HOMER2, a Stereociliary Scaffolding Protein,Is Essential for Normal Hearing in Humans and Mice

Hereditary hearing loss is a clinically and genetically heterogeneous disorder. More than 80 genes have been implicated to date, and with the advent of targeted genomic enrichment and massively parallel sequencing (TGE+MPS) the rate of novel deafness-gene identification has accelerated. Here we report a family segregating post-lingual progressive autosomal dominant non-syndromic hearing loss (ADNSHL). After first excluding plausible variants in known deafness-causing genes using TGE+MPS, we completed whole exome sequencing in three hearing-impaired family members. Only a single variant, p.Arg185Pro in HOMER2, segregated with the hearing-loss phenotype in the extended family. This amino acid change alters a highly conserved residue in the coiled-coil domain of HOMER2 that is essential for protein multimerization and the HOMER2-CDC42 interaction. As a scaffolding protein, HOMER2 is involved in intracellular calcium homeostasis and cytoskeletal organization. Consistent with this function, we found robust expression in stereocilia of hair cells in the murine inner ear and observed that over-expression of mutant p.Pro185 HOMER2 mRNA causes anatomical changes of the inner ear and neuromasts in zebrafish embryos. Furthermore, mouse mutants homozygous for the targeted deletion of Homer2 present with early-onset rapidly progressive hearing loss. These data provide compelling evidence that HOMER2 is required for normal hearing and that its sequence alteration in humans leads to ADNSHL through a dominant-negative mode of action.     

NOX5 in human spermatozoa: expression, function, and regulation

Physiological and pathological processes in spermatozoa involve the production of reactive oxygen species (ROS), but the identity of the ROS-producing enzyme system(s) remains a matter of speculation. We provide the first evidence that NOX5 NADPH oxidase is expressed and functions in human spermatozoa. Immunofluorescence microscopy detected NOX5 protein in both the flagella/neck region and the acrosome. Functionally, spermatozoa exposed to calcium ionophore, phorbol ester, or H(2)O(2) exhibited superoxide anion production, which was blocked by addition of superoxide dismutase, a Ca(2+) chelator, or inhibitors of either flavoprotein oxidases (diphenylene iododonium) or NOX enzymes (GKT136901). Consistent with our previous overexpression studies, we found that H(2)O(2)-induced superoxide production by primary sperm cells was mediated by the non-receptor tyrosine kinase c-Abl. Moreover, the H(V)1 proton channel, which was recently implicated in spermatozoa motility, was required for optimal superoxide production by spermatozoa. Immunoprecipitation experiments suggested an interaction among NOX5, c-Abl, and H(V)1. H(2)O(2) treatment increased the proportion of motile sperm in a NOX5-dependent manner. Statistical analyses showed a pH-dependent correlation between superoxide production and enhanced sperm motility. Collectively, our findings show that NOX5 is a major source of ROS in human spermatozoa and indicate a role for NOX5-dependent ROS generation in human spermatozoa motility.     

A molecular machine biosensor: construction, predictive models and experimental studies

This paper describes the construction, operation and predictive modeling of a molecular machine, functioning as a high sensitivity biosensor. Embedded gramicidin A (gA) ionchannels in a self-assembled tethered lipid bilayer act as biological switches in response to target molecules and provide a signal amplification mechanism that results in high sensitivity molecular detection. The biosensor can be used as a rapid and sensitive point of care diagnostic device in different media such as human serum, plasma and whole blood without the need for pre and post processing steps required in an enzyme-linked immunosorbent assay. The electrical reader of the device provides the added advantage of objective measurement. Novel ideas in the construction of the molecular machine, including fabrication of biochip arrays, and experimental studies of its ability to detect analyte molecules over a wide range of concentrations are presented. Remarkably, despite the complexity of the device, it is shown that the response can be predicted by modeling the analyte fluid flow and surface chemical reactions. The derived predictive models for the sensing dynamics also facilitate determining important variables in the design of a molecular machine such as the ion channel lifetime and diffusion dynamics within the bilayer lipid membrane as well as the bio-molecular interaction rate constants.     

Changes in partial pressures of respiratory gases during submerged voluntary breath hold across odontocetes: is body mass important?

Odontocetes have an exceptional range in body mass spanning 10³ kg across species. Because, size influences oxygen utilization and carbon dioxide production rates in mammals, this lineage likely displays an extraordinary variation in oxygen store management compared to other marine mammal groups. To examine this, we measured changes in the partial pressures of respiratory gases ( P_\textO2 P_{{{\text{O}}_{2} }} , P_\textCO2 P_{{{\text{CO}}_{2} }} ), pH, and lactate in the blood during voluntary, quiescent, submerged breath holds in Pacific white-sided dolphins (Lagenorhynchus obliquidens), bottlenose dolphins (Tursiops truncatus), and a killer whale (Orcinus orca) representing a mass range of 96–3,850 kg. These measurements provided an empirical determination of the effect of body size on the variability in blood biochemistry during breath hold and experimentally determined aerobic dive limits (ADL) within one taxonomic group (odontocetes). For the species in this study, maximum voluntary breath-hold duration was positively correlated with body mass, ranging from 3.5 min in white-sided dolphins to 13.3 min for the killer whale. Variation in breath-hold duration was associated with differences in the rate of change for P_\textO2 P_{{{\text{O}}_{2} }} throughout breath hold; P_\textO2 P_{{{\text{O}}_{2} }} decreased twice as fast for the two smaller species (−0.6 mmHg O₂ min⁻¹) compared to the largest species (−0.3 mmHg O₂ min⁻¹). In contrast, the rate of increase in P_\textCO2 P_{{{\text{CO}}_{2} }} during breath hold was similar across species. These results demonstrate that large body size in odontocetes facilitates increased aerobic breath-hold capacity as mediated by decreased mass-specific metabolic rates (rates of change in P_\textO2 P_{{{\text{O}}_{2} }} served as a proxy for oxygen utilization). Indeed the experimentally determined 5 min ADL for bottlenose dolphins was surpassed by the 13.3 min maximum breath hold of the killer whale, which did not end in a rise in lactate. Rather, breath hold ended voluntarily as respiratory gases and pH fell within a narrow range for both large and small species, likely providing cues for ventilation.     

