26 Related JoVE Articles!
Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples
Institutions: University of Manitoba, University of Manitoba.
Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo
flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.
Medicine, Issue 89, mucosal, immunology, FGT, lavage, cervical, CMC
Design and Implementation of an fMRI Study Examining Thought Suppression in Young Women with, and At-risk, for Depression
Institutions: McMaster University, McMaster University, University of Calgary, McMaster University.
Ruminative brooding is associated with increased vulnerability to major depression. Individuals who regularly ruminate will often try to reduce the frequency of their negative thoughts by actively suppressing them. We aim to identify the neural correlates underlying thought suppression in at-risk and depressed individuals. Three groups of women were studied; a major depressive disorder group, an at-risk group (having a first degree relative with depression) and controls. Participants performed a mixed block-event fMRI paradigm involving thought suppression, free thought and motor control periods. Participants identified the re-emergence of “to-be-suppressed” thoughts (“popping” back into conscious awareness) with a button press. During thought suppression the control group showed the greatest activation of the dorsolateral prefrontal cortex, followed by the at-risk, then depressed group. During the re-emergence of intrusive thoughts compared to successful re-suppression of those thoughts, the control group showed the greatest activation of the anterior cingulate cortices, followed by the at-risk, then depressed group. At-risk participants displayed anomalies in the neural regulation of thought suppression resembling the dysregulation found in depressed individuals. The predictive value of these changes in the onset of depression remains to be determined.
Behavior, Issue 99, Major Depressive Disorder, Risk, Thought Suppression, fMRI, Women, Rumination, Thought Intrusion
The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects
Institutions: University and Bern University Hospital (Inselspital), Kantonsspital Aarau, Boston Children's Hospital, Boston Children's Hospital, University and Bern University Hospital (Inselspital), University Hospital Cologne, Länggasse Bern.
Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.
Medicine, Issue 92,
Subarachnoid hemorrhage, animal models, rabbit, extracorporeal blood shunt, early brain injury, delayed cerebral vasospasm, microsurgery.
Physical, Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites
Institutions: Royal Military College of Canada, Queen's University.
The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with specific functions (e.g.
carbon sequestration, soil quality improvements, or contaminant sorption). In 2013, the International Biochar Initiative (IBI) made publically available their Standardized Product Definition and Product Testing Guidelines (Version 1.1) which set standards for physical and chemical characteristics for biochar. Six biochars made from three different feedstocks and at two temperatures were analyzed for characteristics related to their use as a soil amendment. The protocol describes analyses of the feedstocks and biochars and includes: cation exchange capacity (CEC), specific surface area (SSA), organic carbon (OC) and moisture percentage, pH, particle size distribution, and proximate and ultimate analysis. Also described in the protocol are the analyses of the feedstocks and biochars for contaminants including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), metals and mercury as well as nutrients (phosphorous, nitrite and nitrate and ammonium as nitrogen). The protocol also includes the biological testing procedures, earthworm avoidance and germination assays. Based on the quality assurance / quality control (QA/QC) results of blanks, duplicates, standards and reference materials, all methods were determined adequate for use with biochar and feedstock materials. All biochars and feedstocks were well within the criterion set by the IBI and there were little differences among biochars, except in the case of the biochar produced from construction waste materials. This biochar (referred to as Old biochar) was determined to have elevated levels of arsenic, chromium, copper, and lead, and failed the earthworm avoidance and germination assays. Based on these results, Old biochar would not be appropriate for use as a soil amendment for carbon sequestration, substrate quality improvements or remediation.
Environmental Sciences, Issue 93, biochar, characterization, carbon sequestration, remediation, International Biochar Initiative (IBI), soil amendment
Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis
Institutions: RWTH Aachen University, RWTH Aachen University, RWTH Aachen University.
In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g.
, partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.
Medicine, Issue 96, bile duct ligation, cholestasis, bile obstruction, hepatic fibrosis, inflammation, extracellular matrix, jaundice, mouse
High Precision Zinc Isotopic Measurements Applied to Mouse Organs
Institutions: Université Paris Diderot, Université Paris Diderot.
