JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
Single gene insertion drives bioalcohol production by a thermophilic archaeon.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 11-05-2014
Show Abstract
Hide Abstract
Bioethanol production is achieved by only two metabolic pathways and only at moderate temperatures. Herein a fundamentally different synthetic pathway for bioalcohol production at 70 °C was constructed by insertion of the gene for bacterial alcohol dehydrogenase (AdhA) into the archaeon Pyrococcus furiosus. The engineered strain converted glucose to ethanol via acetate and acetaldehyde, catalyzed by the host-encoded aldehyde ferredoxin oxidoreductase (AOR) and heterologously expressed AdhA, in an energy-conserving, redox-balanced pathway. Furthermore, the AOR/AdhA pathway also converted exogenously added aliphatic and aromatic carboxylic acids to the corresponding alcohol using glucose, pyruvate, and/or hydrogen as the source of reductant. By heterologous coexpression of a membrane-bound carbon monoxide dehydrogenase, CO was used as a reductant for converting carboxylic acids to alcohols. Redirecting the fermentative metabolism of P. furiosus through strategic insertion of foreign genes creates unprecedented opportunities for thermophilic bioalcohol production. Moreover, the AOR/AdhA pathway is a potentially game-changing strategy for syngas fermentation, especially in combination with carbon chain elongation pathways.
Related JoVE Video
Aggregation propensities of superoxide dismutase G93 hotspot mutants mirror ALS clinical phenotypes.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 10-14-2014
Show Abstract
Hide Abstract
Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misassembly and aggregation in cells affected by the motor neuron disease ALS. However, the mechanistic relationship between superoxide dismutase 1 (SOD1) mutations and human disease is controversial, with many hypotheses postulated for the propensity of specific SOD mutants to cause ALS. Here, we experimentally identify distinguishing attributes of ALS mutant SOD proteins that correlate with clinical severity by applying solution biophysical techniques to six ALS mutants at human SOD hotspot glycine 93. A small-angle X-ray scattering (SAXS) assay and other structural methods assessed aggregation propensity by defining the size and shape of fibrillar SOD aggregates after mild biochemical perturbations. Inductively coupled plasma MS quantified metal ion binding stoichiometry, and pulsed dipolar ESR spectroscopy evaluated the Cu(2+) binding site and defined cross-dimer copper-copper distance distributions. Importantly, we find that copper deficiency in these mutants promotes aggregation in a manner strikingly consistent with their clinical severities. G93 mutants seem to properly incorporate metal ions under physiological conditions when assisted by the copper chaperone but release copper under destabilizing conditions more readily than the WT enzyme. Altered intradimer flexibility in ALS mutants may cause differential metal retention and promote distinct aggregation trends observed for mutant proteins in vitro and in ALS patients. Combined biophysical and structural results test and link copper retention to the framework destabilization hypothesis as a unifying general mechanism for both SOD aggregation and ALS disease progression, with implications for disease severity and therapeutic intervention strategies.
Related JoVE Video
Over-reliance on technology may be at the expense of care.
Nurs Stand
PUBLISHED: 10-09-2014
Show Abstract
Hide Abstract
With technology racing ahead and more patient observations being taken using electronic devices rather than paper charts, I would like to warn nurses against becoming reliant solely on the technology.
Related JoVE Video
In Utero Dimethadione Exposure Causes Postnatal Disruption in Cardiac Structure and Function in the Rat.
Toxicol. Sci.
PUBLISHED: 09-19-2014
Show Abstract
Hide Abstract
In utero exposure of rat embryos to dimethadione (DMO), the N-demethylated teratogenic metabolite of the anticonvulsant trimethadione, induces a high incidence of cardiac heart defects including ventricular septal defects (VSDs). The same exposure regimen also leads to in utero cardiac functional deficits, including bradycardia, dysrhythmia, and a reduction in cardiac output (CO) and ejection fraction that persist until parturition (10 days after the final dose). Despite a high rate of spontaneous postnatal VSD closure, we hypothesize that functional sequelae will persist into adulthood. Pregnant Sprague Dawley rats were administered six 300?mg/kg doses of DMO, one every 12?h in mid-pregnancy beginning on the evening of gestation day 8. Postnatal cardiac function was assessed in control (CTL) and DMO-exposed offspring using radiotelemetry and ultrasound at 3 and 11 months of age, respectively. Adult rats exposed to DMO in utero had an increased incidence of arrhythmia, elevated blood pressure and CO, greater left ventricular volume and elevated locomotor activity versus CTL. The mean arterial pressure of DMO-exposed rats was more sensitive to changes in dietary salt load compared with CTL. Importantly, most treated rats had functional deficits in the absence of a persistent structural defect. It was concluded that in utero DMO exposure causes cardiovascular deficits that persist into postnatal life in the rat, despite absence of visible structural anomalies. We speculate this is not unique to DMO, suggesting possible health implications for infants with unrecognized gestational chemical exposures.
Related JoVE Video
Invasive crayfish as vectors of mercury in freshwater food webs of the Pacific Northwest.
Environ. Toxicol. Chem.
PUBLISHED: 09-16-2014
Show Abstract
Hide Abstract
Invasive species are important drivers of environmental change in aquatic ecosystems and can alter habitat characteristics, community composition, and ecosystem energetics. Such changes have important implications for many ecosystem processes, including the bioaccumulation and biomagnification of contaminants through food webs. Mercury concentrations were measured in 2 nonnative and 1 native crayfish species from western Oregon (USA). Nonnative red swamp crayfish had mercury concentrations similar to those in native signal crayfish (0.29?±?0.05?µg/g dry wt and 0.36?±?0.06?µg/g dry wt, respectively), whereas the nonnative ringed crayfish had lower mercury concentrations (0.10?±?0.02?µg/g dry wt) than either of the other species. The mean energy content of muscle was similar between the native signal crayfish and nonnative ringed crayfish but was significantly higher in the nonnative red swamp crayfish. Across species, mercury concentrations were negatively correlated with energy density. Such energetic differences could exacerbate changes in mercury transfer through trophic pathways of food webs, especially via alterations to the growth dynamics of consumers. Thus, it is important to consider the role of energy content in determining effective mercury exposure even when mercury concentrations on a per-unit mass basis do not differ between species. Environ Toxicol Chem 2014;33:2639-2645. Published 2014 Wiley Periodicals, Inc. on behalf of SETAC. This article is a US Government work and as such, is in the public domain in the United States of America.
Related JoVE Video
Phosphate: an old bone molecule but new cardiovascular risk factor.
Br J Clin Pharmacol
PUBLISHED: 09-13-2014
Show Abstract
Hide Abstract
Phosphate handling in the body is complex and involves hormones produced by the bone, the parathyroid gland and the kidneys. Phosphate is mostly found in hydroxyapatite. however recent evidence suggests that phosphate is also a signalling molecule associated with bone formation. Phosphate balance requires careful regulation of gut and kidney phosphate transporters, SLC34 transporter family, but phosphate signalling in osteoblasts and vascular smooth muscle cells is likely mediated by the SLC20 transporter family (PiT1 and PiT2). If not properly regulated, phosphate imblanace could lead to mineral disorders as well as vascular calcification. In chronic kidney disease-mineral bone disorder, hyperphosphataemia has been consistently associated with extra-osseous calcification and cardiovascular disease. This review focuses on the physiological mechanisms involved in phosphate balance and cell signalling (i.e. osteoblasts and vascular smooth muscle cells) as well as pathological consequences of hyperphosphataemia. Finally, conventional as well as new and experimental therapeutics in the treatment of hyperphosphataemia are explored.
Related JoVE Video
Complete Genome Sequence of Pelosinus sp. Strain UFO1 Assembled Using Single-Molecule Real-Time DNA Sequencing Technology.
Genome Announc
PUBLISHED: 09-04-2014
Show Abstract
Hide Abstract
Pelosinus species can reduce metals such as Fe(III), U(VI), and Cr(VI) and have been isolated from diverse geographical regions. Five draft genome sequences have been published. We report the complete genome sequence for Pelosinus sp. strain UFO1 using only PacBio DNA sequence data and without manual finishing.
Related JoVE Video
Calcification of the internal pudendal artery and development of erectile dysfunction in adenine-induced chronic kidney disease: a sentinel of systemic vascular changes.
J Sex Med
PUBLISHED: 08-19-2014
Show Abstract
Hide Abstract
Chronic kidney disease (CKD), erectile dysfunction (ED), and cardiovascular disease share common vascular etiologies and risk factors.
Related JoVE Video
Novel Hydrogen Bioreactor and Detection Apparatus.
Adv. Biochem. Eng. Biotechnol.
PUBLISHED: 07-16-2014
Show Abstract
Hide Abstract
: In vitro hydrogen generation represents a clear opportunity for novel bioreactor and system design. Hydrogen, already a globally important commodity chemical, has the potential to become the dominant transportation fuel of the future. Technologies such as in vitro synthetic pathway biotransformation (SyPaB)-the use of more than 10 purified enzymes to catalyze unnatural catabolic pathways-enable the storage of hydrogen in the form of carbohydrates. Biohydrogen production from local carbohydrate resources offers a solution to the most pressing challenges to vehicular and bioenergy uses: small-size distributed production, minimization of CO2 emissions, and potential low cost, driven by high yield and volumetric productivity. In this study, we introduce a novel bioreactor that provides the oxygen-free gas phase necessary for enzymatic hydrogen generation while regulating temperature and reactor volume. A variety of techniques are currently used for laboratory detection of biohydrogen, but the most information is provided by a continuous low-cost hydrogen sensor. Most such systems currently use electrolysis for calibration; here an alternative method, flow calibration, is introduced. This system is further demonstrated here with the conversion of glucose to hydrogen at a high rate, and the production of hydrogen from glucose 6-phosphate at a greatly increased reaction rate, 157 mmol/L/h at 60 °C.
