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.
Genotyping of Burkholderia mallei from an outbreak of glanders in Bahrain suggests multiple introduction events.
PLoS Negl Trop Dis
PUBLISHED: 09-01-2014
Show Abstract
Hide Abstract
Glanders, caused by the gram-negative bacterium Burkholderia mallei, is a highly infectious zoonotic disease of solipeds causing severe disease in animals and men. Although eradicated from many Western countries, it recently emerged in Asia, the Middle-East, Africa, and South America. Due to its rareness, little is known about outbreak dynamics of the disease and its epidemiology.
Related JoVE Video
Sleep and moral awareness.
J Sleep Res
PUBLISHED: 08-27-2014
Show Abstract
Hide Abstract
The implications of sleep for morality are only starting to be explored. Extending the ethics literature, we contend that because bringing morality to conscious attention requires effort, a lack of sleep leads to low moral awareness. We test this prediction with three studies. A laboratory study with a manipulation of sleep across 90 participants judging a scenario for moral content indicates that a lack of sleep leads to low moral awareness. An archival study of Google Trends data across 6 years highlights a national dip in Web searches for moral topics (but not other topics) on the Monday after the Spring time change, which tends to deprive people of sleep. Finally, a diary study of 127 participants indicates that (within participants) nights with a lack of sleep are associated with low moral awareness the next day. Together, these three studies suggest that a lack of sleep leaves people less morally aware, with important implications for the recognition of morality in others.
Related JoVE Video
Effects of enzyme inducers efavirenz and tipranavir/ritonavir on the pharmacokinetics of the HIV integrase inhibitor dolutegravir.
Eur. J. Clin. Pharmacol.
PUBLISHED: 08-23-2014
Show Abstract
Hide Abstract
Dolutegravir (DTG) is an unboosted, integrase inhibitor for the treatment of HIV infection. Two studies evaluated the effects of efavirenz (EFV) and tipranavir/ritonavir (TPV/r) on DTG pharmacokinetics (PK) in healthy subjects.
Related JoVE Video
Cardiolipin profiles as a potential biomarker of mitochondrial health in diet-induced obese mice subjected to exercise, diet-restriction and ephedrine treatment.
J Appl Toxicol
PUBLISHED: 08-15-2014
Show Abstract
Hide Abstract
Cardiolipin (CL) is crucial for mitochondrial energy metabolism and structural integrity. Alterations in CL quantity or CL species have been associated with mitochondrial dysfunction in several pathological conditions and diseases, including mitochondrial dysfunction-related compound attrition and post-market withdrawal of promising drugs. Here we report alterations in the CL profiles in conjunction with morphology of soleus muscle (SM) and brown adipose tissue (BAT) in diet-induced obese (DIO) mice, subjected to ephedrine treatment (EPH: 200?mg?kg(-1) ?day(-1) orally), treadmill exercise (EX: 10 meters per min, 1?h per day), or dietary restriction (DR: 25% less of mean food consumed by the EX group) for 7?days. Mice from the DR and EPH groups had a significant decrease in percent body weight and reduced fat mass compared with DIO controls. Morphologic alterations in the BAT included brown adipocytes with reduced cytoplasmic lipid droplets and increased cytoplasmic eosinophilia in the EX, DR and EPH groups. Increased cytoplasmic eosinophilia in the BAT was ultrastructurally manifested by increased mitochondrial cristae, fenestration of mitochondrial cristae, increased electron density of mitochondrial matrix, and increased complexity of shape and elongation of mitochondria. Mitochondrial ultrastructural alterations in the SM of the EX and DR groups included increased mitochondrial cristae, cup-shaped mitochondria and mitochondrial degeneration. All four CL species (tri-linoleoyl-mono-docosahexaenoyl, tetralinoleoyl, tri-linoleoyl-mono-oleoyl, and di-linoleoyl-di-oleoyl) were increased in the BAT of the DR and EPH groups and in the SM of the EPH and EX groups. In conclusion, cardiolipin profiling supported standard methods for assessing mitochondrial biogenesis and health, and may serve as a potential marker of mitochondrial dysfunction in preclinical toxicity studies.
Related JoVE Video
A CD40-targeted peptide controls and reverses type 1 diabetes in NOD mice.
Diabetologia
PUBLISHED: 08-08-2014
Show Abstract
Hide Abstract
The CD40-CD154 interaction directs autoimmune inflammation. Therefore, a long-standing goal in the treatment of autoimmune disease has been to control the formation of that interaction and thereby prevent destructive inflammation. Antibodies blocking CD154 are successful in mouse models of autoimmune disease but, while promising when used in humans, unfortunate thrombotic events have occurred, forcing the termination of those studies.
Related JoVE Video
An Outbreak of Respiratory Tularemia Caused by Diverse Clones of Francisella tularensis.
Clin. Infect. Dis.
PUBLISHED: 08-05-2014
Show Abstract
Hide Abstract
The bacterium Francisella tularensis is recognized for its virulence, infectivity, genetic homogeneity, and potential as a bioterrorism agent. Outbreaks of respiratory tularemia, caused by inhalation of this bacterium, are poorly understood. Such outbreaks are exceedingly rare, and F. tularensis is seldom recovered from clinical specimens.
Related JoVE Video
The comparative disposition and metabolism of dolutegravir, a potent HIV-1 integrase inhibitor, in mice, rats, and monkeys.
Xenobiotica
PUBLISHED: 07-19-2014
Show Abstract
Hide Abstract
Abstract 1.? Plasma clearance of dolutegravir, an unboosted HIV-1 integrase inhibitor, was low in rat and monkey (0.23 and 2.12?mL/min/kg, respectively) as was the volume of distribution (0.1 and 0.28?L/kg, respectively) with terminal elimination half-life approximately 6?h. Dolutegravir was rapidly absorbed from oral solution with a high bioavailability in rat and monkey (75.6 and 87.0% respectively), but solubility or dissolution rate limited when administered as suspension. 2.? Dolutegravir was highly bound (>99%) to serum proteins in rat and monkey, similar to binding to plasma and serum proteins in human. Radioactivity was associated with the plasma versus cellular components of blood across all species. 3.? Following oral administration to rats, [(14)C]dolutegravir-related radioactivity was distributed to most tissues, due in part to high permeability; however, because of high plasma protein binding, tissue to blood ratios were low. In mouse, rat and monkey, the absorbed dose was extensively metabolized and secreted into bile, with the majority of the administered radioactivity eliminated in feces within 24?h. 4.? The primary route of metabolism of dolutegravir was through the formation of an ether glucuronide. Additional biotransformation pathways: benzylic oxidation followed by hydrolysis to an N-dealkylated product, glucose conjugation, oxidative defluorination, and glutathione conjugation.
Related JoVE Video
Multiple mutations in the para -sodium channel gene are associated with pyrethroid resistance in Rhipicephalus microplus from the United States and Mexico.
Parasit Vectors
PUBLISHED: 07-09-2014
Show Abstract
Hide Abstract
BackgroundAcaricide resistant Rhipicephalus microplus populations have become a major problem for many cattle producing areas of the world. Pyrethroid resistance in arthropods is typically associated with mutations in domains I, II, III, and IV of voltage-gated sodium channel genes. In R. microplus, known resistance mutations include a domain II change (C190A) in populations from Australia, Africa, and South America and a domain III mutation (T2134A) that only occurs in Mexico and the U.S.MethodsWe investigated pyrethroid resistance in cattle fever ticks from Texas and Mexico by estimating resistance levels in field-collected ticks using larval packet discriminating dose (DD) assays and identifying single nucleotide polymorphisms (SNPs) in the para-sodium channel gene that associated with resistance. We then developed qPCR assays for three SNPs and screened a larger set of 1,488 R. microplus ticks, representing 77 field collections and four laboratory strains, for SNP frequency.ResultsWe detected resistance SNPs in 21 of 68 U.S. field collections and six of nine Mexico field collections. We expected to identify the domain III SNP (T2134A) at a high frequency; however, we only found it in three U.S. collections. A much more common SNP in the U.S. (detected in 19 of 21 field collections) was the C190A domain II mutation, which has never before been reported from North America. We also discovered a novel domain II SNP (T170C) in ten U.S. and two Mexico field collections. The T170C transition mutation has previously been associated with extreme levels of resistance (super-knockdown resistance) in insects. We found a significant correlation (r¿=¿0.81) between the proportion of individuals in field collections that carried any two resistance SNPs and the percent survivorship of F1 larvae from these collections in DD assays. This relationship is accurately predicted by a simple linear regression model (R2¿=¿0.6635).ConclusionsThese findings demonstrate that multiple mutations in the para-sodium channel gene independently associate with pyrethroid resistance in R. microplus ticks, which is likely a consequence of human-induced selection.
Related JoVE Video
Use of an oral stable isotope label to confirm variation in red blood cell mean age that influences HbA1c interpretation.
Am. J. Hematol.
PUBLISHED: 06-29-2014
Show Abstract
Hide Abstract
HbA1c is commonly used to monitor glycemic control. However, there is growing evidence that the relationship between HbA1c and mean blood glucose (MBG) is influenced by variation in red blood cell (RBC) lifespan in hematologically normal individuals. Correction of HbA1c for mean RBC age (MRBC ) requires a noninvasive, accurate, and affordable method to measure RBC survival. In this study, we evaluated whether a stable isotope approach would satisfy these requirements. RBC lifespan and MRBC were determined in a group of nine hematologically normal diabetic and nondiabetic subjects using oral (15) N-glycine to label heme in an age cohort of RBC. The MRBC was 58.7 ± 9.1 (2SD) days and RBC lifespan was 106 ± 21 (2SD) days. This degree of variation (±15 - 20%) is consistent with previous studies using other techniques. In a subset of seven subjects, MRBC determined with the biotin label technique were available from approximately five years prior, and strongly correlated with the stable isotope values (R(2) = 0.79). This study suggests that the MRBC is stable over time but varies substantially among individuals, and supports the importance of its variation in HbA1c interpretation. The characteristics of the stable isotope method support its suitability for studies to directly evaluate the impact of variation in MRBC on the interpretation of HbA1c.
