Changes in ventral respiratory column GABAaR ε- and δ-subunits during hibernation mediate resistance to depression by EtOH and pentobarbital

During hibernation in the 13-lined ground squirrel, Ictidomys tridecemlineatus, the cerebral cortex is electrically silent, yet the brainstem continues to regulate cardiorespiratory function. Previous work showed that neurons in slices through the medullary ventral respiratory column (VRC) but not the cortex are insensitive to high doses of pentobarbital during hibernation, leading to the hypothesis that GABA(A) receptors (GABA(A)R) in the VRC undergo a seasonal modification in subunit composition. To test whether alteration of GABA(A)R subunits are responsible for hibernation-associated pentobarbital insensitivity, we examined an array of subunits using RT-PCR and Western blots and identified changes in ε- and δ-subunits in the medulla but not the cortex. Using immunohistochemistry, we confirmed that during hibernation, the expression of ε-subunit-containing GABA(A)Rs nearly doubles in the VRC. We also identified a population of δ-subunit-containing GABA(A)Rs adjacent to the VRC that were differentially expressed during hibernation. As δ-subunit-containing GABA(A)Rs are particularly sensitive to ethanol (EtOH), multichannel electrodes were inserted in slices of medulla and cortex from hibernating squirrels and EtOH was applied. EtOH, which normally inhibits neuronal activity, excited VRC but not cortical neurons during hibernation. This excitation was prevented by bicuculline pretreatment, indicating the involvement of GABA(A)Rs. We propose that neuronal activity in the VRC during hibernation is unaffected by pentobarbital due to upregulation of ε-subunit-containing GABA(A)Rs on VRC neurons. Synaptic input from adjacent inhibitory interneurons that express δ-subunit-containing GABA(A)Rs is responsible for the excitatory effects of EtOH on VRC neurons during hibernation.     

Differential mortality and the excess burden of end-stage renal disease among First Nations people with diabetes mellitus: a competing-risks analysis

Background:

Diabetes-related end-stage renal disease disproportionately affects indigenous peoples. We explored the role of differential mortality in this disparity.

Methods:

In this retrospective cohort study, we examined the competing risks of end-stage renal disease and death without end-stage renal disease among Saskatchewan adults with diabetes mellitus, both First Nations and non–First Nations, from 1980 to 2005. Using administrative databases of the Saskatchewan Ministry of Health, we developed Fine and Gray subdistribution hazards models and cumulative incidence functions.

Results:

Of the 90 429 incident cases of diabetes, 8254 (8.9%) occurred among First Nations adults and 82 175 (90.9%) among non–First Nations adults. Mean age at the time that diabetes was diagnosed was 47.2 and 61.6 years, respectively (p < 0.001). After adjustment for sex and age at the time of diabetes diagnosis, the risk of end-stage renal disease was 2.66 times higher for First Nations than non–First Nations adults (95% confidence interval [CI] 2.24–3.16). Multivariable analysis with adjustment for sex showed a higher risk of death among First Nations adults, which declined with increasing age at the time of diabetes diagnosis. Cumulative incidence function curves stratified by age at the time of diabetes diagnosis showed greatest risk for end-stage renal disease among those with onset of diabetes at younger ages and greatest risk of death among those with onset of diabetes at older ages.

Interpretation:

Because they are typically younger when diabetes is diagnosed, First Nations adults with this condition are more likely than their non–First Nations counterparts to survive long enough for end-stage renal disease to develop. Differential mortality contributes substantially to ethnicity-based disparities in diabetes-related end-stage renal disease and possibly to chronic diabetes complications. Understanding the mechanisms underlying these disparities is vital in developing more effective prevention and management initiatives.Indigenous peoples experience an excess burden of diabetes-related end-stage renal disease,^1–4 but the reasons for this disparity are incompletely understood. Although the increase in end-stage renal disease among indigenous peoples has paralleled the global emergence of type 2 diabetes mellitus,⁵ disparities in end-stage renal disease among Canada’s First Nations adults persist² after adjustment for elevated prevalence of diabetes.⁶ In an earlier study, we suggested that First Nations adults might be more prone to diabetic nephropathy and might experience more rapid progression to end-stage renal disease.⁷ However, although albuminuria is more prevalent in this population,⁸ affected individuals unexpectedly have a longer average time from diagnosis of diabetes to end-stage renal disease than people from non–First Nations populations.² These findings could be explained by a younger age at the time of diabetes diagnosis⁶ and lower mortality among those with chronic kidney disease.⁸ An age-related survival benefit among First Nations adults with diabetes could lead to longer exposure to the metabolic consequences of diabetes and greater likelihood of end-stage renal disease.Our objective was to examine the contribution of differential mortality to disparities in diabetes-related end-stage renal disease within large populations of indigenous and non-indigenous North Americans. Accordingly, we used competing-risks survival analysis to compare the simultaneous risks of diabetes-related end-stage renal disease and death without end-stage renal disease among First Nations and non–First Nations adults.⁹     

Initial phases of microbial biofilm formation on opaque,innovative anti‐adhesive surfaces using a modular microfluidic system