We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (64
Zn and 70
Zn) which are naturally fractionated between mouse organs. We first show how to dissolve the different organs in order to free the Zn atoms; this step is realized by a mixture of HNO3
. We then purify the zinc atoms from all the other elements, in particular from isobaric interferences (e.g.
, Ni), by anion-exchange chromatography in a dilute HBr/HNO3
medium. These first two steps are performed in a clean laboratory using high purity chemicals. Finally, the isotope ratios are measured by using a multi-collector inductively-coupled-plasma mass-spectrometer, in low resolution. The samples are injected using a spray chamber and the isotopic fractionation induced by the mass-spectrometer is corrected by comparing the ratio of the samples to the ratio of a standard (standard bracketing technique). This full typical procedure produces an isotope ratio with a 50 ppm (2 s.d.) reproducibility.
Chemistry, Issue 99, Zinc, isotopes, MC-ICP-MS, chromatography, mice, purification
Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound
Institutions: University of Toronto, Sunnybrook Research Institute, Mount Sinai Hospital, Toronto.
Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or αv
) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.
Developmental Biology, Issue 97, Micro-ultrasound, Molecular imaging, Mouse embryo, Microbubble, Ultrasound contrast agent, Perfusion
A Microfluidic Chip for ICPMS Sample Introduction
Institutions: ETH Zurich.
This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 µm and from 90 to 7,000 Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.
Bioengineering, Issue 97, mass spectrometry, ICPMS, microfluidics, droplet microfluidics, monodisperse, sample introduction, chip, red blood cells, erythrocytes, single cell analysis
Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability
Institutions: University of New Mexico, University of Wyoming, University of Wyoming, Colorado State University, Colorado State University, California Northstate University.
recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted through ion exchange. The resultant production bleed water (PBW) contains contaminants such as arsenic and other heavy metals. Samples of PBW from an active ISR uranium facility were treated with cupric oxide nanoparticles (CuO-NPs). CuO-NP treatment of PBW reduced priority contaminants, including arsenic, selenium, uranium, and vanadium. Untreated and CuO-NP treated PBW was used as the liquid component of the cell growth media and changes in viability were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human embryonic kidney (HEK 293) and human hepatocellular carcinoma (Hep G2) cells. CuO-NP treatment was associated with improved HEK and HEP cell viability. Limitations of this method include dilution of the PBW by growth media components and during osmolality adjustment as well as necessary pH adjustment. This method is limited in its wider context due to dilution effects and changes in the pH of the PBW which is traditionally slightly acidic however; this method could have a broader use assessing CuO-NP treatment in more neutral waters.
Environmental Sciences, Issue 100, Energy production, uranium in situ recovery, water decontamination, nanoparticles, toxicity, cytotoxicity, in vitro cell culture
Isolation of Leukocytes from the Human Maternal-fetal Interface
Institutions: NICHD/NIH/DHHS, University of Michigan, Michigan State University, Wayne State University, Wayne State University School of Medicine, Wayne State University School of Medicine.
Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro
leukocyte functionality (i.e.
, phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.
), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.
Immunology, Issue 99, Accutase, Decidua Basalis, Decidua Parietalis, Flow Cytometry, Immunophenotyping, Pregnancy
Establishment and Characterization of UTI and CAUTI in a Mouse Model
Institutions: Washington University School of Medicine.
Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli
(UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis
. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis
bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.
Medicine, Issue 100, Escherichia coli, UPEC, Enterococcus faecalis, uropathogenic, catheter, urinary tract infection, IBC, chronic cystitis
Isolation of Neonatal Extrahepatic Cholangiocytes
Institutions: The Children's Hospital of Philadelphia, The Perelman School of Medicine at the University of Pennsylvania.
The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, differences between intra and extrahepatic cholangiocytes, and between neonatal and adult cells, are not well understood.