Related JoVE Video
Diversity of Francisella tularensis subsp. holarctica lineages, China.
Emerging Infect. Dis.
PUBLISHED: 06-26-2014
Show Abstract
Hide Abstract
We analyzed 10 isolates of Francisella tularensis subspecies holarctica from China and assigned them to known clades by using canonical single-nucleotide polymorphisms. We found 4 diverse subtypes, including 3 from the most basal lineage, biovar japonica. This result indicates unprecedented levels of diversity from a single region and suggests new models for emergence.
Related JoVE Video
Francisella tularensis subsp. tularensis group A.I, United States.
Emerging Infect. Dis.
PUBLISHED: 04-24-2014
Show Abstract
Hide Abstract
We used whole-genome analysis and subsequent characterization of geographically diverse strains using new genetic signatures to identify distinct subgroups within Francisella tularensis subsp. tularensis group A.I: A.I.3, A.I.8, and A.I.12. These subgroups exhibit complex phylogeographic patterns within North America. The widest distribution was observed for A.I.12, which suggests an adaptive advantage.
Related JoVE Video
Multisystemic therapy for disruptive behavior problems in youths with autism spectrum disorders: a progress report.
J Marital Fam Ther
PUBLISHED: 04-23-2014
Show Abstract
Hide Abstract
Youths with autism spectrum disorders (ASD) often engage in serious disruptive behaviors that interfere with their ability to successfully manage day-to-day responsibilities and contribute to relationship problems with caregivers, peers, and teachers. Effective treatments are needed to address the factors linked with disruptive behavior problems in this population of youths. Multisystemic therapy (MST) is a comprehensive family- and community-based treatment approach that has been effective with other difficult-to-treat populations of youths and holds promise for youths with ASD. In this article, we review the broad range of factors associated with disruptive behaviors among youths with ASD and discuss how MST interventions can be adapted to address those factors. We also present a framework for our adaptation of the MST model for youths with ASD. This framework includes a recently completed pilot study as well as an ongoing efficacy trial that together have served to identify key interventions for our adaptation of the MST model.
Related JoVE Video
Widespread movement of invasive cattle fever ticks (Rhipicephalus microplus) in southern Texas leads to shared local infestations on cattle and deer.
Parasit Vectors
PUBLISHED: 04-12-2014
Show Abstract
Hide Abstract
Rhipicephalus (Boophilus) microplus is a highly-invasive tick that transmits the cattle parasites (Babesia bovis and B. bigemina) that cause cattle fever. R. microplus and Babesia are endemic in Mexico and ticks persist in the United States inside a narrow tick eradication quarantine area (TEQA) along the Rio Grande. This containment area is threatened by unregulated movements of illegal cattle and wildlife like white-tailed deer (WTD; Odocoileus virginianus).
Related JoVE Video
Invasive potential of cattle fever ticks in the southern United States.
Parasit Vectors
PUBLISHED: 04-05-2014
Show Abstract
Hide Abstract
For >100 years cattle production in the southern United States has been threatened by cattle fever. It is caused by an invasive parasite-vector complex that includes the protozoan hemoparasites Babesia bovis and B. bigemina, which are transmitted among domestic cattle via Rhipicephalus tick vectors of the subgenus Boophilus. In 1906 an eradication effort was started and by 1943 Boophilus ticks had been confined to a narrow tick eradication quarantine area (TEQA) along the Texas-Mexico border. However, a dramatic increase in tick infestations in areas outside the TEQA over the last decade suggests these tick vectors may be poised to re-invade the southern United States. We investigated historical and potential future distributions of climatic habitats of cattle fever ticks to assess the potential for a range expansion.
Related JoVE Video
The economic impact of multisystemic therapy through midlife: a cost-benefit analysis with serious juvenile offenders and their siblings.
J Consult Clin Psychol
PUBLISHED: 03-31-2014
Show Abstract
Hide Abstract
This study investigated the economic benefits of multisystemic therapy (MST) versus individual therapy (IT) using arrest data from 176 serious juvenile offenders and 129 of their closest-in-age siblings who participated, on average, 25 years earlier in a randomized clinical trial (Borduin et al., 1995).
Related JoVE Video
The Effect of Repeated Microwave Irradiation on the Dimensional Stability of a Specific Acrylic Denture Resin.
J Prosthodont
PUBLISHED: 03-18-2014
Show Abstract
Hide Abstract
To determine the dimensional stability of a poly(methyl methacrylate) (PMMA) acrylic resin when subjected to multiple sessions of repeated microwave irradiation at power settings of 700 and 420 W.
Related JoVE Video
Estimating Sympathetic Tone by Recording Subcutaneous Nerve Activity in Ambulatory Dogs.
J. Cardiovasc. Electrophysiol.
PUBLISHED: 03-13-2014
Show Abstract
Hide Abstract
We tested the hypothesis that subcutaneous nerve activity (SCNA) of the thorax correlates with the stellate ganglion nerve activity (SGNA) and can be used to estimate the sympathetic tone.
Related JoVE Video
Component alignment during total knee arthroplasty with use of standard or custom instrumentation: a randomized clinical trial using computed tomography for postoperative alignment measurement.
J Bone Joint Surg Am
PUBLISHED: 03-07-2014
Show Abstract
Hide Abstract
Patient-specific femoral and tibial cutting blocks produced with use of data from preoperative computed tomography (CT) or magnetic resonance imaging (MRI) scans have been employed recently to optimize component alignment in total knee arthroplasty. We report the results of a randomized controlled trial in which CT scans were used to compare postoperative component alignment between patients treated with custom instruments and those managed with traditional instruments.
Related JoVE Video
Defining a new biomarker for the autoimmune component of Multiple Sclerosis: Th40 cells.
J. Neuroimmunol.
PUBLISHED: 02-28-2014
Show Abstract
Hide Abstract
Multiple Sclerosis (MS) is a chronic inflammatory, neurodegenerative disease. Diagnosis is very difficult requiring defined symptoms and multiple CNS imaging. A complicating issue is that almost all symptoms are not disease specific for MS. Autoimmunity is evident, yet the only immune-related diagnostic tool is cerebral-spinal fluid examination for oligoclonal bands. This study addresses the impact of Th40 cells, a pathogenic effector subset of helper T cells, in MS. MS patients including relapsing/remitting MS, secondary progressive MS and primary progressive MS were examined for Th40 cell levels in peripheral blood and, similar to our findings in autoimmune type 1 diabetes, the levels were significantly (p<0.0001) elevated compared to controls including healthy non-autoimmune subjects and another non-autoimmune chronic disease. Classically identified Tregs were at levels equivalent to non-autoimmune controls but the Th40/Treg ratio still predicted autoimmunity. The cohort displayed a wide range of HLA haplotypes including the GWAS identified predictive HLA-DRB1*1501 (DR2). However half the subjects did not carry DR2 and regardless of HLA haplotype, Th40 cells were expanded during disease. In RRMS Th40 cells demonstrated a limited TCR clonality. Mechanistically, Th40 cells demonstrated a wide array of response to CNS associated self-antigens that was dependent upon HLA haplotype. Th40 cells were predominantly memory phenotype producing IL-17 and IFN? with a significant portion producing both inflammatory cytokines simultaneously suggesting an intermediary between Th1 and Th17 phenotypes.
Related JoVE Video
Yersinia pestis and the plague of Justinian 541-543 AD: a genomic analysis.
Lancet Infect Dis
PUBLISHED: 01-28-2014
Show Abstract
Hide Abstract
Yersinia pestis has caused at least three human plague pandemics. The second (Black Death, 14-17th centuries) and third (19-20th centuries) have been genetically characterised, but there is only a limited understanding of the first pandemic, the Plague of Justinian (6-8th centuries). To address this gap, we sequenced and analysed draft genomes of Y pestis obtained from two individuals who died in the first pandemic.
Related JoVE Video
Pro-inflammatory T-lymphocytes rapidly infiltrate into the brain and contribute to neuronal injury following cardiac arrest and cardiopulmonary resuscitation.
J. Neuroimmunol.
PUBLISHED: 01-22-2014
Show Abstract
Hide Abstract
Although inflammatory mechanisms have been linked to neuronal injury following global cerebral ischemia, the presence of infiltrating peripheral immune cells remains understudied. We performed flow cytometry of single cell suspensions obtained from the brains of mice at varying time points after global cerebral ischemia induced by cardiac arrest and cardiopulmonary resuscitation (CA/CPR) to characterize the influx of lymphocytes into the injured brain. We observed that CA/CPR caused a large influx of lymphocytes within 3h of resuscitation that was maintained for the 3day duration of our experiments. Using cell staining flow cytometry we observed that the large majority of infiltrating lymphocytes were CD4(+) T cells. Intracellular stains revealed a large proportion of pro-inflammatory T cells expressing either TNF? or INF?. Importantly, the lack of functional T cells in TCR? knockout mice reduced neuronal injury following CA/CPR, implicating pro-inflammatory T cells in the progression of ischemic neuronal injury. Finally, we made the remarkable observation that the novel CD4(+)CD40(+) (Th40) population of pro-inflammatory T cells that are strongly associated with autoimmunity are present in large numbers in the injured brain. These data indicate that studies investigating the neuro-immune response after global cerebral ischemia should consider the role of infiltrating T cells in orchestrating the acute and sustained immune response.
