Functional organization of TRPC-Ca channels and regulation of calcium microdomains

TRP family of proteins are components of unique cation channels that are activated in response to diverse stimuli ranging from growth factor and neurotransmitter stimulation of plasma membrane receptors to a variety of chemical and sensory signals. This review will focus on members of the TRPC sub-family (TRPC1–TRPC7) which currently appear to be the strongest candidates for the enigmatic Ca²⁺ influx channels that are activated in response to stimulation of plasma membrane receptors which result in phosphatidyl inositol-(4,5)-bisphosphate (PIP₂) hydrolysis, generation of IP₃ and DAG, and IP₃-induced Ca²⁺ release from the intracellular Ca²⁺ store via inositol trisphosphate receptor (IP₃R). Homomeric or selective heteromeric interactions between TRPC monomers generate distinct channels that contribute to store-operated as well as store-independent Ca²⁺ entry mechanisms. The former is regulated by the emptying/refilling of internal Ca²⁺ store(s) while the latter depends on PIP₂ hydrolysis (due to changes in PIP₂ per se or an increase in diacylglycerol, DAG). Although the exact physiological function of TRPC channels and how they are regulated has not yet been conclusively established, it is clear that a variety of cellular functions are controlled by Ca²⁺ entry via these channels. Thus, it is critical to understand how cells coordinate the regulation of diverse TRPC channels to elicit specific physiological functions. It is now well established that segregation of TRPC channels mediated by interactions with signaling and scaffolding proteins, determines their localization and regulation in functionally distinct cellular domains. Furthermore, both protein and lipid components of intracellular and plasma membranes contribute to the organization of these microdomains. Such organization serves as a platform for the generation of spatially and temporally dictated [Ca²⁺]_i signals which are critical for precise control of downstream cellular functions.     

The relative contribution of the small and large intestine to the absorption and metabolism of rutin in man

Tomato juice containing rutin (quercetin-3-rutinoside) was ingested by healthy volunteers and ileostomists. Blood and urine collected over 24 h were analysed by HPLC with photodiode array (PDA) and tandem mass spectrometric detection. Low concentrations of isorhamnetin-3-glucuronide (C_max = 4.3 ± 1.5 nmoles/l) and quercetin-3-glucuronide (C_max = 12 ± 2 nmoles/l) were detected in plasma of healthy subjects. Metabolites appeared in blood after 4 h indicating absorption from the large intestine. Nine metabolites of rutin were detected in urine but with considerable variation in total amount (40 ± 1-4981 ± 115 nmoles over 24 h). No metabolites were detected in plasma or urine of ileostomists and 86 ± 3% of the ingested rutin was recovered in ileal fluid. In subjects with an intact large intestine, but not ileostomists, rutin was catabolised with the appearance of 3,4-dihydroxyphenylacetic acid, 3-methoxy-4-hydroxyphenylacetic acid and 3-hydroxyphenylacetic acid in urine accounting for 22% of rutin intake.     

A multivalent combination of experimental antituberculosis DNA vaccines based on Ag85B and regions of difference antigens

Two candidate DNA vaccines based on the proteins CFP10 and CFP21 encoded by regions of difference (RDs) of Mycobacterium tuberculosis were evaluated individually and in multivalent combination with the immunodominant protein Ag85B for induction of protective immune responses against experimental tuberculosis. Experimental DNA vaccines induced substantial levels of cell-mediated immune responses as indicated by marked lymphocyte proliferation, significant release of the Th1 cytokines IFN-gamma and IL-12 (p40), and predominant cytotoxic T cell activity. High levels of antigen-specific IgG1 and IgG2a antibodies observed in the sera of immunized mice depicted strong humoral responses generated by DNA vaccine constructs. The multivalent combination of three DNA vaccine constructs induced maximal T cell and humoral immune responses. All the experimental vaccines imparted significant protection against challenge with M. tuberculosis H(37)Rv (in terms of colony-forming unit reduction in lungs and spleen) as compared to vector controls. The level of protection exhibited by multivalent DNA vaccine formulation was found to be equivalent to that of Mycobacterium bovis BCG observed both at 4 and 8 weeks post-challenge. These results show the protective potential of the multivalent DNA vaccine formulation used in this study.     

