equal concentration of nitrous acid (HNO₂) and hypochlorous acid (HClO), the HNO₂ will be the main determinant of the pH of the mixture as its K_a (4.6 × 10⁻⁴) is approximately 10,000 times higher than the K_a (2.9 × 10⁻⁸) of HClO. According to Le Chatelier's principle, HClO shows decreased dissociation in the presence of HNO₂.

 Core: Analytical Chemistry

Isolating Nucleic Acids from Yeast

One of the many advantages to using yeast as a model system is that large quantities of biomacromolecules, including nucleic acids (DNA and RNA), can be purified from the cultured cells.

This video will address the steps required to carry out nucleic acid extraction. We will begin by briefly outlining the growth and harvest, and lysis of yeast cells, which are the initial steps common to the isolation of all biomacromolecules. Next, we will discuss two unique purification methods for the separation of nucleic acids: column binding and phase separation. Additionally, we will demonstrate several ways in which these methods are applied in the laboratory, including the preparation of nucleic acids for molecular biology techniques such as PCR and southern blotting, quantification of gene expression in response to environmental stimuli, and purification of large amounts of recombinant proteins.

 Biology I

Basic Mouse Care and Maintenance

Mice (Mus musculus) are small rodents that breed and sexually mature quickly, making them perfectly suited to generating large animal colonies for biological research. As compared to other mammalian species, mice are simple and inexpensive to maintain in the laboratory. Nevertheless, mouse colonies do have specific husbandry needs that are critical to preserving animal health and safety as well as experimental reproducibility.

This video demonstrates standard practices that ensure mice are treated as humanely as possible within the laboratory animal facility, or vivarium. The discussion begins by reviewing a typical mouse housing setup, consisting of a plastic cage equipped with a layer of soft bedding and nesting material. The preformulated food pellets (also known as chow) that comprise the typical mouse diet are also introduced. In order to facilitate experiments performed on mice, safe animal handling practices are demonstrated, including common restraint techniques like “scruffing,” and the strategies used by researchers to keep track of individual mice within the facility. Finally, experimental manipulations of mouse housing and diet are discussed, in addition to one of the most common applications of the scruffing technique — performing injections.

 Biology II

The Morris Water Maze

The Morris water maze is one of the most widely used behavioral tests for studying spatial learning and memory. In the initial phases of this task, rodents must swim to a platform to escape from a pool of water. The platform is then hidden under the water’s surface, so that the animal is required to remember it’s location in order to escape.This simple yet powerful maze design can be used to assay cognitive function, study animal models of neurodegenerative disease, and test potential drug therapies.

This video provides an introduction to the Morris water maze and the principles surrounding its use, including a discussion of the different types of memory tested in the maze, important points to consider when designing and conducting this experiment, and the procedures for setup and running of the test. Several applications of the maze are examined, such as investigating how radiation treatment may lead to memory impairment. Finally, other types of water mazes, such as the 8-arm radial maze, are introduced to show how this paradigm can be adapted to engage different types of memory.

 Neuroscience

Induced Pluripotency

Induced pluripotent stem cells (iPSCs) are somatic cells that have been genetically reprogrammed to form undifferentiated stem cells. Like embryonic stem cells, iPSCs can be grown in culture conditions that promote differentiation into different cell types. Thus, iPSCs may provide a potentially unlimited source of any human cell type, which is a major breakthrough in the field of regenerative medicine. However, more research into the derivation and differentiation of iPSCs is still needed to actually use these cells in clinical practice.

This video first introduces the fundamental principles behind cellular reprogramming, and then demonstrates a protocol for the generation of iPSCs from differentiated mouse embryonic fibroblasts. Finally, it will discuss several experiments in which scientists are improving or applying iPSC generation techniques.

 Developmental Biology

Balance and Coordination Testing

Balance and coordination are critical components involved in the control of movement. Many sensory receptors and neural processing units are required to help individuals maintain balance while performing various activities. Deficits in balance and coordination occur in patients suffering from movement disorders or due to aging. Therefore, scientists are trying to understand the pathophysiology behind these conditions. One way to do that is by using rodent models and testing them on behavioral paradigms such as the rotarod or balance beam.

This video discusses the currently known neurophysiology behind balance and coordination. Then, we go over protocols to run balance tests in rodents using the rotarod and balance beam. Finally, we'll discuss some current studies utilizing these methods to investigate aging, muscular dystrophy and Parkinson's disease.

 Behavioral Science

Solid-Liquid Extraction

Source: Laboratory of Dr. Jay Deiner — City University of New York

Extraction is a crucial step in most chemical analyses. It entails removing the analyte from its sample matrix and passing it into the phase required for spectroscopic or chromatographic identification and quantification. When the sample is a solid and the required phase for analysis is a liquid, the process is called solid-liquid extraction. A simple and broadly applicable form of solid-liquid extraction entails combining the solid with a solvent in which the analyte is soluble. Through agitation, the analyte partitions into the liquid phase, which may then be separated from the solid through filtration. The choice of solvent must be made based on the solubility of the target analyte, and on the balance of cost, safety, and environmental concerns.

 Organic Chemistry

Annexin V and Propidium Iodide Labeling

Staining with annexin V and propidium iodide (PI) provides researchers with a way to identify different types of cell death—either necrosis or apoptosis. This technique relies on two components. The first, annexin V, is a protein that binds certain phospholipids called phosphatidylserines, which normally occur only in the inner, cytoplasm-facing leaflet of a cell’s membrane, but become “flipped” to the outer leaflet during the early stages of apoptosis. The second component is the DNA-binding dye molecule PI, which can only enter cells when their membranes are ruptured—a characteristic of both necrosis and late apoptosis.

This video article begins with a review of the concepts behind annexin V and PI staining, and emphasizes how differential patterns of staining can be used to distinguish between cells progressing down different death pathways. We then review a generalized protocol for this technique, followed by a description of how researchers are currently using annexin V and PI staining to better understand cell death.

 Cell Biology

Reconstitution of Membrane Proteins

Reconstitution is the process of returning an isolated biomolecule to its original form or function. This is particularly useful for studying membrane proteins, which enable important cellular functions and affect the behavior of nearby lipids. To study the function of purified membrane proteins in situ, they must be reconstituted by integrating them into an artificial lipid membrane.

This video introduces membrane protein reconstitution concepts and related procedures, such as protein isolation using detergent, formation of artificial vesicles using lipids, incorporation of the isolated protein into the artificial vesicle, and separation of the detergent from the solution. Finally, two applications are covered: reconstitution of membrane transport proteins and reconstitution of light-harvesting proteins. 

 Biochemistry

Synthetic Biology

This video presents synthetic biology and its role in bioengineering. Synthetic biology refers to the methods used to genetically modify organisms in order to make them capable of producing large quantities of a product. This product could be a protein that the cell already makes or a new protein that has been encoded in a newly-inserted DNA sequence.

Here, we discuss how an organism's genetic material is modified using transformation or transfection. Then, the process is shown in the laboratory, and the applications of the technique discussed.

 Bioengineering

Energy Diagrams, Transition States, and Intermediates

Free-energy diagrams, or reaction coordinate diagrams, are graphs showing the energy changes that occur during a chemical reaction. The reaction coordinate represented on the horizontal axis shows how far the reaction has progressed structurally. Positions along the x-axis close to the reactants have structures resembling the reactants, while positions close to the products resemble the products.  Peaks on the energy diagram represent stable structures with measurable lifetimes, while other points along the graph represent unstable structures that cannot be isolated.

This high-energy unstable structure is called the transition state or activated complex. In this high-energy process, bonds are in the process of being broken and/or formed simultaneously. The structure is so strained that it transitions into new, less strained structures.

George Hammond formulated a principle that relates the nature of a transition state to its location on the reaction diagram. The Hammond Postulate states that a transition state will be structurally and energetically similar to the species nearest to it on the reaction diagram. In the case of an exothermic reaction, the transition state resembles the reactant species, whereas, in the case of an endothermic reaction, the transition state resembles the products. In a multi-step reaction, each step has a transition state and corresponding activation energy. The transition states of such reactions are punctuated with reactive intermediates, which are represented as local minima on the energy diagrams.

Reactive intermediates are products of bonds breaking and cannot be isolated for prolonged periods of time. Some of the most common reactive intermediates in organic chemistry are carbon ions or radicals. Carbocations are electrophiles, and carbanions are nucleophiles. Carbon radicals have only seven valence electrons and may be considered electron deficient; however, they do not, in general, bond to nucleophilic electron pairs, so their chemistry exhibits unique differences from that of conventional electrophiles. Radical intermediates are often called free radicals.

 Core: Organic Chemistry

Nucleophiles

The word “nucleophile” has a Greek root and translates to nucleus-loving. Nucleophiles are either negatively charged or neutral species with a pair of electrons in a high-energy occupied molecular orbital (HOMO). As these species tend to donate electron pairs, nucleophiles are considered Lewis bases as well. Negatively charged species, like OH⁻, Cl⁻, or HS⁻, with one or several pairs of electrons, are typically nucleophiles. Similarly, neutral species such as ammonia, amines, water, and alcohol have non-bonding lone pairs of electrons and can act as nucleophiles. Furthermore, molecules without a lone pair of electrons can still act as nucleophiles, such as alkenes and aromatic rings with bonding π orbitals.

The relative strength of a nucleophile to displace a leaving group in a substitution reaction is called nucleophilicity. The negatively charged species are more nucleophilic than their neutral counterpart species. As an empirical rule, the higher the pK_a of a conjugate acid, the better the nucleophile. For example, the hydroxide ion — a conjugate base of water (pK_a 15.7) is a better nucleophile than the acetate ion — a conjugate base of acetic acid (pK_a~5).

Since nucleophilicity is not the inherent property given to a specific species, it is affected by many factors, including the type of solvent in which the reaction is conducted. In polar protic solvents, high solvation of anions reduces the nucleophile’s availability to participate in substitution reactions.

When comparing halides, fluoride, being the smallest and most electronegative anion, is solvated the strongest, while iodide, the largest and least electronegative ion, is solvated the least. Thus, in polar protic solvents, iodide is the best nucleophile. In polar aprotic solvents, however, anions are “naked” due to poor solvation and can participate freely in a nucleophilic attack. In polar aprotic solvents, the nucleophile’s basicity dictates its nucleophilicity making fluoride the best nucleophile.

Furthermore, the polarizability of atoms affects nucleophilicity. Polarizability describes how easily electrons in the cloud can be distorted. A nucleophile with a large atom has greater polarizability, meaning it can donate a higher electron density to the electrophile compared to a small atom, whose electrons are held more tightly.

 Core: Organic Chemistry

Hydroboration-Oxidation of Alkenes

In addition to the oxymercuration–demercuration method, which converts the alkenes to alcohols with Markovnikov orientation, a complementary hydroboration-oxidation method yields the anti-Markovnikov product. The hydroboration reaction, discovered in 1959 by H.C. Brown, involves the addition of a B–H bond of borane to an alkene giving an organoborane intermediate. The oxidation of this intermediate with basic hydrogen peroxide forms an alcohol.

Borane as a reagent is very reactive, as the boron atom has only six electrons in its valence shell. The unoccupied 2p orbital of the boron is perpendicular to the plane, which is occupied by the boron and the three other hydrogens oriented at an angle of 120°. Thus, borane is electrophilic with its structure resembling a carbocation without any charge.

Due to high reactivity, two borane molecules dimerize such that two hydrogen atoms are partially bonded to two boron atoms with a total of two electrons. Hence, they are called three-center, two-electron bonds. Diborane co-exists in equilibrium with a small amount of borane. 

The electron-deficient borane easily accepts an electron pair from tetrahydrofuran to complete its octet forming a stable borane-ether complex. This is used as a reagent in hydroboration reactions under an inert atmosphere to avoid spontaneous ignition in the air.

Hydroboration Mechanism

The mechanism starts with a borane attacking the π bond at the less substituted and sterically less hindered site of an alkene forming a cyclic transition state. The overall result is a syn-addition of BH₂ and hydrogen across the alkene double bond, producing an alkylborane. The reaction of a second alkene with the alkylborane yields a dialkylborane followed by the addition of a third alkene to produce a trialkylborane.

Oxidation Mechanism

The oxidation begins with the deprotonation of hydrogen peroxide by a hydroxide ion forming a hydroperoxide. The hydroperoxide acts as a nucleophile and attacks the trialkylborane, resulting in an unstable intermediate. This is followed by the migration of an alkyl group from boron to the adjacent oxygen atom, releasing a hydroxide ion. This series of three steps is repeated to convert the remaining trialkylborane into a trialkoxyborane.

The boron atom of the trialkoxyborane is attacked by the nucleophilic hydroxide ion with the subsequent departure of the alkoxide ion neutralizing the formal charge on the boron atom. Finally, protonation of the alkoxide ion gives an alcohol as the final product.

 Core: Organic Chemistry

Electrophilic Addition to Alkynes: Hydrohalogenation

Electrophilic addition of hydrogen halides, HX (X = Cl, Br or I) to alkenes forms alkyl halides as per Markovnikov's rule, where the hydrogen gets added to the less substituted carbon of the double bond. Hydrohalogenation of alkynes takes place in a similar manner, with the first addition of HX forming a vinyl halide and the second giving a geminal dihalide.

Addition of HCl to an Alkyne

Mechanism I – Vinylic carbocation Intermediate

The mechanism begins with a proton transfer from HCl to the alkyne. Here, the π electrons attack the hydrogen atom of hydrogen chloride and displace the chloride ion. This gives a stable secondary vinylic carbocation, which is further attacked by the chloride ion to form a vinyl chloride.

The addition of a second equivalent of hydrogen chloride proceeds with the protonation of vinyl chloride, giving two possible carbocations. In the secondary carbocation the positive charge is delocalized through resonance. As a result, it is more stable and favored over the primary carbocation.

A second nucleophilic attack by the chloride ion gives a geminal dichloride.

Mechanism II – Concerted Termolecular Process

In a vinylic carbocation, the positive charge resides on an electronegative sp-hybridized carbon making it unstable. The second mechanism avoids the formation of this carbocation. Instead, it proceeds via a termolecular (three molecules) process where the alkyne interacts simultaneously with two equivalents of a hydrogen halide like HCl. This leads to a transition state with a partially broken C–C π bond and partially formed C–Cl and C–H σ bonds. The net result is a trans addition of hydrogen from one HCl and a chloride from the other HCl to form a chloroalkene. This further reacts with the displaced HCl to form a geminal dichloride as the final product.

Halogenation of Alkynes with Peroxides

When treated specifically with HBr in the presence of peroxides, terminal alkynes undergo anti-Markovnikov's addition. The Br gets added to the less substituted carbon forming a mixture of E and Z alkenes.

 Core: Organic Chemistry

The Structure of Intermediate Filaments

The intermediate filaments are one of three widely studied cytoskeletal filaments. They are so named as their diameter (10 nm) is in between that of microfilaments (7 nm) and the microtubules (25 nm).  These filaments are highly stable and can remain intact when exposed to high salt concentrations and detergents. These filaments are responsible for providing stability and mechanical support to the cells. They also help in cell adhesion and maintaining tissue integrity.

Intermediate filaments are found in almost all eukaryotic cells except lower eukaryotes like fungi and invertebrates like arthropods. In humans, around 70 genes code for different types of intermediate filament with cell type-specific expression depending on the function they perform. The mutation in these genes can lead to different diseases or disorders in humans, including Werner's syndrome, Alexander's disease, and Charcot-Marie tooth disease.

Intermediate filaments are non-polar with no defined plus or minus ends like microfilaments and microtubules; thus, no molecular motor proteins are known to be associated with them. Unlike microfilaments (F-actins) and microtubules made up of globular proteins, the monomeric units of intermediate filaments are rigid, fibrous rope-like proteins. These monomers vary among the cell types, forming a specific type of intermediate filament depending on their function. However, all monomeric units comprise a conserved alpha-helical central coiled-coil rod domain flanked by the head and tail domains. The conserved alpha-helical rod domain has 310 amino acids divided into three conserved segments with hydrophobic amino acid sequences separated by linkers. The rod domain helps in the lateral association of monomers to form the dimers and subsequently the tetramers, the basic soluble units of the intermediate filaments. The tetramers then assemble into unit-length filaments, which further associate to form the intermediate filaments.

 Core: Cell Biology

Structure and Nomenclature of Thiols and Sulfides

Thiols and sulfides are sulfur analogs of alcohols and ethers, respectively, where the sulfur atom takes the place of the oxygen atom. Thus, thiols are generally represented as RSH, where R is an alkyl substituent and —SH is the functional group. On the other hand, in sulfides, the central sulfur atom is bonded to two hydrocarbon groups on either side. Depending upon the type of group, sulfides can be either symmetrical or asymmetrical. Both thiols and sulfides display a bent geometry, similar to alcohols and ethers. However, the larger size of the sulfur atom deviates the C–S–C and C–S–H bond angles and the C–S and S–H bond lengths from the corresponding bond angle and bond length values of alcohols and ethers.

Generally, common names of thiols consist of the suffix "mercaptan," following the name of the parent alkyl group. In contrast, the IUPAC names of thiols are obtained by adding the suffix "thiol" to the parent alkane name while retaining the letter "e" of the parent alkane. Similarly, the suffix "sulfide" is added to the alkyl groups' names listed in alphabetical order to derive the common names of sulfides. The IUPAC names of sulfides are obtained by first determining the parent chain and prefixing the name of the smaller alkyl group as an alkylthio substituent to the parent alkane name.