Related JoVE Video
Long-term prevention of criminality in siblings of serious and violent juvenile offenders: a 25-year follow-up to a randomized clinical trial of multisystemic therapy.
J Consult Clin Psychol
PUBLISHED: 01-13-2014
Show Abstract
Hide Abstract
Family-based treatment models that have shown effectiveness with juvenile offenders may also lead to reduced criminality in siblings of those offenders. However, the lasting effects of such treatments on siblings have not been evaluated. In the present study, the authors examined criminal outcomes for siblings of serious and violent juvenile offenders who had participated on average 25.0 years earlier in a clinical trial of multisystemic therapy (MST; Borduin et al., 1995).
Related JoVE Video
Insights to Genetic Characterization Tools for Epidemiological Tracking of Francisella tularensis in Sweden.
PLoS ONE
PUBLISHED: 01-01-2014
Show Abstract
Hide Abstract
Tularaemia, caused by the bacterium Francisella tularensis, is endemic in Sweden and is poorly understood. The aim of this study was to evaluate the effectiveness of three different genetic typing systems to link a genetic type to the source and place of tularemia infection in Sweden. Canonical single nucleotide polymorphisms (canSNPs), MLVA including five variable number of tandem repeat loci and PmeI-PFGE were tested on 127 F. tularensis positive specimens collected from Swedish case-patients. All three typing methods identified two major genetic groups with near-perfect agreement. Higher genetic resolution was obtained with canSNP and MLVA compared to PFGE; F. tularensis samples were first assigned into ten phylogroups based on canSNPs followed by 33 unique MLVA types. Phylogroups were geographically analysed to reveal complex phylogeographic patterns in Sweden. The extensive phylogenetic diversity found within individual counties posed a challenge to linking specific genetic types with specific geographic locations. Despite this, a single phylogroup (B.22), defined by a SNP marker specific to a lone Swedish sequenced strain, did link genetic type with a likely geographic place. This result suggests that SNP markers, highly specific to a particular reference genome, may be found most frequently among samples recovered from the same location where the reference genome originated. This insight compels us to consider whole-genome sequencing (WGS) as the appropriate tool for effectively linking specific genetic type to geography. Comparing the WGS of an unknown sample to WGS databases of archived Swedish strains maximizes the likelihood of revealing those rare geographically informative SNPs.
Related JoVE Video
TaqMan real-time PCR assays for single-nucleotide polymorphisms which identify Francisella tularensis and its subspecies and subpopulations.
PLoS ONE
PUBLISHED: 01-01-2014
Show Abstract
Hide Abstract
Francisella tularensis, the etiologic agent of tularemia and a Class A Select Agent, is divided into three subspecies and multiple subpopulations that differ in virulence and geographic distribution. Given these differences, there is a need to rapidly and accurately determine if a strain is F. tularensis and, if it is, assign it to subspecies and subpopulation. We designed TaqMan real-time PCR genotyping assays using eleven single nucleotide polymorphisms (SNPs) that were potentially specific to closely related groups within the genus Francisella, including numerous subpopulations within F. tularensis species. We performed extensive validation studies to test the specificity of these SNPs to particular populations by screening the assays across a set of 565 genetically and geographically diverse F. tularensis isolates and an additional 21 genetic near-neighbor (outgroup) isolates. All eleven assays correctly determined the genetic groups of all 565 F. tularensis isolates. One assay differentiates F. tularensis, F. novicida, and F. hispaniensis from the more genetically distant F. philomiragia and Francisella-like endosymbionts. Another assay differentiates F. tularensis isolates from near neighbors. The remaining nine assays classify F. tularensis-confirmed isolates into F. tularensis subspecies and subpopulations. The genotyping accuracy of these nine assays diminished when tested on outgroup isolates (i.e. non F. tularensis), therefore a hierarchical approach of assay usage is recommended wherein the F. tularensis-specific assay is used before the nine downstream assays. Among F. tularensis isolates, all eleven assays were highly sensitive, consistently amplifying very low concentrations of DNA. Altogether, these eleven TaqMan real-time PCR assays represent a highly accurate, rapid, and sensitive means of identifying the species, subspecies, and subpopulation of any F. tularensis isolate if used in a step-wise hierarchical scheme. These assays would be very useful in clinical, epidemiological, and/or forensic investigations involving F. tularensis.
Related JoVE Video
Polyphyly of Lichen-cryptic Dagger Moths: synonymy of Agriopodes Hampson and description of a new basal acronictine genus, Chloronycta, gen. n. (Lepidoptera, Noctuidae).
Zookeys
PUBLISHED: 01-01-2014
Show Abstract
Hide Abstract
The taxonomic composition and systematic position of Agriopodes Hampson is examined through an integrated approach using adult and larval morphology, biology, and molecular sequence data. The type-species of Agriopodes, Moma fallax Herrich-Schäffer is shown to be derived within the Acronicta grisea Walker species-group; accordingly, Agriopodes is relegated to synonymy under Acronicta Ochsenheimer, syn. n. (Acronictinae). Additionally, molecular markers and morphology show that Agriopodes is not monophyletic: Agriopodes tybo (Barnes) is not closely related to A. fallax nor to Acronicta, and is transferred to a new genus, Chloronycta Schmidt & Anweiler, gen. n. The immature stages of Chloronycta tybo comb. n. are described and illustrated for the first time. Although previously treated as a valid species, we show that Agriopodes geminata (Smith) represents the northern terminus of clinal variation in wing pattern of A. fallax and synonymize A. geminata under A. fallax (syn. n.). The history and identity of Agriopodes corticosa (Boisduval), a nomen dubium, is discussed.
Related JoVE Video
Phylogeography of Bacillus anthracis in the country of Georgia shows evidence of population structuring and is dissimilar to other regional genotypes.
PLoS ONE
PUBLISHED: 01-01-2014
Show Abstract
Hide Abstract
Sequence analyses and subtyping of Bacillus anthracis strains from Georgia reveal a single distinct lineage (Aust94) that is ecologically established. Phylogeographic analysis and comparisons to a global collection reveals a clade that is mostly restricted to Georgia. Within this clade, many groups are found around the country, however at least one subclade is only found in the eastern part. This pattern suggests that dispersal into and out of Georgia has been rare and despite historical dispersion within the country, for at least for one lineage, current spread is limited.
Related JoVE Video
Altered expression of transmembrane mucins, MUC1 and MUC4, in bladder cancer: pathological implications in diagnosis.
PLoS ONE
PUBLISHED: 01-01-2014
Show Abstract
Hide Abstract
Radical changes in both expression and glycosylation pattern of transmembrane mucins have been observed in various malignancies. We and others have shown that MUC1 and MUC4, two transmembrane mucins, play a sentinel role in cell signaling events that drive several epithelial malignancies. In the present study, we investigated the expression profile of MUC1 and MUC4 in the non-neoplastic bladder urothelium, in various malignant neoplasms of bladder and in bladder carcinoma cell lines.
Related JoVE Video
A new cryptic Sympistis from eastern North America revealed by novel larval phenotype and host plant association (Lepidoptera, Noctuidae, Oncocnemidinae).
Zookeys
PUBLISHED: 01-01-2014
Show Abstract
Hide Abstract
A Triosteum-feeding species of Sympistis is described from eastern North America: Sympistis forbesi sp. n. Identity of the new species is most reliably determined from larval morphology and host plant association-both adult scaling and genitalic characters overlap with those of Sympisitis chionanthi, a Chionanthus and Fraxinus feeder.
Related JoVE Video
An alternative role for Foxp3 as an effector T cell regulator controlled through CD40.
J. Immunol.
PUBLISHED: 06-17-2013
Show Abstract
Hide Abstract
The BDC2.5 T cell clone is highly diabetogenic, but the transgenic mouse generated from that clone is surprisingly slow in diabetes development. Although defining pathogenic effector T cells in autoimmunity has been inconsistent, CD4(+) cells expressing the CD40 receptor (Th40 cells) are highly diabetogenic in NOD mice, and NOD.BDC2.5.TCR.Tg mice possess large numbers of these cells. Given the importance of CD40 for pathogenic T cell development, BDC2.5.CD40(-/-) mice were created. Regulatory T cells, CD4(+)CD25(hi)Foxp3(+), develop normally, but pathogenic effector cells are severely reduced in number. Th40 cells from diabetic BDC2.5 mice rapidly induce diabetes in NOD.scid recipients, but Th40 cells from prediabetic mice transfer diabetes very slowly. Demonstrating an important paradigm shift, effector Th40 cells from prediabetic mice are Foxp3(+). As mice age, moving to type 1 diabetes development, Th40 cells lose Foxp3. When Th40 cells that are Foxp3(+) are transferred to NOD.scid recipients, disease is delayed. Th40 cells that are Foxp3(-) rapidly transfer disease. Th40 cells from BDC2.5.CD40(-/-) mice do not transfer disease nor do they lose Foxp3 expression. Mechanistically, Foxp3(+) cells produce IL-17 but do not produce IFN-?, whereas Foxp3(-) Th40 cells produce IFN-? and IL-2. This poses a new consideration for the function of Foxp3, as directly impacting effector T cell function.
Related JoVE Video
Molecular genotyping of Acinetobacter spp. isolated in Arizona, USA, using multilocus PCR and mass spectrometry.