Related JoVE Video
Significance of cartilage endplate within herniated disc tissue.
Eur Spine J
PUBLISHED: 05-27-2014
Show Abstract
Hide Abstract
Disc herniations sometimes contain hyaline cartilage fragments, but their origins and significance are uncertain.
Related JoVE Video
Intact functional fourteen-subunit respiratory membrane-bound [NiFe]-hydrogenase complex of the hyperthermophilic archaeon Pyrococcus furiosus.
J. Biol. Chem.
PUBLISHED: 05-23-2014
Show Abstract
Hide Abstract
The archaeon Pyrococcus furiosus grows optimally at 100 °C by converting carbohydrates to acetate, CO2, and H2, obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H2 production to oxidation of reduced ferredoxin with generation of a sodium ion gradient. MBH is encoded by a 14-gene operon with both hydrogenase and Na(+)/H(+) antiporter modules. Herein a His-tagged MBH was expressed in P. furiosus and the detergent-solubilized complex purified under anaerobic conditions by affinity chromatography. Purified MBH contains all 14 subunits by electrophoretic analysis (13 subunits were also identified by mass spectrometry) and had a measured iron:nickel ratio of 15:1, resembling the predicted value of 13:1. The as-purified enzyme exhibited a rhombic EPR signal characteristic of the ready nickel-boron state. The purified and membrane-bound forms of MBH both preferentially evolved H2 with the physiological donor (reduced ferredoxin) as well as with standard dyes. The O2 sensitivities of the two forms were similar (half-lives of ? 15 h in air), but the purified enzyme was more thermolabile (half-lives at 90 °C of 1 and 25 h, respectively). Structural analysis of purified MBH by small angle x-ray scattering indicated a Z-shaped structure with a mass of 310 kDa, resembling the predicted value (298 kDa). The angle x-ray scattering analyses reinforce and extend the conserved sequence relationships of group 4 enzymes and complex I (NADH quinone oxidoreductase). This is the first report on the properties of a solubilized form of an intact respiratory MBH complex that is proposed to evolve H2 and pump Na(+) ions.
Related JoVE Video
Related JoVE Video
Response to Comment on "Revealing nature's cellulase diversity: the digestion mechanism of Caldicellulosiruptor bescii CelA".
Science
PUBLISHED: 05-10-2014
Show Abstract
Hide Abstract
Gusakov critiques our methodology for comparing the cellulolytic activity of the bacterial cellulase CelA with the fungal cellulase Cel7A. We address his concerns by clarifying some misconceptions, carefully referencing the literature, and justifying our approach to point out that the results from our study still stand.
Related JoVE Video
Mannosylglycerate and di-myo-inositol phosphate have interchangeable roles during adaptation of Pyrococcus furiosus to heat stress.
Appl. Environ. Microbiol.
PUBLISHED: 05-02-2014
Show Abstract
Hide Abstract
Marine hyperthermophiles accumulate small organic compounds, known as compatible solutes, in response to supraoptimal temperatures or salinities. Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at temperatures near 100°C. This organism accumulates mannosylglycerate (MG) and di-myo-inositol phosphate (DIP) in response to osmotic and heat stress, respectively. It has been assumed that MG and DIP are involved in cell protection; however, firm evidence for the roles of these solutes in stress adaptation is still missing, largely due to the lack of genetic tools to produce suitable mutants of hyperthermophiles. Recently, such tools were developed for P. furiosus, making this organism an ideal target for that purpose. In this work, genes coding for the synthases in the biosynthetic pathways of MG and DIP were deleted by double-crossover homologous recombination. The growth profiles and solute patterns of the two mutants and the parent strain were investigated under optimal growth conditions and also at supraoptimal temperatures and NaCl concentrations. DIP was a suitable replacement for MG during heat stress, but substitution of MG for DIP and aspartate led to less efficient growth under conditions of osmotic stress. The results suggest that the cascade of molecular events leading to MG synthesis is tuned for osmotic adjustment, while the machinery for induction of DIP synthesis responds to either stress agent. MG protects cells against heat as effectively as DIP, despite the finding that the amount of DIP consistently increases in response to heat stress in the nine (hyper)thermophiles examined thus far.
Related JoVE Video
Functional phylogenetics reveals contributions of pleiotropic Peptide action to ligand-receptor coevolution.
Sci Rep
PUBLISHED: 04-22-2014
Show Abstract
Hide Abstract
The evolution of peptidergic signaling has been accompanied by a significant degree of ligand-receptor coevolution. Closely related clusters of peptide signaling molecules are observed to activate related groups of receptors, implying that genes encoding these ligands may orchestrate an array of functions, a phenomenon known as pleiotropy. Here we examine whether pleiotropic actions of peptide genes might influence ligand-receptor coevolution. Four test groups of neuropeptides characterized by conserved C-terminal amino acid sequence motifs and their cognate receptors were examined in the red flour beetle (Tribolium castaneum): 1) cardioacceleratory peptide 2b (CAPA); CAPAr, 2) pyrokinin/diapause hormone (PK1/DH); PKr-A, -B, 3) pyrokinin/pheromone biosynthesis activating hormone (PK2/PBAN); PKr-C, and 4) ecdysis triggering hormone (ETH); ETHr-b. Ligand-receptor specificities were established through heterologous expression of receptors in cell-based assays for 9 endogenous ligands. Based on ligand-receptor specificity analysis, we found positive pleiotropism exhibited by ETH on ETHR-b and CAPAr, whereas PK1/DH and CAPA are more highly selective for their respective authentic receptors than would be predicted by phylogenetic analysis. Disparities between evolutionary trees deduced from receptor sequences vs. functional ligand-receptor specificities lead to the conclusion that pleiotropy exhibited by peptide genes influences ligand-receptor coevolution.
Related JoVE Video
Metallomics of two microorganisms relevant to heavy metal bioremediation reveal fundamental differences in metal assimilation and utilization.
Metallomics
PUBLISHED: 04-08-2014
Show Abstract
Hide Abstract
Although as many as half of all proteins are thought to require a metal cofactor, the metalloproteomes of microorganisms remain relatively unexplored. Microorganisms from different environments are likely to vary greatly in the metals that they assimilate, not just among the metals with well-characterized roles but also those lacking any known function. Herein we investigated the metal utilization of two microorganisms that were isolated from very similar environments and are of interest because of potential roles in the immobilization of heavy metals, such as uranium and chromium. The metals assimilated and their concentrations in the cytoplasm of Desulfovibrio vulgaris strain Hildenborough (DvH) and Enterobacter cloacae strain Hanford (EcH) varied dramatically, with a larger number of metals present in Enterobacter. For example, a total of 9 and 19 metals were assimilated into their cytoplasmic fractions, respectively, and DvH did not assimilate significant amounts of zinc or copper whereas EcH assimilated both. However, bioinformatic analysis of their genome sequences revealed a comparable number of predicted metalloproteins, 813 in DvH and 953 in EcH. These allowed some rationalization of the types of metal assimilated in some cases (Fe, Cu, Mo, W, V) but not in others (Zn, Nd, Ce, Pr, Dy, Hf and Th). It was also shown that U binds an unknown soluble protein in EcH but this incorporation was the result of extracellular U binding to cytoplasmic components after cell lysis.
Related JoVE Video
Resident duty hours in the outpatient electronic health record era: inaccuracies and implications.
J Grad Med Educ
PUBLISHED: 04-05-2014
Show Abstract
Hide Abstract
The Accreditation Council for Graduate Medical Education expects resident duty hours to be monitored, yet no previous studies have examined the effect of after-hours electronic health record (EHR) use on resident hours or burnout.
Related JoVE Video
Structure-activity relationship study of sesquiterpene lactones and their semi-synthetic amino derivatives as potential antitrypanosomal products.
Molecules
PUBLISHED: 03-13-2014
Show Abstract
Hide Abstract
Sesquiterpene lactones (STLs) are natural products that have potent antitrypanosomal activity in vitro and, in the case of cynaropicrin, also reduce parasitemia in the murine model of trypanosomiasis. To explore their structure-antitrypanosomal activity relationships, a set of 34 natural and semi-synthetic STLs and amino-STLs was tested in vitro against T. b. rhodesiense (which causes East African sleeping sickness) and mammalian cancer cells (rat bone myoblast L6 cells). It was found that the ?-methylene-?-lactone moiety is necessary for both antitrypanosomal effects and cytotoxicity. Antitrypanosomal selectivity is facilitated by 2-(hydroxymethyl)acrylate or 3,4-dihydroxy-2-methylenebutylate side chains, and by the presence of cyclopentenone rings. Semi-synthetic STL amines with morpholino and dimethylamino groups showed improved in vitro activity over the native STLs. The dimethylamino derivative of cynaropicrin was prepared and tested orally in the T. b. rhodesiense acute mouse model, where it showed reduced toxicity over cynaropicrin, but also lost antitrypanosomal activity.
Related JoVE Video
Why do some intervertebral discs degenerate, when others (in the same spine) do not?
Clin Anat
PUBLISHED: 03-04-2014
Show Abstract
Hide Abstract
This review suggests why some discs degenerate rather than age normally. Intervertebral discs are avascular pads of fibrocartilage that allow movement between vertebral bodies. Human discs have a low cell density and a limited ability to adapt to mechanical demands. With increasing age, the matrix becomes yellowed, fibrous, and brittle, but if disc structure remains intact, there is little impairment in function, and minimal ingrowth of blood vessels or nerves. Approximately half of old lumbar discs degenerate in the sense of becoming physically disrupted. The posterior annulus and lower lumbar discs are most affected, presumably because they are most heavily loaded. Age and genetic inheritance can weaken discs to such an extent that they are physically disrupted during everyday activities. Damage to the endplate or annulus typically decompresses the nucleus, concentrates stress within the annulus, and allows ingrowth of nerves and blood vessels. Matrix disruption progresses by mechanical and biological means. The site of initial damage leads to two disc degeneration "phenotypes": endplate-driven degeneration is common in the upper lumbar and thoracic spine, and annulus-driven degeneration is common at L4-S1. Discogenic back pain can be initiated by tissue disruption, and amplified by inflammation and infection. Healing is possible in the outer annulus only, where cell density is highest. We conclude that some discs degenerate because they are disrupted by excessive mechanical loading. This can occur without trauma if tissues are weakened by age and genetic inheritance. Moderate mechanical loading, in contrast, strengthens all spinal tissues, including discs. Clin. Anat., 2014. © 2014 Wiley Periodicals, Inc.