 Core: Organic Chemistry

Diels–Alder vs Retro-Diels–Alder Reaction: Thermodynamic Factors

The Diels–Alder reaction is thermally reversible, meaning that the reaction reverts to the starting diene and dienophile under suitable temperatures. The forward reaction gives a cyclohexene derivative and is favored at low to medium temperatures. The reverse process, also called retro-Diels–Alder reaction, is a ring-opening process favored at high temperatures.

Thermodynamic factors

The influence of temperature on the spontaneity of a particular reaction can be assessed based on the change in the Gibbs free energy, ΔG. If ΔG is negative, the reaction occurs spontaneously. However, if ΔG is positive, the reaction occurs spontaneously in the opposite direction.

ΔG is a composite of two terms, ΔH and −TΔS. In a Diels–Alder reaction, two stronger σ bonds are formed at the expense of two weaker π bonds, resulting in a negative ΔH. Cycloaddition reactions proceed with a decrease in entropy. Consequently, ΔS is negative, and the term −TΔS is positive. At low temperatures, the sum of the two terms is negative, implying that the forward reaction is spontaneous. In contrast, the sum is positive at high temperatures, indicating that the reverse reaction is spontaneous.

 Core: Organic Chemistry

Conjugated Proteins

 Core: Cell Biology

Lineage Commitment

Commitment is the  process whereby stem cells:

1.  lose their ability to form all cell types and
2. irreversibly change into a specific type.

The multipotent hematopoietic stem cells, (HSCs), differentiate into the multipotent hematopoietic progenitor cells,  (HPCs). The HPCs express many lineage-specific cytokine receptors. Each of these receptors binds specific cytokines, activates distinct signaling pathways, and expresses a particular gene set. The HPCs further differentiate to form committed progenitors, forming either  common myeloid progenitors (CMPs) or common lymphoid progenitors (CLPs). The CMPs and CLPs proliferate, self-renew, and further differentiate into mature blood cells and immune cells depending on the receptors they express and the specific cytokines that bind. For example:

• Thrombopoietin (TpoR)- They help progenitors differentiate into megakaryocytes or platelets.
• Erythropoietin (EpoR)-These receptors promote the development of erythrocytes.
• Macrophage-colony stimulating factor (M-CSFR)-These receptors regulate the formation of macrophages.
• Granulocyte-colony stimulating factor (G-CSFR)- They help HPCs differentiate into granulocytes.
• Interleukin-7 receptor (IL-7R)- They help progenitor cells become lymphocytes.

Thus, lineage commitment helps HSCs lose their multipotency and differentiate into more restricted cell fate.

 Core: Cell Biology

Chromatin Structure and RNA Splicing

 Core: Cell Biology

Mechanism of Ciliary Motion

The ciliary structures were first seen in 1647 by Antonie Leeuwenhoek while observing the protozoans. In lower organisms, these appendages are responsible for cell movement, while in higher organisms, these appendages help in the movement of the extracellular fluids within the body cavities.

The cilia are made up of microtubules in a 9+2 arrangement, with nine microtubule doublet ring bundles, surrounding a pair of central singlet microtubule bundles. The doublet microtubule bundles are connected by nexin protein and axonemal dyneins. Radial spokes connect these outer doublet microtubules to the inner central pair. The coordinated movement of axonemal dyneins is responsible for the characteristic whip-like movement of the cilia. This characteristic ciliary movement is explained by the switch inhibition or switch-point mechanism proposed by Wais-Steider and Satir in 1979. The model suggests that during the ciliary motion, only half of the dyneins are active at a given time, while the other remains inactive. The axonemal dyneins on either side alternately switch between active and inactive forms to propel the ciliary motion. The sliding microtubules within the cilia require energy from ATP hydrolysis within the heavy chain domain of the axonemal dyneins.

Cilia in humans move rhythmically; they constantly remove waste materials such as dust, mucus, and bacteria through the airways, away from the lungs, and toward the mouth. Beating cilia on cells in the female fallopian tubes move egg cells from the ovary towards the uterus. A flagellum is an appendage larger than a cilium and specialized for cell locomotion. In humans, sperms are the only flagellated cell that must propel themselves towards female egg cells during fertilization.

 Core: Cell Biology

The Eukaryotic Promoter Region

 Core: Cell Biology

Protein and Protein Structures

 Core: Cell Biology

Overview of DNA Repair

 Core: Cell Biology

Hedgehog Signaling Pathway

 Core: Cell Biology

Immunoprecipitation

Immunoprecipitation, or IP, is a widely used technique that employs protein-antibody interactions to isolate proteins or protein complexes in their native state for studying protein-protein interactions, quaternary structures, or supramolecular complexes. Various modifications of the technique, including chromatin IP, cross-linking IP, and fluorescence IP, are commonly used.

Chromatin Immunoprecipitation

Chromatin immunoprecipitation, also known as ChIP, is used to study protein-DNA or protein-RNA interactions. It is an important technique for studying crucial cellular processes such as gene transcription, DNA replication, recombination and repair, cell cycle progression, and epigenetics. It is also helpful to identify the in vivo location of binding sites of various transcription factors, histones, and other regulatory proteins.

The major steps in ChIP include fixation, sonication, immunoprecipitation, and analysis. It involves cross-linking the target protein with the DNA using a fixing agent, such as formaldehyde, followed by sonication or enzymatic hydrolysis to obtain smaller chromatin fragments. The technique then utilizes high-affinity antibodies specific against the protein of interest to capture the DNA bound to the protein in an immunoprecipitation reaction. The cross-linking is then reversed using high heat, high salt concentration, and proteinase K to release the DNA from associated proteins. The DNA is further purified to prepare it for analysis.

Cross-linking Immunoprecipitation

Cross-linking immunoprecipitation, or CLIP, identifies the regions of protein binding sites on endogenous RNA by co-precipitating them in the active transcription phase. RNA molecules are cross-linked to proteins to hold them together tightly and prevent their degradation. The procedure for the breakdown and isolation of the complexes is similar to ChIP. This technique is used to study the interaction of RNA with RNA binding proteins and their modifications in various biological systems.

Limitations of IP

Though immunoprecipitation techniques tend to reduce the number of purification steps, it has certain limitations. The antibody binding recombinant bacterial proteins, proteins A or G, conjugated to agarose beads, and antibodies used in the method may undergo non-specific binding, introducing contaminants in the purified protein preparation. Also, the immobilization of antibodies on beads requires optimization and is time-consuming. The washing of the beads after the antigen-antibody complex is critical, but there is a chance of losing the target protein at this step.

 Core: Cell Biology

Immunocytochemistry and Immunohistochemistry

Immunocytochemistry (ICC) and immunohistochemistry (IHC) are techniques that use antibodies to check for specific proteins or antigens in a sample. The technique was first published by Albert Coons in 1941 to detect the presence of pneumococcal antigen in tissue sections from mice infected with Pneumococcus. Immunocytochemistry helps localization of proteins or antigens in individual cells like blood cells, stem cells, etc., while immunohistochemistry does the same for tissue samples.

These techniques can be colorimetric if enzyme-conjugated antibodies are used or if fluorescence-based fluorophore-tagged antibodies localize the proteins or antigens of interest. The colored product obtained via the enzymatic reaction or fluorescence emitted by the fluorophore is then visualized under an optical or immunofluorescence microscope.

These techniques can use primary or secondary antibodies tagged with an enzyme or a fluorophore. If a primary antibody is used, the technique is referred to as direct ICC or IHC, as the conjugated antibodies directly bind to the epitope of the protein or antigen of interest. In indirect, a secondary antibody tagged with the enzyme or fluorophore binds to the primary antibody attached to the epitope of the protein or antigen of interest. The signal emitted upon binding of a secondary antibody is stronger than the signal from a primary antibody.

Both the techniques vary slightly in the steps of sample preparation. In ICC, a monoculture layer of cells is generated, which are then treated with fixing agents like formaldehyde to prevent the enzymatic degradation of the antigens, followed by treatment with detergents like Triton X or Tween 20 to make the cell membrane permeable for the entry of antibodies. Similarly, in IHC, the tissue samples are first treated with a fixing solution, followed by embedding in paraffin wax to prevent damage to fragile tissue. The tissue samples are then sectioned into thin slices and treated with detergent before the introduction of antibodies.

Once the cells or tissue slides are treated with detergent, the antibodies are then added. In direct ICC or IHC, the primary antibody tagged with fluorophore or enzymes is added, and post-incubation, they are visualized under immunofluorescence or optical microscope. In indirect ICC or IHC, the samples are first incubated with the primary antibody, followed by washing to remove unbound antibodies. The secondary antibody is then introduced, which binds with the primary antibody and emits fluorescence or colored product upon adding dye. The samples are now ready for visualization.

 Core: Cell Biology

Positive Regulator Molecules

 Core: Cell Biology

Forces Acting on Chromosomes

 Core: Cell Biology

Cancers Originate from Somatic Mutations in a Single Cell

 Core: Cell Biology

Cancer Therapies

 Core: Cell Biology

Overview of Regeneration and Repair

Regeneration and repair processes are critical in healing damages caused by injury, disease, and aging. In regeneration, the damaged tissue is entirely replaced with new growth that restores the original architecture and function. In contrast, tissue repair usually results in a fixed tissue architecture involving scar formation. Scars generally do not reestablish tissue function and may also exhibit structural abnormalities at the injury site.

Regeneration

All animals have varying degrees of regenerative capabilities, but only some animals exhibit exceptional regeneration, such as generating an entire organ. For example, salamanders have a specialized population of stem cells that plays a significant role in regenerating fully functional limbs and tails. In mammals, only a few adult tissues like the liver, epithelium of the gut, and bone marrow can regenerate after an injury. The planarian flatworms, with a much simpler body structure, are capable of whole-body regeneration; if a planarium is cut into several pieces, each piece can develop into a completely new organism. Similarly, the hydra can regenerate its whole body even from a small fragment.

The regeneration process begins with the crucial step of covering the injury site. The epidermal cells migrate to the injury site, forming a thick covering called the apical epidermal cap. This is followed by clustering of undifferentiated fibroblast cells underneath the cap, forming a blastema. The blastema is a population of stem cells that has the potential to differentiate  into any tissue or organ. The blastema is supplied with oxygen and nutrients via a new blood capillary network, allowing the cells to divide and differentiate.

Repair

The repair mechanism involves four phases: hemostasis, inflammation, proliferation, and remodeling. After an injury, the exposure of collagen— a structural protein in the walls of blood vessels— begins a coagulation cascade and vasoconstriction, thus, minimizing blood loss. During hemostasis, the clot fills the wound bed, providing a temporary matrix for the movement of various cells such as macrophages, neutrophils, and platelets. Platelet degranulation activates the complement cascade that stimulates inflammatory cells to attack the bacteria. During the proliferation phase, various cytokines and growth factors are released into the wound site, signaling the cells to proliferate and heal the wound. The final repair phase involves forming scar tissue that marks the completion of repair.

 Core: Cell Biology

Heat and Free Expansion

The work done by a thermodynamic system depends not only on the initial and final states but also on the intermediate states—that is, on the path. Like work, when heat is added to a thermodynamic system, it undergoes a change of state, and the state attained depends on the path from the initial state to the final state. Consider an ideal gas cylinder fitted with a piston. When the cylinder is heated at a constant temperature, the gas molecules absorb energy and expand slowly in a controlled isothermal manner. This pushes the piston upwards, and gas eventually attains the final volume. Here, the work is done by the gas due to heat expansion on the piston.

The gas can also attain the same final volume through a different process. Consider a cylinder surrounded by insulating walls and divided by a thin, removable partition. The lower compartment is filled with the same amount of gas at the same temperature so that the initial state is the same as mentioned above. When the partition is removed, the gas undergoes a rapid, uncontrolled expansion, with no heat passing through the insulating walls, and reaches the same final volume as in the above case. Here, no work is done by the gas during this expansion as it does not push against anything that moves. This uncontrolled expansion of a gas into the vacuum is called a free expansion.

Experimentally, there is no temperature change under a free expansion of ideal gas. This means that the final state of the gas remains the same. The intermediate states (pressures and volumes) during the transition from the initial to the final state are entirely different in the two cases. As a result, it represents two different paths connecting the same initial and final states.

 Core: Physics

Enzymes And Activation Energy

 Core: Anatomy and Physiology

What Are Membranes?

 Core: Anatomy and Physiology

What Is The Cell Cycle?

 Core: Anatomy and Physiology

Systematic Error: Methodological and Sampling Errors

In the case of systematic errors, the sources can be identified, and the errors can be subsequently minimized by addressing these sources. According to the source, systematic errors can be divided into sampling, instrumental, methodological, and personal errors.

Sampling errors originate from improper sampling methods or the wrong sample population. These errors can be minimized by refining the sampling strategy. Defective instruments or faulty calibrations are the sources of instrumental errors. Periodic calibration of instruments is essential to eliminate such errors.

Method errors occur due to the limitations in an analytical method, the non-ideal behavior of reagents used, and invalid assumptions made while setting up the measurement. These errors can be mitigated using standard reference materials or analysis independent of the existing method but carried out in parallel.

Personal errors arise due to analysts' carelessness or lack of skill. Proper organization of materials and equipment, standardization of protocols, and attention to detail can help minimize these errors. Automated procedures can also be instituted to minimize human handling, reducing personal errors.

Systematic errors can also either be constant errors or proportional errors. The absolute magnitude of constant errors remains the same irrespective of the sample size. These errors can be minimized by increasing the sample size, as the contribution from the constant error relative to a larger sample size is less significant than the same constant error relative to a smaller sample size. On the other hand, a proportional error will increase in magnitude with increasing sample size, hence the term 'proportional.' Increasing the sample size will not help to reduce these types of errors. Using high-precision instruments is one way of reducing proportional errors.

 Core: Analytical Chemistry

Ionic Strength: Overview

The ionic strength of a solution is a quantitative way of expressing the total electrolyte concentration of a solution. This concept was first introduced in 1921 by two American physical chemists, Gilbert N. Lewis and Merle Randall, while describing the activity coefficient of strong electrolytes. During the calculation of ionic strength (I or μ), all the cations and anions are considered. However, the concentration (c) of an ion with a greater charge number (z) has a greater contribution to the total ionic strength because the charge of the ion is squared.

While calculating ionic strength for a salt that will produce multiple equivalents of the same ion on dissociation, we need to account for the contribution from each ion. For example, the ionic strength of 0.1 mol/L Na₂SO₄ can be calculated as follows:

The concentration of Na⁺ is 0.2 mol/L because one molecule of Na₂SO₄ will dissociate to give two Na⁺ ions in solution. The ionic strength of dilute solutions can be calculated easily. However, in a more concentrated solution, the calculation becomes more complex and less accurate, as the salts do not dissociate completely. For example, in an aqueous solution of 0.025 mol/L MgSO₄, 25% to 35% of MgSO₄ exists as the ion pair MgSO₄(aq).

The concept of ionic strength can be further extended to strong and weak acids. Because strong acids dissociate completely in solution, their ionic strengths can be calculated in the same way as those of dissociated salts. For weak acids, the concentration of ionized species can be calculated from the ionization constant value and then used for ionic strength determination. If the acid is very weak and mostly remains non-ionized, its contribution to the total ionic strength of the solution is negligible.

 Core: Analytical Chemistry

Titrimetric Methods: Types and Commonly Used Strategies

In chemistry, titrimetric methods are broadly classified into three types: volumetric, gravimetric, and coulometric. Volumetric titrations involve measuring the volume of a titrant of known concentration that is required to react completely with an analyte. In gravimetric titrations, the standard solution reacts with the analyte to form an insoluble precipitate, which is filtered, dried, and weighed. In coulometric titrations, current is applied to an electrochemical reaction until the reaction is complete, and the charge required for this reaction is calculated to determine the amount or concentration of the reactant(s).

Generally, in titrations, a titrant with a known concentration is added to the analyte until the analyte is consumed, i.e. when the endpoint is reached. In this case, the process is called direct titration. In some cases, the reaction may be too slow for a sharp endpoint to be obtained. In such cases, back titration is used. In a back titration, the analyte is allowed to react with an excess amount of a standard solution, and the remaining quantity of this standard solution is titrated with a second standard solution. The exact amount of the first standard solution required to react with the analyte can then be calculated.

Sometimes, no suitable indicator can be found for either a direct or an indirect (back) titration. In this case, displacement titration can be used, in which the analyte displaces a reagent, usually from a complex, and the displaced reagent is measured by titration.

 Core: Analytical Chemistry

Masking and Demasking Agents

EDTA titrations may necessitate masking and demasking agents to temporarily protect a particular metal ion in a mixture from the EDTA reaction. These agents facilitate the sequential analysis of the metal ions by forming stable complexes with some—but not all—metal ions during certain steps.

There are many masking agents, such as cyanide, fluoride, triethanolamine, thiourea, and 2,3-bis(sulfanyl)propan-1-ol (formerly 2,3-dimercapto-1-propanol), with the masking agent chosen based on the metal ions involved. For example, a cyanide mask is used during the EDTA titration of a lead and cadmium mixture. Cyanide only masks the cadmium ion, so the lead ion still reacts with EDTA.

Demasking agents are used to release the metal ions from masking agents. For example, formaldehyde acts as a demasking agent for cyanide complexes.