Isolation and process parameter optimization of Aspergillus sp. for removal of chromium from tannery effluent

Five morphologically different fungi were isolated from leather tanning effluent in which Aspergillus sp. and Hirsutella sp. had higher potential to remove chromium. The potential of Aspergillus sp. for removal of chromium was evaluated in shake flask culture in different pH, temperature, inoculums size, carbon and nitrogen source. The maximum chromium was removed at pH 6, temperature 30 degrees C, sodium acetate (0.2%) and yeast extract (0.1%). Aspergillus sp. was applied in 2l bioreactor for removal of chromium, and it was observed that 70% chromium was removed after 3 days.     

Gene expression profiles in HPV-immortalized human cervical cells treated with the nicotine-derived carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

Human papillomavirus (HPV) infection is an established etiological factor for cervical cancer. Epidemiological studies suggest that smoking in combination with HPV infection plays a significant role in the etiology of this disease. We have previously shown that the tobacco carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is present in human cervical mucus. Here, we hypothesized that treatment of HPV-16-immortalized human ectocervical cells (Ecto1/E6E7) with NNK would alter the expression of genes involved in cellular transformation. Ecto1/E6E7 cells were treated with water (vehicle control) alone or with 1 μM, 10 μM, and 100 μM of NNK in water for 12 weeks. The colony-forming efficiency increased following NNK treatment; the maximum effect was observed after 12 weeks with 100 μM NNK. Microarray analysis revealed that, independent of the dose of NNK, expression of 30 genes was significantly altered; 22 of these genes showed a dose-response pattern. Genes identified are categorized as immune response (LTB4R), RNA surveillance pathway (SMG1), metabolism (ALDH7A1), genes frequently expressed in later stages of neoplastic development (MT1F), DNA binding (HIST3H3 and CHD1L), and protein biosynthesis (UBA52). Selected genes were confirmed by qRT-PCR. Western blot analysis indicates that phosphorylation of histone 3 at serine 10, a marker of cellular transformation, was up-regulated in cells treated with NNK. This is the first study showing that NNK can alter gene expression that may, in part, account for transformation of HPV-immortalized human cervical cells. The results support previous epidemiological observations that, in addition to HPV, tobacco smoking also plays an important role in the development of cervical cancer.     

Extracellular Ca2+ sensing in salivary ductal cells

Ca(2+) is secreted from the salivary acinar cells as an ionic constituent of primary saliva. Ions such as Na(+) and Cl(-) get reabsorbed whereas primary saliva flows through the salivary ductal system. Although earlier studies have shown that salivary [Ca(2+)] decreases as it flows down the ductal tree into the oral cavity, ductal reabsorption of Ca(2+) remains enigmatic. Here we report a potential role for the G protein-coupled receptor, calcium-sensing receptor (CSR), in the regulation of Ca(2+) reabsorption by salivary gland ducts. Our data show that CSR is present in the apical region of ductal cells where it is co-localized with transient receptor potential canonical 3 (TRPC3). CSR is activated in isolated salivary gland ducts as well as a ductal cell line (SMIE) by altering extracellular [Ca(2+)] or by aromatic amino acid, l-phenylalanine (l-Phe, endogenous component of saliva), as well as neomycin. CSR activation leads to Ca(2+) influx that, in polarized cells grown on a filter support, is initiated in the luminal region. We show that TRPC3 contributes to Ca(2+) entry triggered by CSR activation. Further, stimulation of CSR in SMIE cells enhances the CSR-TRPC3 association as well as surface expression of TRPC3. Together our findings suggest that CSR could serve as a Ca(2+) sensor in the luminal membrane of salivary gland ducts and regulate reabsorption of [Ca(2+)] from the saliva via TRPC3, thus contributing to maintenance of salivary [Ca(2+)]. CSR could therefore be a potentially important protective mechanism against formation of salivary gland stones (sialolithiasis) and infection (sialoadenitis).     

Computational selection of flavonoid compounds as inhibitors against SARS-CoV-2 main protease,RNA-dependent RNA polymerase and spike proteins: A molecular docking study