Related JoVE Video
Conversion of 4-hydroxybutyrate to acetyl coenzyme A and its anapleurosis in the Metallosphaera sedula 3-hydroxypropionate/4-hydroxybutyrate carbon fixation pathway.
Appl. Environ. Microbiol.
PUBLISHED: 02-14-2014
Show Abstract
Hide Abstract
The extremely thermoacidophilic archaeon Metallosphaera sedula (optimum growth temperature, 73°C, pH 2.0) grows chemolithoautotrophically on metal sulfides or molecular hydrogen by employing the 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) carbon fixation cycle. This cycle adds two CO2 molecules to acetyl coenzyme A (acetyl-CoA) to generate 4HB, which is then rearranged and cleaved to form two acetyl-CoA molecules. Previous metabolic flux analysis showed that two-thirds of central carbon precursor molecules are derived from succinyl-CoA, which is oxidized to malate and oxaloacetate. The remaining one-third is apparently derived from acetyl-CoA. As such, the steps beyond succinyl-CoA are essential for completing the carbon fixation cycle and for anapleurosis of acetyl-CoA. Here, the final four enzymes of the 3HP/4HB cycle, 4-hydroxybutyrate-CoA ligase (AMP forming) (Msed_0406), 4-hydroxybutyryl-CoA dehydratase (Msed_1321), crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase (Msed_0399), and acetoacetyl-CoA ?-ketothiolase (Msed_0656), were produced recombinantly in Escherichia coli, combined in vitro, and shown to convert 4HB to acetyl-CoA. Metabolic pathways connecting CO2 fixation and central metabolism were examined using a gas-intensive bioreactor system in which M. sedula was grown under autotrophic (CO2-limited) and heterotrophic conditions. Transcriptomic analysis revealed the importance of the 3HP/4HB pathway in supplying acetyl-CoA to anabolic pathways generating intermediates in M. sedula metabolism. The results indicated that flux between the succinate and acetyl-CoA branches in the 3HP/4HB pathway is governed by 4-hydroxybutyrate-CoA ligase, possibly regulated posttranslationally by the protein acetyltransferase (Pat)/Sir2-dependent system. Taken together, this work confirms the final four steps of the 3HP/4HB pathway, thereby providing the framework for examining connections between CO2 fixation and central metabolism in M. sedula.
Related JoVE Video
Mechanical properties of alginate hydrogels manufactured using external gelation.
J Mech Behav Biomed Mater
PUBLISHED: 01-30-2014
Show Abstract
Hide Abstract
Alginate hydrogels are commonly used in biomedical applications such as scaffolds for tissue engineering, drug delivery, and as a medium for cell immobilisation. Multivalent cations are often employed to create physical crosslinks between carboxyl and hydroxyl moieties on neighbouring polysaccharide chains, creating hydrogels with a range of mechanical properties. This work describes the manufacture and characterisation of sodium alginate hydrogels using the divalent cations Mg(2+), Ca(2+) and Sr(2+) to promote gelation via non-covalent crosslinks. Gelation time and Young?s modulus are characterised as a function of cation and alginate concentrations. The implications of this work towards the use of environmental elasticity to control stem cell differentiation are discussed.
Related JoVE Video
In vitro metabolic engineering of hydrogen production at theoretical yield from sucrose.
Metab. Eng.
PUBLISHED: 01-26-2014
Show Abstract
Hide Abstract
Hydrogen is one of the most important industrial chemicals and will be arguably the best fuel in the future. Hydrogen production from less costly renewable sugars can provide affordable hydrogen, decrease reliance on fossil fuels, and achieve nearly zero net greenhouse gas emissions, but current chemical and biological means suffer from low hydrogen yields and/or severe reaction conditions. An in vitro synthetic enzymatic pathway comprised of 15 enzymes was designed to split water powered by sucrose to hydrogen. Hydrogen and carbon dioxide were spontaneously generated from sucrose or glucose and water mediated by enzyme cocktails containing up to 15 enzymes under mild reaction conditions (i.e. 37°C and atm). In a batch reaction, the hydrogen yield was 23.2mol of dihydrogen per mole of sucrose, i.e., 96.7% of the theoretical yield (i.e., 12 dihydrogen per hexose). In a fed-batch reaction, increasing substrate concentration led to 3.3-fold enhancement in reaction rate to 9.74mmol of H2/L/h. These proof-of-concept results suggest that catabolic water splitting powered by sugars catalyzed by enzyme cocktails could be an appealing green hydrogen production approach.
Related JoVE Video
Teaching tobacco dependence treatment and counseling skills during medical school: rationale and design of the Medical Students helping patients Quit tobacco (MSQuit) group randomized controlled trial.
Contemp Clin Trials
PUBLISHED: 01-20-2014
Show Abstract
Hide Abstract
Physician-delivered tobacco treatment using the 5As is clinically recommended, yet its use has been limited. Lack of adequate training and confidence to provide tobacco treatment is cited as leading reasons for limited 5A use. Tobacco dependence treatment training while in medical school is recommended, but is minimally provided. The MSQuit trial (Medical Students helping patients Quit tobacco) aims to determine if a multi-modal and theoretically-guided tobacco educational intervention will improve tobacco dependence treatment skills (i.e. 5As) among medical students.
Related JoVE Video
Deletion of acetyl-CoA synthetases I and II increases production of 3-hydroxypropionate by the metabolically-engineered hyperthermophile Pyrococcus furiosus.
Metab. Eng.
PUBLISHED: 01-11-2014
Show Abstract
Hide Abstract
The heterotrophic, hyperthermophilic archaeon Pyrococcus furiosus is a new addition to the growing list of genetically-tractable microorganisms suitable for metabolic engineering to produce liquid fuels and industrial chemicals. P. furiosus was recently engineered to generate 3-hydroxypropionate (3-HP) from CO? and acetyl-CoA by the heterologous-expression of three enzymes from the CO? fixation cycle of the thermoacidophilic archaeon Metallosphaera sedula using a thermally-triggered induction system. The acetyl-CoA for this pathway is generated from glucose catabolism that in wild-type P. furiosus is converted to acetate with concurrent ATP production by the heterotetrameric (????) acetyl-CoA synthetase (ACS). Hence ACS in the engineered 3-HP production strain (MW56) competes with the heterologous pathway for acetyl-CoA. Herein we show that strains of MW56 lacking the ?-subunit of either of the two ACSs previously characterized from P. furiosus (ACSI and ACSII) exhibit a three-fold increase in specific 3-HP production. The ?ACSI? strain displayed only a minor defect in growth on either maltose or peptides, while no growth defect on these substrates was observed with the ?ACSII? strain. Deletion of individual and multiple ACS subunits was also shown to decrease CoA release activity for several different CoA ester substrates in addition to acetyl-CoA, information that will be extremely useful for future metabolic engineering endeavors in P. furiosus.
Related JoVE Video
Inflammation in rat pregnancy inhibits spiral artery remodeling leading to fetal growth restriction and features of preeclampsia.
J. Exp. Med.
PUBLISHED: 01-06-2014
Show Abstract
Hide Abstract
Fetal growth restriction (FGR) and preeclampsia (PE) are often associated with abnormal maternal inflammation, deficient spiral artery (SA) remodeling, and altered uteroplacental perfusion. Here, we provide evidence of a novel mechanistic link between abnormal maternal inflammation and the development of FGR with features of PE. Using a model in which pregnant rats are administered low-dose lipopolysaccharide (LPS) on gestational days 13.5-16.5, we show that abnormal inflammation resulted in FGR mediated by tumor necrosis factor-? (TNF). Inflammation was also associated with deficient trophoblast invasion and SA remodeling, as well as with altered uteroplacental hemodynamics and placental nitrosative stress. Moreover, inflammation increased maternal mean arterial pressure (MAP) and was associated with renal structural alterations and proteinuria characteristic of PE. Finally, transdermal administration of the nitric oxide (NO) mimetic glyceryl trinitrate prevented altered uteroplacental perfusion, LPS-induced inflammation, placental nitrosative stress, renal structural and functional alterations, increase in MAP, and FGR. These findings demonstrate that maternal inflammation can lead to severe pregnancy complications via a mechanism that involves increased maternal levels of TNF. Our study provides a rationale for the use of antiinflammatory agents or NO-mimetics in the treatment and/or prevention of inflammation-associated pregnancy complications.
Related JoVE Video
Antitrypanosomal structure-activity-relationship study of synthetic cynaropicrin derivatives.
Bioorg. Med. Chem. Lett.
PUBLISHED: 01-02-2014
Show Abstract
Hide Abstract
Cynaropicrin is a guaianolide sesquiterpene lactone with a 5-7-5 tricyclic skeleton, four exo-olefins, and two hydroxyl groups. Recently, it was found that the compound is a potent in vitro and in vivo inhibitor of the protozoan parasite Trypanosoma brucei, which causes human African trypanosomiasis (HAT; sleeping sickness). In this Letter, chemical derivatization of cynaropicrin and the structure-activity-relationship (SAR) study against T. brucei is described.
Related JoVE Video
Characterization of ten heterotetrameric NDP-dependent acyl-CoA synthetases of the hyperthermophilic archaeon Pyrococcus furiosus.