 Core: Analytical Chemistry

Inductively Coupled Plasma–Mass Spectrometry (ICP–MS): Overview

In inductively coupled plasma–mass spectrometry (ICP–MS), an inductively coupled plasma (ICP) torch is used as an atomizer and ionizer. Solid samples are dissolved and volatilized before being introduced into the high-temperature argon plasma, while solution samples are nebulized and passed through the high-temperature argon plasma. Plasma dissociates the analytes and ionizes their component atoms to form a mixture of positive ions and molecular species. The positive ions are then passed on to the mass spectrometer and analyzed by their m/z values. Since an ICP source operates at atmospheric pressure and a mass spectrometer requires a vacuum, the plasma is passed through an interfacial region, reducing the pressure from 1 bar to around 10⁻⁹ bar. The interfacial region consists of two metallic cones (a sampler cone and a skimmer cone), an extraction lens, and a collision cell. The sampler cone and skimmer cone have narrow orifices allowing only a small amount of plasma to pass through. The high negative voltage of the extraction lens allows only the positive ions to enter the collision cell. The collision cell reduces the range of kinetic energies of the ions and directs the ion beam into the quadrupole mass analyzer. ICP–MS is a useful technique in elemental analysis due to its low detection limits, high selectivity, and excellent sensitivity.

 Core: Analytical Chemistry

Gravimetry: Inorganic And Organic Precipitating Agents

In gravimetry, the precipitant is chosen carefully to obtain a pure solid that can be easily filtered. Common inorganic precipitants can be used to determine several cations and anions. In some cases, the formation of the same precipitate can be used to determine the cation and the anion. For example, the reaction of barium and chromate ions to give barium chromate is used to determine both barium and chromate. However, precipitates such as hydroxides, oxalates, and metal ammonium phosphates are first converted to a weighable form. Precipitation methods can also be applied to determine organic functional groups such as organic halides, carbonyl, alkoxy groups, aromatic nitro, azo, and phosphate.

Organic precipitants are usually more selective than their inorganic counterparts and yield sparingly soluble precipitates with high molecular masses. A small number of analyte ions will yield a large amount of precipitate. For example, sodium tetraphenylborate is a near-specific precipitant for potassium and ammonium ions, yielding ionic precipitates. Several organic precipitants contain multiple functional groups that can bond with the cation to generate five- or six-membered rings called chelates. Typical chelating agents include 8-hydroxyquinoline and cupferron.

 Core: Analytical Chemistry

Functions of Smooth Muscles

Smooth muscles are an important type of muscle tissue that plays a vital role in the involuntary movements of internal organs. For example, they help regulate the movement of food through the gut and the flow of blood through the circulatory system.

Function of visceral smooth muscles

Visceral smooth muscle is found in the walls of all hollow organs, except the heart, and is a key player in the involuntary movements that drive the functioning of these internal organs. This tissue is arranged in layers of tightly packed fibers that communicate via gap junctions, enabling them to contract synchronously as a single unit. For example, smooth muscle in the intestine is organized into two primary layers: the longitudinal and circular layers. The longitudinal layers run along the length of the intestine, and their contraction shortens the overall length of the intestinal tract. Meanwhile, the circular layers wrap around the circumference and facilitate constriction and dilation of the inner cavity. The alternating contraction and relaxation of these layers result in peristalsis that helps propel the contents and ensures thorough mixing and breakdown, optimizing nutrient absorption and digestion.

Function of multi-unit smooth muscles

While many organs rely on the coordinated activity of visceral smooth muscle, certain areas, like the eye and walls of large arteries, contain multi-unit smooth muscle fibers. These fibers operate independently, each with its own nerve supply, allowing for precise and localized control. In large pulmonary arteries, for instance, multi-unit smooth muscles adjust the diameter of the lumen to direct blood flow to well-ventilated regions of the lung, enhancing oxygenation and adjusting to the body's changing needs.

Overall, smooth muscles in various organs demonstrate incredible adaptability and precision in their functions. For example, in the respiratory system, smooth muscles adjust the airway diameter, increasing or decreasing airflow in response to the body's oxygen demands. In the vascular system, these muscles regulate blood pressure and flow by altering the diameter of blood vessels.

 Core: Anatomy and Physiology

Muscles that Move the Forearm

The muscles that move the forearms can be divided into four groups: forearm flexors, forearm extensors, forearm pronators, and forearm supinators. The flexors and extensors act on the elbow joint, while the pronators and supinators act on the radioulnar joints.

Forearm Flexors

The biceps brachii, brachialis, and brachioradialis are forearm flexors. The biceps brachii is made up of two heads. Its long head originates at the supraglenoid tubercle of the scapula, whereas that of the short head is at the coracoid process of the scapula. The heads merge and insert at radial tuberosity. A fibrous membrane known as bicipital aponeurosis emerges from the distal end of the biceps brachii and inserts into the deep fascia of the forearm. The anterior surface of the humerus is the origin site for the brachialis muscle. It inserts into the ulnar tuberosity and coronoid process of the ulna. The brachioradialis muscle originates on the lateral border of the distal end of the humerus and inserts on the radius, superior to the styloid process.

Forearm Extensors

The triceps brachii, and anconeus are forearm extensors. The triceps brachii is a three-headed muscle. Its long head originates on the infraglenoid tubercle. The lateral head originates from the lateral and posterior surface of the humerus. The posterior surface of the humerus is also the origin point for the medial head. Together, the three heads are inserted at the olecranon of the ulna. Finally, the anconeus originates from the humerus at the lateral epicondyle and inserts on the olecranon and superior portion of the ulnar shaft.

Pronators and Supinators

Pronator teres and pronator quadratus are forearm pronators. The pronator teres originate from the medial epicondyle of the humerus and the coronoid process of the ulna. It inserts on the mid-lateral surface of the radius. The pronator quadratus originates on the distal shaft of the ulna and inserts onto the distal part of the radius. The supinator muscle supinates the forearm. It originates from the lateral epicondyle of the humerus and the ridge near the radial notch of the ulna. It inserts on the lateral surface of the proximal one-third of the radius.

 Core: Anatomy and Physiology

Propagation of Action Potentials

The propagation of an action potential refers to the process by which a nerve impulse, or "action potential," travels along a neuron.

Neurons (nerve cells) have a resting membrane potential, with a slightly negative charge inside compared to outside. This is maintained by ion channels, such as sodium (Na⁺) and potassium (K⁺⁾ channels, which control the flow of ions. When a stimulus, like a touch or a signal from another neuron, triggers the neuron, sodium channels open, allowing sodium ions to rush into the neuron, causing depolarization.

If the depolarization is strong enough and reaches a certain threshold, it triggers an action potential. The initiation of an action potential occurs at the axon's beginning, or the initial segment, where a high concentration of voltage-gated Na⁺ channels allows a swift depolarization. As the depolarization advances along the axon, more Na⁺ channels open, facilitating the spread of the action potential. This is achieved as Na⁺ ions flow inwards, progressively depolarizing the cell membrane.

However, the Na⁺ channels become inactivated at peak depolarization, rendering them unopenable for a brief period, known as the absolute refractory period. As a result, any depolarization attempting to reverse direction is null, ensuring that the action potential's propagation is towards the axon terminals, thereby preserving neuronal polarity.

This propagation method applies to unmyelinated axons. In myelinated axons, the process differs. The depolarization spreads optimally due to the absence of constant Na⁺ channel opening along the axon segment. The precise placement of nodes ensures the membrane remains sufficiently depolarized at the next node.

Propagation in unmyelinated axons, known as continuous conduction, is slower due to the constant influx of Na⁺. In contrast, myelinated axons exhibit saltatory conduction - a faster method as the action potential leaps node to node, renewing the depolarized membrane. Furthermore, the speed of conduction can be influenced by the axon's diameter, a concept known as resistance.

 Core: Anatomy and Physiology

Diencephalon: Thalamus and Information Relay

The thalamus, often called “the gateway to the cerebral cortex,” is vital in processing and directing sensory and motor signals throughout the brain. Almost all inputs destined for the cerebral cortex, except for olfactory signals, are relayed through the thalamus. The thalamus is  a sophisticated relay station, channeling information from various brain regions to the cerebral cortex, as well as a filter, prioritizing certain signals over others based on current physiological states or needs.

The thalamus comprises several groups of paired nuclei, each having a specific role in relaying information, ensuring that signals reach their intended destinations accurately and efficiently. The paired nuclei include the following:

Anterior Nucleus: This nucleus serves as a conduit for information from the hypothalamus to the limbic system, a collection of structures involved in emotion, memory, and arousal. This pathway is essential for integrating emotional content with sensory experiences.

Medial Nuclei: Information received from the limbic system is further processed and relayed by the medial nuclei, facilitating the emotional and cognitive responses to sensory inputs.

Lateral Geniculate Nucleus: Specialized in relaying visual information, the lateral geniculate nucleus directs signals from the eyes to the visual cortex. This process is fundamental for the perception and interpretation of visual stimuli.

Medial Geniculate Nucleus: Comparable to its lateral counterpart, the medial geniculate nucleus focuses on auditory information, channeling sound signals from the ear to the auditory cortex. This relay is crucial for hearing and the cognitive processing of sounds.

Ventral Posterior Nucleus: This nucleus conveys somatosensory information, such as touch and pain, from the body to the cerebral cortex. It ensures that sensory experiences are accurately perceived and interpreted.

Lateral Dorsal Nucleus: The lateral dorsal nucleus plays a role in expressing emotions and facilitates the integration of emotional states with sensory experiences.

Lateral Posterior and Pulvinar Nuclei: These nuclei integrate sensory information, helping to combine different sensory inputs for a cohesive perception of the environment.

Beyond sensory information, the thalamus is instrumental in relaying motor impulses. Signals from the basal nuclei, which are involved in controlling movement, are relayed through the ventral anterior nuclei. Additionally, impulses originating from both the basal nuclei and the cerebellum, a region responsible for coordination and balance, are directed through the ventral lateral nuclei. This coordination ensures that motor functions are executed smoothly and accurately. The thalamus ensures the brain responds appropriately to internal and external stimuli by directing sensory and motor signals to the appropriate cortical areas.

 Core: Anatomy and Physiology

Spinal Nerves: Plexus II

The plexuses of the lower body include the lumbar, sacral, and coccygeal plexuses, which innervate the abdomen, pelvis, legs, and coccygeal region. These plexuses control the transmission of sensory information and coordinate motor functions of the lower body.

The Lumbar Plexus

The lumbar plexus is situated within the lumbar region of the back and is primarily formed by the first four lumbar spinal nerves (L1 to L4). This plexus extends its branches into several nerves, including the iliohypogastric, ilioinguinal, genitofemoral, lateral femoral cutaneous, femoral, and obturator nerves. These nerves provide sensory and motor innervation to the muscles and skin of the abdomen, pelvis, and parts of the lower limbs. The lumbar plexus facilitates movements and transmits sensory information from these areas, enabling functions such as walking, posture maintenance, and the protection of abdominal organs.

The Sacral Plexus

Within the pelvis, the sacral plexus is formed by the nerves spanning from the fourth lumbar (L4) to the fourth sacral (S4) segments. This plexus gives rise to several vital nerves, including the pudendal, superior and inferior gluteal, posterior femoral cutaneous, and sciatic nerves. Notably, the sciatic nerve is recognized as the thickest and longest nerve in the body, extending down to the lower leg and foot. This nerve is a common source of pain and discomfort when irritated or compressed — a condition called sciatica. The sacral plexus primarily provides sensory and motor innervation to the pelvis and legs, overseeing functions such as leg movement, pelvic organ control, and sensation in the lower extremities.

Due to the substantial overlap in the nerves originating from the lumbar and sacral plexuses, these two networks are sometimes collectively referred to as the lumbosacral plexus. This combined structure highlights the complexity of neural pathways and their essential roles in bodily operations.

The Coccygeal Plexus

Though the smallest among the three, the coccygeal plexus plays a specific role in innervating the skin over the coccygeal area. It is located at the very base of the spine around the coccyx and primarily comprises the anococcygeal nerve. Despite its limited size and scope compared to the lumbar and sacral plexuses, the coccygeal plexus is crucial for transmitting sensory information from the coccygeal region, contributing to the overall sensory map of the lower body.

 Core: Anatomy and Physiology

Higher Mental Functions of the Brain: Language

Language is a system of communication that allows the expression of thoughts, ideas, and feelings. The brain processes language in both hemispheres.

Language formation and comprehension take place in the dominant hemisphere. The dominant hemisphere is responsible for understanding the meaning of spoken, written, or sign language, as well as the ability to communicate. For most people, the left hemisphere is the dominant one. The right hemisphere, then, gives tone and emotional context to the language.

Language processing in the left hemisphere happens in two areas. Wernicke's area, also known as the "language comprehension center," is located in the brain's left temporal lobe and is responsible for understanding language. This area interprets incoming speech from verbal, gestures, and written sources. Broca's area, also known as the "speech production center," is located in the brain's left frontal lobe and is responsible for producing language. It sends signals to the motor cortex that then initiates movements of muscles involved in speech.

The neural pathways involved in perceiving and vocalizing a specific word operate as follows:

• • First, information about the word is conveyed to Wernicke's area.
  •  If the word is written, Wernicke's area receives input about the word from the primary visual area.
  • If the word is spoken, Wernicke's area receives input about the word from the primary auditory area.
• • Upon receiving the information, Wernicke's area translates the written or spoken word into the corresponding thought.
• • Next, for the individual to respond to the spoken or written word or a gesture, Wernicke's area transmits information about the word to Broca's area.
• • Broca's area then receives this input and develops a motor pattern to activate the necessary muscles for articulating the word.
• • The motor pattern is conveyed from Broca's area to the primary motor area, which triggers the appropriate muscle contractions required for speech.
• • Ultimately, the coordinated contraction of the speech muscles allows the word to be spoken.

 Core: Anatomy and Physiology

Photoreceptors and Visual Pathways

At the molecular level, visual signals trigger transformations in photopigment molecules, resulting in changes in the photoreceptor cell's membrane potential. The photon's energy level is denoted by its wavelength, with each specific wavelength of visible light associated with a distinct color. The spectral range of visible light, classified as electromagnetic radiation, spans from 380 to 720 nm. Electromagnetic radiation wavelengths exceeding 720 nm fall under the infrared category, whereas those below 380 nm are classified as ultraviolet radiation. Blue light corresponds to a wavelength of 380 nm, while dark red light corresponds to a wavelength of 720 nm. Other colors lie at varying points within this wavelength spectrum, from red to blue.

Opsin pigments, in fact, are transmembrane proteins integrated with a cofactor named retinal. This retinal is a constituent of vitamin A and a hydrocarbon molecule. The significant biochemical alteration in the extensive hydrocarbon chain of the retinal molecule is triggered when a photon impacts it. This specific process, known as photoisomerization, transitions some of the double-bonded carbons inside the chain from a cis to a trans configuration owing to the photon interaction. Before the photon interaction, the flexible double-bonded carbons of the retinal are in the cis conformation, leading to the formation of a molecule known as 11-cis-retinal. The double-bonded carbons assume the trans-conformation when a photon impacts the molecule, forming an all-trans-retinal characterized by a straight hydrocarbon chain.

The visual transduction process within the retina commences with the alteration in the retinal structure in photoreceptors. This leads to the activation of retinal and opsin proteins, which stimulate a G protein. The activated G protein then modifies the photoreceptor cell's membrane potential, causing a decrease in the release of neurotransmitters into the retina's outer synaptic layer. This state continues until the retinal molecule reverts to its original shape, the 11-cis-retinal form - a process referred to as bleaching. If a substantial amount of photopigments undergo bleaching, the retina transmits data as if contrasting visual inputs are being received. Afterimages, typically observed as negative-type images, are a common occurrence following exposure to an intense flash of light. A series of enzymatic alterations facilitate the photoisomerization reversal process, thus enabling the reactivation of the retinal in response to additional light energy.

Opsins exhibit specific sensitivity to particular light wavelengths. The rod photopigment, rhodopsin, exhibits peak sensitivity to light that has a wavelength of 498 nm. On the other hand, three color opsins are optimally responsive to wavelengths of 564 nm, 534 nm, and 420 nm, which approximately align with the primary colors—red, green, and blue. Rhodopsin found in rods demonstrates a higher sensitivity to light than cone opsins; this means that rods contribute to vision under dim light conditions while cones contribute under brighter conditions. In normal sunlight, rhodopsin is continuously bleached, and cones remain active. Conversely, in a dimly lit room, the light intensity is insufficient to stimulate cone opsins, making vision entirely reliant on rods. In fact, rods have such a high sensitivity to light that a solitary photon can trigger an action potential in a rod's corresponding RGC.

Cone opsins, differentiated by their sensitivity to distinct light wavelengths, furnish the ability to perceive color. By analyzing the responses of the three unique cone types, our brain distills color data from what we see. Consider, for instance, a bright blue light with a wavelength near 450 nm. This would cause minimal stimulation of the "red" cones, slight activation of the "green" cones, and significant stimulation of the "blue" cones. The brain computes this differential activation of the cones and interprets the color as blue. However, under dim light conditions, cones are ineffectual, and rods, which cannot discern color, dominate. As a result, our vision in low light is essentially monochromatic, meaning everything appears in varying shades of gray in a dark room.

Some common eye disorders:

Color blindness, clinically known as achromatopsia, is a condition characterized by a deficiency in distinguishing colors. This disorder usually results from an inherited defect in the retina's cones (light-sensitive cells). Symptoms may include difficulty distinguishing between colors or shades of colors.

Night blindness, medically referred to as nyctalopia or hemeralopia, is a disorder that affects an individual's ability to see in low light or at night. Causes can range from vitamin A deficiency to underlying diseases such as retinitis pigmentosa. Individuals with this disorder experience difficulties with night-time vision or adjusting to dim lighting.