An outbreak of Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has been recognized as a global health concern. Since, no specific antiviral drug is proven effective for treatment against COVID-19, identification of new therapeutics is an urgent need. In this study, flavonoid compounds were analyzed for its inhibitory potential against important protein targets of SARS-CoV-2 using computational approaches. Virtual docking was performed for screening of flavonoid compounds retrieved from PubChem against the main protease of SARS-CoV-2 using COVID-19 docking server. The cut off of dock score was set to >?9 kcal/mol and screened compounds were individually docked against main protease, RNA-dependent RNA polymerase, and spike proteins using AutoDock 4.1 software. Finally, lead flavonoid compounds were subjected to ADMET analysis. A total of 458 flavonoid compounds were virtually screened against main protease target and 36 compounds were selected based on the interaction energy value >?9 kcal/mol. Furthermore, these compounds were individually docked against protein targets and top 10 lead compounds were identified. Among the lead compounds, agathisflavone showed highest binding energy value of ?8.4 kcal/mol against main protease, Albireodelphin showed highest dock score of ?9.8 kcal/mol and ?11.2 kcal/mol against RdRp, and spike proteins, respectively. Based on the high dock score and ADMET properties, top 5 lead molecules such as Albireodelphin, Apigenin 7-(6″-malonylglucoside), Cyanidin-3-(p-coumaroyl)-rutinoside-5-glucoside, Delphinidin 3-O-beta-D-glucoside 5-O-(6-coumaroyl-beta-D-glucoside) and (-)-Maackiain-3-O-glucosyl-6″-O-malonate were identified as potent inhibitors against main protease, RdRp, and spike protein targets of SARS-CoV-2. These all compounds are having non-carcinogenic and non-mutagenic properties. This study finding suggests that the screened compounds include Albireodelphin, Apigenin 7-(6″-malonylglucoside), Cyanidin-3-(p-coumaroyl)-rutinoside-5-glucoside, Delphinidin 3-O-beta-D-glucoside 5-O-(6-coumaroyl-beta-D-glucoside) and (-)-Maackiain-3-O-glucosyl-6″-O-malonate could be the potent inhibitors of SARS-CoV-2 targets.     

Continuous fill intermittent decant type sequencing batch reactor application to upgrade the UASB treated sewage

The performance of continuous flow intermittent decant type sequencing batch (CFID) reactor treating the effluent of an UASB reactor treating domestic wastewater and operated at 8 h hydraulic retention time (HRT) was investigated. The CFID was operated at three different HRTs (22, 8 and 6 h) and three different dissolved oxygen (DO) patterns (<0.5, 2.5–3.5 and 3.5–4.5 mg/L). The highest effluent quality was observed at the 8 h HRT and 2.5–3.5 mg/L DO concentration. At this operational condition, the average BOD, TSS, ammonia nitrogen and fecal coliform removal efficiencies were 83, 90, 74 and 99 %, respectively. The CFID is a promising post-treatment option for existing UASB systems, with a final effluent quality that comply with receiving water and effluent reuse criteria.     

Sedentary Behaviour and Physical Activity in South Asian Women: Time to Review Current Recommendations?

Objective

Our aims were to describe activity and sedentary behaviours in urban Asian women, with dysglycaemia (diagnosed at recruitment), and without dysglycaemia and examine the relative contribution of these parameters to their glycaemic status.

Methods

2800 urban women (30–45 years) were selected by random cluster sampling and screened for dysglycaemia for a final sample of 272 newly diagnosed, drug naive dysglycaemic and 345 normoglycaemic women. Physical activity and sedentary behaviours were assessed by the International Physical Activity Questionnaire (IPAQ). Demographic data, diet and anthropometry were recorded. Logistic regression analysis assessed contribution of all parameters to dysglycaemia and exposure attributable fractions were calculated.

Results

The mean energy expenditure on walking (2648.5±1023.7 MET-min/week) and on moderate and vigorous physical activity (4342.3±1768.1 MET-min/week) for normoglycemic women and dysglycaemic women (walking;1046.4±728.4 MET-min/week, moderate and vigorous physical activity; 1086.7±1184.4 MET-min/week) was above the recommended amount of physical activity per week. 94.3% of women spent >1000 MET-minutes/week on activity. Mean sitting and TV time for normoglycaemic and dysglycaemic women were 154.3±62.8, 38.4±31.9, 312.6±116.7 and 140.2±56.5 minutes per day respectively. Physical activity and sedentary behaviour contributed to dysglycaemia after adjustment for family history, diet, systolic blood pressure and Body Mass Index. Exposure attributable fractions for dysglycaemia were; lower physical activity: 78%, higher waist circumference: 94%, and TV viewing time: 85%.

Conclusions

Urban South Asian women are at risk of dysglycaemia at lower levels of sedentary behaviour and greater physical activity than western populations, indicating the need for re-visiting current physical activity guidelines for South Asians.