Archaea
PUBLISHED: 01-01-2014
Show Abstract
Hide Abstract
The hyperthermophilic archaeon Pyrococcus furiosus grows by fermenting peptides and carbohydrates to organic acids. In the terminal step, acyl-CoA synthetase (ACS) isoenzymes convert acyl-CoA derivatives to the corresponding acid and conserve energy in the form of ATP. ACS1 and ACS2 were previously purified from P. furiosus and have ? 2 ? 2 structures but the genome contains genes encoding three additional ?-subunits. The ten possible combinations of ? and ? genes were expressed in E. coli and each resulted in stable and active ? 2 ? 2 isoenzymes. The ?-subunit of each isoenzyme determined CoA-based substrate specificity and between them they accounted for the CoA derivatives of fourteen amino acids. The ?-subunit determined preference for adenine or guanine nucleotides. The GTP-generating isoenzymes are proposed to play a role in gluconeogenesis by producing GTP for GTP-dependent phosphoenolpyruvate carboxykinase and for other GTP-dependent processes. Transcriptional and proteomic data showed that all ten isoenzymes are constitutively expressed indicating that both ATP and GTP are generated from the metabolism of most of the amino acids. A phylogenetic analysis showed that the ACSs of P. furiosus and other members of the Thermococcales are evolutionarily distinct from those found throughout the rest of biology, including those of other hyperthermophilic archaea.
Related JoVE Video
Revealing natures cellulase diversity: the digestion mechanism of Caldicellulosiruptor bescii CelA.
Science
PUBLISHED: 12-21-2013
Show Abstract
Hide Abstract
Most fungi and bacteria degrade plant cell walls by secreting free, complementary enzymes that hydrolyze cellulose; however, some bacteria use large enzymatic assemblies called cellulosomes, which recruit complementary enzymes to protein scaffolds. The thermophilic bacterium Caldicellulosiruptor bescii uses an intermediate strategy, secreting many free cellulases that contain multiple catalytic domains. One of these, CelA, comprises a glycoside hydrolase family 9 and a family 48 catalytic domain, as well as three type III cellulose-binding modules. In the saccharification of a common cellulose standard, Avicel, CelA outperforms mixtures of commercially relevant exo- and endoglucanases. From transmission electron microscopy studies of cellulose after incubation with CelA, we report morphological features that suggest that CelA not only exploits the common surface ablation mechanism driven by general cellulase processivity, but also excavates extensive cavities into the surface of the substrate. These results suggest that natures repertoire of cellulose digestion paradigms remain only partially discovered and understood.
Related JoVE Video
Engineering Hydrogen Gas Production from Formate in a Hyperthermophile by Heterologous Production of an 18-Subunit Membrane-Bound Complex.
J. Biol. Chem.
PUBLISHED: 12-07-2013
Show Abstract
Hide Abstract
Biohydrogen gas has enormous potential as a source of reductant for the microbial production of biofuels, but its low solubility and poor gas mass transfer rates are limiting factors. These limitations could be circumvented by engineering biofuel production in microorganisms that are also capable of generating H2 from highly soluble chemicals such as formate, which can function as an electron donor. Herein the model hyperthermophile, Pyrococcus furiosus, which grows optimally near 100 degree Celsius by fermenting sugars to produce H2, has been engineered to also efficiently convert formate to H2. Using a bacterial artificial chromosome vector, the 16.9 kb 18-gene cluster encoding the membrane-bound, respiratory formate hydrogen lyase complex of Thermococcus onnurineus was inserted into the P. furiosus chromosome and expressed as a functional unit. This enabled P. furiosus to utilize formate as well as sugars as an H2 source, and to do so at both 80 and 95 degrees Celsius, near the optimum growth temperature of the donor (T. onnurineus) and engineered host (P. furiosus), respectively. This accomplishment also demonstrates the versatility of P. furiosus for metabolic engineering applications.
Related JoVE Video
Usefulness of the ACGME resident survey in the NAS: a view from Internal Medicine Program Directors.
Am. J. Med.
PUBLISHED: 10-21-2013
Show Abstract
Hide Abstract
Annual data reporting for residency programs in the Next Accreditation System (NAS) includes new components, including changes in the annual resident survey. Program directors from multiple specialties have expressed concerns regarding the survey related to residents understanding of the questions, accuracy of the data, and use of the results by the Accreditation Council for Graduate Medical Education (ACGME). We surveyed internal medicine program directors to ascertain their opinions of the ACGME resident survey in order to better inform policymakers as residency programs implement the NAS. 272 program directors responded. Most program directors disagreed with the changes in the resident survey, did not believe there was sufficient transparency in its use, and believe they should receive aggregate responses from their residents. A majority of program directors used resident survey data to secure program resources. Program directors do not seem to find it overly burdensome to have residents complete the survey, with 41% finding it "easy" to assure completion. The 2013 changes in the annual resident survey as part of the NAS were not generally accepted by program directors in internal medicine. Nonetheless, program directors continue to use survey results to support the program. How the survey results will be incorporated into accreditation decisions is a significant concern among internal medicine program directors.
Related JoVE Video
Effect of modest pay-for-performance financial incentive on time-to-discharge summary dictation among medical residents.
Qual Manag Health Care
PUBLISHED: 10-04-2013
Show Abstract
Hide Abstract
Evaluate the effect of a modest financial incentive on time-to-discharge summary dictation among medicine residents.
Related JoVE Video
Hemodynamic and behavioral differences after administration of meloxicam, buprenorphine, or tramadol as analgesics for telemeter implantation in mice.
J. Am. Assoc. Lab. Anim. Sci.
PUBLISHED: 09-18-2013
Show Abstract
Hide Abstract
Cannulation of the common carotid artery for chronic, continuous radiotelemetric recording of aortic hemodynamic properties in mice is a highly invasive recovery surgery. Radiotelemetric recording, by its continuous nature, gives the most accurate measurements of hemodynamic variables in experimental animals, and is widely used in the study of cardiovascular diseases including hypertension. The American Heart Association has recommended data acquisition by radiotelemetric recording but did not provide guidelines regarding postoperative analgesic support. We assessed hemodynamic parameters, locomotor activity, food intake, and weight loss in radiotransmitter-implanted CD1 female mice receiving analgesic support during the first 48 h after surgery. The efficacy of analgesic support from the NSAID meloxicam was compared with that of the widely used opioid agonist buprenorphine and the related compound, tramadol. Meloxicam-treated mice recovered lost body weight more rapidly than did tramadol-or buprenorphine-treated mice. Furthermore, meloxicam-treated mice maintained circadian rhythm after surgery and had tighter regulation of mean arterial pressure than did tramadol- or buprenorphine-treated mice. Meloxicam was also superior with regard to food intake, locomotor activity, and limiting variance in hemodynamic parameters. This study indicates that when compared with buprenorphine and tramadol, meloxicam should be the postoperative analgesic of choice for radiotelemeter implantation in mice.
Related JoVE Video
Degradation of high loads of crystalline cellulose and of unpretreated plant biomass by the thermophilic bacterium Caldicellulosiruptor bescii.
Bioresour. Technol.
PUBLISHED: 09-05-2013
Show Abstract
Hide Abstract
The thermophilic bacterium Caldicellulosiruptor bescii grows at 78°C on high concentrations (200gL(-1)) of both crystalline cellulose and unpretreated switchgrass, while low concentrations (<20gL(-1)) of acid-pretreated switchgrass inhibit growth. Degradation of crystalline cellulose, but not that of unpretreated switchgrass, was limited by nitrogen and vitamin (folate) availability. Under optimal conditions, C. bescii solubilized approximately 60% of the crystalline cellulose and 30% of the unpretreated switchgrass using initial substrate concentrations of 50gL(-1). Further fermentation of crystalline cellulose and of switchgrass was inhibited by organic acid end-products and by a specific inhibitor of C. bescii growth that did not affect other thermophilic bacteria, respectively. Soluble mono- and oligosaccharides, organic acids, carbon dioxide, and microbial biomass, quantitatively accounted for the crystalline cellulose and plant biomass carbon utilized. C. bescii therefore degrades industrially-relevant concentrations of lignocellulosic biomass that have not undergone pretreatment thereby demonstrating its potential utility in biomass conversion.
Related JoVE Video
Low voltage-activated calcium channels gate transmitter release at the dorsal root ganglion sandwich synapse.
J. Physiol. (Lond.)
PUBLISHED: 09-02-2013
Show Abstract
Hide Abstract
A subpopulation of dorsal root ganglion (DRG) neurons are intimately attached in pairs and separated solely by thin satellite glial cell membrane septa. Stimulation of one neuron leads to transglial activation of its pair by a bi-, purinergic/glutamatergic synaptic pathway, a transmission mechanism that we term sandwich synapse (SS) transmission. Release of ATP from the stimulated neuron can be attributed to a classical mechanism involving Ca(2+) entry via voltage-gated calcium channels (CaV) but via an unknown channel type. Specific blockers and toxins ruled out CaV1, 2.1 and 2.2. Transmission was, however, blocked by a moderate depolarization (-50 mV) or low-concentration Ni(2+) (0.1 mM). Transmission persisted using a voltage pulse to -40 mV from a holding potential of -80 mV, confirming the involvement of a low voltage-activated channel type and limiting the candidate channel type to either CaV3.2 or a subpopulation of inactivation- and Ni(2+)-sensitive CaV2.3 channels. Resistance of the neuron calcium current and SS transmission to SNX482 argue against the latter. Hence, we conclude that inter-somatic transmission at the DRG SS is gated by CaV3.2 type calcium channels. The use of CaV3 family channels to gate transmission has important implications for the biological function of the DRG SS as information transfer would be predicted to occur not only in response to action potentials but also to sub-threshold membrane voltage oscillations. Thus, the SS synapse may serve as a homeostatic signalling mechanism between select neurons in the DRG and could play a role in abnormal sensation such as neuropathic pain.