Cataracts, a common eye disorder especially among older adults, are characterized by clouding of the normally transparent eye lens. This can result in blurred vision, similar to looking through a fogged-up window. Most cataracts develop slowly over time and can eventually interfere with vision.

Glaucoma is another severe eye condition where the optic nerve, which sends images to the brain, gets damaged due to increased pressure in the eye. It can lead to vision loss if left untreated. The most common type of glaucoma, open-angle glaucoma, often has no symptoms other than gradual vision loss.

 Core: Anatomy and Physiology

Hormones of the Pituitary Gland

The small, pea-sized pituitary gland is located at the base of the brain. It is crucial in regulating various bodily functions, from growth to reproduction. The gland is divided into the anterior lobe and the posterior lobe. The secretory cell clusters in the pars distalis of the anterior pituitary lobe are controlled by hypothalamic regulators and synthesize six primary hormones.

The most abundantly secreted hormone from the anterior lobe is the growth hormone, which controls overall growth by regulating the production of insulin-like growth factors from the liver, bones, and muscles. Also, it plays a vital role in regulating metabolism by balancing fat and glucose usage for energy generation.

Another group of hormones, tropins, are secreted by the anterior lobe, which controls the secretion of hormones by other endocrine glands. For example, the thyroid-stimulating hormone is a tropin regulating the hormones of the thyroid gland, while the adrenocorticotropic hormone controls cortisol release from the adrenal glands.

The luteinizing hormone, LH, and follicle-stimulating hormone, FSH, in both sexes control the functioning of reproductive organs. These hormones regulate the menstrual cycle in females and testosterone production in males. Lastly, prolactin stimulates breast milk production in lactating mothers.

 Core: Anatomy and Physiology

Voltage-gated Ion Channels

 Core: Anatomy and Physiology

Power and Energy

The power and energy delivered to an element are subjects of great significance in the field of electrical engineering. It is a well-known fact that a 100-watt light bulb emits more light than a 60-watt one. Therefore, power and energy calculations play a crucial role in the analysis of electrical circuits.

Power, defined as the time rate of expending or absorbing energy, is quantified in units called watts (W). The relation between power and energy is mathematically given as

where "p" represents the power in watts (W), "w" denotes the energy in joules (J), and "t" signifies the time in seconds (s).

The power associated with the current passing through an element within a circuit is the product of the voltage across that element and the current flowing through it. When the current enters the circuit element at the positive terminal of the voltage and exits at the negative terminal, it adheres to the passive convention. In this convention, the voltage propels a positive charge in the same direction as indicated by the current. Consequently, the power computed by multiplying the element voltage by the element current represents the power received by the element. This power is occasionally referred to as "the power absorbed by the element" or "the power dissipated by the element." Importantly, the power received by an element can assume either positive or negative values, contingent upon the actual values of the element's voltage and current.

On the other hand, when the passive convention is not adhered to, the current enters the circuit element at the negative terminal of the voltage and exits at the positive terminal. In this case, the voltage drives a positive charge in the direction opposite to that indicated by the current. Therefore, when the element voltage and current do not comply with the passive convention, the power computed by multiplying these values represents the power supplied by the element. Similar to received power, supplied power can also be positive or negative, depending on the specific values of the element's voltage and current.

The relationship between the power received by an element and the power supplied by that same element is expressed as

When the element voltage and current align with the passive convention, the energy received by an element is given by

Energy is defined as the capacity to perform work and is expressed in joules (J).

 Core: Electrical Engineering

Integrator and Differentiator

Op-amp circuits have significant applications in various fields, including automotive engineering. One such application is cruise control systems in cars, where op-amp circuits are integral for maintaining a constant speed. In these systems, op-amps function as both integrators and differentiators.

An integrator within an op-amp circuit produces an output directly proportional to the integral of the input signal. This is achieved by replacing the feedback resistor in a typical inverting amplifier circuit with a capacitor, resulting in an ideal integrator. An equation relating output and input voltages is derived by applying Kirchhoff's current law and utilizing current-voltage relationships for resistors and capacitors. When integrated, this equation demonstrates that the output voltage corresponds to the integral of the input signal.

Conversely, a differentiator within an op-amp circuit yields an output proportional to the input signal's rate of change. Achieving this involves replacing the input resistor with a capacitor in a standard inverting amplifier, creating a differentiator circuit. An equation linking output and input voltages is established by applying Kirchhoff's current law and employing current-voltage relations. In this case, the equation indicates that the output voltage is proportional to the derivative of the input signal.

It is worth noting that these op-amp circuits are valuable in energy storage applications and are often designed using resistors and capacitors due to their compactness and cost-effectiveness. While integrators are widely employed in analog computers and various applications, differentiators are less common in practice due to their tendency to amplify electrical noise, making them electronically unstable.

 Core: Electrical Engineering

Series RLC Circuit without Source

Within the field of electrical circuits, source-free RLC circuits present an intriguing domain. These circuits comprise a series arrangement of a resistor, inductor, and capacitor, operating independently of external energy sources. Their initiation hinges upon utilizing the initial energy stored within the capacitor and inductor to instigate their functionality. Their mathematical equation, a second-order differential equation, sets these circuits apart. This equation captures how the circuit's components interact, forming the basis for understanding its behavior.

The resistor in this circuit plays a significant role by dissipating energy, leading to an exponential solution for the differential equation. Substituting this solution yields a quadratic equation, and the two roots of this equation hold special significance. These roots are the circuit's natural frequencies and are instrumental in describing its natural response.

Expressed in terms of the damping factor and resonant frequency, these roots provide insights into the circuit's behavior. If the damping factor surpasses the resonant frequency, the circuit exhibits an overdamped response with distinct real roots. When the damping factor equals the resonant frequency, a critically damped response ensues, characterized by equal roots. Finally, if the damping factor falls short of the resonant frequency, the circuit enters an underdamped state with complex roots.

Various response scenarios within source-free RLC circuits offer an intriguing and valuable aspect of circuit analysis. Further exploration of each case provides a comprehensive understanding of their behavior and practical applications in electrical circuits.

 Core: Electrical Engineering

Superposition Theorem for AC Circuits

Consider encountering a circuit in a steady state where all its inputs are sinusoidal, yet they do not all possess the same frequency. Such a circuit is not classified as an alternating current (AC) circuit, and consequently, its currents and voltages will not exhibit sinusoidal behavior. However, this circuit can be analyzed using the principle of superposition.

The principle of superposition stipulates that the output of a linear circuit with several concurrent inputs is equivalent to the cumulative outputs when each input operates independently. The inputs to the circuit are the voltages from the independent voltage sources and the currents from the independent current sources.

When all inputs except one are set to zero, the remaining inputs become 0-V voltage sources and 0-A current sources. Given that 0-V voltage sources equate to short circuits and 0-A current sources correspond to open circuits, the sources linked to the other inputs are replaced by open or short circuits. What remains is a steady-state circuit with a single sinusoidal input, which qualifies as an AC circuit and is analyzed using phasors and impedances.

Hence, the principle of superposition is employed to transform a circuit with multiple sinusoidal inputs at varying frequencies into several separate circuits, each with a singular sinusoidal input. Each of these AC circuits is then analyzed using phasors and impedances to determine its sinusoidal output. The aggregate of these sinusoidal outputs will coincide with the output of the initial circuit.

 Core: Electrical Engineering

Position and Displacement

 Core: Mechanical Engineering

Titration of Polyprotic Acids with a Strong Base

Titration of a polyprotic acid, which contains multiple ionizable protons, involves distinct dissociation steps, each with its own dissociation constant (K_a). Each successive K_a is weaker than the previous one. In the titration of a polyprotic acid like sulfurous acid with a strong base such as sodium hydroxide, the base first neutralizes the initial ionizable proton, forming an intermediate species (e.g., hydrogen sulfite ions). This step's titration curve resembles that of a weak monoprotic acid, with a half-equivalence point where the pH equals the first pK_a. As the titration continues, an additional base neutralizes the second ionizable proton. This process requires twice the amount of base for complete neutralization, leading to another half-equivalence point and an equivalence point in the basic region. This pattern extends to other polyprotic acids, such as triprotic phosphoric acid, which will have three equivalence points. The number of equivalence points in the titration curve of a weak polyprotic acid corresponds to the number of ionizable protons, provided there's a significant difference between the K_a values of these protons.

 Core: Analytical Chemistry

Yeast Reproduction

Saccharomyces cerevisiae is a species of yeast that is an extremely valuable model organism. Importantly, S. cerevisiae is a unicellular eukaryote that undergoes many of the same biological processes as humans. This video provides an introduction to the yeast cell cycle, and explains how S. cerevisiae reproduces both asexually and sexually Yeast reproduce asexually through a process known as budding. In contrast, yeast sometimes participate in sexual reproduction, which is important because it introduces genetic variation to a population. During environmentally stressful conditions, S. cerevisiae will undergo meiosis and form haploid spores that are released when environmental conditions improve. During sexual reproduction, these haploid spores fuse, ultimately forming a diploid zygote. In the lab, yeast can be genetically manipulated to further understand the genetic regulation of the cell cycle, reproduction, aging, and development. Therefore, scientists study the reproduction of yeast to gain insight into processes that are important in human biology.

 Biology I

Development and Reproduction of the Laboratory Mouse

Successful breeding of the laboratory mouse (Mus musculus) is critical to the establishment and maintenance of a productive animal colony. Additionally, mouse embryos are frequently studied to answer questions about developmental processes. A wide variety of genetic tools now exist for regulating gene expression during mouse embryonic and postnatal development, which can help scientists to understand more about heritable diseases affecting human development.

This video provides an introduction to the reproduction and development of mice. In addition to clarifying the terminology used to describe developmental progression, the presentation reviews key stages of the mouse life cycle. First, major development events that take place in utero are described, with special attention given to the unique layout of early rodent embryos. Next, husbandry protocols are provided for postnatal mice, or pups, including the process of weaning, or removal of pups from their mother's cage. Since males and females must be separated at this stage to prevent unscheduled mating, the demonstration also reveals how to determine mouse sex. Subsequently, instructions are given for carrying out controlled mouse breeding, including screening for the copulatory plug, which is useful for precisely timed embryonic development. Finally, the video highlights strategies used to investigate the complex processes that govern mouse development, including the generation of genetically altered “knockout” mice.

 Biology II

fMRI: Functional Magnetic Resonance Imaging

Functional magnetic resonance imaging (fMRI) is a non-invasive neuroimaging technique used to investigate human brain function and cognition in both healthy individuals and populations with abnormal brain states. Functional MRI utilizes a magnetic resonance signal to detect changes in blood flow that are coupled to neuronal activation when a specific task is performed. This is possible because hemoglobin within the blood has different magnetic properties depending on whether or not it is bound to oxygen. When a certain task is performed, there is an influx of oxygenated blood to brain regions responsible for that function, and this influx can then be detected with specific MRI scan parameters. This phenomenon is termed the blood oxygen level ependent (BOLD) effect, and can be used to create maps of brain activity.

This video begins with a brief overview of how MRI and fMRI signal is obtained. Then, basic experimental design is reviewed, which involves first setting up a stimulus presentation that is specifically designed to test the function that will be mapped. Next, key steps involved in performing the fMRI scan are introduced, including subject safety and setting up at the scanner. Commonly used steps for data processing are then presented, including pre-processing and statistical analysis with the general linear model. Finally, some specific applications of fMRI are reviewed, such as investigations into abnormal function in psychological disorders, and combining fMRI with complimentary imaging modalities, such as diffusion tensor imaging (DTI).

 Neuroscience

An Introduction to Organogenesis

Organogenesis is the process by which organs arise from one of three germ layers during the later stages of embryonic development. Researchers studying organogenesis want to better understand the genetic programs, cell-cell interactions, and mechanical forces involved in this process. Ultimately, scientists hope to use this knowledge to create therapies and artificial organs that will help treat human diseases.

This video offers a comprehensive overview of organogenesis, including historical highlights starting with breakthrough studies done in the 1800s. Next, key questions asked by developmental biologists are introduced, followed by a discussion of how tissue transplantations, imaging, and in vitro culture techniques can be used to answer these queries. Finally, we describe how these methods are currently being employed in developmental biology laboratories.

 Developmental Biology

Assessing Dexterity with Reaching Tasks

Reaching tasks are employed in behavioral neuroscience to investigate motor learning and forelimb dexterity. Much like human hands, rodents have dexterous forepaws, which are necessary for executing coordinated and precise motor movements. Experimenters may utilize food rewards to train rodents to reach and for testing their reaching abilities. These tasks help behavioral neuroscientist in understanding how CNS injuries, such as a stroke, may impair reaching ability and dexterity in humans.

This video begins by discussing the principles and neurobiology of forelimb use in rodents, and then explains a protocol on how to conduct reaching experiments using different types of food rewards. Applications section reviews studies that involve reaching and food handling in animal models of CNS injury.

 Behavioral Science

An Overview of Genetic Analysis

An organism’s physical traits, or phenotype, are a product of its genotype, which is the combination of alleles (gene variants) inherited from its parents. To varying degrees, genes interact with each other and environmental factors to generate traits. The distribution of alleles and traits within a population is influenced by a number of factors, including natural selection, migration, and random genetic drift.

In this video, JoVE introduces some of the foundational discoveries in genetics, from Gregor Mendel’s elucidation of the genetic basis of inheritance, to how natural processes affect allele distributions within populations, to the modern synthesis of biology that brought together Mendelian genetics and Darwinian evolution. We then review the questions asked by geneticists today regarding how genes influence traits, and some of the main tools used to answer these questions. Finally, several applications of techniques such as genetic crosses, screens and evolution experiments will be presented.

 Genetics

Separation of Mixtures via Precipitation

Source: Laboratory of Dr. Ana J. García-Sáez — University of Tübingen

Most samples of interest are mixtures of many different components. Sample preparation, a key step in the analytical process, removes interferences that may affect the analysis. As such, developing separation techniques is an important endeavor not just in academia, but also in industry. 

One way to separate mixtures is to use their solubility properties. In this short paper, we will deal with aqueous solutions. The solubility of a compound of interest depends on (1) ionic strength of solution, (2) pH, and (3) temperature. By manipulating with these three factors, a condition in which the compound is insoluble can be used to remove the compound of interest from the rest of the sample.¹

 Organic Chemistry

The TUNEL Assay

One of the hallmarks of apoptosis is the nuclear DNA fragmentation by nucleases. These enzymes are activated by caspases, the family of proteins that execute the cell death program. TUNEL assay is a method that takes advantage of this feature to detect apoptotic cells. In this assay, an enzyme called terminal deoxynucleotidyl transferase catalyzes the addition of dUTP nucleotides to the free 3’ ends of fragmented DNA. By using dUTPs that are labeled with chemical tags that can produce fluorescence or color, apoptotic cells can be specifically identified.

JoVE’s video on the TUNEL assay begins by discussing how this technique can be used to detect apoptotic cells. We then go through a general protocol for performing TUNEL assays on tissue sections and visualizing the results using fluorescence microscopy. Finally, several applications of the assay to current research will be covered.

 Cell Biology

Electrophoretic Mobility Shift Assay (EMSA)

The electrophoretic mobility shift assay (EMSA) is a biochemical procedure used to elucidate binding between proteins and nucleic acids. In this assay a radiolabeled nucleic acid and test protein are mixed. Binding is determined via gel electrophoresis which separates components based on mass, charge, and conformation.

This video shows the concepts of EMSA and a general procedure, including gel and protein preparation, binding, electrophoresis, and detection. Applications covered in this video include the analysis of chromatin-remodeling enzymes, a modified EMSA that incorporates biontinylation, and the study of binding sites of bacterial response regulators.

 Biochemistry

Batch and Continuous Bioreactors

Bioreactors are used to grow organisms in large volumes, thereby enabling the production of mass quantities of the target product. These reactors can be batch reactors, which contain all of the components needed for cell growth, or continuous reactors, which have inlet and outlet ports allowing for the addition of fresh growth media and the removal of cell waste.

This video presents batch and continuous reactors and demonstrates the use of bioreactors to grow bacteria in the laboratory. Finally, this video considers how these reactors are used in the bioengineering field to produce products such as protein therapeutics or even beer.

 Bioengineering

Conformations of Ethane and Propane

In an organic molecule, free rotation about the carbon-carbon single bond results in energetically different conformers of the molecule. Due to this rotation, called the internal rotation, ethane has two major conformations — staggered and eclipsed.

Staggered conformation is a low energy and more stable conformation with the C-H bonds on the front carbon placed at 60°dihedral angles relative to the C-H bonds on the back carbon, leading to a reduced torsional strain. In staggered ethane, the bonding molecular orbital of one C-H bond interacts with the antibonding molecular orbital of the other, thereby further stabilizing the conformation. The rotation of farther carbon while keeping the carbon nearer to the observer stationary generates an infinite number of conformers. At  0° dihedral angles, the C-H groups cover one another to form an eclipsed conformation. This conformation has about 12 kJ/mol more torsional strain than the staggered conformation and hence is less stable. Ethane molecules rapidly interconvert between several staggered states while passing through the higher energy eclipsed states. The molecular collisions provide the energy required to cross this torsional barrier.

Similar to ethane, propane also has two major conformers: the stable staggered conformer (low energy) and the unstable eclipsed conformer (higher energy).

 Core: Organic Chemistry

Electrophiles

This lesson explains the definition, classification, and characteristic features of an electrophile that are key features of nucleophilic substitution reactions. An analysis of their charge and orbital picture helps understand their reactivity for seeking electrons. Electrophiles can be classified into positive and neutral species. Other classes include free radicals and polar functional groups.

