Discrimination task reveals differences in neural bases of tinnitus and hearing impairment

We investigated auditory perception and cognitive processing in individuals with chronic tinnitus or hearing loss using functional magnetic resonance imaging (fMRI). Our participants belonged to one of three groups: bilateral hearing loss and tinnitus (TIN), bilateral hearing loss without tinnitus (HL), and normal hearing without tinnitus (NH). We employed pure tones and frequency-modulated sweeps as stimuli in two tasks: passive listening and active discrimination. All subjects had normal hearing through 2 kHz and all stimuli were low-pass filtered at 2 kHz so that all participants could hear them equally well. Performance was similar among all three groups for the discrimination task. In all participants, a distributed set of brain regions including the primary and non-primary auditory cortices showed greater response for both tasks compared to rest. Comparing the groups directly, we found decreased activation in the parietal and frontal lobes in the participants with tinnitus compared to the HL group and decreased response in the frontal lobes relative to the NH group. Additionally, the HL subjects exhibited increased response in the anterior cingulate relative to the NH group. Our results suggest that a differential engagement of a putative auditory attention and short-term memory network, comprising regions in the frontal, parietal and temporal cortices and the anterior cingulate, may represent a key difference in the neural bases of chronic tinnitus accompanied by hearing loss relative to hearing loss alone.     

Large-scale screening of a targeted Enterococcus faecalis mutant library identifies envelope fitness factors

Spread of antibiotic resistance among bacteria responsible for nosocomial and community-acquired infections urges for novel therapeutic or prophylactic targets and for innovative pathogen-specific antibacterial compounds. Major challenges are posed by opportunistic pathogens belonging to the low GC% gram-positive bacteria. Among those, Enterococcus faecalis is a leading cause of hospital-acquired infections associated with life-threatening issues and increased hospital costs. To better understand the molecular properties of enterococci that may be required for virulence, and that may explain the emergence of these bacteria in nosocomial infections, we performed the first large-scale functional analysis of E. faecalis V583, the first vancomycin-resistant isolate from a human bloodstream infection. E. faecalis V583 is within the high-risk clonal complex 2 group, which comprises mostly isolates derived from hospital infections worldwide. We conducted broad-range screenings of candidate genes likely involved in host adaptation (e.g., colonization and/or virulence). For this purpose, a library was constructed of targeted insertion mutations in 177 genes encoding putative surface or stress-response factors. Individual mutants were subsequently tested for their i) resistance to oxidative stress, ii) antibiotic resistance, iii) resistance to opsonophagocytosis, iv) adherence to the human colon carcinoma Caco-2 epithelial cells and v) virulence in a surrogate insect model. Our results identified a number of factors that are involved in the interaction between enterococci and their host environments. Their predicted functions highlight the importance of cell envelope glycopolymers in E. faecalis host adaptation. This study provides a valuable genetic database for understanding the steps leading E. faecalis to opportunistic virulence.     

PEGylation potentiates the effectiveness of an antagonistic peptide that targets the EphB4 receptor with nanomolar affinity

