Increased sequence diversity coverage improves detection of HIV-specific T cell responses

The accurate identification of HIV-specific T cell responses is important for determining the relationship between immune response, viral control, and disease progression. HIV-specific immune responses are usually measured using peptide sets based on consensus sequences, which frequently miss responses to regions where test set and infecting virus differ. In this study, we report the design of a peptide test set with significantly increased coverage of HIV sequence diversity by including alternative amino acids at variable positions during the peptide synthesis step. In an IFN-gamma ELISpot assay, these "toggled" peptides detected HIV-specific CD4(+) and CD8(+) T cell responses of significantly higher breadth and magnitude than matched consensus peptides. The observed increases were explained by a closer match of the toggled peptides to the autologous viral sequence. Toggled peptides therefore afford a cost-effective and significantly more complete view of the host immune response to HIV and are directly applicable to other variable pathogens.     

Effects of implant design parameters on fluid convection, potentiating third-body debris ingress into the bearing surface during THA impingement/subluxation

Aseptic loosening from polyethylene wear debris is the leading cause of failure for metal-on-polyethylene total hip implants. Third-body debris ingress to the bearing space results in femoral head roughening and acceleration of polyethylene wear. How third-body particles manage to enter the bearing space between the closely conforming articulating surfaces of the joint is not well understood. We hypothesize that one such mechanism is from convective fluid transport during subluxation of the total hip joint. To test this hypothesis, a three-dimensional (3D) computational fluid dynamics (CFD) model was developed and validated, to quantify fluid ingress into the bearing space during a leg-cross subluxation event. The results indicated that extra-articular joint fluid could be drawn nearly to the pole of the cup with even very small separations of the femoral head (<0.60mm). Debris suspended near the equator of the cup at the site of maximum fluid velocity just before the subluxation began could be transported to within 11 degrees from the cup pole. Larger head diameters resulted in increased fluid velocity at all sites around the entrance to the gap compared to smaller head sizes, with fluid velocity being greatest along the anterosuperolateral cup edge, for all head sizes. Fluid pathlines indicated that suspended debris would reach similar angular positions in the bearing space regardless of head size. Increased inset of the femoral head into the acetabular cup resulted both in higher fluid velocity and in transport of third-body debris further into the bearing space.     

Four independent mutations in the feline fibroblast growth factor 5 gene determine the long-haired phenotype in domestic cats

To determine the genetic regulation of "hair length" in the domestic cat, a whole-genome scan was performed in a multigenerational pedigree in which the "long-haired" phenotype was segregating. The 2 markers that demonstrated the greatest linkage to the long-haired trait (log of the odds > or = 6) flanked an estimated 10-Mb region on cat chromosome B1 containing the Fibroblast Growth Factor 5 (FGF5) gene, a candidate gene implicated in regulating hair follicle growth cycle in other species. Sequence analyses of FGF5 in 26 cat breeds and 2 pedigrees of nonbreed cats revealed 4 separate mutations predicted to disrupt the biological activity of the FGF5 protein. Pedigree analyses demonstrated that different combinations of paired mutant FGF5 alleles segregated with the long-haired phenotype in an autosomal recessive manner. Association analyses of more than 380 genotyped breed and nonbreed cats were consistent with mutations in the FGF5 gene causing the long-haired phenotype in an autosomal recessive manner. In combination, these genomic approaches demonstrated that FGF5 is the major genetic determinant of hair length in the domestic cat.     

Hypoxia suppresses astrocyte glutamate transport independently of amyloid formation

Sustained hypoxia alters the expression of numerous proteins and predisposes individuals to Alzheimer’s disease (AD). We have previously shown that hypoxia in vitro alters Ca²⁺ homeostasis in astrocytes and promotes increased production of amyloid β peptides (Aβ) of AD. Indeed, alteration of Ca²⁺ homeostasis requires amyloid formation. Here, we show that electrogenic glutamate uptake by astrocytes is suppressed by hypoxia (1% O₂, 24 h) in a manner that is independent of amyloid β peptide formation. Thus, hypoxic suppression of glutamate uptake and expression levels of glutamate transporter proteins EAAT1 and EAAT2 were not mimicked by exogenous application of amyloid β peptide, or by prevention of endogenous amyloid peptide formation (using inhibitors of either β or γ secretase). Thus, dysfunction in glutamate homeostasis in hypoxic conditions is independent of Aβ production, but will likely contribute to neuronal damage and death associated with AD following hypoxic events.     

An integrated view of oxidative stress in aging: basic mechanisms, functional effects, and pathological considerations

Aging is an inherently complex process that is manifested within an organism at genetic, molecular, cellular, organ, and system levels. Although the fundamental mechanisms are still poorly understood, a growing body of evidence points toward reactive oxygen species (ROS) as one of the primary determinants of aging. The "oxidative stress theory" holds that a progressive and irreversible accumulation of oxidative damage caused by ROS impacts on critical aspects of the aging process and contributes to impaired physiological function, increased incidence of disease, and a reduction in life span. While compelling correlative data have been generated to support the oxidative stress theory, a direct cause-and-effect relationship between the accumulation of oxidatively mediated damage and aging has not been strongly established. The goal of this minireview is to broadly describe mechanisms of in vivo ROS generation, examine the potential impact of ROS and oxidative damage on cellular function, and evaluate how these responses change with aging in physiologically relevant situations. In addition, the mounting genetic evidence that links oxidative stress to aging is discussed, as well as the potential challenges and benefits associated with the development of anti-aging interventions and therapies.     