While a positive electrophile, like a proton, reacts due to its vacant, low-energy 1s orbital, the other positive electrophiles, like carbocations, are reactive due to their vacant p orbital.

On the other hand, neutral electrophiles, analogous to Lewis acids, possess empty p orbitals that can accept electrons from the nucleophile to generate stable complexes. An electrophilic center can often be formed in a neutral molecule due to the electron-withdrawing inductive effect in the presence of a more electronegative substituent attached to the molecular chain. This explains the partial positive charge on the carbon atom in a carbonyl group. 

In the context of a chemical reaction, a comprehensive picture of the electron transfer in this process is necessary. A nucleophile deposits its electrons into the lower energy antibonding π orbital of the electrophile. In contrast, the dipole of the σ bond forces the nucleophilic electrons to move into the lower energy antibonding σ orbital, resulting in bond breaking. These phenomena are often explained using examples of carbonyl groups and HCl. Typically, the lowest occupied molecular orbitals (LUMOs) in organic electrophiles are antibonding orbitals with low energy, as they are associated with electronegative atoms. These happen to be either π* orbitals or σ* orbitals.

Some molecules, such as halogens, also make good electrophiles. Here, despite the absence of a dipole, a poor overlap between the atomic orbitals of the two halides weakens the bond making it more prone to a nucleophile attack. This leads to the other classification of strong versus weak electrophiles, covered in later lessons. Usually, molecules with a single or double bond linked to an electronegative atom like O, N, Cl, or Br make good electrophiles.

 Core: Organic Chemistry

Regioselectivity and Stereochemistry of Hydroboration

A significant aspect of hydroboration–oxidation is the regio- and stereochemical outcome of the reaction.

Hydroboration proceeds in a concerted fashion with the attack of borane on the π bond, giving a cyclic four-centered transition state. The –BH₂ group is bonded to the less substituted carbon and –H to the more substituted carbon. The concerted nature requires the simultaneous addition of –H and –BH₂ across the same face of the alkene giving syn stereochemistry.

The observed preference in regioselectivity can be explained on the basis of steric and electronic factors.

In the transition state, the larger part of the reagent (–BH₂) is bonded to the less substituted carbon, thereby minimizing the steric tension. This results in a less crowded low-energy transition state, which is more stable than Markovnikov's transition state.

Further, the addition of borane can result in a partial positive charge on either of the two carbons. However, a partial positive charge on the more substituted carbon is highly favorable, as it gives a more stable transition state. Hence, in order to achieve this, –BH₂ must be placed at the less substituted carbon resulting in an anti-Markovnikov orientation.

The second part of the reaction is the oxidation of the product obtained from hydroboration.

The migration of the alkyl group in this mechanism occurs with retention of configuration as it transfers with the electron pairs without reconstructing the tetrahedral geometry of the migrating carbon.

Since the reaction is stereospecific, it is essential to recognize the number of chiral centers formed. If one chiral center is formed, both enantiomers are obtained, as syn addition can occur from either face of the alkene with equal probability. However, if two chiral centers are formed, the syn addition dictates which pair of enantiomers is predominantly obtained.

 Core: Organic Chemistry

Alkynes to Aldehydes and Ketones: Acid-Catalyzed Hydration

Introduction

Analogous to alkenes, alkynes also undergo acid-catalyzed hydration. While the addition of water to an alkene gives an alcohol, hydration of alkynes produces different products such as aldehydes and ketones.       

Since the rate of acid-catalyzed hydration of alkynes is much slower than alkenes, a mercuric salt like mercuric sulfate (HgSO₄) is usually added to facilitate the reaction. Hydration of terminal alkynes follows Markovnikov's rule; however, for internal alkynes, the addition of water is non-regioselective.

Mechanism

The mechanism begins with a nucleophilic attack by the alkyne π bond on the Hg²⁺ ion resulting in the formation of a cyclic mercurinium ion intermediate. A second nucleophilic attack by water on the more substituted carbon forms an organomercuric enol that rapidly converts into a stable keto form via keto-enol tautomerism. Protonation of the keto intermediate followed by the loss of an Hg²⁺ ion yields the enol form of the product. The final step proceeds with the tautomerization of the enol to the desired ketone.

Keto-Enol Tautomerism

Unlike alkenes, acid-catalyzed hydration of alkynes is irreversible. This is because the enol intermediate formed during the hydration of alkynes is unstable and rapidly isomerizes to a more stable keto form. The chemical equilibrium that exists between the two forms is referred to as keto-enol tautomerism. Since the C=O bond is considerably stronger than the C=C bond, the equilibrium favors the keto isomer. Keto-enol tautomerism is characterized by the migration of a proton and the change in the location of a double bond.

Acid-catalyzed tautomerization is a two-step process:

Step 1: Addition of proton  across the enol double bond

Step 2: Loss of a proton to yield the keto form

Example

Acid-catalyzed hydration of 1-propyne initially forms the less stable enol isomer, propen-2-ol, which tautomerizes into a more stable keto product, propan-2-one.

Hydration of Terminal And Internal Alkynes

Acid-catalyzed hydration is most useful for terminal and symmetrical internal alkynes because they form only one final product. In contrast, unsymmetrical internal alkynes yield a mixture of products that need to be separated. This lowers the overall yield and makes the process less efficient.

 Core: Organic Chemistry

Structure and Nomenclature of Alcohols and Phenols

Overview

Alcohols are one of the most important functional groups in organic chemistry. The name of alcohol comes from the hydrocarbon from which it is derived. Alcohols are organic molecules containing the functional hydroxyl or –OH group directly bonded to carbon. Phenols have an OH group directly attached to a benzene ring. While alcohols are colorless, phenol is a white crystalline compound with a characteristic "hospital smell" odor.

As with other organic compounds, alcohols and phenols are named by formal and standard systems. The most adopted system is the International Union of Pure and Applied Chemistry (IUPAC).

The IUPAC names of the alcohols are derived by adding the suffix 'ol' to the name of the parent alkane. Phenols, on the other hand, are named as hydroxy derivatives of benzene. 'Phenol' is used as the parent name rather than benzene.

Like all organic compounds, alcohols and phenols have several commercial applications. For instance, ethanol is the main ingredient in alcoholic beverages like wine or beer. Phenols are widely used as antiseptics and as disinfectants.

Naming Alcohols

The table below shows the classification and nomenclature of some alcohols and phenols.

Skeletal Structures	IUPAC Name	Common Name
	1-butanol	n-butyl alcohol
	2-butanol	sec-butyl alcohol
	2-methyl-2-butanol	t-amyl alcohol
	2-bromo-4-chlorocyclopentanol	-
	2-propen-1-ol	allyl alcohol
	3-cyclohexen-1-ol	-
	1,2-ethanediol	ethylene glycol
	1,2,3-propanetriol	glycerol
	phenol	phenol
	benzene-1,3-diol	resorcinol
	2-methylphenol	o-cresol
	3-methylphenol	m-cresol
	4-methylphenol	p-cresol

 Core: Organic Chemistry

Preparation and Reactions of Thiols

Thiols are prepared using the hydrosulfide anion as a nucleophile in a nucleophilic substitution reaction with alkyl halides. For instance, bromobutane reacts with sodium hydrosulfide to give butanethiol.

This reaction fails because the thiol product can undergo a second nucleophilic substitution reaction in the presence of an excess alkyl halide to generate a sulfide as a by-product.

This limitation can be overcome by using thiourea as the nucleophile. The reaction first produces an alkyl isothiourea salt as an intermediate, which forms thiol as a final product upon hydrolysis with an aqueous base.

Thiols can readily oxidize to disulfides, sulfinic acid, and sulfonic acid. The oxidation of thiols to disulfides can even occur in the presence of atmospheric air. Thus, the high susceptibility of thiols to undergo air oxidation necessitates the storage of thiols in an inert atmosphere. Oxidation of thiols to disulfides can also be accomplished using reagents like molecular bromine or iodine in the presence of a base. Disulfides, however, can be easily reduced back to thiols by treatment with reducing agents such as HCl in the presence of zinc. Notably, oxidation of thiols to disulfides is a redox reaction. The interconversion between thiols and disulfides is ascribed to the bond strength of the S–S bond, which is approximately half the strength of other covalent bonds.

 Core: Organic Chemistry

Overview of Cell Death

Cell death is an essential process where the body gets rid of old or damaged cells. Cell proliferation and death need to be balanced, as an imbalance between the two may lead to cancer or autoimmune diseases.

Cell death was observed in the early 19th century, but there was no experimental evidence to prove it. In 1842, Carl Vogt first discovered cell death in a metamorphic toad; however, it was not termed ‘cell death.’ Scientists discovered different cell death pathways only in the 20th century with advancements in microscopy and histology.

While apoptosis, autophagy, and necrosis are the most commonly known types of death,  some other types are necroptosis, pyroptosis, ferroptosis,entosis, and paraptosis. These types differ in their pathways toward cell death.

Necroptosis is a combination of apoptosis and necrosis. It begins when a ligand binds to death receptors, activating the receptor-interacting protein kinases-1 (RIPK-1) instead of caspase-8,  generally activated during apoptosis. Further, many intermediate proteins are activated, causing cell swelling and lysis. Pyroptosis is a caspase-mediated cell death that involves caspase-1 or caspase-11, required for the maturation of inflammatory cytokines.

In ferroptosis, iron-dependent lipid peroxidation occurs, accumulating reactive oxygen species that eventually induce cell death. Prominent morphological features of ferroptosis include shrinkage of mitochondria and decreased cristae. During entosis, a cell gets internalized into the neighboring cell with the help of Rho GTPase and is then degraded by the lysosomal enzymes.

Paraptosis is activated when the insulin-like growth factor 1 receptor is overexpressed, triggering events leading to cell death. Paraptosis mainly occurs in neurons, and the paraptotic pathway is also being explored in apoptosis-resistant cancer cells. Anoikis is a programmed apoptotic cell death occurring in cells that get detached from the extracellular matrix. These cells follow the extrinsic or intrinsic pathways of apoptosis.

 Core: Cell Biology

Amyloid Fibrils

 Core: Cell Biology

Regulation of Hematopoietic Stem Cells

All blood and immune cells are produced from the multipotent hematopoietic stem cells (HSCs) by the process of hematopoiesis. However, they all have a limited life span. In addition, many are depleted in immune surveillance or combatting an injury or infection. This makes blood one of the most regenerative tissues. Hematopoiesis helps replenish these blood and immune cells, restoring the body's normal functioning. However, overproduction of blood and immune cells can make them cancerous or activate autoimmune responses. Thus, hematopoiesis needs to be a tightly regulated process.

Regulation of hematopoiesis helps determine whether the HSCs stay quiescent or undergo any other fate such as self-renewal, differentiation, or apoptosis. Several soluble or membrane-bound factors such as cytokines or colony-stimulating factors (CSFs) regulate hematopoiesis.  For example, stem cell factors or steel produced by the bone marrow stroma make contact with c-kit receptors on quiescent HSCs, providing them with survival signals and HSC maintenance. The CSFs are inflammatory cytokines produced by fibroblasts and macrophages in response to allergies and bacterial or parasitic infections. CSFs regulate the rate of HSC proliferation and the number of cell division cycles they must undergo before differentiating into progenitor cells. This ensures the human body possesses an adequate reserve of stem cells to maintain all blood cell types.  CSFs also induce the progenitors to commit to lineage-specific blood and immune cells and perform their functions. They assist with rapid differentiation into specific immune cells and elicit an immediate immune response until the infection is cleared. In the absence of CSFs, HSCs undergo apoptosis.

 Core: Cell Biology

Alternative RNA Splicing

 Core: Cell Biology

Disassembly of Intermediate Filaments

Intermediate filaments (IFs) do not undergo spontaneous disassembly. Enzymes, kinases, and phosphatases add and remove phosphates from specific sites to regulate their disassembly. The IF concentration in the cytoplasm also regulates the disassembly. If the concentration crosses a threshold, it activates the protein kinases in the vicinity, allowing the phosphorylation of IFs.

Keratin proteins, found at the cell periphery near cell junctions, undergo a cycle of assembly and disassembly. In Type III and IV intermediate filaments, the phosphorylation of the N-terminal head domain by the secondary messenger-dependent kinase proteins influences the phosphorylation of the C-terminal tail, that aids in their disassembly.

During mitosis, the transition from prophase to pro-phase results in the nuclear lamins, vimentin, and glial fibrillary acidic proteins undergoing site-specific phosphorylation by Rho kinase, Cdk1, Aurora-B, and PAK1, resulting in disassembly. The phosphorylation of lamins A, B, and C leads to depolymerization of the filaments into lamin dimers, further leading to nuclear membrane disintegration. The lamins remain attached to the disintegrated membrane through their C-terminal prenylation. The removal of phosphates through phosphatase leads to the reassembly of the lamin meshwork during the telophase.

 Core: Cell Biology

Co-activators and Co-repressors

 Core: Cell Biology

Chemistry of the Cell

 Core: Cell Biology

Base Excision Repair

 Core: Cell Biology

NF-kB-dependent Signaling Pathway

 Core: Cell Biology

Tagging and Fusion Proteins

Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and purification processes. Affinity tags, epitope tags, reporter tags, fluorescent tags, and self-splicing intein tags, are just a few of the various protein tags available.

Glutathione S-transferase Tag

Glutathione S-transferase (GST) is a 211 amino acid protein commonly employed to tag recombinant proteins. An expression vector comprising the gene of interest and the DNA sequence for GST is used for expression in a suitable host, such as E.coli. The recombinant protein can be tagged with GST at either its N-terminal or C-terminal. The GST-tag also increases the solubility of the fusion protein compared to the non-tagged native protein. Since GST is an enzyme, it has high binding specificity to its substrate, glutathione. This substrate specificity is used to purify GST-tagged proteins by affinity chromatography using a matrix of glutathione-coated beads.

Tag Cleavage and Self-splicing

While protein tags allow the target protein to be purified, they might hinder further protein analysis. In such cases, the tags are cleaved using proteolytic enzymes. Since these proteases cleave only at specific sites, fusion proteins are designed with such cleavage sites between the target protein and tag. Another method uses self-splicing protein segments, called inteins, that splice the tags from the target protein without additional enzymes. In this method, an intein segment is also recombined into the fusion protein, positioned between the tag and target protein. These inteins self-splice only under certain conditions such as the presence of thiol compounds or specific pH and temperature. Thus, splicing can be specifically induced after purification of the fusion protein to obtain the pure target protein for further analysis.

 Core: Cell Biology

Confocal Fluorescence Microscopy

Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore, very useful for examining thick specimens such as biofilms, which can be examined alive and unfixed.

The confocal microscopes use two pinholes—illumination, and emission pinhole, to modulate the laser beam to obtain clear, crisp images. The laser passes through the illumination pinhole and gets reflected by the dichroic mirror to scan the sample surface. An emission pinhole confocal with the illumination plane; focuses the emitted light reaching the detector. It eliminates light from non-focused z-planes reaching the detector to obtain high contrast two-dimensional images called the optical sections. A computer software program then merges optical sections from different focal planes to reconstruct a three-dimensional image.

Two types of confocal microscope are widely used based on their method of scanning the samples; laser scanning (LSCM) and spinning disc laser (SDLM) microscopy. In LSCM, a point laser scans each focal plane across the sample and collects the emitted fluorescence through a pinhole in detectors. These two-dimensional images, called the optical sections, can be stacked to reconstruct the three-dimensional image. In contrast, SDLM consists of two linked spinning disks with hundreds of pinholes. It allows rapid scanning of the sample surface at different planes and faster image capturing.

Limitations of confocal microscopy

In confocal microscopy, the limited wavelengths of light in the lasers are a disadvantage. The traditional fluorescence microscopes offer a wide range of illumination wavelengths, using mercury or xenon arc lamps as their illumination sources. The high intensity of the laser in early confocal microscopes was damaging for the cells, which has been overcome to a great extent in the multiphoton microscope systems.

 Core: Cell Biology

Inhibition of CDK Activity

 Core: Cell Biology

Separation of Sister Chromatids

 Core: Cell Biology

Tumor Progression

 Core: Cell Biology

Targeted Cancer Therapies

 Core: Cell Biology

Whole Body Regeneration

Regeneration is the process of restoring injured or lost tissues, organs, or body parts. While simpler organisms generally show greater ability to regenerate their whole body, few complex animals show similarly exceptional regeneration. For example, planarian flatworms have a unique regenerative potential making them a popular study organism among biologists to understand the mechanisms of whole body regeneration. Other organisms, such as hydra, also show extreme regeneration potential; even when cut into several pieces, each piece can turn into an individual organism.

Planarian Stem Cells

Planarians are among the few animals that maintain pluripotent stem cells throughout life. First described in the 1800s, these pluripotent stem cells are now called neoblasts. Neoblasts are essential for the regeneration of the endoderm, mesoderm, and ectoderm, thus, facilitating whole body regeneration. These neoblasts are present throughout the planarian body, including the space surrounding the gut. As much as 30 percent of cells in planaria are neoblasts. Thus, a planarian worm can regenerate a whole body even when cut into small pieces.

Regeneration Polarity in Planarians

Regeneration requires the restoration of complex anatomical structures and specific integration of organs within the body through precise control of the organs’ size, location, and identity. When a planarian flatworm is amputated transversely, two fragments are generated — a fragment containing the head that regenerates a tail; and a tail fragment that regenerates the head. The location along the anterior-posterior axis determines whether the tissue differentiates into the head or tail. The Wnt proteins are a major determinant of this axis, with high Wnt expression in the anterior cells and low Wnt expression in the posterior cells. At the wound site, posterior cells express the Wnt1 gene, which triggers tail regeneration. In contrast, anterior cells express the notum gene, which inhibits the Wnt signaling and facilitates regeneration of the head.