Related JoVE Video
Secretory competence in a gateway endocrine cell conferred by the nuclear receptor ?FTZ-F1 enables stage-specific ecdysone responses throughout development in Drosophila.
Dev. Biol.
PUBLISHED: 08-27-2013
Show Abstract
Hide Abstract
Hormone-induced changes in gene expression initiate periodic molts and metamorphosis during insect development. Successful execution of these developmental steps depends upon successive phases of rising and falling 20-hydroxyecdysone (20E) levels, leading to a cascade of nuclear receptor-driven transcriptional activity that enables stage- and tissue-specific responses to the steroid. Among the cellular processes associated with declining steroids is acquisition of secretory competence in endocrine Inka cells, the source of ecdysis triggering hormones (ETHs). We show here that Inka cell secretory competence is conferred by the orphan nuclear receptor ?FTZ-F1. Selective RNA silencing of ?ftz-f1 in Inka cells prevents ETH release, causing developmental arrest at all stages. Affected larvae display buttoned-up, the ETH-null phenotype characterized by double mouthparts, absence of ecdysis behaviors, and failure to shed the old cuticle. During the mid-prepupal period, individuals fail to translocate the air bubble, execute head eversion and elongate incipient wings and legs. Those that escape to the adult stage are defective in wing expansion and cuticle sclerotization. Failure to release ETH in ?ftz-f1 silenced animals is indicated by persistent ETH immunoreactivity in Inka cells. Arrested larvae are rescued by precisely-timed ETH injection or Inka cell-targeted ?FTZ-F1 expression. Moreover, premature ?ftz-f1 expression in these cells also results in developmental arrest. The Inka cell therefore functions as a "gateway cell", whose secretion of ETH serves as a key downstream physiological output enabling stage-specific responses to 20E that are required to advance through critical developmental steps. This secretory function depends on transient and precisely timed ?FTZ-F1 expression late in the molt as steroids decline.
Related JoVE Video
Thermophilic lignocellulose deconstruction.
FEMS Microbiol. Rev.
PUBLISHED: 08-20-2013
Show Abstract
Hide Abstract
Thermophilic microorganisms are attractive candidates for conversion of lignocellulose to biofuels because they produce robust, effective, carbohydrate-degrading enzymes and survive under harsh bioprocessing conditions that reflect their natural biotopes. However, no naturally occurring thermophile is known that can convert plant biomass into a liquid biofuel at rates, yields and titers that meet current bioprocessing and economic targets. Meeting those targets requires either metabolically engineering solventogenic thermophiles with additional biomass-deconstruction enzymes or engineering plant biomass degraders to produce a liquid biofuel. Thermostable enzymes from microorganisms isolated from diverse environments can serve as genetic reservoirs for both efforts. Because of the sheer number of enzymes that are required to hydrolyze plant biomass to fermentable oligosaccharides, the latter strategy appears to be the preferred route and thus has received the most attention to date. Thermophilic plant biomass degraders fall into one of two categories: cellulosomal (i.e. multienzyme complexes) and noncellulosomal (i.e. free enzyme systems). Plant-biomass-deconstructing thermophilic bacteria from the genera Clostridium (cellulosomal) and Caldicellulosiruptor (noncellulosomal), which have potential as metabolic engineering platforms for producing biofuels, are compared and contrasted from a systems biology perspective.
Related JoVE Video
Activities of psilostachyin A and cynaropicrin against Trypanosoma cruzi in vitro and in vivo.
Antimicrob. Agents Chemother.
PUBLISHED: 08-12-2013
Show Abstract
Hide Abstract
In vitro and in vivo activities against Trypanosoma cruzi were evaluated for two sesquiterpene lactones: psilostachyin A and cynaropicrin. Cynaropicrin had previously been shown to potently inhibit African trypanosomes in vivo, and psilostachyin A had been reported to show in vivo effects against T. cruzi, albeit in another test design. In vitro data showed that cynaropicrin was more effective than psilostachyin A. Ultrastructural alterations induced by cynaropicrin included shedding events, detachment of large portions of the plasma membrane, and vesicular bodies and large vacuoles containing membranous structures, suggestive of parasite autophagy. Acute toxicity studies showed that one of two mice died at a cynaropicrin dose of 400 mg/kg of body weight given intraperitoneally (i.p.). Although no major plasma biochemical alterations could be detected, histopathology demonstrated that the liver was the most affected organ in cynaropicrin-treated animals. Although cynaropicrin was as effective as benznidazole against trypomastigotes in vitro, the treatment (once or twice a day) of T. cruzi-infected mice (up to 50 mg/kg/day cynaropicrin) did not suppress parasitemia or protect against mortality induced by the Y and Colombiana strains. Psilostachyin A (0.5 to 50 mg/kg/day given once a day) was not effective in the acute model of T. cruzi infection (Y strain), reaching 100% animal mortality. Our data demonstrate that although it is very promising against African trypanosomes, cynaropicrin does not show efficacy compared to benznidazole in acute mouse models of T. cruzi infection.
Related JoVE Video
Antiprotozoal screening of 60 South African plants, and the identification of the antitrypanosomal germacranolides schkuhrin I and II.
Planta Med.
PUBLISHED: 08-08-2013
Show Abstract
Hide Abstract
Two hundred and seven extracts were prepared from sixty plants from South Africa and screened for in vitro activity against Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani, and Plasmodium falciparum. For the 21 extracts which inhibited the growth of one or more parasites with more than 95 % at 10 µg/mL, the IC50 values against all four protozoal parasites and cytotoxic IC50 values against L6 myoblasts were determined. Amongst the most notable results are the activities of Psoralea pinnata (IC50 of 0.15 µg/mL), Schkuhria pinnata (2.04 µg/mL), and Vernonia mespilifolia (1.01 µg/mL) against Trypansoma brucei rhodesiense. HPLC-based activity profiling was used to identify the active constituents in the extracts, and the germacranolide sesquiterpene lactones schkuhrin I and II from S. pinnata, and cynaropicrin from V. mespilifolia were identified, with IC50 values of 0.9, 1.5, and 0.23 µM, respectively.
Related JoVE Video
Gestational hypertension in atrial natriuretic peptide knockout mice and the developmental origins of salt-sensitivity and cardiac hypertrophy.
Regul. Pept.
PUBLISHED: 07-24-2013
Show Abstract
Hide Abstract
To determine the effect of gestational hypertension on the developmental origins of blood pressure (BP), altered kidney gene expression, salt-sensitivity and cardiac hypertrophy (CH) in adult offspring.
Related JoVE Video
Vitamin K status in chronic kidney disease.
Nutrients
PUBLISHED: 07-11-2013
Show Abstract
Hide Abstract
The purpose of this review is to summarize the research to date on vitamin K status in chronic kidney disease (CKD). This review includes a summary of the data available on vitamin K status in patients across the spectrum of CKD as well as the link between vitamin K deficiency in CKD and bone dynamics, including mineralization and demineralization, as well as ectopic mineralization. It also describes two current clinical trials that are underway evaluating vitamin K treatment in CKD patients. These data may inform future clinical practice in this population.
Related JoVE Video
Cardiovascular disease in an adenine-induced model of chronic kidney disease: the temporal link between vascular calcification and haemodynamic consequences.
J. Hypertens.
PUBLISHED: 07-09-2013
Show Abstract
Hide Abstract
Medial vascular calcification is highly prevalent in chronic kidney disease (CKD), and it is a risk factor for mortality. This study characterizes the time course and the link between calcification of major arteries, changes in blood pressure (BP) and cardiac growth in experimental CKD.
Related JoVE Video
Increased chondrocyte apoptosis is associated with progression of osteoarthritis in spontaneous Guinea pig models of the disease.
Int J Mol Sci
PUBLISHED: 06-20-2013
Show Abstract
Hide Abstract
Osteoarthritis (OA) is the most common joint disease characterised by degradation of articular cartilage and bone remodelling. For almost a decade chondrocyte apoptosis has been investigated as a possible mechanism of cartilage damage in OA, but its precise role in initiation and/or progression of OA remains to the determined. The aim of this study is to determine the role of chondrocyte apoptosis in spontaneous animal models of OA. Right tibias from six male Dunkin Hartley (DH) and Bristol Strain 2 (BS2) guinea pigs were collected at 10, 16, 24 and 30 weeks of age. Fresh-frozen sections of tibial epiphysis were microscopically scored for OA, and immunostained with caspase-3 and TUNEL for apoptotic chondrocytes. The DH strain had more pronounced cartilage damage than BS2, especially at 30 weeks. At this time point, the apoptotic chondrocytes were largely confined to the deep zone of articular cartilage (AC) with a greater percentage in the medial side of DH than BS2 (DH: 5.7%, 95% CI: 4.2-7.2), BS2: 4.8%, 95% CI: 3.8-5.8), p > 0.05). DH had a significant progression of chondrocyte death between 24 to 30 weeks during which time significant changes were observed in AC fibrillation, proteoglycan depletion and overall microscopic OA score. A strong correlation (p ? 0.01) was found between chondrocyte apoptosis and AC fibrillation (r = 0.3), cellularity (r = 0.4) and overall microscopic OA scores (r = 0.4). Overall, the rate of progression in OA and apoptosis over the study period was greater in the DH (versus BS2) and the medial AC (versus lateral). Chondrocyte apoptosis was higher at the later stage of OA development when the cartilage matrix was hypocellular and highly fibrillated, suggesting that chondrocyte apoptosis is a late event in OA.
Related JoVE Video
Cynaropicrin targets the trypanothione redox system in Trypanosoma brucei.
Bioorg. Med. Chem.
