Gustatory sensory cells express a receptor responsive to protein breakdown products (GPR92)

The ingestion of dietary protein is of vital importance for the maintenance of fundamental physiological processes. The taste modality umami, with its prototype stimulus, glutamate, is considered to signal the protein content of food. Umami was thought to be mediated by the heterodimeric amino acid receptor, T1R1 + T1R3. Based on knockout studies, additional umami receptors are likely to exist. In addition to amino acids, certain peptides can also elicit and enhance umami taste suggesting that protein breakdown products may contribute to umami taste. The recently deorphanized peptone receptor, GPR92 (also named GPR93; LPAR5), is expressed in gastric enteroendocrine cells where it responds to protein hydrolysates. Therefore, it was of immediate interest to investigate if the receptor GPR92 is expressed in gustatory sensory cells. Using immunohistochemical approaches we found that a large population of cells in murine taste buds was labeled with an GPR92 antibody. A molecular phenotyping of GPR92 cells revealed that the vast majority of GPR92-immunoreactive cells express PLCβ2 and can therefore be classified as type II cells. More detailed analyses have shown that GPR92 is expressed in the majority of T1R1-positive taste cells. These results indicate that umami cells may respond not only to amino acids but also to peptides in protein hydrolysates.     

Biodegradation of feather keratin with a PEGylated protease of Chryseobacterium gleum

Present study deals with the covalent modification of keratinolytic protease of Chryseobacterium gleum with higher enzyme activity, improved stability, non-immunogenicity and reusability. Protease of C. gleum showing feather degradation ability was modified by covalent attachment to polyethylene glycol. This modification culminated the change in electrophoretic mobility of protease in acrylamide gel. The modified enzyme showed 1.4 times more catalytic activity with better stability than native in aqueous system containing whole feathers as keratin. It showed improved pH, thermal, storage and solvent stability with a broadened range of pH (7–9) and temperature (25–50 °C) than native. The differentiation between modified and native enzyme was authenticated through UV–vis spectroscopy, SEM, XRD, FTIR and DSC. This modification of protease proved to be non-immunogenic in rats. The enzyme extracted after first run could be used for several cycles which clearly demonstrated its reusability in catalytic bioprocess of keratin degradation.     

Receptor specific crosstalk and modulation of signaling upon heterodimerization between β1-adrenergic receptor and somatostatin receptor-5

In the present study we describe heterodimerization, trafficking, coupling to adenylyl cyclase and signaling in HEK-293 cells cotransfected with human-somatostatin receptor 5 (hSSTR5) and β₁-adrenergic receptor (β₁AR). hSSTR5/β₁AR exists as heterodimers in basal conditions which was further enhanced upon synergistic activation of both receptors. Activation of either β₁AR or hSSTR5 displayed dissociation of heterodimerization. In cotransfectants, β₁AR effect on cAMP was predominant; however, blocking β₁AR with antagonist resulted in 60% inhibition of forskolin-stimulated cAMP in the presence of hSSTR5 agonists. cAMP/PKA pathway in cotransfected cells was regulated in receptor-specific manner, in contrast, the status of pERK1/2 and pPI3K/AKT was predominantly regulated by hSSTR5. The expression levels of phosphorylated NFAT remained unchanged indicating blockade of calcineurin-mediated dephosphorylation and nuclear translocation of NFAT, the process predominantly regulated by pJNK in SSTR5 dependent manner. Taken together, the functional consequences of results described here might have relevance in the cardiovascular system where SSTR and AR subtypes play important roles.     

Natural terpenes prevent mitochondrial dysfunction, oxidative stress and release of apoptotic proteins during nimesulide-hepatotoxicity in rats