 Core: Cell Biology

Heat Capacities of an Ideal Gas I

Heat capacity is the ratio of heat absorbed by the substance corresponding to its temperature change. It is also called thermal capacity and the SI unit of heat capacity is J/K. Whereas, specific heat capacity is defined as the amount of heat necessary to change the temperature of 1 kg of a substance by 1 K and is also called massic heat capacity. Its SI unit is J/kg⋅K.

Molar heat capacity quantifies the ratio of the amount of heat added (or removed) to increase (or decrease) the temperature of 1 mole of a substance by 1 K, either measured under a constant volume or constant pressure. Molar heat capacity is one of the characteristics of a substance and is also called molar specific heat. Its SI unit is J/mol·K. Measuring the molar heat capacity at constant volume is the easiest. The system acquires infinite number of possible heat capacities if neither the pressure nor the volume is constant. In most cases, the molar heat capacity at constant pressure (C_P) is higher than the molar heat capacity at constant volume (C_V). For example, the air has 40% greater C_P than C_V.

 Core: Physics

Introduction to Mechanisms of Enzyme Catalysis

 Core: Anatomy and Physiology

Membrane Proteins

 Core: Anatomy and Physiology

Interphase

 Core: Anatomy and Physiology

Random Error

Random or indeterminate errors originate from various uncontrollable variables, such as variations in environmental conditions, instrument imperfections, or the inherent variability of the phenomena being measured. Usually, these errors cannot be predicted, estimated, or characterized because their direction and magnitude often vary in magnitude and direction even during consecutive measurements. As a result, they are difficult to eliminate. However, the aggregate effect of these errors can be approximately characterized by enumerating the frequencies of observations in a large dataset. Characterizing the collective effect of these errors helps with statistical analyses. Take a large data set of temperature measurements in London, for instance. We can plot the magnitude of temperature vs. the frequency of occurrence, and if the variations (or errors) in the temperature are truly random, we will obtain a normal distribution curve, also known as the Gaussian curve. This curve allows us to apply the mathematical laws of probability to estimate the mean value and the deviation from the mean value, also known as the standard deviation. From there, we can perform tests to eliminate outliers and answer questions about the dataset.

 Core: Analytical Chemistry

Ionic Strength: Effects on Chemical Equilibria

The addition of an inert ionic compound increases the solubility of a sparingly soluble salt. For example, adding potassium nitrate to a saturated solution of calcium sulfate significantly enhances the solubility of calcium sulfate. Le Châtelier's principle cannot predict this shift in the equilibrium. Instead, this could be explained in terms of changes in the effective concentration of the ions in solution in the presence of added inert salt.

In this solution, the primary cation—the calcium ion—is surrounded by the primary anion—the sulfate ion—which in turn is surrounded by calcium ions, forming ionic atmospheres around each ion. Additionally, the primary cation and anion are surrounded by the oppositely charged ions of the added inert salt. The ions from the inert compound in the ionic atmosphere causes the net charge on the primary ions—calcium and sulfate, in this case—to decrease, reducing the  frequency of precipitation. This shifts the equilibrium towards the dissociated ion, increasing the solubility of the sparingly soluble salt. This phenomenon is termed the salt effect, electrolyte effect, or diverse ion effect.

The salt effect is highly dependent on the ionic strength of the solution. With an increase in the ionic strength of the solution, more ions diffuse in the ionic atmosphere, causing the net charge on the primary ion to be even lower, facilitating greater dissociation of the salt. Additionally, the charge on the ions constituting the sparingly soluble salt affects the extent of the salt effect. For example, the solubility of doubly charged ions, such as those constituting barium sulfate, is influenced more than the solubility of singly charged ions, such as those constituting silver chloride, by the same concentration of potassium nitrate.

 Core: Analytical Chemistry

Classification of Titrimetric Analysis Based on Reaction Types

Titrimetric analysis in solution chemistry involves measuring the volume of solutions and is often called volumetric analysis. The standard solution of known concentration in the burette is called the titrant, whereas the solution of unknown concentration in the flask is called the analyte, or titrand. Titrimetric analyses can be classified into four types based on the reactions between the titrant and analyte.

Titrations between an acid and a base lead to neutralization reactions that form water molecules. This is called an acid-base titration. For example, the titration of a sodium hydroxide standard solution with the analyte hydrochloric acid generates water, leaving behind the acidically neutral sodium and chloride ions. In the second type of titrimetric analysis – complexometric titrations, metal ions such as silver and mercury come together with electron donors such as cyanide and chloride to form complexes. The third type is precipitation titration, in which the titrant reacts with the analyte to form an insoluble product. For instance, the titration between chloride ion and silver nitrate solution will produce the insoluble silver chloride. In the last type of titrimetric analysis, redox reactions are used to determine the amount or concentration of an analyte. In this case, the oxidation states of the titrant and the analyte change as a result of electron transference between the two. The standard solution can be an oxidizing or a reducing agent, and the end point can be detected with indicators or changes in the electrical signals.

 Core: Analytical Chemistry

Precipitation Titration: Overview

Precipitation titration involves the reaction of a titrant and an analyte to generate an insoluble precipitate. While precipitation titration uses various precipitating agents, silver nitrate is the most common precipitating reagent; titrations involving Ag⁺ are called argentometric titrations. Usually, the endpoint in a precipitation titration can be detected by visual indicators.

A precipitation titration curve demonstrates the change in concentration of the titrant or analyte upon adding the titrant. Titration curves are plotted with the volume of the analyte on the x-axis and the p function of the analyte or titrant concentration on the y-axis. A titration curve has three significant regions: before, at, and after the equivalence point. Before the equivalence point, the analyte concentration is in excess. The concentration of the unreacted analyte is calculated from a ratio of the moles of excess analyte in the solution to the total volume of the solution.

At the equivalence point, a stoichiometric amount of titrant has reacted with the analyte to form the precipitate (e.g., AgCl). However, some redissolution of the precipitate gives equal concentrations of Ag⁺ (titrant) and Cl⁻ (analyte). The analyte concentration can be estimated from the square root of the solubility product.

Beyond the equivalence point, the solution contains excess Ag⁺. Here, the analyte concentration can be estimated using the solubility expression, where the concentration of Ag⁺ is obtained from the ratio of moles of excess titrant to the total volume.

 Core: Analytical Chemistry

Atomic Nuclei: Types of Nuclear Relaxation

Nuclear relaxation restores the equilibrium population imbalance and can occur via spin–lattice or spin–spin mechanisms, which are first-order exponential decay processes.

In spin–lattice or longitudinal relaxation, the excited spins exchange energy with the surrounding lattice as they return to the lower energy level. Among several mechanisms that contribute to spin–lattice relaxation, magnetic dipolar interactions are significant. Here, the excited nucleus transfers energy to a nearby magnetic dipole, usually a tumbling proton.

Spin–lattice relaxation occurs to restore the longitudinal magnetization to its equilibrium value and is characterized by the time constant, T₁, which indicates the average lifetime of a nucleus in the excited state. T₁ is also called the dipolar or dipole–dipole relaxation time and can range from 0.01 to 100 seconds for liquids. The value of T₁ depends on the factors such as the type of nucleus, the location of a nucleus within a molecule, the size of the molecule, and temperature.

Transverse relaxation, also called spin–spin relaxation, occurs when precessing nuclei fall out of phase, resulting in magnetization decay. Transverse relaxation is influenced by static dipolar fields and is usually faster than longitudinal relaxation. The relaxation times observed in typical NMR experiments range from 0.1 to 10 seconds. Additionally, the spin-lattice relaxation time, T₁, depends on the applied magnetic field, while T₂ is independent of it.

While the relaxation process is essential to prevent saturation and obtain a detectable signal, it also affects the intensity of the NMR signals. Generally, the intensity of the NMR signal is affected by T₁ relaxation, whereas shorter T₂ results in broadened NMR signals.

 Core: Analytical Chemistry

Inductively Coupled Plasma-Mass Spectrometry (ICP-MS): Interferences

Inductively coupled plasma–mass spectrometry (ICP–MS) is a highly selective and sensitive technique for accurate elemental analysis. Though the analysis of ICP–MS mass spectra is comparatively straightforward, it is affected by spectroscopic and non-spectroscopic interferences. Spectroscopic interferences arise when the plasma contains ionic species with an m/z value the same as the analyte ion. Spectroscopic interference can be categorized as isobaric, polyatomic ions, and refractory oxide ion interferences.

Isobaric interference occurs when the inductively-coupled plasma contains isobaric species of analytes. These species are different nuclides with the same mass number and so have the same m/z values as the analyte, assuming the same charge. For example, ⁴⁰Ar⁺ and ⁵⁸Fe⁺ generally overlap the peaks for ⁴⁰Ca⁺ and ⁵⁸Ni⁺, respectively. Quadrupole-based mass instruments, having a resolution of less than 1 u, are substantially affected by isobaric interference. However, it can be minimized using higher resolution instruments.

Another spectroscopic interference is observed when various plasma components interact with matrix or atmospheric components to form polyatomic species, which may undergo further fragmentation to form molecular ions having the same m/z value as the analyte ion. ⁴⁰ArH⁺, ¹⁶O₂⁺, and ⁴⁰ArO⁺ are some potential interferents. A blank correction or using a different analyte isotope removes such interferences.

Another critical interference occurs when several analyte and matrix components form oxides and hydroxides whose peaks overlap with the analyte ions. The formation of oxides and hydroxides depends on various experimental factors, like the injector flow rate, the sample orifice size, the sampler skimmer spacing, etc. These interferences are more challenging to eliminate than isobaric and polyatomic ion interferences.

Non-spectroscopic interferences mainly include the matrix effect. It is associated with a high concentration of matrix species—generally, 500 to 1000 mg/mL—causing analyte signal reduction. Specific experimental conditions enhance the analyte signal. Matrix interferences can be eliminated by incorporating an internal standard with mass and ionization potential close to the analyte.

 Core: Analytical Chemistry

The Muscular System

The muscular system is essential to the body's overall structure and function, playing a crucial role in movement, stability, and internal processes. It consists of three distinct types of muscle tissue: the skeletal, the smooth, and the cardiac muscles.

1. Skeletal Muscles: These muscles are under our conscious control and enable movement. They are attached to bones by tendons, and their contraction and relaxation allow us to move. Additionally, they generate heat during physical activity, helping maintain body temperature.
2. Smooth Muscles: Unlike skeletal muscles, smooth muscles operate without conscious control. They are located in the walls of hollow organs such as the stomach, intestines, blood vessels, and airways. The contraction of these muscles aids in processes like digestion, by propelling food through the gastrointestinal tract.
3. Cardiac Muscle: Found exclusively in the heart, this type of muscle contracts rhythmically and continuously, pumping blood throughout the body without fatigue.

The Musculoskeletal System

As the name suggests, the musculoskeletal system is a complex network of muscles and bones. It is critical in facilitating movement and providing structural support to the body.

The skeletal component of the system is primarily made up of bones and associated elements like cartilage, tendons, and ligaments. Bones provide our bodies with basic structure and shape and protect vital organs. For instance, the skull shields the brain, while the ribcage safeguards the heart and lungs. The cartilage, tendons, and ligaments further aid mobility and stability by connecting bones and cushioning joints.

The muscular component, on the other hand, consists of various types of muscle tissues. These include skeletal muscles, which are directly attached to bones and are responsible for voluntary movements such as walking or lifting; smooth muscles, which control involuntary actions like digestion and blood flow; and cardiac muscle, found only in the heart, which pumps blood throughout the body. Together, the muscular and skeletal components work harmoniously to allow locomotor functions.

 Core: Anatomy and Physiology

Muscles of the Forearm that Move the Hand and Fingers

The muscles of the forearm that move the wrist, hand, and digits are numerous and diverse. They can be classified into two groups based on their location and function — the anterior and posterior compartment muscles.

Anterior Compartment

The anterior compartment muscles originate from the humerus. They primarily function as flexors and are also known as flexor muscles. They typically insert on the carpals, metacarpals, and phalanges. The superficial layer includes the flexor carpi radialis, palmaris longus, and flexor carpi ulnaris. Below these lies the flexor digitorum superficialis, one of the largest superficial muscles in the forearm, which plays a crucial role in flexing the digits. There are two muscles in the deep anterior compartment. The flexor pollicis longus is responsible for flexing the distal phalanx of the thumb. The flexor digitorum profundus, however, ends in four tendons that insert into the distal phalanges of the fingers.

Posterior Compartment

The posterior compartment muscles originate from the humerus and function primarily as extensors. The superficial layer includes the extensor carpi radialis longus, extensor carpi radialis brevis, extensor digitorum, extensor digiti minimi, and extensor carpi ulnaris. The extensor digitorum occupies a significant portion of the posterior surface of the forearm and divides into four tendons that insert into the middle and distal phalanges of the fingers. The extensor carpi ulnaris is located medially in this group. In the deep posterior compartment are four muscles — the abductor pollicis longus, extensor pollicis brevis, extensor pollicis longus, and extensor indicis. These muscles play a crucial role in extending the thumb and fingers.

The Retinaculum

The tendons of these forearm muscles that attach to the wrist or continue into the hand are held close to the bones by strong fasciae. Tendon sheaths surround the tendons, providing protection and reducing friction. At the wrist, the deep fascia forms fibrous bands called retinacula, which hold the tendons in place. The flexor retinaculum is located on the palmar surface of the carpal bones, forming the carpal tunnel where the median nerve and tendons of the flexor digitorum superficialis, flexor digitorum profundus, and flexor pollicis longus pass through. On the dorsal surface of the carpal bones, the extensor retinaculum holds the extensor tendons of the wrist and digits.

 Core: Anatomy and Physiology

Overview of Synapses

A synapse is a specialized structure where two neurons connect, allowing them to pass an electrical or chemical signal to another neuron. It is the point of communication between neurons. The term "synapse" is derived from the Greek word "synapsis," which means "conjunction." The entire process of neural communication revolves around the synapse. When activated, a neuron releases chemicals known as neurotransmitters into the synapse. These neurotransmitters cross the synapse and bind to receptors on the receiving neuron, triggering a response. This is how neurons 'talk' to each other.

The significance of synapses in neurobiology is immense. They play a crucial role in the formation of memories and learning. This is due to a phenomenon called synaptic plasticity, which refers to the ability of synapses to strengthen or weaken over time, depending on the amount of activity they experience. This adaptability enables one to learn new things and form new memories.

There are two main types of synapses: electrical and chemical synapses. Electrical synapses allow direct, rapid communication between cells through structures called gap junctions. They are often found in systems that require fast, synchronized activity, for example, in the heart muscle. Chemical synapses, on the other hand, are slower but allow for more complex communication because different neurotransmitters can elicit different responses. They are the most common type of synapse in the human brain.

There are distinct variations between chemical and electrical synapses. In chemical synapses, the transmission of signals relies on the release of neurotransmitter molecules, resulting in a delay of approximately one millisecond between when the axon potential reaches the presynaptic terminal and when the neurotransmitter prompts the opening of postsynaptic ion channels. It is noteworthy that this communication is unidirectional.

Contrarily, electrical synapses facilitate near-instantaneous signaling, which is crucial for synapses involved in reflexes. Some electrical synapses even allow bidirectional signaling. Furthermore, electrical synapses exhibit greater reliability as they are less prone to blockage. These synapses play a crucial role in synchronizing the electrical activity of a group of neurons. For instance, electrical synapses in the thalamus are believed to regulate slow-wave sleep, and their disruption can lead to seizures.

In conclusion, electrical synapses are essential for the proper functioning of many neural circuits. They enable faster information transmission and greater reliability compared to chemical synapses, making them a key component in the processing of signals in the nervous system.

The gap junction is another type of specialized contact between neurons that allows ions and small molecules to pass directly from one neuron to another.

 Core: Anatomy and Physiology

Diencephalon: Hypothalamus and Coordination

The hypothalamus is a small yet highly complex and essential brain region that plays a crucial role in regulating various bodily functions. Anatomically, it is located at the base of the brain, just above the brainstem and below the thalamus, forming part of the limbic system.

The hypothalamus interacts with other brain regions, including the pituitary gland, through a direct physical connection called the hypothalamic-pituitary axis. The hypothalamus receives somatic and visceral inputs and regulates various physiological activities within the body. It is divided into the mammillary, tuberal, supraoptic, and preoptic regions. 

The mammillary region comprises the mammillary bodies and the posterior hypothalamic nuclei. This region acts as a relay station in the olfactory pathway and facilitates body temperature regulation.

The tuberal region includes the stalk-like infundibulum, connecting the pituitary gland to the hypothalamus, plus the dorsomedial, ventromedial, and arcuate nuclei. This region contains satiety centers and regulates the pituitary gland's activity.

The supraoptic region contains four important nuclei: paraventricular, supraoptic, anterior hypothalamic, and suprachiasmatic. This region is involved in critical biological functions, such as producing oxytocin and vasopressin and regulating circadian rhythms.

The preoptic region is located at the anterior part of the hypothalamus and contains the medial and lateral preoptic nuclei. This area plays a crucial role in thermoregulation and controlling various bodily autonomic functions.

 Core: Anatomy and Physiology

Cranial and Spinal Meninges

The cranial and spinal meninges are complex protective structures surrounding the central nervous system (CNS), consisting of the brain and spinal cord. These meninges consist of the dura mater, the arachnoid mater, and the pia mater. They protect the CNS, provide structural support, and aid in circulating cerebrospinal fluid (CSF).

