Ectonucleoside Triphosphate Diphosphohydrolase-3 Antibody Targets Adult Human Pancreatic β Cells for In Vitro and In Vivo Analysis

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Weighted average regression and environmental calibration as a tool for quantifying climate-driven changes in vegetation

Aims Studies of the climatic responses of plant assemblages via vegetation-based environmental reconstructions by weighted averaging (WA) regression and calibration are a recent development in modern vegetation ecology. However, the performance of this technique for plot-based vegetation datasets has not been rigorously tested. We assess the estimation accuracy of the WA approach by comparing results, mainly the root mean square error of prediction (RMSEP) of WA regressions for six different vegetation datasets (total species, high-frequency species and low-frequency species as both abundance and incidence) each from two sites.     

Structural insights into the catalytic mechanism of 5-methylthioribose 1-phosphate isomerase

5-Methylthioribose 1-phosphate isomerase (M1Pi) is a crucial enzyme involved in the universally conserved methionine salvage pathway (MSP) where it is known to catalyze the conversion of 5-methylthioribose 1-phosphate (MTR-1-P) to 5-methylthioribulose 1-phosphate (MTRu-1-P) via a mechanism which remains unspecified till date. Furthermore, although M1Pi has a discrete function, it surprisingly shares high structural similarity with two functionally non-related proteins such as ribose-1,5-bisphosphate isomerase (R15Pi) and the regulatory subunits of eukaryotic translation initiation factor 2B (eIF2B). To identify the distinct structural features that lead to divergent functional obligations of M1Pi as well as to understand the mechanism of enzyme catalysis, the crystal structure of M1Pi from a hyperthermophilic archaeon Pyrococcus horikoshii OT3 was determined. A meticulous structural investigation of the dimeric M1Pi revealed the presence of an N-terminal extension and a hydrophobic patch absent in R15Pi and the regulatory α-subunit of eIF2B. Furthermore, unlike R15Pi in which a kink formation is observed in one of the helices, the domain movement of M1Pi is distinguished by a forward shift in a loop covering the active-site pocket. All these structural attributes contribute towards a hydrophobic microenvironment in the vicinity of the active site of the enzyme making it favorable for the reaction mechanism to commence. Thus, a hydrophobic active-site microenvironment in addition to the availability of optimal amino-acid residues surrounding the catalytic residues in M1Pi led us to propose its probable reaction mechanism via a cis-phosphoenolate intermediate formation.     

Naringin prevents ultraviolet‐B radiation‐induced oxidative damage and inflammation through activation of peroxisome proliferator‐activated receptor γ in mouse embryonic fibroblast (NIH‐3T3) cells

The present study, we investigate the preventive role of naringin, a dietary flavonoid, against ultraviolet‐B (UVB) radiation (280‐320 nm) induced oxidative damage and inflammatory responses in mouse embryonic fibroblast cell lines (NIH‐3T3). In this study, 20 mJ/cm ² of UVB radiation induces cell cytotoxicity, reactive oxygen species (ROS) generation, DNA damage, and antioxidants depletion in NIH‐3T3 cells. Treatment with naringin (60 µM) prior UVB exposure prevented the cell cytotoxicity, ROS generation, DNA damage, and antioxidants depletion in NIH‐3T3 cells. Furthermore, naringin prevents UVB‐induced mitogen‐activated protein kinase families and nuclear factor‐κB (NF‐κB)‐mediated activation of inflammatory factors, that is TNF‐α, IL‐6, IL‐10, and COX‐2 in NIH‐3T3 cells. Peroxisome proliferator‐activated receptor γ (PPARγ) is an anti‐inflammatory agent and it suppressed the UVB‐mediated oxidative and inflammatory responses. In this study, naringin activates PPARγ and prevents inflammatory biomarkers in NIH‐3T3 cells. Thus, naringin prevents UVB‐mediated inflammation and oxidative damage in NIH‐3T3 cells probably over controlling NF‐κB expression and activation of PPARγ.     