Methods for the isolation of cholangiocytes from intrahepatic bile ducts are well established1-4
. Isolation of extrahepatic ductal cells, especially from the neonate, has not yet been described, although this would be of great benefit in understanding the differences between distinct cholangiocyte populations and in studying diseases such as biliary atresia that appear to target the extrahepatic ducts. Described here is an optimized technique to isolate both neonatal and adult mouse extrahepatic bile duct cells. This technique yields a pure cell population with minimal contamination from mesenchymal cells like fibroblasts.
This method is based on the removal of the extrahepatic ducts and gallbladder, followed by meticulous dissection and scraping to remove fat and fibroblast layers. Structures are embedded in thick layers of collagen and cultured for approximately 3 weeks to allow outgrowth of cholangiocytes in monolayers, which can then be trypsinized and re plated for experimental use.
Medicine, Issue 88, Bile Ducts, Bile Ducts, Extrahepatic, Common Bile Duct, Bile Duct Diseases, Cell culture, bile duct, biliary atresia, Liver, gallbladder, fibrosis
Laser-induced Breakdown Spectroscopy: A New Approach for Nanoparticle's Mapping and Quantification in Organ Tissue
Institutions: CNRS - Université Lyon 1, CNRS - Université Lyon 1, CNRS - Université Lyon 1.
Emission spectroscopy of laser-induced plasma was applied to elemental analysis of biological samples. Laser-induced breakdown spectroscopy (LIBS) performed on thin sections of rodent tissues: kidneys and tumor, allows the detection of inorganic elements such as (i) Na, Ca, Cu, Mg, P, and Fe, naturally present in the body and (ii) Si and Gd, detected after the injection of gadolinium-based nanoparticles. The animals were euthanized 1 to 24 hr after intravenous injection of particles. A two-dimensional scan of the sample, performed using a motorized micrometric 3D-stage, allowed the infrared laser beam exploring the surface with a lateral resolution less than 100 μm. Quantitative chemical images of Gd element inside the organ were obtained with sub-mM sensitivity. LIBS offers a simple and robust method to study the distribution of inorganic materials without any specific labeling. Moreover, the compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of the same biological tissue with different types of response: elemental, molecular, or cellular.
Physics, Issue 88, Microtechnology, Nanotechnology, Tissues, Diagnosis, Inorganic Chemistry, Organic Chemistry, Physical Chemistry, Plasma Physics, laser-induced breakdown spectroscopy, nanoparticles, elemental mapping, chemical images of organ tissue, quantification, biomedical measurement, laser-induced plasma, spectrochemical analysis, tissue mapping
Transabdominal Ultrasound for Pregnancy Diagnosis in Reeves' Muntjac Deer
Institutions: Colorado State University.
Reeves' muntjac deer (Muntiacus reevesi
) are a small cervid species native to southeast Asia, and are currently being investigated as a potential model of prion disease transmission and pathogenesis. Vertical transmission is an area of interest among researchers studying infectious diseases, including prion disease, and these investigations require efficient methods for evaluating the effects of maternal infection on reproductive performance. Ultrasonographic examination is a well-established tool for diagnosing pregnancy and assessing fetal health in many animal species1-7
, including several species of farmed cervids8-19
, however this technique has not been described in Reeves' muntjac deer. Here we describe the application of transabdominal ultrasound to detect pregnancy in muntjac does and to evaluate fetal growth and development throughout the gestational period. Using this procedure, pregnant animals were identified as early as 35 days following doe-buck pairing and this was an effective means to safely monitor the pregnancy at regular intervals. Future goals of this work will include establishing normal fetal measurement references for estimation of gestational age, determining sensitivity and specificity of the technique for diagnosing pregnancy at various stages of gestation, and identifying variations in fetal growth and development under different experimental conditions.
Medicine, Issue 83, Ultrasound, Reeves' muntjac deer, Muntiacus reevesi, fetal development, fetal growth, captive cervids
Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools
Institutions: Brigham and Women's Hospital.
Cellular Biology, Issue 8, microfluidics, diagnostics, capture, blood, HIV, bioengineering
An Experimental Paradigm for the Prediction of Post-Operative Pain (PPOP)
Institutions: University of Washington School of Medicine.