J. Med. Microbiol.
PUBLISHED: 06-05-2013
Show Abstract
Hide Abstract
Acinetobacter spp. are a diverse group of Gram-negative bacteria frequently implicated in nosocomial infections. Genotypic methods have been instrumental in studying Acinetobacter, but few offer high resolution, rapid turnaround time, technical ease and high inter-laboratory reproducibility, which has hampered understanding of disease incidence, transmission patterns and diversity within this genus. Here, we further evaluated multilocus PCR electrospray ionization/mass spectrometry (PCR/ESI-MS), a method that is simple and robust, and provides both species characterization and strain-level resolution of Acinetobacter spp. on a single platform. We examined 125 Acinetobacter isolates from 21 hospitals, laboratories and medical centres spanning four counties in Arizona, USA, using PCR/ESI-MS. We compared PCR/ESI-MS with an in-house amplified fragment length polymorphism (AFLP) genotyping scheme. PCR/ESI-MS demonstrated that Acinetobacter spp. from Arizonan hospitals had similar species and strain distributions to other US civilian hospitals. Furthermore, we showed that the PCR/ESI-MS and AFLP genotypes were highly congruent, with the former having the advantages of robust inter-laboratory reproducibility, rapid turnaround time and simple experimental set-up and data analysis. PCR/ESI-MS is an effective and high-throughput platform for strain typing of Acinetobacter baumannii and for identification of other Acinetobacter spp., including the emerging nosocomial pathogens Acinetobacter pittii and Acinetobacter nosocomialis.
Related JoVE Video
Retrospective analysis of Salmonella, Campylobacter, Escherichia coli, and Enterococcus in animal feed ingredients.
Foodborne Pathog. Dis.
PUBLISHED: 05-21-2013
Show Abstract
Hide Abstract
The presence and antimicrobial susceptibility of foodborne pathogens and indicator organisms in animal feed are not well understood. In this study, a total of 201 feed ingredient samples (animal byproducts, n=122; plant byproducts, n=79) were collected in 2002 and 2003 from representative rendering plants and the oilseed (or cereal grain) industry across the United States. The occurrence and antimicrobial susceptibility of four bacterial genera (Salmonella, Campylobacter, Escherichia coli, and Enterococcus) were determined. Salmonella isolates were further characterized by serotyping and pulsed-field gel electrophoresis (PFGE). None of the samples yielded Campylobacter or E. coli O157:H7, whereas Salmonella, generic E. coli, and Enterococcus were present in 22.9%, 39.3%, and 86.6% of samples, respectively. A large percentage (47.8%) of Salmonella-positive samples harbored two serovars, and the vast majority (88.4%) of Enterococcus isolates were E. faecium. Animal byproducts had a significantly higher Salmonella contamination rate (34.4%) than plant byproducts (5.1%) (p<0.05). Among 74 Salmonella isolates recovered, 27 serovars and 55 PFGE patterns were identified; all were pan-susceptible to 17 antimicrobials tested. E. coli isolates (n=131) demonstrated similar susceptibility to these antimicrobials except for tetracycline (15.3% resistance), sulfamethoxazole (7.6%), streptomycin (4.6%), ampicillin (3.8%), and nalidixic acid (1.5%). Enterococcus isolates (n=362) were also resistant to five of 17 antimicrobials tested, ranging from 1.1% to penicillin to 14.6% to tetracycline. Resistance rates were generally higher among isolates recovered from animal byproducts. Taken together, our findings suggest that diverse populations of Salmonella, E. coli, and Enterococcus are commonly present in animal feed ingredients, but antimicrobial resistance is not common. Future large-scale studies to monitor these pathogenic and indicator organisms in feed commodities is warranted.
Related JoVE Video
Metabolism, excretion, and mass balance of the HIV-1 integrase inhibitor dolutegravir in humans.
Antimicrob. Agents Chemother.
PUBLISHED: 05-13-2013
Show Abstract
Hide Abstract
The pharmacokinetics, metabolism, and excretion of dolutegravir, an unboosted, once-daily human immunodeficiency virus type 1 integrase inhibitor, were studied in healthy male subjects following single oral administration of [(14)C]dolutegravir at a dose of 20 mg (80 ?Ci). Dolutegravir was well tolerated, and absorption of dolutegravir from the suspension formulation was rapid (median time to peak concentration, 0.5 h), declining in a biphasic fashion. Dolutegravir and the radioactivity had similar terminal plasma half-lives (t1/2) (15.6 versus 15.7 h), indicating metabolism was formation rate limited with no long-lived metabolites. Only minimal association with blood cellular components was noted with systemic radioactivity. Recovery was essentially complete (mean, 95.6%), with 64.0% and 31.6% of the dose recovered in feces and urine, respectively. Unchanged dolutegravir was the predominant circulating radioactive component in plasma and was consistent with minimal presystemic clearance. Dolutegravir was extensively metabolized. An inactive ether glucuronide, formed primarily via UGT1A1, was the principal biotransformation product at 18.9% of the dose excreted in urine and the principal metabolite in plasma. Two minor biotransformation pathways were oxidation by CYP3A4 (7.9% of the dose) and an oxidative defluorination and glutathione substitution (1.8% of the dose). No disproportionate human metabolites were observed.
Related JoVE Video
Dysfunctional resident lung mesenchymal stem cells contribute to pulmonary microvascular remodeling.
Pulm Circ
PUBLISHED: 05-11-2013
Show Abstract
Hide Abstract
Pulmonary vascular remodeling and oxidative stress are common to many adult lung diseases. However, little is known about the relevance of lung mesenchymal stem cells (MSCs) in these processes. We tested the hypothesis that dysfunctional lung MSCs directly participate in remodeling of the microcirculation. We employed a genetic model to deplete extracellular superoxide dismutase (EC-SOD) in lung MSCs coupled with lineage tracing analysis. We crossed (floxp)sod3 and mT/mG reporter mice to a strain expressing Cre recombinase under the control of the ABCG2 promoter. We demonstrated In vivo that depletion of EC-SOD in lung MSCs resulted in their contribution to microvascular remodeling in the smooth muscle actin positive layer. We further characterized lung MSCs to be multipotent vascular precursors, capable of myofibroblast, endothelial and pericyte differentiation in vitro. EC-SOD deficiency in cultured lung MSCs accelerated proliferation and apoptosis, restricted colony-forming ability, multilineage differentiation potential and promoted the transition to a contractile phenotype. Further studies correlated cell dysfunction to alterations in canonical Wnt/?-catenin signaling, which were more evident under conditions of oxidative stress. Our data establish that lung MSCs are a multipotent vascular precursor population, a population which has the capacity to participate in vascular remodeling and their function is likely regulated in part by the Wnt/?-catenin signaling pathway. These studies highlight an important role for microenviromental regulation of multipotent MSC function as well as their potential to contribute to tissue remodeling.
Related JoVE Video
Yersinia pestis DNA from skeletal remains from the 6(th) century AD reveals insights into Justinianic Plague.
PLoS Pathog.
PUBLISHED: 05-01-2013
Show Abstract
Hide Abstract
Yersinia pestis, the etiologic agent of the disease plague, has been implicated in three historical pandemics. These include the third pandemic of the 19(th) and 20(th) centuries, during which plague was spread around the world, and the second pandemic of the 14(th)-17(th) centuries, which included the infamous epidemic known as the Black Death. Previous studies have confirmed that Y. pestis caused these two more recent pandemics. However, a highly spirited debate still continues as to whether Y. pestis caused the so-called Justinianic Plague of the 6(th)-8(th) centuries AD. By analyzing ancient DNA in two independent ancient DNA laboratories, we confirmed unambiguously the presence of Y. pestis DNA in human skeletal remains from an Early Medieval cemetery. In addition, we narrowed the phylogenetic position of the responsible strain down to major branch 0 on the Y. pestis phylogeny, specifically between nodes N03 and N05. Our findings confirm that Y. pestis was responsible for the Justinianic Plague, which should end the controversy regarding the etiology of this pandemic. The first genotype of a Y. pestis strain that caused the Late Antique plague provides important information about the history of the plague bacillus and suggests that the first pandemic also originated in Asia, similar to the other two plague pandemics.
Related JoVE Video
Draft Genome Sequences of Two Bulgarian Bacillus anthracis Strains.
Genome Announc
PUBLISHED: 04-20-2013
Show Abstract
Hide Abstract
Bacillus anthracis strains previously isolated from Bulgaria form a unique subcluster within the A1.a cluster that is typical for isolates from southeastern Europe. Here, we report the draft genome sequences of two Bulgarian B. anthracis strains belonging to the A branch (A.Br.)008/009 canonical single nucleotide polymorphism (SNP) group of the major A branch.
Related JoVE Video
Geometric mouse variation: implications to the axial ulnar loading protocol and animal specific calibration.
J Biomech
PUBLISHED: 04-19-2013
Show Abstract
Hide Abstract
Large variations in axial ulnar load strain calibration results suggest that animal-specific calibrations may be necessary. However, the optimal set of geometric measures for performing an animal-specific calibration are not known, potentially as a result of confounding effects associated with experimentally introduced variation. The purpose of this study was to characterize the inherent variability of ulnar geometric measures known to influence periosteal midshaft strain during axial ulnar exogenous loading, and to further quantify the relationship between the variance of those geometric measures and periosteal strain during axial loading. Thirty-nine right mouse forelimbs were scanned with microCT. Seven geometric measures that influence periosteal strain resulting from a combined axial and bending loading were computed and used to estimate animal-specific strains on the periosteal midshaft. Animal specific strains were estimated using a theoretical model based on the generalized flexure formula. The predicted mean and standard deviation of the simulated midshaft strain gauge measurement resulting from the inter-animal geometric differences was -985 ± 148 ??/N. The complete beam bending term associated with bending about the I(min) axis accounted for 89% of the variance and reduced the residual RMSE to 50.4 ??. Eccentricity associated with the axial loading contributed a substantial portion of variation to the computed strain suggesting that calibration procedures to account for animal differences should incorporate that variable. The method developed in this study provides a relatively simple procedure for computing animal-specific strains using microCT scan data, without the need of a load/strain calibration study or computationally intensive finite element models.