PUBLISHED: 06-14-2013
Show Abstract
Hide Abstract
In mice cynaropicrin (CYN) potently inhibits the proliferation of Trypanosoma brucei-the causative agent of Human African Trypanosomiasis-by a so far unknown mechanism. We hypothesized that CYNs ?,?-unsaturated methylene moieties act as Michael acceptors for glutathione (GSH) and trypanothione (T(SH)2), the main low molecular mass thiols essential for unique redox metabolism of these parasites. The analysis of this putative mechanism and the effects of CYN on enzymes of the T(SH)2 redox metabolism including trypanothione reductase, trypanothione synthetase, glutathione-S-transferase, and ornithine decarboxylase are shown. A two step extraction protocol with subsequent UPLC-MS/MS analysis was established to quantify intra-cellular CYN, T(SH)2, GSH, as well as GS-CYN and T(S-CYN)2 adducts in intact T. b. rhodesiense cells. Within minutes of exposure to CYN, the cellular GSH and T(SH)2 pools were entirely depleted, and the parasites entered an apoptotic stage and died. CYN also showed inhibition of the ornithine decarboxylase similar to the positive control eflornithine. Significant interactions with the other enzymes involved in the T(SH)2 redox metabolism were not observed. Alongside many other biological activities sesquiterpene lactones including CYN have shown antitrypanosomal effects, which have been postulated to be linked to formation of Michael adducts with cellular nucleophiles. Here the interaction of CYN with biological thiols in a cellular system in general, and with trypanosomal T(SH)2 redox metabolism in particular, thus offering a molecular explanation for the antitrypanosomal activity is demonstrated. At the same time, the study provides a novel extraction and analysis protocol for components of the trypanosomal thiol metabolism.
Related JoVE Video
Tunable microwave signal generator with an optically-injected 1310 nm QD-DFB laser.
Opt Express
PUBLISHED: 05-15-2013
Show Abstract
Hide Abstract
Tunable microwave signal generation with frequencies ranging from below 1 GHz to values over 40 GHz is demonstrated experimentally with a 1310 nm Quantum Dot (QD) Distributed-Feedback (DFB) laser. Microwave signal generation is achieved using the period 1 dynamics induced in the QD DFB under optical injection. Continuous tuning in the positive detuning frequency range of the quantum dots unique stability map is demonstrated. The simplicity of the experimental configuration offers promise for novel uses of these nanostructure lasers in Radio-over-Fiber (RoF) applications and future mobile networks.
Related JoVE Video
Roles of patch characteristics, drought frequency, and restoration in long-term trends of a widespread amphibian.
Conserv. Biol.
PUBLISHED: 04-06-2013
Show Abstract
Hide Abstract
Despite the high profile of amphibian declines and the increasing threat of drought and fragmentation to aquatic ecosystems, few studies have examined long-term rates of change for a single species across a large geographic area. We analyzed growth in annual egg-mass counts of the Columbia spotted frog (Rana luteiventris) across the northwestern United States, an area encompassing 3 genetic clades. On the basis of data collected by multiple partners from 98 water bodies between 1991 and 2011, we used state-space and linear-regression models to measure effects of patch characteristics, frequency of summer drought, and wetland restoration on population growth. Abundance increased in the 2 clades with greatest decline history, but declined where populations are considered most secure. Population growth was negatively associated with temporary hydroperiods and landscape modification (measured by the human footprint index), but was similar in modified and natural water bodies. The effect of drought was mediated by the size of the water body: populations in large water bodies maintained positive growth despite drought, whereas drought magnified declines in small water bodies. Rapid growth in restored wetlands in areas of historical population declines provided strong evidence of successful management. Our results highlight the importance of maintaining large areas of habitat and underscore the greater vulnerability of small areas of habitat to environmental stochasticity. Similar long-term growth rates in modified and natural water bodies and rapid, positive responses to restoration suggest pond construction and other forms of management can effectively increase population growth. These tools are likely to become increasingly important to mitigate effects of increased drought expected from global climate change. Papeles de las Características del Fragmento, Frecuencia de Sequía y Restauración en las Tendencias a Largo Plazo de un Anfibio Ampliamente Distribuido.
Related JoVE Video
Exploiting microbial hyperthermophilicity to produce an industrial chemical, using hydrogen and carbon dioxide.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 03-25-2013
Show Abstract
Hide Abstract
Microorganisms can be engineered to produce useful products, including chemicals and fuels from sugars derived from renewable feedstocks, such as plant biomass. An alternative method is to use low potential reducing power from nonbiomass sources, such as hydrogen gas or electricity, to reduce carbon dioxide directly into products. This approach circumvents the overall low efficiency of photosynthesis and the production of sugar intermediates. Although significant advances have been made in manipulating microorganisms to produce useful products from organic substrates, engineering them to use carbon dioxide and hydrogen gas has not been reported. Herein, we describe a unique temperature-dependent approach that confers on a microorganism (the archaeon Pyrococcus furiosus, which grows optimally on carbohydrates at 100°C) the capacity to use carbon dioxide, a reaction that it does not accomplish naturally. This was achieved by the heterologous expression of five genes of the carbon fixation cycle of the archaeon Metallosphaera sedula, which grows autotrophically at 73°C. The engineered P. furiosus strain is able to use hydrogen gas and incorporate carbon dioxide into 3-hydroxypropionic acid, one of the top 12 industrial chemical building blocks. The reaction can be accomplished by cell-free extracts and by whole cells of the recombinant P. furiosus strain. Moreover, it is carried out some 30°C below the optimal growth temperature of the organism in conditions that support only minimal growth but maintain sufficient metabolic activity to sustain the production of 3-hydroxypropionate. The approach described here can be expanded to produce important organic chemicals, all through biological activation of carbon dioxide.
Related JoVE Video
Regulation of iron metabolism by Pyrococcus furiosus.
J. Bacteriol.
PUBLISHED: 03-15-2013
Show Abstract
Hide Abstract
Iron is an essential element for the hyperthermophilic archaeon Pyrococcus furiosus, and many of its iron-containing enzymes have been characterized. How iron assimilation is regulated, however, is unknown. The genome sequence contains genes encoding two putative iron-responsive transcription factors, DtxR and Fur. Global transcriptional profiles of the dtxR deletion mutant (?DTXR) and the parent strain under iron-sufficient and iron-limited conditions indicated that DtxR represses the expression of the genes encoding two putative iron transporters, Ftr1 and FeoAB, under iron-sufficient conditions. Under iron limitation, DtxR represses expression of the gene encoding the iron-containing enzyme aldehyde ferredoxin oxidoreductase and a putative ABC-type transporter. Analysis of the dtxR gene sequence indicated an incorrectly predicted translation start site, and the corrected full-length DtxR protein, in contrast to the truncated version, specifically bound to the promoters of ftr1 and feoAB, confirming its role as a transcription regulator. Expression of the gene encoding Ftr1 was dramatically upregulated by iron limitation, but no phenotype was observed for the ?FTR1 deletion mutant under iron-limited conditions. The intracellular iron concentrations of ?FTR1 and the parent strain were similar, suggesting that under the conditions tested, Ftr1 is not an essential iron transporter despite its response to iron. In contrast to DtxR, the Fur protein appears not to be a functional regulator in P. furiosus, since it did not bind to the promoters of any of the iron-regulated genes and the deletion mutant (?FUR) revealed no transcriptional responses to iron availability. DtxR is therefore the key iron-responsive transcriptional regulator in P. furiosus.
Related JoVE Video
The structure and mode of action of Caldicellulosiruptor bescii family 3 pectate lyase in biomass deconstruction.
Acta Crystallogr. D Biol. Crystallogr.
PUBLISHED: 03-09-2013
Show Abstract
Hide Abstract
The unique active site of the Caldicellulosiruptor bescii family 3 pectate lyase catalytic module (PL3-cat) has been structurally described and synergistic digestion studies with C. bescii cellulase A have been performed on unpretreated biomass. The X-ray structure of PL3-cat was determined at 1.6 Å resolution (PDB entry 4ew9) in complex with the products of trigalacturonic acid. Comparison with family 1 pectate lyase (PL1) structures shows that the active site of the PL3 catalytic module is considerably different. However, on superimposing the identical sugar rings at the -2 subsites conserved interactions could be identified. Interestingly, only one catalytic residue, the lysine that donates the proton to the carboxylate group in the ?-elimination reaction of PL1 (Lys108 in PL3-cat), is conserved in PL3 and there is no arginine to abstract the proton from the C5 carbon of the galactouronate ring. This suggests that the reaction mechanism of PL3 requires different catalytic residues. Most interestingly, comparison with other proton-abstraction reactions reveals that in PL3 the ?-proton is abstracted by a lysine, in a striking similarity to enolases. These observations led us to propose that in PL3-cat Lys108 is the catalytic base, Glu84 is the catalytic acid and an acidified water molecule completes the anti ?-elimination reaction by protonating the O4 atom of the substrate. Also, our digestion experiments with unpretreated switchgrass show that the loadings of C. bescii cellobiohydrolase A (CelA) can be lowered by the addition of PL3 to the reaction mixture. This result suggests that PL3 can significantly improve the deconstruction of unpretreated biomass by allowing other enzymes to better access their preferred substrates.
Related JoVE Video
Biological conversion of carbon dioxide and hydrogen into liquid fuels and industrial chemicals.
Curr. Opin. Biotechnol.
PUBLISHED: 02-18-2013
Show Abstract
Hide Abstract
Non-photosynthetic routes for biological fixation of carbon dioxide into valuable industrial chemical precursors and fuels are moving from concept to reality. The development of electrofuel-producing microorganisms leverages techniques in synthetic biology, genetic and metabolic engineering, as well as systems-level multi-omic analysis, directed evolution, and in silico modeling. Electrofuel processes are being developed for a range of microorganisms and energy sources (e.g. hydrogen, formate, electricity) to produce a variety of target molecules (e.g. alcohols, terpenes, alkenes). This review examines the current landscape of electrofuel projects with a focus on hydrogen-utilizing organisms covering the biochemistry of hydrogenases and carbonic anhydrases, kinetic and energetic analyses of the known carbon fixation pathways, and the state of genetic systems for current and prospective electrofuel-producing microorganisms.