Nimesulide, an anti-inflammatory and analgesic drug, is reported to cause severe hepatotoxicity. In this study, molecular mechanisms involved in deranged oxidant-antioxidant homeostasis and mitochondrial dysfunction during nimesulide-induced hepatotoxicity and its attenuation by plant derived terpenes, camphene and geraniol has been explored in male Sprague-Dawley rats. Hepatotoxicity due to nimesulide (80 mg/kg BW) was evident from elevated SGPT, SGOT, bilirubin and histo-pathological changes. Antioxidants and key redox enzymes (iNOS, mtNOS, Cu/Zn-SOD, Mn-SOD, GPx and GR) were altered significantly as assessed by their mRNA expression, Immunoblot analysis and enzyme activities. Redox imbalance along with oxidative stress was evident from decreased NAD(P)H and GSH (56% and 74% respectively; P<0.001), increased superoxide and secondary ROS/RNS generation along with oxidative damage to cellular macromolecules. Nimesulide reduced mitochondrial activity, depolarized mitochondria and caused membrane permeability transition (MPT) followed by release of apoptotic proteins (AIF; apoptosis inducing factor, EndoG; endonuclease G, and Cyto c; cytochrome c). It also significantly activated caspase-9 and caspase-3 and increased oxidative DNA damage (level of 8-Oxoguanine glycosylase; P<0.05). A combination of camphene and geraniol (CG; 1:1), when pre-administered in rats (10 mg/kg BW), accorded protection against nimesulide hepatotoxicity in vivo, as evident from normalized serum biomarkers and histopathology. mRNA expression and activity of key antioxidant and redox enzymes along with oxidative stress were also normalized due to CG pre-treatment. Downstream effects like decreased mitochondrial swelling, inhibition in release of apoptotic proteins, prevention of mitochondrial depolarization along with reduction in oxidized NAD(P)H and increased mitochondrial electron flow further supported protective action of selected terpenes against nimesulide toxicity. Therefore CG, a combination of natural terpenes prevented nimesulide induced cellular damage and ensuing hepatotoxicity.     

Impact of multi-targeted antiretroviral treatment on gut T cell depletion and HIV reservoir seeding during acute HIV infection

Background

Limited knowledge exists on early HIV events that may inform preventive and therapeutic strategies. This study aims to characterize the earliest immunologic and virologic HIV events following infection and investigates the usage of a novel therapeutic strategy.

Methods and Findings

We prospectively screened 24,430 subjects in Bangkok and identified 40 AHI individuals. Thirty Thais were enrolled (8 Fiebig I, 5 Fiebig II, 15 Fiebig III, 2 Fiebig IV) of whom 15 completed 24 weeks of megaHAART (tenofovir/emtricitabine/efavirenz/raltegravir/maraviroc). Sigmoid biopsies were completed in 24/30 at baseline and 13/15 at week 24.At baseline, the median age was 29 years and 83% were MSM. Most were symptomatic (87%), and were infected with R5-tropic (77%) CRF01_AE (70%). Median CD4 was 406 cells/mm³. HIV RNA was 5.5 log₁₀ copies/ml. Median total blood HIV DNA was higher in Fiebig III (550 copy/10⁶ PBMC) vs. Fiebig I (8 copy/10⁶ PBMC) (p = 0.01) while the median %CD4+CCR5+ gut T cells was lower in Fiebig III (19%) vs. Fiebig I (59%) (p = 0.0008).After 24 weeks of megaHAART, HIV RNA levels of <50 copies were achieved in 14/15 in blood and 13/13 in gut. Total blood HIV DNA at week 0 predicted reservoir size at week 24 (p<0.001). Total HIV DNA declined significantly and was undetectable in 3 of 15 in blood and 3 of 7 in gut. Frequency of CD4+CCR5+ gut T cells increased from 41% at baseline to 64% at week 24 (p>0.050); subjects with less than 40% at baseline had a significant increase in CD4+CCR5+ T cells from baseline to week 24 (14% vs. 71%, p = 0.02).

Conclusions

Gut T cell depletion and HIV reservoir seeding increases with progression of AHI. MegaHAART was associated with immune restoration and reduced reservoir size. Our findings could inform research on strategies to achieve HIV drug-free remission.     

Cholesterol assimilation and biotransformation by Lactobacillus helveticus

Lactobacillus helveticus, grown at 37°C in MRS medium supplemented with 3 mM cholesterol, assimilated all the cholesterol in 42 h having 68 U mg⁻¹ of intracellular cholesterol oxidase activity. The strain transformed 1 g cholesterol to 0.05 g of androsta-1, 4-diene-3, 17-dione and 0.04 g of androst-4-ene-3, 17 dione within 48 h at 37°C with extracellular cholesterol oxidase activity at 12 U mg⁻¹ and intracellular oxidase at 0.5 U mg⁻¹.     