Many women undergo cesarean delivery without problems, however some experience significant pain after cesarean section. Pain is associated with negative short-term and long-term effects on the mother. Prior to women undergoing surgery, can we predict who is at risk for developing significant postoperative pain and potentially prevent or minimize its negative consequences? These are the fundamental questions that a team from the University of Washington, Stanford University, the Catholic University in Brussels, Belgium, Santa Joana Women's Hospital in São Paulo, Brazil, and Rambam Medical Center in Israel is currently evaluating in an international research collaboration. The ultimate goal of this project is to provide optimal pain relief during and after cesarean section by offering individualized anesthetic care to women who appear to be more 'susceptible' to pain after surgery.
A significant number of women experience moderate or severe acute post-partum pain after vaginal and cesarean deliveries. 1
Furthermore, 10-15% of women suffer chronic persistent pain after cesarean section. 2
With constant increase in cesarean rates in the US 3
and the already high rate in Brazil, this is bound to create a significant public health problem. When questioning women's fears and expectations from cesarean section, pain during and after it is their greatest concern. 4
Individual variability in severity of pain after vaginal or operative delivery is influenced by multiple factors including sensitivity to pain, psychological factors, age, and genetics. The unique birth experience leads to unpredictable requirements for analgesics, from 'none at all' to 'very high' doses of pain medication. Pain after cesarean section is an excellent model to study post-operative pain because it is performed on otherwise young and healthy women. Therefore, it is recommended to attenuate the pain during the acute phase because this may lead to chronic pain disorders. The impact of developing persistent pain is immense, since it may impair not only the ability of women to care for their child in the immediate postpartum period, but also their own well being for a long period of time.
In a series of projects, an international research network is currently investigating the effect of pregnancy on pain modulation and ways to predict who will suffer acute severe pain and potentially chronic pain, by using simple pain tests and questionnaires in combination with genetic analysis. A relatively recent approach to investigate pain modulation is via the psychophysical measure of Diffuse Noxious Inhibitory Control (DNIC). This pain-modulating process is the neurophysiological basis for the well-known phenomenon of 'pain inhibits pain' from remote areas of the body. The DNIC paradigm has evolved recently into a clinical tool and simple test and has been shown to be a predictor of post-operative pain.5
Since pregnancy is associated with decreased pain sensitivity and/or enhanced processes of pain modulation, using tests that investigate pain modulation should provide a better understanding of the pathways involved with pregnancy-induced analgesia and may help predict pain outcomes during labor and delivery. For those women delivering by cesarean section, a DNIC test performed prior to surgery along with psychosocial questionnaires and genetic tests should enable one to identify women prone to suffer severe post-cesarean pain and persistent pain. These clinical tests should allow anesthesiologists to offer not only personalized medicine to women with the promise to improve well-being and satisfaction, but also a reduction in the overall cost of perioperative and long term care due to pain and suffering. On a larger scale, these tests that explore pain modulation may become bedside screening tests to predict the development of pain disorders following surgery.
JoVE Medicine, Issue 35, diffuse noxious inhibitory control, DNIC, temporal summation, TS, psychophysical testing, endogenous analgesia, pain modulation, pregnancy-induced analgesia, cesarean section, post-operative pain, prediction
A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia
Institutions: Universite de Montreal, Universite de Montreal, Universite de Montreal.
There are several lines of evidence supporting the role of de novo
mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo
mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo
mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1
and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo
mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2
. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo
mutations. This is the case for autism and schizophrenia3
. The male-to-female ratio of mutation rate is estimated at about 4–6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo
mutations would more frequently come from males, particularly older males4
. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo
mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo
mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.
Medicine, Issue 52, de novo mutation, complex diseases, schizophrenia, autism, rare variations, DNA sequencing
Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae
Institutions: Irell & Manella Graduate School of Biological Sciences, City of Hope Comprehensive Cancer Center and Beckman Research Institute, University of Southern California, Norris Comprehensive Cancer Center.
Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every eukaryotic genome. This process is an important mechanism for generating diversity between and within organisms1-3
. The human genome consists of approximately 40% repetitive sequence of retrotransposon origin, including a variety of LINEs and SINEs4
. Exchange events between these repetitive elements can lead to genome rearrangements, including translocations, that can disrupt gene dosage and expression that can result in autoimmune and cardiovascular diseases5
, as well as cancer in humans6-9
Exchange between repetitive elements occurs in a variety of ways. Exchange between sequences that share perfect (or near-perfect) homology occurs by a process called homologous recombination (HR). By contrast, non-homologous end joining (NHEJ) uses little-or-no sequence homology for exchange10,11
. The primary purpose of HR, in mitotic cells, is to repair double-strand breaks (DSBs) generated endogenously by aberrant DNA replication and oxidative lesions, or by exposure to ionizing radiation (IR), and other exogenous DNA damaging agents.
In the assay described here, DSBs are simultaneously created bordering recombination substrates at two different chromosomal loci in diploid cells by a galactose-inducible HO-endonuclease (Figure 1
). The repair of the broken chromosomes generates chromosomal translocations by single strand annealing (SSA), a process where homologous sequences adjacent to the chromosome ends are covalently joined subsequent to annealing. One of the substrates, his3-Δ3'
, contains a 3' truncated HIS3
allele and is located on one copy of chromosome XV at the native HIS3
locus. The second substrate, his3-Δ5'
, is located at the LEU2
locus on one copy of chromosome III, and contains a 5' truncated HIS3
allele. Both substrates are flanked by a HO endonuclease recognition site that can be targeted for incision by HO-endonuclease. HO endonuclease recognition sites native to the MAT
locus, on both copies of chromosome III, have been deleted in all strains. This prevents interaction between the recombination substrates and other broken chromosome ends from interfering in the assay. The KAN-MX
-marked galactose-inducible HO endonuclease expression cassette is inserted at the TRP1
locus on chromosome IV. The substrates share 311 bp or 60 bp of the HIS3
coding sequence that can be used by the HR machinery for repair by SSA. Cells that use these substrates to repair broken chromosomes by HR form an intact HIS3
allele and a tXV::III chromosomal translocation that can be selected for by the ability to grow on medium lacking histidine (Figure 2A
). Translocation frequency by HR is calculated by dividing the number of histidine prototrophic colonies that arise on selective medium by the total number of viable cells that arise after plating appropriate dilutions onto non-selective medium (Figure 2B
). A variety of DNA repair mutants have been used to study the genetic control of translocation formation by SSA using this system12-14
Genetics, Issue 55, translocation formation, HO-endonuclease, Genomic Southern blot, Chromosome blot, Pulsed-field gel electrophoresis, Homologous recombination, DNA double-strand breaks, Single-strand annealing
Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer
Institutions: The University of Newcastle.
Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4
, midbrain compression, neurological deficit or increased cerebral infarct2,4
. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7
. Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8
. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9
. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury.
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10
and solid state catheters are the most frequently used11,12,13,14,15
. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1
). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain.
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13
that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw.
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.
Medicine, Issue 62, Neuroscience, brain, rat, intracranial pressure, epidural, fibre-optic transducer, ischemic injury
Automated Midline Shift and Intracranial Pressure Estimation based on Brain CT Images
Institutions: Virginia Commonwealth University, Virginia Commonwealth University Reanimation Engineering Science (VCURES) Center, Virginia Commonwealth University, Virginia Commonwealth University, Virginia Commonwealth University.
In this paper we present an automated system based mainly on the computed tomography (CT) images consisting of two main components: the midline shift estimation and intracranial pressure (ICP) pre-screening system. To estimate the midline shift, first an estimation of the ideal midline is performed based on the symmetry of the skull and anatomical features in the brain CT scan. Then, segmentation of the ventricles from the CT scan is performed and used as a guide for the identification of the actual midline through shape matching. These processes mimic the measuring process by physicians and have shown promising results in the evaluation. In the second component, more features are extracted related to ICP, such as the texture information, blood amount from CT scans and other recorded features, such as age, injury severity score to estimate the ICP are also incorporated. Machine learning techniques including feature selection and classification, such as Support Vector Machines (SVMs), are employed to build the prediction model using RapidMiner. The evaluation of the prediction shows potential usefulness of the model. The estimated ideal midline shift and predicted ICP levels may be used as a fast pre-screening step for physicians to make decisions, so as to recommend for or against invasive ICP monitoring.