Related JoVE Video
Step scaling and behaviour selection in a constrained set of manual material handling transfers.
Ergonomics
PUBLISHED: 04-04-2013
Show Abstract
Hide Abstract
Predictive biomechanical analysis of manual material handling (MMH) transfers is dependent on accurate prediction of foot locations relative to the task. Previous studies have classified common acyclic stepping patterns used during those transfer tasks, but the influence of walking distance prior to the transfer is not well understood. Twenty men and women performed transfers for a minimum of six different delivery distance conditions. The number of steps used by the participants ranged from two to seven. A theoretical framework for idealised step-scaling strategies is proposed and compared with the experimental data. The maximum observed increase in step length prior to delivery was 1.43 times the nominal step length calculated for each participant. The data suggest that although participants can scale their steps to facilitate the use of a single terminal stance at the transfer, the majority of participants chose to utilise a combination of stepping strategies if the preferred contralateral lead foot strategy could not be easily implemented.
Related JoVE Video
Consistency among musculoskeletal models: caveat utilitor.
Ann Biomed Eng
PUBLISHED: 04-01-2013
Show Abstract
Hide Abstract
Musculoskeletal simulation software and model repositories have broadened the user base able to perform musculoskeletal analysis and have facilitated in the sharing of models. As the recognition of musculoskeletal modeling continues to grow as an engineering discipline, the consistency in results derived from different models and software is becoming more critical. The purpose of this study was to compare eight models from three software packages and evaluate differences in quadriceps moment arms, predicted muscle forces, and predicted tibiofemoral contact forces for an idealized knee-extension task spanning -125 to +10° of knee extension. Substantial variation among models was observed for the majority of aspects evaluated. Differences among models were influenced by knee angle, with better agreement of moment arms and tibiofemoral joint contact force occurring at low to moderate knee flexion angles. The results suggest a lack of consistency among models and that output differences are not simply an artifact of naturally occurring inter-individual differences. Although generic musculoskeletal models can easily be scaled to consistent limb lengths and use the same muscle recruitment algorithm, the results suggest those are not sufficient conditions to produce consistent muscle or joint contact forces, even for simplified models with no potential of co-contraction.
Related JoVE Video
CD40 interacts directly with RAG1 and RAG2 in autoaggressive T cells and Fas prevents CD40-induced RAG expression.
Cell. Mol. Immunol.
PUBLISHED: 03-25-2013
Show Abstract
Hide Abstract
CD4(+) T cells expressing CD40 (Th40 cells) constitute a pathogenic T-cell subset that is necessary and sufficient to transfer autoimmune disease. We have previously demonstrated that CD40 signals peripheral Th40 cells to induce RAG1 and RAG2 expression, proteins necessary for the expression of T-cell receptor (TCR), leading to TCR revision. The dependency of TCR expression in the thymus on RAG proteins has long been known. However, despite numerous publications, there is controversy as to whether TCR expression can be altered in the periphery, post-thymic selective pressures. Therefore, a better understanding of TCR expression in primary peripheral cells is needed. We now show that the CD40 protein itself interacts with RAG1 and RAG2 as well as with Ku70 and translocates to the nucleus in Th40 cells. This indicates that the CD40 molecule is closely involved in the mechanism of TCR expression in the periphery. In addition, Fas signals act as a silencing mechanism for CD40-induced RAGs and prevent CD40 translocation to the nucleus. It will be important to further understand the involvement of CD40 in peripheral TCR expression and how TCR revision impacts auto-antigen recognition in order to effectively target and tolerize autoaggressive T cells in autoimmune disease.
Related JoVE Video
Historical changes in northeastern US bee pollinators related to shared ecological traits.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 03-04-2013
Show Abstract
Hide Abstract
Pollinators such as bees are essential to the functioning of terrestrial ecosystems. However, despite concerns about a global pollinator crisis, long-term data on the status of bee species are limited. We present a long-term study of relative rates of change for an entire regional bee fauna in the northeastern United States, based on >30,000 museum records representing 438 species. Over a 140-y period, aggregate native species richness weakly decreased, but richness declines were significant only for the genus Bombus. Of 187 native species analyzed individually, only three declined steeply, all of these in the genus Bombus. However, there were large shifts in community composition, as indicated by 56% of species showing significant changes in relative abundance over time. Traits associated with a declining relative abundance include small dietary and phenological breadth and large body size. In addition, species with lower latitudinal range boundaries are increasing in relative abundance, a finding that may represent a response to climate change. We show that despite marked increases in human population density and large changes in anthropogenic land use, aggregate native species richness declines were modest outside of the genus Bombus. At the same time, we find that certain ecological traits are associated with declines in relative abundance. These results should help target conservation efforts focused on maintaining native bee abundance and diversity and therefore the important ecosystems services that they provide.
Related JoVE Video
Metabolite structure analysis by high-resolution MS: supporting drug-development studies.
Bioanalysis
PUBLISHED: 02-19-2013
Show Abstract
Hide Abstract
Effective characterization of drug metabolites in complex biological matrices is facilitated by mass spectrometers with high resolving power, mass accuracy and sensitivity. This review begins with an overview of high-resolution MS terminology and the different types of instrumentation that are currently available. Metabolite structure analysis offers unique challenges and, therefore, the different types of approaches used to solve problems are highlighted through specific examples. Overall, this review describes the value that high-resolution MS brings to drug-metabolism studies.
Related JoVE Video
A decade of plague in Mahajanga, Madagascar: insights into the global maritime spread of pandemic plague.
MBio
PUBLISHED: 02-14-2013
Show Abstract
Hide Abstract
A cluster of human plague cases occurred in the seaport city of Mahajanga, Madagascar, from 1991 to 1999 following 62 years with no evidence of plague, which offered insights into plague pathogen dynamics in an urban environment. We analyzed a set of 44 Mahajanga isolates from this 9-year outbreak, as well as an additional 218 Malagasy isolates from the highland foci. We sequenced the genomes of four Mahajanga strains, performed whole-genome sequence single-nucleotide polymorphism (SNP) discovery on those strains, screened the discovered SNPs, and performed a high-resolution 43-locus multilocus variable-number tandem-repeat analysis of the isolate panel. Twenty-two new SNPs were identified and defined a new phylogenetic lineage among the Malagasy isolates. Phylogeographic analysis suggests that the Mahajanga lineage likely originated in the Ambositra district in the highlands, spread throughout the northern central highlands, and was then introduced into and became transiently established in Mahajanga. Although multiple transfers between the central highlands and Mahajanga occurred, there was a locally differentiating and dominant subpopulation that was primarily responsible for the 1991-to-1999 Mahajanga outbreaks. Phylotemporal analysis of this Mahajanga subpopulation revealed a cycling pattern of diversity generation and loss that occurred during and after each outbreak. This pattern is consistent with severe interseasonal genetic bottlenecks along with large seasonal population expansions. The ultimate extinction of plague pathogens in Mahajanga suggests that, in this environment, the plague pathogen niche is tenuous at best. However, the temporary large pathogen population expansion provides the means for plague pathogens to disperse and become ecologically established in more suitable nonurban environments.
Related JoVE Video
Genetic characterization of Theileria equi infecting horses in North America: evidence for a limited source of U.S. introductions.
Parasit Vectors
PUBLISHED: 02-05-2013
Show Abstract
Hide Abstract
Theileria equi is a tick-borne apicomplexan hemoparasite that causes equine piroplasmosis. This parasite has a worldwide distribution but the United States was considered to be free of this disease until recently.
Related JoVE Video
Systematic review and consensus guidelines for environmental sampling of Burkholderia pseudomallei.
PLoS Negl Trop Dis
PUBLISHED: 01-28-2013
Show Abstract
Hide Abstract
Burkholderia pseudomallei, a Tier 1 Select Agent and the cause of melioidosis, is a Gram-negative bacillus present in the environment in many tropical countries. Defining the global pattern of B. pseudomallei distribution underpins efforts to prevent infection, and is dependent upon robust environmental sampling methodology. Our objective was to review the literature on the detection of environmental B. pseudomallei, update the risk map for melioidosis, and propose international consensus guidelines for soil sampling.
Related JoVE Video
Accurate and rapid identification of the Burkholderia pseudomallei near-neighbour, Burkholderia ubonensis, using real-time PCR.
PLoS ONE
PUBLISHED: 01-01-2013
Show Abstract
Hide Abstract
Burkholderia ubonensis is an environmental bacterium belonging to the Burkholderia cepacia complex (Bcc), a group of genetically related organisms that are associated with opportunistic but generally nonfatal infections in healthy individuals. In contrast, the near-neighbour species Burkholderia pseudomallei causes melioidosis, a disease that can be fatal in up to 95% of cases if left untreated. B. ubonensis is frequently misidentified as B. pseudomallei from soil samples using selective culturing on Ashdowns medium, reflecting both the shared environmental niche and morphological similarities of these species. Additionally, B. ubonensis shows potential as an important biocontrol agent in B. pseudomallei-endemic regions as certain strains possess antagonistic properties towards B. pseudomallei. Current methods for characterising B. ubonensis are laborious, time-consuming and costly, and as such this bacterium remains poorly studied. The aim of our study was to develop a rapid and inexpensive real-time PCR-based assay specific for B. ubonensis. We demonstrate that a novel B. ubonensis-specific assay, Bu550, accurately differentiates B. ubonensis from B. pseudomallei and other species that grow on selective Ashdowns agar. We anticipate that Bu550 will catalyse research on B. ubonensis by enabling rapid identification of this organism from Ashdowns-positive colonies that are not B. pseudomallei.