Hydrogen storage in C3Ti complex using quantum chemical methods and molecular dynamics simulations

The hydrogen storage capacity of C(3)Ti and C(3)Ti(+) complex was studied using second order M?ller-Plesset (MP2) and density functional theory (DFT) methods with different exchange and correlation functionals. Four and five H(2) molecules can be adsorbed on C(3)Ti and C(3)Ti(+) complex respectively at all the levels of theory used. This corresponds to the gravimetric H(2) uptake capacity of 8.77 and 10.73 wt % for the former and the latter respectively. The nature of interactions between different molecules in H(2) adsorbed complexes is also studied using many-body analysis approach. In the case of C(3)Ti(4H(2)) complex, total five-body interactions is negligible whereas for C(3)Ti(+)(5H(2)) relaxation energy is negligible. All the many-body energies have significant contribution to the binding energy of a respective complex. Atom-centered density matrix propagation molecular dynamics simulations were carried out using different methods to confirm whether H(2) molecules remain adsorbed on C(3)Ti and C(3)Ti(+) complex at room temperature. Adsorption Gibbs free energies show that four and five H(2) molecule adsorption on C(3)Ti and C(3)Ti(+) at room temperature is energetically favorable and unfavorable respectively using MP2 as well as DFT methods used here. H(2) adsorption is thermodynamically favorable over a wide range of temperature on the C(3)Ti than C(3)Ti(+)complex.     

Tonic activity of Galpha-gustducin regulates taste cell responsivity

The taste-selective G protein, α-gustducin (α-gus) is homologous to α-transducin and activates phosphodiesterase (PDE) in vitro. α-Gus-knockout mice are compromized to bitter, sweet and umami taste stimuli, suggesting a central role in taste transduction. Here, we suggest a different role for Gα-gus. In taste buds of α-gus-knockout mice, basal (unstimulated) cAMP levels are high compared to those of wild-type mice. Further, H-89, a cAMP-dependent protein kinase inhibitor, dramatically unmasks responses to the bitter tastant denatonium in gus-lineage cells of knockout mice. We propose that an important role of α-gus is to maintain cAMP levels tonically low to ensure adequate Ca²⁺ signaling.     

Changes in haemolymph constituents of American bollworm, Helicoverpa armigera (Hübner), infected with nucleopolyhedrovirus

Six types of haemocytes viz., prohaemocytes, plasmatocytes (round, fusiform, vermiform and spindle shaped), granular cells, spherule cells, oenocytoids and adipohaemocytes were found in the haemolymph of larvae of American bollworm H. armigera. The total and differential haemocyte counts (THC and DHC) in H. armigera haemolymph were affected by nucleopolyhedrovirus (NPV) treatment. There was a general decrease in THC in response to NPV treatment in both young and old larvae. However the decrease was more apparent in 5 and 8 day old larvae than in 10 day old larvae. The differential haemocytes showed less of granular cells and more of spherule cells and prohaemocytes in the old larvae. Plasmatocytes and granular cells in 10 day old larvae initially phagocytosed polyhedra; however, disintegrated after 3 to 4 hr. The haemolymph of NPV treated larvae melanized slowly particularly in old larvae. Phenoloxidase (PO) activity decreased positively with granular cells and oenocytoids in 10 day old treated larvae. Cellular fraction had high level of PO activity, which was transferred to plasma in response to NPV infection in the older larvae. The role of NPV pathogenesis vis-à-vis immunity in insect is discussed.     

Binary neural network training algorithms based on linear sequential learning

A key problem in Binary Neural Network learning is to decide bigger linear separable subsets. In this paper we prove some lemmas about linear separability. Based on these lemmas, we propose Multi-Core Learning (MCL) and Multi-Core Expand-and-Truncate Learning (MCETL) algorithms to construct Binary Neural Networks. We conclude that MCL and MCETL simplify the equations to compute weights and thresholds, and they result in the construction of simpler hidden layer. Examples are given to demonstrate these conclusions.