Cranial Meninges

These meningeal layers cover the cranium. The dura mater is the outermost layer of cranial meninges. It is a thick and durable membrane of dense collagen fibers divided into two layers — the periosteal layer, which attaches to the skull's inner surface, and the meningeal layer. The dura mater also forms dural venous sinuses responsible for draining blood from the brain.

The arachnoid mater is the middle layer. It is a delicate membrane present beneath the dura mater. The subdural space, which contains a small amount of serous fluid, separates the arachnoid mater from the dura mater. The arachnoid mater also has thin strands called trabeculae that connect to the pia mater.

The innermost layer is the pia mater. It is a thin and highly vascularized membrane closely adhering to the surface of the spinal cord and brain. The pia mater contributes to the formation of the blood-brain barrier.

Spinal Meninges

The spinal dura mater is the outermost layer of the spinal meninges. It consists of dense collagen fibers. The dura mater consists of only one layer, known as the meningeal layer. It is separated from the vertebrae by the epidural space, which houses fat and blood vessels.

Beneath the dura mater is the spinal arachnoid mater. The delicate middle layer is separated from the dura mater by the serous fluid-filled subdural space. The subarachnoid space, a CSF-filled space, separates the arachnoid mater from the pia mater.

The spinal pia mater is a thin, vascularized membrane closely adhering to the spinal cord's surface. It extends as a filament called the filum terminale that anchors the spinal cord to the coccyx.

 Core: Anatomy and Physiology

Smooth Muscle Contraction

Smooth muscle contraction is a complex process vital for various bodily functions, from maintaining blood vessel tension to facilitating the movement of food through the digestive tract. Unlike striated muscles, smooth muscle contraction begins more slowly and lasts longer.

The onset of contraction is triggered by an increase in calcium ions within the sarcoplasm, similar to the process in striated muscle. However, smooth muscles have a relatively smaller reservoir of the sarcoplasmic reticulum, the primary calcium storage site in striated muscles. Instead, calcium ions infiltrate the smooth muscle sarcoplasm from both the interstitial fluid and the sarcoplasmic reticulum. The absence of transverse tubules, replaced by structures called caveolae in smooth muscle, means that calcium takes longer to reach the central filaments, contributing to the slower onset of contraction typical of smooth muscles.

In the sarcoplasm, the regulatory protein calmodulin binds these calcium ions, activating myosin light chain kinases or MLCK enzymes. These enzymes alter the conformation of myosin heads in the thick filaments through phosphorylation. The ATPase activity of the myosin heads increases, preparing them to form cross-bridges with actin filaments. The myosin ATPases hydrolyze ATP to slide actin filaments over myosin filaments, resulting in a muscle contraction. Unlike skeletal muscles, the calcium removal from smooth muscle sarcoplasm is slow, which keeps the cross-bridges engaged longer, leading to sustained contraction and delaying relaxation.

Smooth muscle also differs from skeletal muscle in that its thick and thin filaments are not arranged into sarcomeres, conferring the ability to stretch considerably while maintaining contractile functionality. When stretched, smooth muscle initially contracts and increases tension but soon undergoes a stress-relaxation response, allowing the muscle to lengthen while reducing tension. This phenomenon, called plasticity, allows the smooth muscle to contract over a range of lengths four times greater than skeletal muscle. Plasticity is crucial in organs like the stomach and bladder, which must accommodate varying volumes without significantly altering internal pressure.

 Core: Anatomy and Physiology

Olfactory Receptors: Location and Structure

The process of olfaction, also known as the sense of smell, is a sophisticated chemical response system. The specialized sensory neurons that facilitate this process, known as olfactory receptor neurons, are situated in an upper segment of the nasal cavity, known as the olfactory epithelium. Olfactory sensory neurons are bipolar, with their dendrites extending from the epithelium's apex into the mucus that lines the nasal cavity. Airborne molecules, when inhaled, traverse the olfactory epithelium, dissolving into the mucus. These molecules, called odorants, bind to specific proteins that maintain their mucus solubility and aid in their transport toward the olfactory dendrites. The odorant-protein complexes bind to receptor proteins within the olfactory dendrites' cellular membrane. The receptor proteins are G protein–coupled and generate a graded membrane potential in the olfactory neurons.

The olfactory neuron's axon originates from the epithelial layer's basal surface, traversing an olfactory foramen in the ethmoid bone's cribriform plate and then projects into the brain. The collection of these axons, termed the olfactory tract, interfaces with the olfactory bulb on the frontal lobe's ventral surface. As a result, these axons bifurcate, undertaking diverse paths to various brain locales. Some axons converge on the cerebrum, particularly the primary olfactory cortex in the temporal lobe's inferior and medial regions. Conversely, other target structures are nested within the limbic system and hypothalamus, facilitating the linkage of odors with enduring memory and emotional reactions. An example of this phenomenon is the evocation of emotional recollections by certain scents, such as the aroma of food native to one's place of origin. Notably, olfaction is the singular sensory modality bypassing a synapse in the thalamus before interfacing with the cerebral cortex. This profound interconnection between the olfactory system and the cerebral cortex elucidates why odors can serve as formidable catalysts for memory and emotion.

The respiratory epithelial tissue, including olfactory neurons, may be susceptible to damage from noxious airborne substances. Consequently, olfactory neural cells within the respiratory epithelium undergo periodic regeneration, during which the axons of the newly formed neurons must establish suitable connections within the olfactory bulb. These emerging axons guide their growth pathway by following existing axons in situ within the cranial nerve.

Anosmia: The Impairment of Olfactory Function

The olfactory nerve, pivotal for the perception of smell, may witness a degradation or a complete loss due to severe facial trauma, a scenario frequently observed in vehicular accidents. This particular affliction is referred to as 'anosmia.' The relative movement of the frontal lobe and the ethmoid bone could result in the severing of olfactory tract axons. Individuals engaged in professional combat sports are often susceptible to anosmia due to constant facial and cranial injuries. Moreover, certain medications, notably antibiotics, have the potential to induce anosmia through the extermination of all olfactory neurons simultaneously. The absence of axons within the olfactory nerve implies that the axons from newly generated olfactory neurons lack a pathway to their respective connections in the olfactory bulb. Anosmia can also be transient due to inflammation resulting from respiratory infections or allergies.

Anosmia can diminish the gustatory experience by rendering food tasteless. Individuals with compromised olfactory capacity may necessitate augmented levels of spices and seasonings to detect flavor in their food. There is a potential link between anosmia and mild depressive states, as the diminished pleasure derived from food could potentially instigate a pervasive sense of melancholy.

The olfactory neurons' regenerative capacity diminishes, leading to age-associated anosmia. This can elucidate the heightened use of salt among older adults compared to younger individuals. However, escalated sodium consumption can augment blood volume and arterial pressure, subsequently escalating the probability of cardiovascular diseases among the older demographic.

 Core: Anatomy and Physiology

The Thyroid Gland

The thyroid gland is a small, butterfly-shaped gland located in the neck and covers the anterior surface of the trachea. The gland has two lateral lobes connected by a thin tissue mass called the isthmus. Internally, each lobe comprises many small spherical structures known as thyroid follicles, surrounded by a network of blood vessels.

The follicles have a central cavity lined by simple cuboidal to squamous epithelial cells called follicular cells. These cells produce the glycoprotein thyroglobulin and store it in the central colloid. Thyroglobulin acts as a precursor to triiodothyronine or T3 and thyroxine or T4, which are thyroid hormones essential for maintaining the body's metabolic rate and energy production. Additionally, the parafollicular or C cells scattered between the follicles are responsible for producing the hormone calcitonin, which regulates calcium metabolism.

 Core: Anatomy and Physiology

Mechanically-gated Ion Channels

 Core: Anatomy and Physiology

Electric Circuit Elements

Circuit elements are the basic building blocks of an electric circuit. Essentially, an electric circuit is the interconnection of these elements. Within electric circuits, one can find two types of elements: passive and active. Active elements have the ability to generate energy, whereas passive elements do not. Passive elements include components like resistors, capacitors, and inductors, while active elements typically encompass generators, batteries, and operational amplifiers.

The most crucial active elements are voltage or current sources, which generally provide power to the connected circuit. There are numerous ways to categorize circuit elements. For instance, distinguishing linear models from nonlinear models is crucial because circuits composed entirely of linear circuit elements are simpler to analyze than those with some nonlinear elements.

An element or circuit is considered linear if the element's excitation and response meet certain conditions. Both superposition and homogeneity properties are satisfied by a linear element. Consider a scenario where the excitation is the current (i) and the response is the voltage (v). If the element is subjected to a current (i₁), it delivers a response (v₁). Similarly, when the element is exposed to another current (i₂), it elicits a response (v₂).

Any circuit element that fails to meet either the superposition or the homogeneity principle is classified as nonlinear. Thermistors are examples of such nonlinear elements.

 Core: Electrical Engineering

Cascaded Op Amps

Operational amplifiers (op-amps) are versatile electronic components that can be interconnected in a cascade - one after another in a linear sequence. This cascading is possible due to their infinite input resistance and zero output resistance, allowing them to maintain their input-output relationships even when connected in series.

In a cascaded system, each op-amp is referred to as a stage. The output of one stage drives the input of the subsequent stage. As the input signal passes through each stage, it is amplified by the gain of that stage. The resulting overall gain of the cascaded system is equal to the product of the gains of each stage.

This cascading approach offers several advantages. By distributing the total gain among multiple stages, each stage has less gain than if a single op-amp was used. However, since the gain-bandwidth product remains constant for each op-amp, reducing the gain at each stage effectively increases the bandwidth at that stage. This leads to an overall increase in the bandwidth of the cascaded system.

Cascaded op-amps find extensive use in tuned RF amplifiers found within television circuits. These cascaded amplifiers amplify weak signals and improve impedance matching at the input and output, delivering high-quality audio and video signals to the viewers.

However, designing a cascaded op-amp circuit requires careful consideration. The individual gains must be set such that they do not saturate the signal in the various stages of the cascade. Signal saturation can distort the output, leading to poor performance.

 Core: Electrical Engineering

Types of Responses of Series RLC Circuits

A second-order differential equation characterizes a source-free series RLC circuit, marking its distinct mathematical representation. The complete solution of this equation is a blend of two unique solutions, each linked to the circuit's roots expressed in terms of the damping factor and resonant frequency.

When the damping factor surpasses the resonant frequency, both roots are real and negative, leading to an overdamped response. In this scenario, the circuit's reaction gradually decays over time.

When the damping factor matches the resonant frequency, the second-order differential equation simplifies to a first-order equation with an exponential solution. The natural response follows a pattern of peaking at its time constant and then decaying to zero, signifying critical damping.

For situations where the damping factor is less than the resonant frequency, complex roots emerge, characterized by the damped natural frequency. Euler's formula simplifies the complete response to sine and cosine functions, resulting in an underdamped and oscillatory natural response with a time period proportional to the damped natural frequency.

These different response behaviors illustrate the significance of source-free RLC circuits in circuit analysis, offering intriguing insights into electrical circuit behavior and applications.

 Core: Electrical Engineering

Op Amp AC Circuits

Within an audio system, the filter circuit plays a pivotal role in processing the amplified audio signal from an amplifier. Its primary function is significantly attenuating signal components with lower frequencies, thereby shaping the audio output. This circuit's operations are examined, focusing on the fundamental filter configuration. This configuration involves an operational amplifier arranged in an inverting setup coupled with resistors (R₁ and R₂) and a capacitor (C₁).

When faced with a known input signal, the challenge lies in determining the resultant output signal. The first step involves calculating the capacitor's impedance, which is achieved by employing the angular frequency derived from the time-domain expression of the input voltage.

As a result, the analysis transitions into the frequency domain, where the input signal is represented in polar form alongside the impedance components Z₁ and Z₂. Z₂ relates explicitly to the parallel combination of capacitor C₁ and resistor R₂. The core of the analysis rests on applying Kirchhoff's current law and Ohm's law at a specific node in the circuit, thereby formulating a nodal equation for an ideal op-amp. This equation, when rearranged, reveals a critical insight: the ratio of the output to the input voltage is inversely proportional to the ratio of impedances.

The known and calculated values are skillfully substituted into this equation to unveil the output voltage in polar form. The outcome, representing the output voltage, can then be transformed into the time domain, providing a comprehensive understanding of the filter circuit's response to the input signal. For the analysis, ideal op-amp properties are often assumed, including the principle that no current enters either of its input terminals and that the voltage across its input terminals remains zero. 

 Core: Electrical Engineering

Position and Displacement Vectors

 Core: Mechanical Engineering

Buffers: Overview

Buffers play a crucial role in stabilizing the pH of a solution by mitigating the effects of small amounts of added acid or base. They consist of a weak acid and its conjugate base or a weak base and its conjugate acid. A solution of acetic acid and sodium acetate is an example of a buffer that consists of a weak acid and its salt: CH₃COOH (aq) + CH₃COONa (aq). An example of a buffer that consists of a weak base and its salt is a solution of ammonia and ammonium chloride: NH₃ (aq) + NH₄Cl (aq).

This combination prevents significant pH changes as long as the buffer's capacity is not exceeded. For example, human blood uses a carbonic acid-bicarbonate buffer system to maintain its pH near 7.4. In a buffer, the weak acid component neutralizes added bases by reacting with hydroxide ions, while the conjugate base neutralizes added acids by reacting with hydronium ions.

 Core: Analytical Chemistry

C. elegans Chemotaxis Assay

Chemotaxis is a process in which cells or organisms move in response to a chemical stimulus. In nature, chemotaxis is important for organisms to sense and move toward food sources and move away from stimuli that may be toxic or harmful. Chemotaxis is also important at the cellular level. For example, chemotaxis is required for the movement of sperm toward an egg prior to fertilization. In the lab, chemotaxis is frequently examined in the nematode, C. elegans, which is known to migrate towards food sources in soil, but away from toxins such as heavy metals, substances with a low pH, and detergents. This video demonstrates how to perform a chemotaxis assay, which includes preparing the chemotaxis plates and the worms, running the assay, and analyzing the data. Then, we discuss examples of how chemotaxis assays can be used in C. elegans as a tool to understand learning and memory, olfactory adaptation, and neurological disease such as Alzheimer"s disease. Chemotaxis experiments in C. elegans have near-limitless possibilities for learning more about the cellular and genetic mechanisms of many biological processes, and may lead to a greater understanding of human biology, development, and disease.

 Biology I

Mouse Genotyping

Even though the human genome was mapped over 10 years ago, scientists are still far from understanding the function of every human gene! One way to evaluate how a gene functions is to disrupt the sequence encoding it and then evaluate the impact of this change (the phenotype) on the animal’s biology. This approach is commonly used in the mouse (Mus musculus), since it shares a high degree of genetic similarity with humans. To track the animals bearing genetic changes over several generations, it is necessary to screen the DNA of each mouse in a process known as genotyping.

This video provides an overview of the theory and practice behind genotyping mice. The discussion begins with the basic principles of mouse genetics, including a review of the terms homozygote, heterozygote, wildtype, mutant, and transgenic. Next, step-by-step instructions are supplied for extracting and purifying genomic DNA from mouse tissue. Examples are provided demonstrating how to interpret genotyping results, as well as how to keep track of mice with the desired genotype. Finally, some representative applications of the genotyping procedure will be presented in order to demonstrate why this common technique is so essential to mouse research.

 Biology II

An Introduction to Cellular and Molecular Neuroscience

Cellular and molecular neuroscience is one of the newest and fastest growing subdisciplines in neuroscience. By investigating the influences of genes, signaling molecules, and cellular morphology, researchers in this field uncover crucial insights into normal brain development and function, as well as the root causes of many pathological conditions.

This video introduction to the fascinating world of cellular and molecular neuroscience begins with a timeline of landmark studies, from the discovery of DNA in 1953 to more recent breakthroughs like the cloning of ion channels. Next, key questions in the field are introduced, such as how genes influence neuron activity and how the nervous system is modified by experience. This is followed by brief descriptions of some prominent methods used to analyze genetic material in neurons, manipulate expression of genes, and visualize neurons and their parts. Finally, several applications of molecular and cellular neuroscience are presented to demonstrate how cellular and molecular approaches can be used to profile neuron populations and explore their functions.

 Neuroscience

Fate Mapping

Fate mapping is a technique used to understand how embryonic cells divide, differentiate, and migrate during development. In classic fate mapping experiments, cells in different areas of an embryo are labeled with a chemical dye and then tracked to determine which tissues or structures they form. Technological improvements now allow for individual cells to be marked and traced throughout embryonic development and adulthood.

This video reviews the concepts behind fate mapping, and then details a fate mapping protocol in zebrafish using photoactivatable fluorescent proteins. Finally, specific applications and modifications of this unique technique are discussed.

 Developmental Biology

An Introduction to Reward and Addiction

Consequences play a major role in controlling our behavior. If the consequence is a reward, then it encourages the associated behavior. Rewards can come in many forms such as a pleasant feeling, money, or food. However, sometimes an individual engages in compulsive behavior despite of negative consequences, and this state is known as addiction. Administration of addictive substances is neurochemically rewarding, which ultimately causes a loss of control in limiting the intake. Scientists aim to better understand the mechanisms behind these concepts and subsequently develop new therapies for treating substance abuse disorders.

JoVE's introduction to reward and addiction explains the neuroanatomical components of the reward pathway. This is followed by some of the important questions asked by behavioral researchers such as how does our brain chemistry change in response to drug use. Prominent methods section reviews some of the tools being employed in the field, like self-administration protocols. Finally, the video discusses example experiments conducted in labs interested in investigating reward and addiction.