The EphB4 receptor tyrosine kinase together with its preferred ligand, ephrin-B2, regulates a variety of physiological and pathological processes, including tumor progression, pathological forms of angiogenesis, cardiomyocyte differentiation and bone remodeling. We previously reported the identification of TNYL-RAW, a 15 amino acid-long peptide that binds to the ephrin-binding pocked of EphB4 with low nanomolar affinity and inhibits ephrin-B2 binding. Although ephrin-B2 interacts promiscuously with all the EphB receptors, the TNYL-RAW peptide is remarkably selective and only binds to EphB4. Therefore, this peptide is a useful tool for studying the biological functions of EphB4 and for imaging EphB4-expressing tumors. Furthermore, TNYL-RAW could be useful for treating pathologies involving EphB4-ephrin-B2 interaction. However, the peptide has a very short half-life in cell culture and in the mouse blood circulation due to proteolytic degradation and clearance by the kidneys and reticuloendothelial system. To overcome these limitations, we have modified TNYL-RAW by fusion with the Fc portion of human IgG1, complexation with streptavidin or covalent coupling to a 40 KDa branched polyethylene glycol (PEG) polymer. These modified forms of TNYL-RAW all have greatly increased stability in cell culture, while retaining high binding affinity for EphB4. Furthermore, PEGylation most effectively increases peptide half-life in vivo. Consistent with increased stability, submicromolar concentrations of PEGylated TNYL-RAW effectively impair EphB4 activation by ephrin-B2 in cultured B16 melanoma cells as well as capillary-like tube formation and capillary sprouting in co-cultures of endothelial and epicardial mesothelial cells. Therefore, PEGylated TNYL-RAW may be useful for inhibiting pathological forms of angiogenesis through a novel mechanism involving disruption of EphB4-ephrin-B2 interactions between endothelial cells and supporting perivascular mesenchymal cells. Furthermore, the PEGylated peptide is suitable for other cell culture and in vivo applications requiring prolonged EphB4 receptor targeting.     

Crystallographic and kinetic evidence of allostery in a trypsin-like protease

Protein allostery is based on the existence of multiple conformations in equilibrium linked to distinct functional properties. Although evidence of allosteric transitions is relatively easy to identify by functional studies, structural detection of a pre-existing equilibrium between alternative conformations remains challenging even for textbook examples of allosteric proteins. Kinetic studies show that the trypsin-like protease thrombin exists in equilibrium between two conformations where the active site is either collapsed (E*) or accessible to substrate (E). However, structural demonstration that the two conformations exist in the same enzyme construct free of ligands has remained elusive. Here we report the crystal structure of the thrombin mutant N143P in the E form, which complements the recently reported structure in the E* form, and both the E and E* forms of the thrombin mutant Y225P. The side chain of W215 moves 10.9 ? between the two forms, causing a displacement of 6.6 ? of the entire 215-217 segment into the active site that in turn opens or closes access to the primary specificity pocket. Rapid kinetic measurements of p-aminobenzamidine binding to the active site confirm the existence of the E*-E equilibrium in solution for wild-type and the mutants N143P and Y225P. These findings provide unequivocal proof of the allosteric nature of thrombin and lend strong support to the recent proposal that the E*-E equilibrium is a key property of the trypsin fold.     

Gender-specific effects of exercise on cardiac pathology in Na+/H+ exchanger overexpressing mice

The Na(+)/H(+) exchanger isoform 1 (NHE1) has been implicated in various cardiac pathologies including ischemia/reperfusion damage to the myocardium and cardiac hypertrophy. It is known that NHE1 levels increase in cardiac disease and we have recently demonstrated that expression of an activated NHE1 protein promotes cardiac hypertrophy in the mouse myocardium. We examined the gender-specific effects of exercise in combination with elevated cardiac expression of NHE1 on the myocardium in mice. Control mice and transgenic mice expressing elevated levels of wild type NHE1 and activated NHE1 were examined. There were gender-specific differences in the effects of NHE1 with exercise. Exercised wild type male mice showed a tendency toward increased heart weight. This was not apparent in female mice expressing elevated NHE1 levels. In some transgenic female mice, there was a significant decrease in the size of the exercised hearts, which was different from what occurred with male mice. Body weight was maintained in exercised control and transgenic male mice; however, it decreased in female mice with exercise more so in transgenic female mice expressing elevated levels of NHE1. Female mice expressing activated NHE1 had elevated HW/BW ratios compared to males, and this was exaggerated by exercise. These results suggest that gender-specific activation of NHE1 may be critical and that NHE1 plays a more critical role in promoting some types of hypertrophy in females in comparison with males.     