Related JoVE Video
Climate-associated phenological advances in bee pollinators and bee-pollinated plants.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 12-05-2011
Show Abstract
Hide Abstract
The phenology of many ecological processes is modulated by temperature, making them potentially sensitive to climate change. Mutualistic interactions may be especially vulnerable because of the potential for phenological mismatching if the species involved do not respond similarly to changes in temperature. Here we present an analysis of climate-associated shifts in the phenology of wild bees, the most important pollinators worldwide, and compare these shifts to published studies of bee-pollinated plants over the same time period. We report that over the past 130 y, the phenology of 10 bee species from northeastern North America has advanced by a mean of 10.4 ± 1.3 d. Most of this advance has taken place since 1970, paralleling global temperature increases. When the best available data are used to estimate analogous rates of advance for plants, these rates are not distinguishable from those of bees, suggesting that bee emergence is keeping pace with shifts in host-plant flowering, at least among the generalist species that we investigated.
Related JoVE Video
Population differences in host immune factors may influence survival of Gunnisons prairie dogs (Cynomys gunnisoni) during plague outbreaks.
J. Wildl. Dis.
PUBLISHED: 11-22-2011
Show Abstract
Hide Abstract
Over the past 40 yr, epizootics of plague (Yersinia pestis) in northern Arizona have reduced populations of the Gunnisons prairie dog (Cynomys gunnisoni), with the exception of a large population found in the Aubrey Valley (AV). To examine potential mechanisms accounting for their survival, we collected prairie dog serum samples in 2005-2006 from AV and a neighboring population near Seligman (SE), Arizona. We quantified gene expression at 58 diverse immune proteins using a multiplexed enzyme-linked immunosorbent assay panel. We found a subset of proteins important in coagulation and inflammation (tissue factor [TF], calbindin [Cal], and thrombopoietin [TPO]) and T-cell responses (CD40L and CD40) that were present in AV at levels two to eight times greater than SE. These results suggest that AV and SE animals might differ in their ability to mount an immune response.
Related JoVE Video
MALDI imaging mass spectrometry: bridging biology and chemistry in drug development.
Bioanalysis
PUBLISHED: 11-15-2011
Show Abstract
Hide Abstract
Our understanding of drug tissue distribution impacts a number of areas in drug development, including: pharmacology, pharmacokinetics, safety, drug-drug interactions, transport and metabolism. Despite their extensive use, autoradiography and tissue homogenate LC-MS analysis have limitations in providing a comprehensive assessment of tissue distributions. In the case of autoradiography, it is the inability to distinguish between parent drug and drug metabolites. In LC-MS analysis of tissue homogenate, all tissue localization information is lost. The emerging technique of MALDI imaging mass spectrometry has the capability to distinguish between parent and metabolites while maintaining spatial distribution in tissues. In this article, we will review the MALDI imaging MS methodology as applied to drug development and provide examples highlighting the impact of this important technique in drug development.
Related JoVE Video
Isolation & characterization of Hoechst(low) CD45(negative) mouse lung mesenchymal stem cells.
J Vis Exp
PUBLISHED: 11-09-2011
Show Abstract
Hide Abstract
Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.
Related JoVE Video
Towards a rapid molecular diagnostic for melioidosis: Comparison of DNA extraction methods from clinical specimens.
J. Microbiol. Methods
PUBLISHED: 09-27-2011
Show Abstract
Hide Abstract
Optimising DNA extraction from clinical samples for Burkholderia pseudomallei Type III secretion system real-time PCR in suspected melioidosis patients confirmed that urine and sputum are useful diagnostic samples. Direct testing on blood remains problematic; testing DNA extracted from plasma was superior to DNA from whole blood or buffy coat.
Related JoVE Video
Tularemia in Alaska, 1938 - 2010.
Acta Vet. Scand.
PUBLISHED: 09-26-2011
Show Abstract
Hide Abstract
Tularemia is a serious, potentially life threatening zoonotic disease. The causative agent, Francisella tularensis, is ubiquitous in the Northern hemisphere, including Alaska, where it was first isolated from a rabbit tick (Haemophysalis leporis-palustris) in 1938. Since then, F. tularensis has been isolated from wildlife and humans throughout the state. Serologic surveys have found measurable antibodies with prevalence ranging from < 1% to 50% and 4% to 18% for selected populations of wildlife species and humans, respectively. We reviewed and summarized known literature on tularemia surveillance in Alaska and summarized the epidemiological information on human cases reported to public health officials. Additionally, available F. tularensis isolates from Alaska were analyzed using canonical SNPs and a multi-locus variable-number tandem repeats (VNTR) analysis (MLVA) system. The results show that both F. t. tularensis and F. t. holarctica are present in Alaska and that subtype A.I, the most virulent type, is responsible for most recently reported human clinical cases in the state.
Related JoVE Video
Resistance to plague among black-tailed prairie dog populations.
Vector Borne Zoonotic Dis.
PUBLISHED: 09-16-2011
Show Abstract
Hide Abstract
In some rodent species frequently exposed to plague outbreaks caused by Yersinia pestis, resistance to the disease has evolved as a population trait. As a first step in determining if plague resistance has developed in black-tailed prairie dogs (Cynomys ludovicianus), animals captured from colonies in a plague-free region (South Dakota) and two plague-endemic regions (Colorado and Texas) were challenged with Y. pestis at one of three doses (2.5, 250, or 2500 mouse LD50s). South Dakota prairie dogs were far more susceptible to plague than Colorado and Texas prairie dogs (p<0.001), with a mortality rate of nearly 100% over all doses. Colorado and Texas prairie dogs were quite similar in their response, with overall survival rates of 50% and 60%, respectively. Prairie dogs from these states were heterogeneous in their response, with some animals dying at the lowest dose (37% and 20%, respectively) and some surviving even at the highest dose (29% and 40%, respectively). Microsatellite analysis revealed that all three groups were distinct genetically, but further studies are needed to establish a genetic basis for the observed differences in plague resistance.
Related JoVE Video
A tight coupling between ??Y97 and ??F200 of the GABA(A) receptor mediates GABA binding.
J. Neurochem.
PUBLISHED: 08-31-2011
Show Abstract
Hide Abstract
The GABA(A) receptor is an oligopentameric chloride channel that is activated via conformation changes induced upon the binding of the endogenous ligand, GABA, to the extracellular inter-subunit interfaces. Although dozens of amino acid residues at the ?/? interface have been implicated in ligand binding, the structural elements that mediate ligand binding and receptor activation are not yet fully described. In this study, double-mutant cycle analysis was employed to test for possible interactions between several arginines (??R67, ??R120, ??R132, and ??R207) and two aromatic residues (??Y97 and ??F200) that are present in the ligand-binding pocket and are known to influence GABA affinity. Our results show that neither ??R67 nor ??R120 is functionally coupled to either of the aromatics, whereas a moderate coupling exists between ??R132 and both aromatic residues. Significant functional coupling between ??R207 and both ??Y97 and ??F200 was found. Furthermore, we identified an even stronger coupling between the two aromatics, ??Y97 and ??F200, and for the first time provided direct evidence for the involvement of ??Y97 and ??F200 in GABA binding. As these residues are tightly linked, and mutation of either has similar, severe effects on GABA binding and receptor kinetics, we believe they form a single functional unit that may directly coordinate GABA.
Related JoVE Video
Three arginines in the GABAA receptor binding pocket have distinct roles in the formation and stability of agonist- versus antagonist-bound complexes.
Mol. Pharmacol.
PUBLISHED: 07-15-2011
Show Abstract
Hide Abstract
Binding of the agonist GABA to the GABA(A) receptor causes channel gating, whereas competitive antagonists that bind at the same site do not. The details of ligand binding are not well understood, including which residues interact directly with ligands, maintain the structure of the binding pocket, or transduce the action of binding into opening of the ion channel gate. Recent work suggests that the amine group of the GABA molecule may form a cation-? bond with residues in a highly conserved "aromatic box" within the binding pocket. Although interactions with the carboxyl group of GABA remain unknown, three positively charged arginines (?(1)Arg67, ?(1)Arg132, and ?(2)Arg207) just outside of the aromatic box are likely candidates. To explore their roles in ligand binding, we individually mutated these arginines to alanine and measured the effects on microscopic ligand binding/unbinding rates and channel gating. The mutations ?(1)R67A or ?(2)R207A slowed agonist binding and sped unbinding with little effect on gating, demonstrating that these arginines are critical for both formation and stability of the agonist-bound complex. In addition, ?(1)R67A sped binding of the antagonist 2-(3-carboxypropyl)-3-amino-6-(4 methoxyphenyl)pyridazinium bromide (SR-95531), indicating that this arginine poses a barrier to formation of the antagonist-bound complex. In contrast, ?(2)R207A and ?(1)R132A sped antagonist unbinding, indicating that these arginines stabilize the antagonist-bound state. ?(1)R132A also conferred a new long-lived open state, indicating that this arginine influences the channel gate. Thus, each of these arginines plays a unique role in determining interactions with agonists versus antagonists and with the channel gate.
Related JoVE Video
Diversity of 16S-23S rDNA internal transcribed spacer (ITS) reveals phylogenetic relationships in Burkholderia pseudomallei and its near-neighbors.