Medicine, Issue 74, Biomedical Engineering, Molecular Biology, Neurobiology, Biophysics, Physiology, Anatomy, Brain CT Image Processing, CT, Midline Shift, Intracranial Pressure Pre-screening, Gaussian Mixture Model, Shape Matching, Machine Learning, traumatic brain injury, TBI, imaging, clinical techniques
Detection of Architectural Distortion in Prior Mammograms via Analysis of Oriented Patterns
Institutions: University of Calgary , University of Calgary .
We demonstrate methods for the detection of architectural distortion in prior mammograms of interval-cancer cases based on analysis of the orientation of breast tissue patterns in mammograms. We hypothesize that architectural distortion modifies the normal orientation of breast tissue patterns in mammographic images before the formation of masses or tumors. In the initial steps of our methods, the oriented structures in a given mammogram are analyzed using Gabor filters and phase portraits to detect node-like sites of radiating or intersecting tissue patterns. Each detected site is then characterized using the node value, fractal dimension, and a measure of angular dispersion specifically designed to represent spiculating patterns associated with architectural distortion.
Our methods were tested with a database of 106 prior mammograms of 56 interval-cancer cases and 52 mammograms of 13 normal cases using the features developed for the characterization of architectural distortion, pattern classification via
quadratic discriminant analysis, and validation with the leave-one-patient out procedure. According to the results of free-response receiver operating characteristic analysis, our methods have demonstrated the capability to detect architectural distortion in prior mammograms, taken 15 months (on the average) before clinical diagnosis of breast cancer, with a sensitivity of 80% at about five false positives per patient.
Medicine, Issue 78, Anatomy, Physiology, Cancer Biology, angular spread, architectural distortion, breast cancer, Computer-Assisted Diagnosis, computer-aided diagnosis (CAD), entropy, fractional Brownian motion, fractal dimension, Gabor filters, Image Processing, Medical Informatics, node map, oriented texture, Pattern Recognition, phase portraits, prior mammograms, spectral analysis
Diffusion Tensor Magnetic Resonance Imaging in the Analysis of Neurodegenerative Diseases
Institutions: University of Ulm.
Diffusion tensor imaging (DTI) techniques provide information on the microstructural processes of the cerebral white matter (WM) in vivo
. The present applications are designed to investigate differences of WM involvement patterns in different brain diseases, especially neurodegenerative disorders, by use of different DTI analyses in comparison with matched controls.
DTI data analysis is performed in a variate fashion, i.e.
voxelwise comparison of regional diffusion direction-based metrics such as fractional anisotropy (FA), together with fiber tracking (FT) accompanied by tractwise fractional anisotropy statistics (TFAS) at the group level in order to identify differences in FA along WM structures, aiming at the definition of regional patterns of WM alterations at the group level. Transformation into a stereotaxic standard space is a prerequisite for group studies and requires thorough data processing to preserve directional inter-dependencies. The present applications show optimized technical approaches for this preservation of quantitative and directional information during spatial normalization in data analyses at the group level. On this basis, FT techniques can be applied to group averaged data in order to quantify metrics information as defined by FT. Additionally, application of DTI methods, i.e.
differences in FA-maps after stereotaxic alignment, in a longitudinal analysis at an individual subject basis reveal information about the progression of neurological disorders. Further quality improvement of DTI based results can be obtained during preprocessing by application of a controlled elimination of gradient directions with high noise levels.
In summary, DTI is used to define a distinct WM pathoanatomy of different brain diseases by the combination of whole brain-based and tract-based DTI analysis.