Related JoVE Video
Nuclear magnetic resonance metabonomic profiling using tO2PLS.
Anal. Chim. Acta
PUBLISHED: 02-12-2013
Show Abstract
Hide Abstract
Blood plasma collected from adult fish (black bream, Sparidae) exposed to a dose of 5 mg kg(-1) 17?-estradiol underwent metabonomic profiling using nuclear magnetic resonance (NMR). An extension of the orthogonal 2 projection to latent structure (O2PLS) analysis, tO2PLS, was proposed and utilized to classify changes between the control and experimental metabolic profiles. As a bidirectional modeling tool, O2PLS examines the (variable) commonality between two different data blocks, and extracts the joint correlations as well as the unique variations present within each data block. tO2PLS is a proposed matrix transposition of O2PLS to allow for commonality between experiments (spectral profiles) to be observed, rather than between sample variables. tO2PLS analysis highlighted two potential biomarkers, trimethylamine-N-oxide (TMAO) and choline, that distinguish between control and 17?-estradiol exposed fish. This study presents an alternative way of examining spectroscopic (metabolite) data, providing a method for the visual assessment of similarities and differences between control and experimental spectral features in large data sets.
Related JoVE Video
High-yield production of dihydrogen from xylose by using a synthetic enzyme cascade in a cell-free system.
Angew. Chem. Int. Ed. Engl.
PUBLISHED: 01-29-2013
Show Abstract
Hide Abstract
Let enzymes work: H2 was produced from xylose and water in one reactor containing 13 enzymes (red). By using a novel polyphosphate xylulokinase (XK), xylose was converted into H2 and CO2 with approaching 100?% of the theoretical yield. The findings suggest that cell-free biosystems could produce H2 from biomass xylose at low cost. Xu5P = xylulose 5-phosphate, G6P = glucose 6-phosphate.
Related JoVE Video
Case study of the Swiss flora for prior phytochemical and biological investigations.
J. Nat. Prod.
PUBLISHED: 01-29-2013
Show Abstract
Hide Abstract
Estimates in the literature as to what extent the worlds higher plant species have been studied chemically or for bioactivity are contradictory and range from 0.5% to >12%. In this survey, a model to make credible estimates of the extent of their study is proposed and is exemplified by applying it in a case study of plants native to Switzerland. Using a widely available database (SciFinder Scholar), 454?535 literature references for the 2677 native Swiss plant species were retrieved. It was determined that 55% of these species have been investigated phytochemically and 28% for biological activity. The influence of factors such as commonness, growth form, habitat, medicinal use, and reported toxicity on the extent to which different plant groups have been studied is analyzed. The predictive value of random sampling of subsets of plants is compared to the study of the entire Swiss flora, to show that a credible estimate of the extent of prior studies can be achieved with just 5% of these species.
Related JoVE Video
Insulin resistance is associated with Fibroblast Growth Factor-23 in stage 3-5 chronic kidney disease patients.
J. Diabetes Complicat.
PUBLISHED: 01-24-2013
Show Abstract
Hide Abstract
To determine the associations between insulin resistance, fibroblast growth factor 23 (FGF-23), and coronary artery calcification (CAC) in chronic kidney disease (CKD) patients.
Related JoVE Video
Dietary vitamin K and therapeutic warfarin alter the susceptibility to vascular calcification in experimental chronic kidney disease.
Kidney Int.
PUBLISHED: 01-23-2013
Show Abstract
Hide Abstract
The leading cause of death in patients with chronic kidney disease (CKD) is cardiovascular disease, with vascular calcification being a key modifier of disease progression. A local regulator of vascular calcification is vitamin K. This ?-glutamyl carboxylase substrate is an essential cofactor in the activation of several extracellular matrix proteins that inhibit calcification. Warfarin, a common therapy in dialysis patients, inhibits the recycling of vitamin K and thereby decreases the inhibitory activity of these proteins. In this study, we sought to determine whether modifying vitamin K status, either by increasing dietary vitamin K intake or by antagonism with therapeutic doses of warfarin, could alter the development of vascular calcification in male Sprague-Dawley rats with adenine-induced CKD. Treatment of CKD rats with warfarin markedly increased pulse pressure and pulse wave velocity, as well as significantly increased calcium concentrations in the thoracic aorta (3-fold), abdominal aorta (8-fold), renal artery (4-fold), and carotid artery (20-fold). In contrast, treatment with high dietary vitamin K1 increased vitamin K tissue concentrations (10-300-fold) and blunted the development of vascular calcification. Thus, vitamin K has an important role in modifying mechanisms linked to the susceptibility of arteries to calcify in an experimental model of CKD.
Related JoVE Video
Male reproductive health disorders among Aboriginal and Torres Strait Islander men: a hidden problem?
Med. J. Aust.
PUBLISHED: 01-22-2013
Show Abstract
Hide Abstract
To better understand help-seeking behaviours and reproductive health disorders among Aboriginal and Torres Strait Islander men.
Related JoVE Video
Metallochaperones regulate intracellular copper levels.
PLoS Comput. Biol.
PUBLISHED: 01-17-2013
Show Abstract
Hide Abstract
Copper (Cu) is an important enzyme co-factor that is also extremely toxic at high intracellular concentrations, making active efflux mechanisms essential for preventing Cu accumulation. Here, we have investigated the mechanistic role of metallochaperones in regulating Cu efflux. We have constructed a computational model of Cu trafficking and efflux based on systems analysis of the Cu stress response of Halobacterium salinarum. We have validated several model predictions via assays of transcriptional dynamics and intracellular Cu levels, discovering a completely novel function for metallochaperones. We demonstrate that in addition to trafficking Cu ions, metallochaperones also function as buffers to modulate the transcriptional responsiveness and efficacy of Cu efflux. This buffering function of metallochaperones ultimately sets the upper limit for intracellular Cu levels and provides a mechanistic explanation for previously observed Cu metallochaperone mutation phenotypes.
Related JoVE Video
Ribosomal DNA analyses reveal greater sequence variation in Polymyxa species than previously thought and indicate the possibility of new ribotype-host-virus associations.
Environ Microbiol Rep
PUBLISHED: 01-13-2013
Show Abstract
Hide Abstract
Polymyxa species transmit viruses to many important crops. They are poorly understood obligate parasites occupying a distinct position in the Tree of Life. To better understand the potential for spread of Polymyxa-vectored diseases, ribosomal DNA was analysed from isolates covering a wide range of geographical locations, virus associations and hosts. Internal transcribed spacer 2 structure analysis indicated that Polymyxa graminis isolates could represent many species and there was more sequence variation within the known subgroups (ribotypes) than previously described. In cereal crops and soils from temperate climates Polymyxa isolates were usually ribotype I or II, but their host specificities or preferences were unclear. For the first time, there was evidence that ribotype I (in addition to ribotype II) could transmit SBWMV/SBCMV. Different ribotypes often occurred together in the same soil or plant. New hosts were identified for particular ribotypes, including the first detection of the sugar beet-infecting Polymyxa betae, in wheat. Unexpectedly, ribotype III-like sequences, usually restricted to crops in the tropics, were found in wheat from the USA. P.?betae isolates showed limited variation (??2%) and the recent change in susceptibility of sugar beet varieties to BNYVV in the USA is unlikely to be due to changes in P.?betae.
Related JoVE Video
The risk of bleeding with duloxetine treatment in patients who use nonsteroidal anti-inflammatory drugs (NSAIDs): analysis of placebo-controlled trials and post-marketing adverse event reports.
Drug Healthc Patient Saf
PUBLISHED: 01-01-2013
Show Abstract
Hide Abstract
To assess the safety of duloxetine with regards to bleeding-related events in patients who concomitantly did, versus did not, use nonsteroidal anti-inflammatory drugs (NSAIDs), including aspirin.
Related JoVE Video
Trends in amphibian occupancy in the United States.
PLoS ONE
PUBLISHED: 01-01-2013
Show Abstract
Hide Abstract
Though a third of amphibian species worldwide are thought to be imperiled, existing assessments simply categorize extinction risk, providing little information on the rate of population losses. We conducted the first analysis of the rate of change in the probability that amphibians occupy ponds and other comparable habitat features across the United States. We found that overall occupancy by amphibians declined 3.7% annually from 2002 to 2011. Species that are Red-listed by the International Union for Conservation of Nature (IUCN) declined an average of 11.6% annually. All subsets of data examined had a declining trend including species in the IUCN Least Concern category. This analysis suggests that amphibian declines may be more widespread and severe than previously realized.
Related JoVE Video
Analysis of maternal and fetal cardiovascular systems during hyperglycemic pregnancy in the nonobese diabetic mouse.
Biol. Reprod.
PUBLISHED: 01-01-2013
Show Abstract
Hide Abstract
Preconception or gestationally induced diabetes increases morbidities and elevates long-term cardiovascular disease risks in women and their children. Spontaneously hyperglycemic (d)-NOD/ShiLtJ female mice, a type 1 diabetes model, develop bradycardia and hypotension after midpregnancy compared with normoglycemic, age- and gestational day (GD)-matched control (c-NOD) females. We hypothesized that onset of the placental circulation at GD 9-10 and rapid fetal growth from GD 14 correlate with aberrant hemodynamic outcomes in d-NOD females. To develop further gestational time-course correlations between maternal cardiac and renal parameters, high-frequency ultrasonography was applied to d- and c-NOD mice (virgin and at GD 8-16). Cardiac output and left ventricular (LV) mass increased in c-NOD but not in d-NOD mice. Ultrasound and postmortem histopathology showed overall greater LV dilation in d-NOD than in c-NOD mice at mid to late gestation. These changes suggest blunted remodeling and altered functional adaptation of d-NOD hearts. Umbilical cord ultrasounds revealed lower fetal heart rates from GD 12 and lower umbilical flow velocities at GD 14 and GD 16 in d-NOD versus c-NOD pregnancies. From GD 14 to GD 16, d-NOD fetal losses exceeded c-NOD fetal losses. Similar aberrant responses in pregnancies of women with diabetes may elevate postpartum maternal and child cardiovascular risk, particularly if mothers lack adequate prenatal care or have poor glycemic control during gestation.