Social learning spreads knowledge about dangerous humans among American crows

Individuals face evolutionary trade-offs between the acquisition of costly but accurate information gained firsthand and the use of inexpensive but possibly less reliable social information. American crows (Corvus brachyrhynchos) use both sources of information to learn the facial features of a dangerous person. We exposed wild crows to a novel 'dangerous face' by wearing a unique mask as we trapped, banded and released 7-15 birds at five study sites near Seattle, WA, USA. An immediate scolding response to the dangerous mask after trapping by previously captured crows demonstrates individual learning, while an immediate response by crows that were not captured probably represents conditioning to the trapping scene by the mob of birds that assembled during the capture. Later recognition of dangerous masks by lone crows that were never captured is consistent with horizontal social learning. Independent scolding by young crows, whose parents had conditioned them to scold the dangerous mask, demonstrates vertical social learning. Crows that directly experienced trapping later discriminated among dangerous and neutral masks more precisely than did crows that learned through social means. Learning enabled scolding to double in frequency and spread at least 1.2 km from the place of origin over a 5 year period at one site.     

Sudden expansion of a single brown bear maternal lineage across northern continental Eurasia after the last ice age: a general demographic model for mammals?

The brown bear has proved a useful model for studying Late Quaternary mammalian phylogeography. However, information is lacking from northern continental Eurasia, which constitutes a large part of the species' current distribution. We analysed mitochondrial DNA sequences (totalling 1943 bp) from 205 bears from northeast Europe and Russia in order to characterize the maternal phylogeography of bears in this region. We also estimated the formation times of the sampled brown bear lineages and those of its extinct relative, the cave bear.
Four closely related haplogroups belonging to a single mitochondrial subclade were identified in northern continental Eurasia. Several haplotypes were found throughout the whole study area, while one haplogroup was restricted to Kamchatka. The haplotype network, estimated divergence times and various statistical tests indicated that bears in northern continental Eurasia recently underwent a sudden expansion, preceded by a severe bottleneck. This brown bear population was therefore most likely founded by a small number of bears that were restricted to a single refuge area during the last glacial maximum. This pattern has been described previously for other mammal species and as such may represent one general model for the phylogeography of Eurasian mammals. Bayesian divergence time estimates are presented for different brown and cave bear clades. Moreover, our results demonstrate the extent of substitution rate variation occurring throughout the phylogenetic tree, highlighting the need for appropriate calibration when estimating divergence times.     

Control of positive end-expiratory pressure (PEEP) for small animal ventilators

Background  

The positive end-expiratory pressure (PEEP) for the mechanical ventilation of small animals is frequently obtained with water seals or by using ventilators developed for human use. An alternative mechanism is the use of an on-off expiratory valve closing at the moment when the alveolar pressure is equal to the target PEEP. In this paper, a novel PEEP controller (PEEP-new) and the PEEP system of a commercial small-animal ventilator, both based on switching an on-off valve, are evaluated.     

Modulation of the RNA binding and protein processing activities of poliovirus polypeptide 3CD by the viral RNA polymerase domain

To study the role of the RNA polymerase domain (3D) in the proteinase substrate recognition and RNA binding properties of poliovirus polypeptide 3CD, we generated recombinant 3C and 3CD polypeptides and purified them to near homogeneity. By using these purified proteins in in vitro cleavage assays with structural and non-structural viral polyprotein substrates, we found that 3CD processes the poliovirus structural polyprotein precursor (P1) 100 to 1000 times more efficiently than 3C processes P1. We also found that trans-cleavage of other 3CD molecules and sites within the non-structural P3 precursor is more efficiently mediated by 3CD than 3C. However, 3C and 3CD appear to be equally efficient in the processing of a non-structural polyprotein precursor, 2C3AB. Four mutated 3CD polyproteins with site-directed lesions in the 3D domain of the proteinase were analyzed for their ability to process viral polyprotein precursors and to form a ternary complex with RNA sequences encoded in the 5' terminus of the viral genome. Analysis of mutated 3CD polypeptides revealed that specific mutations within the 3D amino acid sequences of 3CD confer differential effects on 3CD activity. All four mutated 3CD proteins tested were able to process the P1 structural precursor with wild type or near wild type efficiency. However, three of the mutated enzymes demonstrated an impaired ability to process some sites within the P3 non-structural precursor, relative to wild type 3CD. One of the mutant 3CD polypeptides, 3CD-3DK127A, also displayed a defect in its ability to form a ternary ribonucleoprotein complex with poliovirus 5' RNA sequences.