PLoS ONE
PUBLISHED: 07-04-2011
Show Abstract
Hide Abstract
Length polymorphisms within the 16S-23S ribosomal DNA internal transcribed spacer (ITS) have been described as stable genetic markers for studying bacterial phylogenetics. In this study, we used these genetic markers to investigate phylogenetic relationships in Burkholderia pseudomallei and its near-relative species. B. pseudomallei is known as one of the most genetically recombined bacterial species. In silico analysis of multiple B. pseudomallei genomes revealed approximately four homologous rRNA operons and ITS length polymorphisms therein. We characterized ITS distribution using PCR and analyzed via a high-throughput capillary electrophoresis in 1,191 B. pseudomallei strains. Three major ITS types were identified, two of which were commonly found in most B. pseudomallei strains from the endemic areas, whereas the third one was significantly correlated with worldwide sporadic strains. Interestingly, mixtures of the two common ITS types were observed within the same strains, and at a greater incidence in Thailand than Australia suggesting that genetic recombination causes the ITS variation within species, with greater recombination frequency in Thailand. In addition, the B. mallei ITS type was common to B. pseudomallei, providing further support that B. mallei is a clone of B. pseudomallei. Other B. pseudomallei near-neighbors possessed unique and monomorphic ITS types. Our data shed light on evolutionary patterns of B. pseudomallei and its near relative species.
Related JoVE Video
Increased gene sampling strengthens support for higher-level groups within leaf-mining moths and relatives (Lepidoptera: Gracillariidae).
BMC Evol. Biol.
PUBLISHED: 06-24-2011
Show Abstract
Hide Abstract
Researchers conducting molecular phylogenetic studies are frequently faced with the decision of what to do when weak branch support is obtained for key nodes of importance. As one solution, the researcher may choose to sequence additional orthologous genes of appropriate evolutionary rate for the taxa in the study. However, generating large, complete data matrices can become increasingly difficult as the number of characters increases. A few empirical studies have shown that augmenting genes even for a subset of taxa can improve branch support. However, because each study differs in the number of characters and taxa, there is still a need for additional studies that examine whether incomplete sampling designs are likely to aid at increasing deep node resolution. We target Gracillariidae, a Cretaceous-age (~100 Ma) group of leaf-mining moths to test whether the strategy of adding genes for a subset of taxa can improve branch support for deep nodes. We initially sequenced ten genes (8,418 bp) for 57 taxa that represent the major lineages of Gracillariidae plus outgroups. After finding that many deep divergences remained weakly supported, we sequenced eleven additional genes (6,375 bp) for a 27-taxon subset. We then compared results from different data sets to assess whether one sampling design can be favored over another. The concatenated data set comprising all genes and all taxa and three other data sets of different taxon and gene sub-sampling design were analyzed with maximum likelihood. Each data set was subject to five different models and partitioning schemes of non-synonymous and synonymous changes. Statistical significance of non-monophyly was examined with the Approximately Unbiased (AU) test.
Related JoVE Video
A new 13C breath test to detect vitamin B12 deficiency: a prevalent and poorly diagnosed health problem.
J Breath Res
PUBLISHED: 06-23-2011
Show Abstract
Hide Abstract
Vitamin B12 deficiency is emerging as a growing public health problem. The most commonly used diagnostic tests are limited in accuracy, sensitivity, and are non-specific for B12 deficiency. The aim of this study was to develop a simple B12 breath test (BBT) to more accurately evaluate vitamin B12 status as an alternative to the most common diagnostic test, serum B12 levels. The breath test is based on the metabolism of sodium 1-(13)C-propionate to (13)CO(2) which requires B12 as a cofactor. We initially compared the BBT to current B12 diagnostic methods in 58 subjects. Subjects also received a second BBT 1-3 days after initial testing to evaluate reproducibility of results. Propionate dosage, fasting times, and collection periods were compared, respectively. The dose of sodium 1-(13)C-propionate (10-50 mg) gave equivalent results while an 8 h fast was essential. Statistical analysis revealed that breath collection times could be reduced to just a baseline and 10 and 20 min following propionate dosing. We also measured the incidence of B12 deficiency with the BBT in 119 patients with chronic pancreatitis, Crohns disease, small intestinal bacterial overgrowth, and subjects over 65 years of age. The BBT results agreed with previous publications showing a higher incidence of B12 deficiency in these patients. The BBT may provide clinicians with a non-invasive, accurate, reliable, and reproducible diagnostic test to detect vitamin B12 deficiency.
Related JoVE Video
A new Zanclognatha from eastern North America and a preliminary key to the larvae of the genus (Lepidoptera, Erebidae, Herminiinae).
Zookeys
PUBLISHED: 06-21-2011
Show Abstract
Hide Abstract
The adult of a widespread but previously undescribed species of Zanclognatha Lederer is described from eastern North America. Images of the mature larva and life history datafor Zanclognathadentatasp. n. are included, along with a preliminary key to the larvae of ten eastern North American Zanclognatha species.
Related JoVE Video
Human polymorphisms in the glutathione transferase zeta 1/maleylacetoacetate isomerase gene influence the toxicokinetics of dichloroacetate.
J Clin Pharmacol
PUBLISHED: 06-03-2011
Show Abstract
Hide Abstract
Dichloroacetate (DCA), a chemical relevant to environmental science and allopathic medicine, is dehalogenated by the bifunctional enzyme glutathione transferase zeta (GSTz1)/maleylacetoacetate isomerase (MAAI), the penultimate enzyme in the phenylalanine/tyrosine catabolic pathway. The authors postulated that polymorphisms in GSTz1/MAAI modify the toxicokinetics of DCA. GSTz1/MAAI haplotype significantly affected the kinetics and biotransformation of 1,2-¹³C-DCA when it was administered at either environmentally (µg/kg/d) or clinically (mg/kg/d) relevant doses. GSTz1/MAAI haplotype also influenced the urinary accumulation of potentially toxic tyrosine metabolites. Atomic modeling revealed that GSTz1/MAAI variants associated with the slowest rates of DCA metabolism induced structural changes in the enzyme homodimer, predicting protein instability or abnormal protein-protein interactions. Knowledge of the GSTz1/MAAI haplotype can be used prospectively to identify individuals at potential risk of DCAs adverse side effects from environmental or clinical exposure or who may exhibit aberrant amino acid metabolism in response to dietary protein.
Related JoVE Video
Epidemiological tracking and population assignment of the non-clonal bacterium, Burkholderia pseudomallei.
PLoS Negl Trop Dis
PUBLISHED: 05-30-2011
Show Abstract
Hide Abstract
Rapid assignment of bacterial pathogens into predefined populations is an important first step for epidemiological tracking. For clonal species, a single allele can theoretically define a population. For non-clonal species such as Burkholderia pseudomallei, however, shared allelic states between distantly related isolates make it more difficult to identify population defining characteristics. Two distinct B. pseudomallei populations have been previously identified using multilocus sequence typing (MLST). These populations correlate with the major foci of endemicity (Australia and Southeast Asia). Here, we use multiple Bayesian approaches to evaluate the compositional robustness of these populations, and provide assignment results for MLST sequence types (STs). Our goal was to provide a reference for assigning STs to an established population without the need for further computational analyses. We also provide allele frequency results for each population to enable estimation of population assignment even when novel STs are discovered. The ability for humans and potentially contaminated goods to move rapidly across the globe complicates the task of identifying the source of an infection or outbreak. Population genetic dynamics of B. pseudomallei are particularly complicated relative to other bacterial pathogens, but the work here provides the ability for broad scale population assignment. As there is currently no independent empirical measure of successful population assignment, we provide comprehensive analytical details of our comparisons to enable the reader to evaluate the robustness of population designations and assignments as they pertain to individual research questions. Finer scale subdivision and verification of current population compositions will likely be possible with genotyping data that more comprehensively samples the genome. The approach used here may be valuable for other non-clonal pathogens that lack simple group-defining genetic characteristics and provides a rapid reference for epidemiologists wishing to track the origin of infection without the need to compile population data and learn population assignment algorithms.
Related JoVE Video
Phylogeography and molecular epidemiology of Yersinia pestis in Madagascar.
PLoS Negl Trop Dis
PUBLISHED: 05-18-2011
Show Abstract
Hide Abstract
Plague was introduced to Madagascar in 1898 and continues to be a significant human health problem. It exists mainly in the central highlands, but in the 1990s was reintroduced to the port city of Mahajanga, where it caused extensive human outbreaks. Despite its prevalence, the phylogeography and molecular epidemiology of Y. pestis in Madagascar has been difficult to study due to the great genetic similarity among isolates. We examine island-wide geographic-genetic patterns based upon whole-genome discovery of SNPs, SNP genotyping and hypervariable variable-number tandem repeat (VNTR) loci to gain insight into the maintenance and spread of Y. pestis in Madagascar.
Related JoVE Video
Deriving tissue density and elastic modulus from microCT bone scans.