 Behavioral Science

Genetic Crosses

To dissect genetic processes or create organisms with novel suites of traits, scientists can perform genetic crosses, or the purposeful mating of two organisms. The recombination of parental genetic material in the offspring allows researchers to deduce the functions, interactions, and locations of genes.

This video will examine how genetic crosses were influential in developing Mendel's three laws of inheritance, which form the basis of our understanding of genetics. One genetic crossing technique that was first developed for single-celled organisms such as yeast, known as tetrad analysis, will then be presented in detail, followed by some examples of how this classical tool is used in genetic studies today.

 Genetics

Using Differential Scanning Calorimetry to Measure Changes in Enthalpy

Source: Laboratory of Dr. Terry Tritt — Clemson University

Differential Scanning Calorimetry (DSC) is a method of thermodynamic analysis based on heat-flux method, wherein a sample material (enclosed in a pan) and an empty reference pan are subjected to identical temperature conditions. The energy difference that is required to maintain both the pans at the same temperature, owing to the difference in the heat capacities of the sample and the reference pan, is recorded as a function of temperature. This energy released or absorbed is a measure of the enthalpy change (ΔΗ) of the sample with respect to the reference pan.

 General Chemistry

An Introduction to Cell Metabolism

In cells, critical molecules are either built by joining together individual units like amino acids or nucleotides, or broken down into smaller components. Respectively, the reactions responsible for this are referred to as anabolic and catabolic. These reactions require or produce energy typically in the form of a “high-energy” molecule called ATP. Together, these processes make up “Cell Metabolism,” and are hallmarks of healthy, living cells.

JoVE’s introduction to cell metabolism briefly reviews the rich history of this field, ranging from early studies on photosynthesis to more recent discoveries pertaining to energy production in all cells. This is followed by a discussion of some key questions asked by scientists studying metabolism, and common methods that they apply to answer these questions. Finally, we’ll explore how current researchers are studying alterations in metabolism that accompany metabolic disorders, or that occur following exposure to environmental stressors.

 Cell Biology

Co-Immunoprecipitation and Pull-Down Assays

Co-immunoprecipitation (CoIP) and pull-down assays are closely related methods to identify stable protein-protein interactions. These methods are related to immunoprecipitation, a method for separating a target protein bound to an antibody from unbound proteins. In CoIP, an antibody-bound protein is itself bound to another protein that does not bind with the antibody, this is followed by a separation process that preserves the protein-protein complex. The difference in pull-down assays is that affinity-tagged bait proteins replace antibodies, and affinity chromatography is used to isolate protein-protein complexes.

This video explains CoIP, pull-down assays, and their implementation in the laboratory. A step-by-step protocol for each technique is covered, including the reagents, apparatus, and instruments used to purify and analyze bound proteins. Additionally, the applications section of this video describes a procedure to study how myxovirus proteins inhibit influenza nucleoprotein, an investigation into the role of calcium ions in calmodulin via a pull-down assay, and a modified pull-down assay for characterizing transient protein interactions.

 Biochemistry

Overview of Biosensing

Biosensors are devices that use a wide range of biological processes and physical properties in order to detect either a biological molecule, such as a protein or cell, or a non-biological molecule, such as a chemical component or contaminant. This interdisciplinary field utilizes electrical, optical, electrochemical, or even mechanical properties to detect the presence of the target molecule.

This video introduces the field of biosensing, and reviews common types of biosensor technologies. This video also discusses key challenges in the field, and provides insight into how biosensors are used in the field.

 Bioengineering

Conformations of Butane

Unlike ethane and propane that have only two major conformations, butane has more than two conformers. The staggered form of butane in which the bulky methyl groups on the two carbons are placed on opposite sides, that is, at a dihedral angle of 180°, is the lowest energy, most stable form — called the anti conformer. This conformation is stabilized due to the absence of steric repulsion between the largely spaced out methyl groups. The other two staggered conformations are degenerate and have 3.8 kJ/mol more energy than the anti conformation, emerging from the steric repulsion between the methyl groups that are now positioned at 60° dihedral angles. These unfavorable steric interactions are known as gauche interactions, and the conformers, as such, are called gauche conformers.

The totally eclipsed butane, due to the two eclipsing methyl groups at 0° dihedral angle, has the highest energy (19 kJ/mol) and is the most unstable form. The other two eclipsed conformations — with two CH₃-H eclipses and one H-H eclipse — are degenerate, each with an energy cost of 16 kJ/mol.

 Core: Organic Chemistry

S_N1 Reaction: Mechanism

Kinetic studies of ionization of a tertiary halide in a protic solvent suggest that only the substrate participates in the rate-determining step (slow step). The nucleophile is involved only after the slowest step. The S_N1 reaction takes place in a multiple-step mechanism. 

Firstly, the haloalkane ionizes to generate a carbocation intermediate and a halide ion. This heterolytic cleavage is highly endothermic with large activation energy. The ionization of the substrate, facilitated by a polar protic solvent, is the slowest of all steps, making it the rate-determining step of an S_N1 reaction. The ions formed are stabilized through solvation. In the second step, the reactive carbocation intermediate behaves as a strong electrophile and is attacked by the nucleophilic solvent molecule that quickly donates an electron pair to generate an oxonium ion. This process is exothermic. In the third step, the solvent abstracts a proton from the oxonium ion to yield the final nucleophilic substituted product. 

Thus, the S_N1 reaction consists of two core steps for substitution and an additional step of proton loss. The mechanism further suggests that several factors such as the stability of the carbocation, the nature of the leaving group, and the nature of the solvent used, favor the S_N1 mechanism. 

 Core: Organic Chemistry

Oxidation of Alkenes: Syn Dihydroxylation with Osmium Tetraoxide

Alkenes are converted to 1,2-diols or glycols through a process called dihydroxylation. It involves the addition of two hydroxyl groups across the double bond with two different stereochemical approaches, namely anti and syn. Dihydroxylation using osmium tetroxide progresses with syn stereochemistry.

Syn Dihydroxylation Mechanism

The reaction comprises a two-step mechanism. It begins with the addition of osmium tetroxide across the alkene double bond in a concerted manner forming a five-membered cyclic osmate ester as an intermediate, which can be isolated and characterized. Osmium tetroxide is electrophilic in nature, serving as a strong oxidizing agent. It accepts an electron pair from the alkene π bond undergoing a reduction from +VIII to +VI.

In the next step,  the cyclic osmate ester reacts with a reducing agent like sodium bisulfite that cleaves the Os–O bond producing a cis-glycol with retention of the syn stereochemistry of the two newly formed C–O bonds.

A major drawback of the method is the use of toxic and expensive osmium tetroxide. To overcome this, osmium tetroxide is often used as a catalyst along with the co-oxidants like N-methylmorpholine N-oxide (NMO) or tert-butyl hydroperoxide (TBHP). The co-oxidants reoxidize the osmium +VI species to +VIII, thereby regenerating osmium tetroxide for further oxidation of the remaining alkenes.

Stereochemical Outcome

As oxidation of alkenes using osmium tetroxide is a stereospecific syn addition process, the two oxygens of osmium tetroxide are simultaneously added to the same face of the alkene π bond. Based on this, dihydroxylation of (E)-hex-3-ene produces a pair of enantiomers, while (Z)-hex-3-ene gives a meso compound.

Sharpless Asymmetric Dihydroxylation

Interestingly, Karl Barry Sharpless developed an enantioselective method for syn dihydroxylation of alkenes, for which he was awarded the Nobel prize. This method is known as Sharpless asymmetric dihydroxylation that is carried out using osmium tetroxide, a stoichiometric amount of the co-oxidant, and a chiral amine ligand. 

 Core: Organic Chemistry

Alkynes to Aldehydes and Ketones: Hydroboration-Oxidation

Introduction

One of the convenient methods for the preparation of aldehydes and ketones is via hydration of alkynes. Hydroboration-oxidation of alkynes is an indirect hydration reaction in which an alkyne is treated with borane followed by oxidation with alkaline peroxide to form an enol that rapidly converts into an aldehyde or a ketone. Terminal alkynes form aldehydes, whereas internal alkynes give ketones as the final product.

Mechanism

The hydroboration-oxidation reaction is a two-step process. It begins with the hydroboration step, which involves a concerted syn addition of BH₃ across the carbon–carbon triple bond to form an alkenylborane. The concerted nature of the reaction also accounts for the anti-Markovnikov regiochemistry, where the BH₂ group adds to the less substituted carbon and H to the more substituted carbon of the triple bond.

 Three successive hydroboration reactions convert an alkene into a trialkenylborane intermediate. The second part of the sequence is oxidation, where the trialkenylborane is treated with alkaline hydrogen peroxide to form an enol. The enol eventually converts into a stable carbonyl product via keto-enol tautomerism.

Hydroboration of Alkynes with Disubstituted Boranes

Unlike alkenes, hydroboration of alkynes does not stop at the first addition of BH₃. This is because alkynes have two π bonds, each capable of reacting with BH₃. The first addition forms an organoborane, which is an alkene derivative that can react further with another equivalent of BH₃.

Terminal alkynes being less hindered than internal alkynes are more susceptible to a second BH₃ addition. With internal alkynes, the addition of BH₃ stops after the first stage and proceeds in a direction to give the trialkenylborane.

 Nevertheless, hydroboration of terminal alkynes can be stopped at the first step by using bulky disubstituted boranes (R₂BH) such as disiamylborane and 9-BBN instead of BH₃.

The first addition of the bulky reagent forms a sterically hindered alkenylborane that resists any further additions and helps in the efficient conversion of alkynes to stable carbonyl compounds.

 Core: Organic Chemistry

Physical Properties of Alcohols and Phenols

Alcohols are organic compounds in which a hydroxy group is attached to a saturated carbon. Phenols are a class of alcohols containing a hydroxy group attached to an aromatic ring. The physical properties of the alcohols and phenols are influenced by hydrogen bonding due to the oxygen–hydrogen dipole in the hydroxy functional group and dispersion forces between alkyl or aryl regions of alcohol and phenol molecules.

Alcohols possess a higher boiling point than aliphatic hydrocarbons of similar molecular weights due to intermolecular hydrogen bonding. As in hydrocarbons, the dispersion forces are the reason for the higher boiling point upon increasing carbon chain length.

Hydrogen bonding between the hydroxy group and water facilitates the solubility of alcohols in water. However, water solubility also depends on the length of the alkyl or nonpolar region of the molecule. Alcohols with an alkyl region of up to three carbon atoms are miscible with water. As the chain length increases, the increased surface area of the nonpolar region hinders solvation by the water molecules.

The solubility of branched alcohols is higher than that of linear alcohols of similar molecular weight. Branching reduces the surface area for intermolecular interactions between nonpolar regions; hence, the hydrophobic nonpolar region is smaller. Because of the weaker intermolecular interactions, the boiling points of branched alcohols are lower than the corresponding linear alcohols.

Multiple sites for hydrogen bonding in one molecule increase the boiling point; therefore, diols and amino alcohols possess higher boiling points and better water solubility than alcohols.

Compared to linear alcohols, cyclic alcohols can only exist in a limited number of conformations due to steric restrictions. The increased intermolecular interactions that arise from the close packing of cyclic alcohol in the liquid phase results in a higher boiling point as compared to that of a linear alcohol.

Intermolecular hydrogen bonds also play a role in defining phenols' high boiling point and solubility in water. The boiling point of phenol is higher than that of the corresponding aliphatic alcohol due to the close packing of phenol molecules, facilitated by π–π stacking interactions between the large, planar aromatic rings. Close-packed aromatic rings increase the surface area of the nonpolar region in the liquid phase and limit the solubility of phenol (9.3 g in 100 g H₂O). However, this solubility is higher than that of alcohols with a similar molecular weight due to the increased polarity of the oxygen–hydrogen bond dipole induced by adjacent electron-withdrawing aromatic rings.

Structure	Name	Molecular weight (g/mol)	Boiling point (oC)	Solubility (g/100 g H2O)
	1-Butanol	74	118	9.1
	Isobutanol	74	108	10
	tert-Butanol	74	83	miscible (∞)
	Pentane	72	36	insoluble
	Propane-1,2-diol	76	188	miscible (∞)
	1-Hexanol	102	156	0.6
	Cyclohexanol	100	162	3.6
	Phenol	94	182	9.3
	Toluene	92	110	insoluble

Alcohols are widely used as antiseptics due to their antibacterial properties. Isopropanol or ethanol is the major component of hand sanitizer. An ideal antibacterial agent should have a significant nonpolar region or alkyl region that can penetrate the cell membranes of microorganisms and destroy them. At the same time, it should have high solubility in the transport medium, which is water. In smaller alcohols, the optimal balance between these two conditions is fulfilled.

 Core: Organic Chemistry

Preparation and Reactions of Sulfides

Sulfides are the sulfur analog of ethers, just as thiols are the sulfur analog of alcohol. Like ethers, sulfides also consist of two hydrocarbon groups bonded to the central sulfur atom. Depending upon the type of groups present, sulfides can be symmetrical or asymmetrical. Symmetrical sulfides can be prepared via an S_N2 reaction between 2 equivalents of an alkyl halide and one equivalent of sodium sulfide.

Asymmetrical sulfides can be synthesized by treating thiols with an alkyl halide and a base. The reaction follows an S_N2 pathway and proceeds via the thiolate ion intermediate. This reaction is the sulfur analog of Williamson ether synthesis and prefers methyl, primary, and secondary alkyl halides but not tertiary alkyl halides. Sulfides can readily oxidize to sulfoxide and sulfones.

Treatment of sulfide with one equivalent of hydrogen peroxide at room temperature yields sulfoxide, which upon further oxidation with a peroxy acid yields a sulfone. However, 2 equivalents of hydrogen peroxide oxidize sulfide directly to sulfone.

Dimethyl sulfoxide (DMSO) and tetramethylene sulfone are common examples of sulfoxides and sulfones, respectively. They both are excellent dipolar aprotic solvents.

 Core: Organic Chemistry

Apoptosis

Apoptosis is a combination of two Greek words, 'apo' and 'ptosis,' meaning separation and falling off, respectively. Hippocrates used this word to describe gangrene, which was caused due to bandaging of fractured bones. Apoptosis was distinguished from necrosis in 1970 when John Kerr reported observations of morphological changes occurring during apoptosis. During one experiment, he observed that the disruption of blood supply to the liver tissue resulted in a size reduction of the tissue. After examining the tissue under an electron microscope, he observed that the hepatocytes had shrunk and the chromatin had condensed. In 1980, Wyllie established the relationship between DNA degradation and apoptosis.

Apoptosis allows a cell to die in a controlled manner that prevents the release of potentially damaging molecules from inside the cell to the extracellular space. Many internal checkpoints monitor a cell's health. If abnormalities are observed, a cell can spontaneously initiate apoptosis. However, the cell's standard checks and balances fail in some cases, such as during viral infection or uncontrolled cell division due to cancer. External signaling can also initiate apoptosis. For example, most normal animal cells have receptors that interact with the extracellular matrix, a network of glycoproteins that provides structural support for cells in an organism. The binding of cellular receptors to the extracellular matrix initiates a signaling cascade within the cell. However, if the cell moves away from the extracellular matrix, the signaling ceases, and the cell undergoes apoptosis. This system prevents cells from traveling through the body and proliferating out of control, as happens with tumor cells that metastasize.

Regulated apoptosis is essential to maintain normal physiology and tissue homeostasis. Dysregulated apoptosis in cells leads to various diseases, such as cancer, Alzheimer's, and cardiovascular diseases. Dysregulated apoptosis can also cause increased apoptosis, as observed during various autoimmune diseases like Hashimoto's thyroiditis, systemic lupus erythematosus, and rheumatoid arthritis.

Some portion of this text is adapted from Openstax, Biology 2e, Section: 9.3

 Core: Cell Biology

Ligand Binding Sites

 Core: Cell Biology

Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are adult stem cells that can differentiate into most connective tissue cell types, except for hematopoietic cells, depending upon the source of MSCs. For example, bone-marrow-derived MSCs (BM-MSCs) can differentiate into osteocytes, hepatocytes, and pancreatic and neuronal cells. MSCs can be isolated from various sources such as bone marrow, placenta, adipose tissue, teeth, and Wharton’s jelly, a gelatinous substance in the umbilical cord. The ease of their access and in vitro expansion and stability have made MSCs the choice of cells in regenerative medicine.

MSCs also show anti-inflammatory and immunomodulatory properties. Immunomodulation is the ability to activate or suppress the immune system to respond to an infection. Immunomodulation is facilitated by extracellular vesicles (EVs) released from MSCs. EVs are reported to efficiently carry molecules such as nucleic acids, proteins, and other paracrine signaling molecules to recipient cells. The EVs can also cross the blood-brain barrier and effectively trigger immune responses. Hence, MSCs and EVs are being investigated for their use as therapeutic agents to treat diseases such as Rheumatoid arthritis and other autoimmune disorders.

While MSCs are explored for their therapeutic potential in repair and regeneration, MSCs also show pro-tumorigenic properties. MSCs secrete growth factors that induce angiogenesis and facilitate tumor growth and invasion, especially when close to other tumor cells. Despite this challenge, MSCs are used as therapeutic agents for cancer treatment. MSCs can inhibit pathways related to the tumor cell cycle or release cytotoxic factors to induce programmed cell death in cancer cells. Thus, MSCs are a ‘double-edged sword’ in cancer treatment.

 Core: Cell Biology