Medicine, Issue 77, Neuroscience, Neurobiology, Molecular Biology, Biomedical Engineering, Anatomy, Physiology, Neurodegenerative Diseases, nuclear magnetic resonance, NMR, MR, MRI, diffusion tensor imaging, fiber tracking, group level comparison, neurodegenerative diseases, brain, imaging, clinical techniques
Measuring Frailty in HIV-infected Individuals. Identification of Frail Patients is the First Step to Amelioration and Reversal of Frailty
Institutions: University of Arizona, University of Arizona.
A simple, validated protocol consisting of a battery of tests is available to identify elderly patients with frailty syndrome. This syndrome of decreased reserve and resistance to stressors increases in incidence with increasing age. In the elderly, frailty may pursue a step-wise loss of function from non-frail to pre-frail to frail. We studied frailty in HIV-infected patients and found that ~20% are frail using the Fried phenotype using stringent criteria developed for the elderly1,2
. In HIV infection the syndrome occurs at a younger age.
HIV patients were checked for 1) unintentional weight loss; 2) slowness as determined by walking speed; 3) weakness as measured by a grip dynamometer; 4) exhaustion by responses to a depression scale; and 5) low physical activity was determined by assessing kilocalories expended in a week's time. Pre-frailty was present with any two of five criteria and frailty was present if any three of the five criteria were abnormal.
The tests take approximately 10-15 min to complete and they can be performed by medical assistants during routine clinic visits. Test results are scored by referring to standard tables. Understanding which of the five components contribute to frailty in an individual patient can allow the clinician to address relevant underlying problems, many of which are not evident in routine HIV clinic visits.
Medicine, Issue 77, Infection, Virology, Infectious Diseases, Anatomy, Physiology, Molecular Biology, Biomedical Engineering, Retroviridae Infections, Body Weight Changes, Diagnostic Techniques and Procedures, Physical Examination, Muscle Strength, Behavior, Virus Diseases, Pathological Conditions, Signs and Symptoms, Diagnosis, Musculoskeletal and Neural Physiological Phenomena, HIV, HIV-1, AIDS, Frailty, Depression, Weight Loss, Weakness, Slowness, Exhaustion, Aging, clinical techniques
Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice
Institutions: University Health Network, University Health Network, University Health Network.
experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.
Medicine, Issue 79, Liver Neoplasms, Hepatectomy, animal models, hepatocellular carcinoma, xenograft, cancer, liver, subcutaneous, intrahepatic, orthotopic, mouse, human, immunodeficient
A Murine Model of Subarachnoid Hemorrhage
Institutions: University of Munich Medical Center.
In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.
Medicine, Issue 81, Nervous System Diseases, Subarachnoid hemorrhage (SAH), mouse model, filament perforation, intracranial pressure monitoring, blood distribution, surgical technique
Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae
Institutions: Utah State University.
Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages include high potential yield, use of non-arable land and integration with waste streams. The nutrient requirements of a large-scale microalgae production system will require the coupling of cultivation systems with industrial waste resources, such as carbon dioxide from flue gas and nutrients from wastewater. Inorganic contaminants present in these wastes can potentially lead to bioaccumulation in microalgal biomass negatively impact productivity and limiting end use. This study focuses on the experimental evaluation of the impact and the fate of 14 inorganic contaminants (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Se, Sn, V and Zn) on Nannochloropsis salina
growth. Microalgae were cultivated in photobioreactors illuminated at 984 µmol m-2
and maintained at pH 7 in a growth media polluted with inorganic contaminants at levels expected based on the composition found in commercial coal flue gas systems. Contaminants present in the biomass and the medium at the end of a 7 day growth period were analytically quantified through cold vapor atomic absorption spectrometry for Hg and through inductively coupled plasma mass spectrometry for As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Sb, Se, Sn, V and Zn. Results show N. salina
is a sensitive strain to the multi-metal environment with a statistical decrease in biomass yieldwith the introduction of these contaminants. The techniques presented here are adequate for quantifying algal growth and determining the fate of inorganic contaminants.
Environmental Sciences, Issue 101, algae, heavy metals, Nannochloropsis salina, photobioreactor, flue gas, inductively coupled plasma mass spectrometry, ICPMS, cold vapor atomic absorption spectrometry, CVAAS