Bone
PUBLISHED: 04-18-2011
Show Abstract
Hide Abstract
Tissue level density and elastic modulus are intrinsic properties that can be used to quantify bone material and analyses incorporating those quantities have been used to evaluate bone on a macroscopic scale. Micro-computed tomography (microCT) technology has been used to construct tissue level finite element models to simulate macroscopic fracture strength, however, a single method for assigning voxel-specific tissue density and elastic modulus based on those data has not been universally accepted. One method prevalent in the literature utilizes an empirical relationship that derives tissue stiffness as a function of bone calcium content weight fraction. To derive calcium content weight fraction from microCT scans, a measure of tissue density is required and a constant value is traditionally used. However, experimental data suggest a non-trivial amount of tissue heterogeneity suggesting a constant tissue density may not be appropriate. A theoretical derivation for determining the relationship between voxel-specific tissue density and microCT scan data (i.e., microCT derived tissue mineral density (TMD), mgHA/cm(3)) and bone constituent properties is proposed. Constant model parameters used in the derivation include the density of water, ash, and organics (i.e., bone constituents) and the volume fraction of the organics constituent. The effect of incorporating the theoretically derived tissue density (instead of a constant value) in determining voxel-specific elastic modulus resulted in a maximum observed increase of 12GPa (5.9GPa versus 17.9GPa, for the constant value and derived tissue density formulations, respectively) for a measured TMD of 1.02gHA/cm(3). Average and bounding quantities for the four constant model parameters were defined from the literature and the influence of those values on the derived tissue density and elastic modulus relationships were also evaluated. The theoretical relationships of tissue density and elastic modulus, with the average constant model parameters applied, were consistent with previously published empirical relationships derived from experimental data. Tissue density as a function of microCT TMD was formulated as a linear relationship and the density of water and ash was shown to solely influence the proportionality (i.e., slope) between those values. The density of water and organics (i.e., collagen) and the volume fraction of the organics constituent were shown to influence the constant offset (intercept) between tissue density and TMD with no influence from ash density. Incorporating tissue density heterogeneity into the derivation of elastic modulus resulted in a significant increase in predicted modulus (for microCT TMD ranges observed for healthy tissue) as compared to when a constant tissue density was used. The presented approach provides a novel method for deriving tissue-level bone material properties and quantifies the effect of assuming tissue homogeneity when calculating elastic modulus (when using a prevalent method in the literature) from microCT scan data.
Related JoVE Video
Molecular investigations of a locally acquired case of melioidosis in Southern AZ, USA.
PLoS Negl Trop Dis
PUBLISHED: 04-01-2011
Show Abstract
Hide Abstract
Melioidosis is caused by Burkholderia pseudomallei, a Gram-negative bacillus, primarily found in soils in Southeast Asia and northern Australia. A recent case of melioidosis in non-endemic Arizona was determined to be the result of locally acquired infection, as the patient had no travel history to endemic regions and no previous history of disease. Diagnosis of the case was confirmed through multiple microbiologic and molecular techniques. To enhance the epidemiological analysis, we conducted several molecular genotyping procedures, including multi-locus sequence typing, SNP-profiling, and whole genome sequence typing. Each technique has different molecular epidemiologic advantages, all of which provided evidence that the infecting strain was most similar to those found in Southeast Asia, possibly originating in, or around, Malaysia. Advancements in new typing technologies provide genotyping resolution not previously available to public health investigators, allowing for more accurate source identification.
Related JoVE Video
Upper urinary tract carcinoma in Lynch syndrome cases.
J. Urol.
PUBLISHED: 03-21-2011
Show Abstract
Hide Abstract
Patients with Lynch syndrome are much more likely to have generally rare upper urinary tract urothelial carcinoma but not bladder urothelial carcinoma. While the risk has been quantified, to our knowledge there is no description of how this population of patients with Lynch syndrome and upper urinary tract cancer differs from the general population with upper urinary tract cancer.
Related JoVE Video
Biology and systematics of the New World Phyllocnistis Zeller leafminers of the avocado genus Persea (Lepidoptera, Gracillariidae).
Zookeys
PUBLISHED: 03-07-2011
Show Abstract
Hide Abstract
Four New World species of Phyllocnistis Zeller are described from serpentine mines in Persea (Family Lauraceae). Phyllocnistis hyperpersea,new species, mines the upper leaf surfaces of avocado, Persea americana Mill., and red bay, Persea borbonia (L.) Spreng. and ranges over much of the southeastern United States into Central America. Phyllocnistis subpersea,new species, mines the underside and occasionally upper sides of new leaves of Persea borbonia in southeastern United States. Phyllocnistis longipalpa, new species, known only from southern Florida also mines the undersides of new leaves of Persea borbonia. Phyllocnistis perseafolia,new species, mines both leaf surfaces and possibly fruits of Persea americana in Colombia, South America. As in all known species of Phyllocnistis, the early instars are subepidermal sapfeeders in young (not fully hardened) foliage, and the final instar is an extremely specialized, nonfeeding larval form, whose primary function is to spin the silken cocoon, at the mine terminus, prior to pupation. Early stages are illustrated and described for three of the species. The unusual morphology of the pupae, particularly the frontal process of the head, is shown to be one of the most useful morphological sources of diagnostic characters for species identification of Phyllocnistis. COI barcode sequence distances are provided for the four proposed species and a fifth, undescribed species from Costa Rica.
Related JoVE Video
Phylogeography of Francisella tularensis subspecies holarctica from the country of Georgia.
BMC Microbiol.
PUBLISHED: 03-04-2011
Show Abstract
Hide Abstract
Francisella tularensis, the causative agent of tularemia, displays subspecies-specific differences in virulence, geographic distribution, and genetic diversity. F. tularensis subsp. holarctica is widely distributed throughout the Northern Hemisphere. In Europe, F. tularensis subsp. holarctica isolates have largely been assigned to two phylogenetic groups that have specific geographic distributions. Most isolates from Western Europe are assigned to the B.Br.FTNF002-00 group, whereas most isolates from Eastern Europe are assigned to numerous lineages within the B.Br.013 group. The eastern geographic extent of the B.Br.013 group is currently unknown due to a lack of phylogenetic knowledge about populations at the European/Asian juncture and in Asia. In this study, we address this knowledge gap by describing the phylogenetic structure of F. tularensis subsp. holarctica isolates from the country of Georgia, and by placing these isolates into a global phylogeographic context.
Related JoVE Video
Evaluation of a chimeric (uPA+/+)/SCID mouse model with a humanized liver for prediction of human metabolism.
Xenobiotica
PUBLISHED: 03-04-2011
Show Abstract
Hide Abstract
A model that predicts human metabolism and disposition of drug candidates would be of value in early drug development. In this study, a chimeric (uPA+/+)/SCID mouse model was evaluated with three structurally distinct compounds (GW695634, a benzophenone, SB-406725, a tetrahydroisoquinoline and GW823093, a fluoropyrrolidine) for which human metabolism and disposition was characterized. Human metabolite profiles in plasma and/or urine were compared to those of chimeric (uPA+/+)/SCID and control CD-1 or (uPA+/+)/SCID) mice. GW695634 and SB-406725 exhibited primarily hepatic metabolism and were chosen as probes to assess which human metabolites would likely circulate systemically. GW823093 exhibited a combination of hepatic and extrahepatic metabolism such that renal excretion of drug-related material was ~2-fold greater in humans than in mice, and thus chosen as a probe to assess if the chimeric (uPA+/+)/SCID mouse would predict the urinary excretion of human metabolites. We observed that human metabolism and disposition was well represented for GW695634, somewhat represented for GW823093 and minimally represented for SB-406725. Collectively, the results of this and other studies suggest that while limitations for prediction of human metabolism and disposition exist, humanized chimeric mouse models can potentially represent informative new tools in drug discovery and development.
Related JoVE Video
The pathology of bleomycin-induced fibrosis is associated with loss of resident lung mesenchymal stem cells that regulate effector T-cell proliferation.
Stem Cells
PUBLISHED: 02-12-2011
Show Abstract
Hide Abstract
Tissue-resident mesenchymal stem cells (MSCs) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis, and tumor formation. Here, we define a population of resident lung MSCs (luMSCs) that function to regulate the severity of bleomycin injury via modulation of the T-cell response. Bleomycin-induced loss of these endogenous luMSCs and elicited fibrosis (pulmonary fibrosis), inflammation, and pulmonary arterial hypertension (PAH). Replacement of resident stem cells by administration of isolated luMSCs attenuated the bleomycin-associated pathology and mitigated the development of PAH. In addition, luMSC modulated a decrease in numbers of lymphocytes and granulocytes in bronchoalveolar fluid and demonstrated an inhibition of effector T-cell proliferation in vitro. Global gene expression analysis indicated that the luMSCs are a unique stromal population differing from lung fibroblasts in terms of proinflammatory mediators and profibrotic pathways. Our results demonstrate that luMSCs function to protect lung integrity after injury; however, when endogenous MSCs are lost, this function is compromised illustrating the importance of this novel population during lung injury. The definition of this population in vivo in both murine and human pulmonary tissue facilitates the development of a therapeutic strategy directed at the rescue of endogenous cells to facilitate lung repair during injury.
Related JoVE Video
A state-dependent salt-bridge interaction exists across the ?/? intersubunit interface of the GABAA receptor.
Mol. Pharmacol.
PUBLISHED: 01-05-2011
Show Abstract
Hide Abstract
The GABA(A) receptor is a multisubunit protein that transduces the binding of a neurotransmitter at an intersubunit interface into the opening of a central ion channel. The structural components that mediate the steps involved in this action are poorly defined. A large amount of work has focused on clarifying the specific functions and interactions of residues believed to surround the GABA binding pocket. Here, we explored two charged residues (?(2)Asp163 and ?(1)Arg120), which have been suggested by homology models to participate in a salt-bridge interaction. When mutated to alanine, both single mutants, as well as the double mutant, increase EC(50-GABA), decrease the GABA binding rate, and accelerate deactivation and GABA unbinding rates. Double-mutant cycle analysis demonstrates that the effects of each alanine mutation on the GABA binding rate were additive and independent. In contrast, a significant coupling energy was found during an analysis of deactivation time constants. Using kinetic modeling, we further demonstrated that the GABA unbinding rates, in particular, are strongly coupled. These data suggest that ?(2)Asp163 and ?(1)Arg120 form a state-dependent salt bridge, interacting when GABA is bound to the receptor but not when the receptor is in the unbound state.
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.