Biofilms can cause numerous problems, hence it is important to understand their formation on surfaces in order to develop resistant materials and avoidance strategies. Therefore, information is required regarding adhesion processes on surfaces generally and innovative anti‐adhesive coatings in particular. Our flow cell system allows biofilms to be monitored in continuous flow conditions, without removing material for postflow imaging. The shown laminar flow ensures the maintenance of highly controlled conditions for biofilm growth. However, carried simulations of the oxygen demands of Escherichia coli cultivated as biofilms under the chosen regime indicate that conditions may become anaerobic, at least at the outlet of the flow cell, after a certain period of time. We report data on the biofouling tendencies on coatings generated with the help of direct laser interference patterning on stainless steel surfaces. Data were estimated from images acquired by fluorescence microscopy. Differences between patterned and unpatterned surfaces were not found, which is in accordance with the attachment point theory. Nevertheless, it is particularly important to elucidate in future studies the behavior of microorganisms during their attachment and the effects of variables of potentially sensitive surfaces (such as hydrophobicity, nanotopography, and charge) on their adhesion.     

Mutagenesis of the fusion peptide-like domain of hepatitis C virus E1 glycoprotein: involvement in cell fusion and virus entry

Background  

Envelope (E) glycoprotein E2 of the hepatitis C virus (HCV) mediates binding of the virus to target cell receptors. Nevertheless, the precise role of E1 in viral entry remains elusive.     

Epsin 1 is Involved in Recruitment of Ubiquitinated EGF Receptors into Clathrin-Coated Pits

Epsin consists of an epsin NH₂-terminal homology domain that promotes interaction with phospholipids, several AP-2-binding sites, two clathrin-binding sequences and several Eps15 homology domain-binding motifs. Epsin additionally possesses ubiquitin-interacting motifs (UIMs) and has been demonstrated to bind ubiquitinated cargo. We therefore investigated whether epsin promoted clathrin-mediated endocytosis of the ubiquitinated EGF receptor (EGFR). By immunoprecipitation, we found that epsin 1 interacted with ubiquitinated EGFR and that functional UIMs were essential for complex formation. Furthermore, RNA interference-mediated knockdown of epsin 1 was found to inhibit internalization of the EGFR, while having no effect on endocytosis of the transferrin receptor. Additionally, upon knockdown of epsin 1, translocation of the EGFR to central parts of clathrin-coated pits was inhibited. This supports the contention that epsin 1 promotes endocytosis of the ubiquitinated EGFR.     

Protein markers for insulin-producing beta cells with higher glucose sensitivity

Background and Methodology

Pancreatic beta cells show intercellular differences in their metabolic glucose sensitivity and associated activation of insulin production. To identify protein markers for these variations in functional glucose sensitivity, rat beta cell subpopulations were flow-sorted for their level of glucose-induced NAD(P)H and their proteomes were quantified by label-free data independent alternate scanning LC-MS. Beta cell-selective proteins were also identified through comparison with rat brain and liver tissue and with purified islet alpha cells, after geometrical normalization using 6 stably expressed reference proteins.

Principal Findings

All tissues combined, 943 proteins were reliably quantified. In beta cells, 93 out of 467 quantifiable proteins were uniquely detected in this cell type; several other proteins presented a high molar abundance in beta cells. The proteome of the beta cell subpopulation with high metabolic and biosynthetic responsiveness to 7.5 mM glucose was characterized by (i) an on average 50% higher expression of protein biosynthesis regulators such as 40S and 60S ribosomal constituents, NADPH-dependent protein folding factors and translation elongation factors; (ii) 50% higher levels of enzymes involved in glycolysis and in the cytosolic arm of the malate/aspartate-NADH-shuttle. No differences were noticed in mitochondrial enzymes of the Krebs cycle, beta-oxidation or respiratory chain.

Conclusions

Quantification of subtle variations in the proteome using alternate scanning LC-MS shows that beta cell metabolic glucose responsiveness is mostly associated with higher levels of glycolytic but not of mitochondrial enzymes.     

Mycoflora and mycotoxins natural occurrence in corn from entre rios province, Argentina

Corn samples were collected in 1999 from three departments of Entre Réos province, Argentina, and were surveyed for mould contamination and natural occurrence ofFusarium mycotoxins, ochratoxin A and aflatoxins.Fusarium verticillioides was the most prevalent fungal species recorded at all departments. Zearalenone, deoxynivalenol and ochratoxin A were not found in any samples. Only one of the 52 corn samples analysed was contaminated with aflatoxin B₁ (17 μg/kg). Fumonisin B₁ was found in 58 % of samples (range of positive samples: 47– 3,347 μg/kg), fumonisin B₂ in 33.0 % (range of positive samples: 23–537 μg/kg) and fumonisin B₃ in 25.0 % (range of positive samples: 24–287 μg/kg) of them. This is the first report on the natural occurrence of mycotoxins in corn from Entre Ríos province, Argentina. Levels of fumonisins were lower than detected in other Argentinian provinces.