Development of the swimbladder and its innervation in the zebrafish, Danio rerio

Many teleosts including zebrafish, Danio rerio, actively regulate buoyancy with a gas-filled swimbladder, the volume of which is controlled by autonomic reflexes acting on vascular, muscular, and secretory effectors. In this study, we investigated the morphological development of the zebrafish swimbladder together with its effectors and innervation. The swimbladder first formed as a single chamber, which inflated at 1-3 days posthatching (dph), 3.5-4 mm body length. Lateral nerves were already present as demonstrated by the antibody zn-12, and blood vessels had formed in parallel on the cranial aspect to supply blood to anastomotic capillary loops as demonstrated by Tie-2 antibody staining. Neuropeptide Y-(NPY-) like immunoreactive (LIR) fibers appeared early in the single-chambered stage, and vasoactive intestinal polypeptide (VIP)-LIR fibers and cell bodies developed by 10 dph (5 mm). By 18 dph (6 mm), the anterior chamber formed by evagination from the cranial end of the original chamber; both chambers then enlarged with the ductus communicans forming a constriction between them. The parallel blood vessels developed into an arteriovenous rete on the cranial aspect of the posterior chamber and this region was innervated by zn-12-reactive fibers. Tyrosine hydroxylase- (TH-), NPY-, and VIP-LIR fibers also innervated this area and the lateral posterior chamber. Innervation of the early anterior chamber was also demonstrated by VIP-LIR fibers. By 25-30 dph (8-9 mm), a band of smooth muscle formed in the lateral wall of the posterior chamber. Although gas in the swimbladder increased buoyancy of young larvae just after first inflation, our results suggest that active control of the swimbladder may not occur until after the formation of the two chambers and subsequent development and maturation of vasculature, musculature and innervation of these structures at about 28-30 dph.     

Masters of efficiency

Manufacturing jobs may be shifting from the large drug companies to contract organizations as firms re-evaluate their strengths. But scientists with analytical skills and an eye for efficiency can find a job transforming materials into medicines.     

Differential effect of HOE642 on two separate monovalent cation transporters in the human red cell membrane.

Residual K(+) fluxes in red blood cells can be stimulated in conditions of low ionic strength. Previous studies have identified both the non-selective, voltage-dependent cation (NSVDC) channel and the K(+)(Na(+))/H(+) exchanger as candidate pathways mediating this effect, although it is possible that these pathways represent different modes of operation of a single system. In the present study the effects of HOE642, recently characterised as an inhibitor of the K(+)(Na(+))/H(+) exchanger, on NSVDC has been determined to clarify this question. Radioisotope flux measurements and conductance determinations showed that HOE642 exerted differential effects on the NSVDC channel and the K(+)(Na(+))/H(+) exchanger, confirming that the salt loss observed in low ionic strength solutions represents contributions from at least two independent ion transport pathways. The findings are discussed in the context of red blood cell apoptosis (eryptosis) and disease.     

Stopped-flow fluorescence resonance energy transfer for analysis of nucleosome dynamics

Macromolecular assemblies and machines undergo large-scale conformational changes as essential features of their normal function. Modern stopped-flow instrumentation and biotechnology combine to provide a powerful tool for characterizing the rates and natures of these conformational changes. Standard commercially available instruments provide extraordinary sensitivity and speed, allowing analysis of millisecond or longer timescale processes, with concentrations as low as a few nanomolar and volumes of just a few hundred microliters. One can now place specific dyes anywhere desired on a nucleic acid, and often on a protein as well. This ability allows the use of fluorescence resonance energy transfer experiments for detailed conformational analyses, even as the system is evolving rapidly over time following the initiation of a reaction. This approach is ideally suited for analysis of intrinsic properties of chromatin and of the machines that control chromatin assembly, disassembly, and function.     

Phylogenetic relationships within an endemic group of Malagasy 'assassin spiders' (Araneae, Archaeidae): ancestral character reconstruction, convergent evolution and biogeography

The phylogenetic relationships in an endemic group of Malagasy 'assassin spiders' (Araneae, Archaeidae: Eriauchenius) called the gracilicollis group, are inferred from mitochondrial 12S, 16S and COI DNA sequence data. Archaeid spiders of Madagascar have evolved varying degrees of elongation in the cephalic area. These molecular data support the monophyly of the gracilicollis group. The evolution of the cephalic area is examined by performing an ancestral character reconstruction on this character, which reveals that the cephalic area is elongating independently. The biogeography of the gracilicollis group reveals an east-west split of the clade on Madagascar.