A p.C343S missense mutation in PJVK causes progressive hearing loss

Mutations in PJVK, encoding Pejvakin, cause autosomal recessive nonsyndromic hearing loss in humans at the DFNB59 locus on chromosome 2q31.2. Pejvakin is involved in generating auditory and neural signals in the inner ear. We have identified a consanguineous Pakistani family segregating sensorineural progressive hearing loss as a recessive trait, consistent with linkage to DFNB59. We sequenced PJVK and identified a novel missense mutation, c.1028G>C in exon 7 (p.C343S) co-segregating with the phenotype in the family. The p.C343 residue is fully conserved among orthologs from different vertebrate species. We have also determined that mutations in PJVK are not a common cause of hearing loss in families with moderate to severe hearing loss in Pakistan. This is the first report of PJVK mutation in a Pakistani family and pinpoints an important residue for PJVK function.     

An analysis of 332 fatalities infected with pandemic 2009 influenza A (H1N1) in Argentina

Background

The apparent high number of deaths in Argentina during the 2009 pandemic led to concern that the influenza A H1N1pdm disease was different there. We report the characteristics and risk factors for influenza A H1N1pdm fatalities.

Methods

We identified laboratory-confirmed influenza A H1N1pdm fatalities occurring during June-July 2009. Physicians abstracted data on age, sex, time of onset of illness, medical history, clinical presentation at admission, laboratory, treatment, and outcomes using standardize questionnaires. We explored the characteristics of fatalities according to their age and risk group.

Results

Of 332 influenza A H1N1pdm fatalities, 226 (68%) were among persons aged <50 years. Acute respiratory failure was the leading cause of death. Of all cases, 249 (75%) had at least one comorbidity as defined by Advisory Committee on Immunization Practices. Obesity was reported in 32% with data and chronic pulmonary disease in 28%. Among the 40 deaths in children aged <5 years, chronic pulmonary disease (42%) and neonatal pathologies (35%) were the most common co-morbidities. Twenty (6%) fatalities were among pregnant or postpartum women of which only 47% had diagnosed co-morbidities. Only 13% of patients received antiviral treatment within 48 hours of symptom onset. None of children aged <5 years or the pregnant women received antivirals within 48 h of symptom onset. As the pandemic progressed, the time from symptom-onset to medical care and to antiviral treatment decreased significantly among case-patients who subsequently died (p<0.001).

Conclusion

Persons with co-morbidities, pregnant and who received antivirals late were over-represented among influenza A H1N1pdm deaths in Argentina, though timeliness of antiviral treatment improved during the pandemic.     

A crucial role of Flagellin in the induction of airway mucus production by Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen involved in nosocomial infections. Flagellin is a P. aeruginosa virulence factor involved in host response to this pathogen. We examined the role of flagellin in P. aeruginosa-induced mucus secretion. Using a mouse model of pulmonary infection we showed that PAK, a wild type strain of P. aeruginosa, induced airway mucus secretion and mucin muc5ac expression at higher levels than its flagellin-deficient mutant (ΔFliC). PAK induced expression of MUC5AC and MUC2 in both human airway epithelial NCI-H292 cell line and in primary epithelial cells. In contrast, ΔFliC infection had lower to no effect on MUC5AC and MUC2 expressions. A purified P. aeruginosa flagellin induced MUC5AC expression in parallel to IL-8 secretion in NCI-H292 cells. Accordingly, ΔFliC mutant stimulated IL-8 secretion at significantly lower levels compared to PAK. Incubation of NCI-H292 cells with exogenous IL-8 induced MUC5AC expression and pre-incubation of these cells with an anti-IL-8 antibody abrogated flagellin-mediated MUC5AC expression. Silencing of TLR5 and Naip, siRNA inhibited both flagellin-induced MUC5AC expression and IL-8 secretion. Finally, inhibition of ERK abolished the expression of both PAK- and flagellin-induced MUC5AC. We conclude that: (i) flagellin is crucial in P. aeruginosa-induced mucus hyper-secretion through TLR5 and Naip pathways; (ii) this process is mediated by ERK and amplified by IL-8. Our findings help understand the mechanisms involved in mucus secretion during pulmonary infectious disease induced by P. aeruginosa, such as in cystic fibrosis.