Related JoVE Video
Perinatal iron deficiency combined with a high-fat diet causes obesity and cardiovascular dysregulation.
Endocrinology
PUBLISHED: 12-30-2011
Show Abstract
Hide Abstract
Consumption of a high-fat Western diet (WD) and the resultant obesity is linked to a number of chronic pathologies, including cardiovascular dysregulation. The purpose of the present study was to determine whether perinatal iron deficiency (PID) added to the consumption of a WD would precipitate an obese phenotype with exacerbated metabolic and cardiovascular outcomes in adult offspring. Female Sprague Dawley rats were fed either a control (225 mg/kg Fe) or an iron-restricted diet (3-10 mg/kg Fe) prior to and throughout gestation. At birth, all dams were fed an iron-replete diet. At weaning, offspring were fed a normal diet or WD for up to 21 wk. Hemodynamics and locomotor activity were assessed by radiotelemetry starting at 15 wk of age. Iron restriction during pregnancy caused severe anemia in dams and offspring, resulting in 15% lower birth weights in the offspring. PID offspring fed the WD had greater caloric intake and exhibited reduced locomotor activity compared with their normal diet-fed littermates; no such effects were observed in normal iron control offspring. Despite having a similar effect on serum lipid profiles, consumption of the WD had a greater impact on body weight in the PID group, and this weight gain was due largely to visceral adipose tissue accumulation. A significant correlation between visceral adipose tissue weight and mean arterial pressure was observed in the PID offspring but not in controls. These observations demonstrate that PID predisposes offspring to an enhanced response to WD characterized by increased fat accumulation and cardiovascular dysregulation.
Related JoVE Video
Engineering hyperthermophilic archaeon Pyrococcus furiosus to overproduce its cytoplasmic [NiFe]-hydrogenase.
J. Biol. Chem.
PUBLISHED: 12-07-2011
Show Abstract
Hide Abstract
The cytoplasmic hydrogenase (SHI) of the hyperthermophilic archaeon Pyrococcus furiosus is an NADP(H)-dependent heterotetrameric enzyme that contains a nickel-iron catalytic site, flavin, and six iron-sulfur clusters. It has potential utility in a range of bioenergy systems in vitro, but a major obstacle in its use is generating sufficient amounts. We have engineered P. furiosus to overproduce SHI utilizing a recently developed genetic system. In the overexpression (OE-SHI) strain, transcription of the four-gene SHI operon was under the control of a strong constitutive promoter, and a Strep-tag II was added to the N terminus of one subunit. OE-SHI and wild-type P. furiosus strains had similar rates of growth and H(2) production on maltose. Strain OE-SHI had a 20-fold higher transcription of the polycistronic hydrogenase mRNA encoding SHI, and the specific activity of the cytoplasmic hydrogenase was ?10-fold higher when compared with the wild-type strain, although the expression levels of genes encoding processing and maturation of SHI were the same in both strains. Overexpressed SHI was purified by a single affinity chromatography step using the Strep-tag II, and it and the native form had comparable activities and physical properties. Based on protein yield per gram of cells (wet weight), the OE-SHI strain yields a 100-fold higher amount of hydrogenase when compared with the highest homologous [NiFe]-hydrogenase system previously reported (from Synechocystis). This new P. furiosus system will allow further engineering of SHI and provide hydrogenase for efficient in vitro biohydrogen production.
Related JoVE Video
Radiation dose associated with renal failure mortality: a potential pathway to partially explain increased cardiovascular disease mortality observed after whole-body irradiation.
Radiat. Res.
PUBLISHED: 12-07-2011
Show Abstract
Hide Abstract
Whole-body and thoracic ionizing radiation exposure are associated with increased cardiovascular disease (CVD) risk. In atomic bomb survivors, radiation dose is also associated with increased hypertension incidence, suggesting that radiation dose may be associated with chronic renal failure (CRF), thus explaining part of the mechanism for increased CVD. Multivariate Poisson regression was used to evaluate the association of radiation dose with various definitions of chronic kidney disease (CKD) mortality in the Life Span Study (LSS) of atomic bomb survivors. A secondary analysis was performed using a subsample for whom self-reported information on hypertension and diabetes, the two biggest risk factors for CRF, had been collected. We found a significant association between radiation dose and only our broadest definition of CRF among the full cohort. A quadratic dose excess relative risk model [ERR/Gy(2) = 0.091 (95% CI: 0.05, 0.198)] fit minimally better than a linear model. Within the subsample, association was also observed only with the broadest CRF definition [ERR/Gy(2) = 0.15 (95% CI: 0.02, 0.32)]. Adjustment for hypertension and diabetes improved model fit but did not substantially change the ERR/Gy(2) estimate, which was 0.17 (95% CI: 0.04, 0.35). We found a significant quadratic dose relationship between radiation dose and possible chronic renal disease mortality that is similar in shape to that observed between radiation and incidence of hypertension in this population. Our results suggest that renal dysfunction could be part of the mechanism causing increased CVD risk after whole-body irradiation, a hypothesis that deserves further study.
Related JoVE Video
Screening and HPLC-Based Activity Profiling for New Antiprotozoal Leads from European Plants.
Sci Pharm
PUBLISHED: 11-14-2011
Show Abstract
Hide Abstract
Based on a survey of remedies used in Renaissance Europe to treat malaria, we prepared and screened a library of 254 extracts from 61 plants for antiplasmodial activity in vitro. HPLC-based activity profiling was performed for targeted identification of active constituents in extracts. One of the most remarkable results was the identification of onopordopicrin, a germacranolide sesquiterpene lactone isolated from Arctium nemorosum as a potent inhibitor of P. falciparum with an IC(50) of 6.9 ?M. It was tested similarly against Trypanosoma brucei rhodesiense, the parasite which causes African sleeping sickness. With an IC(50) of 0.37 ?M, onopordopicrin was one of the most potent natural products reported so far. Cytotoxicity was determined against rat myoblast L6 cells (IC(50): 3.06).
Related JoVE Video
Characterization of the vasculature supplying the genital tissues in female rats.
J Sex Med
PUBLISHED: 10-24-2011
Show Abstract
Hide Abstract
The internal pudendal arteries are the key resistance vessels controlling the peripheral circulatory component of sexual responses in both male and females. Previous studies in the male rat demonstrated that this vessel has markedly heightened susceptibility to vascular damage compared with other vessels in the body. Evidence suggests that the female may also be susceptible to vascular pathologies contributing to sexual dysfunction.
Related JoVE Video
Deletion strains reveal metabolic roles for key elemental sulfur-responsive proteins in Pyrococcus furiosus.
J. Bacteriol.
PUBLISHED: 09-30-2011
Show Abstract
Hide Abstract
Transcriptional and enzymatic analyses of Pyrococcus furiosus previously indicated that three proteins play key roles in the metabolism of elemental sulfur (S(0)): a membrane-bound oxidoreductase complex (MBX), a cytoplasmic coenzyme A-dependent NADPH sulfur oxidoreductase (NSR), and sulfur-induced protein A (SipA). Deletion strains, referred to as MBX1, NSR1, and SIP1, respectively, have now been constructed by homologous recombination utilizing the uracil auxotrophic COM1 parent strain (?pyrF). The growth of all three mutants on maltose was comparable without S(0), but in its presence, the growth of MBX1 was greatly impaired while the growth of NSR1 and SIP1 was largely unaffected. In the presence of S(0), MBX1 produced little, if any, sulfide but much more acetate (per unit of protein) than the parent strain, demonstrating that MBX plays a critical role in S(0) reduction and energy conservation. In contrast, comparable amounts of sulfide and acetate were produced by NSR1 and the parent strain, indicating that NSR is not essential for energy conservation during S(0) reduction. Differences in transcriptional responses to S(0) in NSR1 suggest that two sulfide dehydrogenase isoenzymes provide a compensatory NADPH-dependent S(0) reduction system. Genes controlled by the S(0)-responsive regulator SurR were not as highly regulated in MBX1 and NSR1. SIP1 produced the same amount of acetate but more sulfide than the parent strain. That SipA is not essential for growth on S(0) indicates that it is not required for detoxification of metal sulfides, as previously suggested. A model is proposed for S(0) reduction by P. furiosus with roles for MBX and NSR in bioenergetics and for SipA in iron-sulfur metabolism.
Related JoVE Video
Application of colloid probe atomic force microscopy to the adhesion of thin films of viscous and viscoelastic silicone fluids.
Langmuir
PUBLISHED: 08-26-2011
Show Abstract
Hide Abstract
The adhesive characteristics of thin films (0.2-2 ?m) of linear poly(dimethylsiloxane) (PDMS) liquids with a wide range of molecular weights have been measured using an atomic force microscope with a colloid probe (diameters 5 and 12 ?m) for different separation velocities. The data were consistent with a residual film in the contact region having a thickness of ?6 nm following an extended dwell time before separation of the probe. It was possible to estimate the maximum adhesive force as a function of the capillary number, Ca, by applying existing theoretical models based on capillary interactions and viscous flow except at large values of Ca in the case of viscoelastic fluids, for which it was necessary to develop a nonlinear viscoelastic model. The compliance of the atomic force microscope colloid beam was an important factor in governing the retraction velocity of the probe and therefore the value of the adhesive force, but the inertia of the beam and viscoelastic stress overshoot effects were not significant in the range of separation velocities investigated.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.