Red panda fine‐scale habitat selection along a Central Himalayan longitudinal gradient

Red panda Ailurus fulgens, an endangered habitat specialist, inhabits a narrow distribution range in bamboo abundance forests along mountain slopes in the Himalaya and Hengduan Mountains. However, their habitat use may be different in places with different longitudinal environmental gradients, climatic regimes, and microclimate. This study aimed to determine the habitat variables affecting red panda distribution across different longitudinal gradients through a multivariate analysis. We studied habitat selection patterns along the longitudinal gradient in Nepal's Himalaya which is grouped into the eastern, central, and western complexes. We collected data on red panda presence and habitat variables (e.g., tree richness, canopy cover, bamboo abundance, water availability, tree diameter, tree height) by surveys along transects throughout the species’ potential range. We used a multimodal inference approach with a generalized linear model to test the relative importance of environmental variables. Although the study showed that bamboo abundance had a major influence, habitat selection was different across longitudinal zones. Both canopy cover and species richness were unimportant in eastern Nepal, but their influence increased progressively toward the west. Conversely, tree height showed a decreasing influence on habitat selection from Eastern to Western Nepal. Red panda's habitat selection revealed in this study corresponds to the uneven distribution of vegetation assemblages and the dry climatic gradient along the eastern‐western Himalayas which could be related to a need to conserve energy and thermoregulate. This study has further highlighted the need of importance of bamboo conservation and site‐specific conservation planning to ensure long‐term red panda conservation.     

A protocol for culturing Drosophila melanogaster stage 9 egg chambers for live imaging

This protocol describes a method for the dissection of egg chambers from intact Drosophila females and culture conditions that permit live imaging of them, with a particular emphasis on stage 9. This stage of development is characterized by oocyte growth and patterning, outer follicle cell rearrangement and migration of border cells. Although in vitro culture of egg chambers of later developmental stages has long been possible, until recently stage 9 egg chambers could only be kept alive for short periods, did not develop normally, and border cell migration failed entirely. We have established culture conditions that support overall egg chamber development including border cell migration in vitro. This protocol makes possible direct observation of molecular and cellular dynamics in both wild-type and mutant egg chambers, and opens the door to testing of pharmacological inhibitors and the use of biosensors. The entire protocol takes approximately 24 h while the preparation of egg chambers for live imaging requires only 15-20 min.     

Rapid in vitro multiplication of Drosera indica L.: a vulnerable, medicinally important insectivorous plant

An efficient protocol is described for rapid in vitro multiplication of the vulnerable medicinal herb Drosera indica L. by enhanced axillary bud proliferation from shoot tips as explants. In order to standardize in vitro multiplication of D. indica, the effects of different strengths of Murashige and Skoog (MS) medium (1/4, 1/3, 1/2 and full strength), different percentages of sucrose (1, 2 and 3%), various pH (3.7, 4.7, 5.7 and 6.7) and MS basal medium fortified with different concentrations of zeatin (Z), kinetin (KN) (0.1, 0.5, 1.0 and 2.0 mg/l) and 6-benzylaminopurine (BA) (0.01, 0.05 and 0.1 mg/l) were tried. Multiple shoot production was independent of different strengths of MS, various percentages of sucrose and also when pH was altered. Although the number of multiple shoots developed on MS medium supplemented with Z (0.1, 0.5, 1.0 and 2.0 mg/l), KN (0.5 and 1.0 mg/l) and BA (0.1 mg/l) separately was high, the maximum number was observed on MS fortified with Z (0.5 mg/l) and KN (0.5 mg/l), respectively, which clearly depicts that there is not much difference comparatively with a variation in hormone concentration in case of Z. High cytokinin concentrations resulted in retardation of shoot growth. Rooting was best achieved on MS basal medium. This protocol could be useful for production of large biomass within 6 weeks for plumbagin bioprospection and long term in vitro conservation.     

Structure and function analysis of CaMdr1p, a major facilitator superfamily antifungal efflux transporter protein of Candida albicans: identification of amino acid residues critical for drug/H+ transport

We have cloned and overexpressed multidrug transporter CaMdr1p as a green fluorescent protein-tagged protein to show its capability to extrude drug substrates. The drug extrusion was sensitive to pH and energy inhibitors and displayed selective substrate specificity. CaMdr1p has a unique and conserved antiporter motif, also called motif C [G(X6)G(X3)GP(X2)GP(X2)G], in its transmembrane segment 5 (TMS 5). Alanine scanning of all the amino acids of the TMS 5 by site-directed mutagenesis highlighted the importance of the motif, as well as that of other residues of TMS 5, in drug transport. The mutant variants of TMS 5 were placed in four different categories. The first category had four residues, G244, G251, G255, and G259, which are part of the conserved motif C, and their substitution with alanine resulted in increased sensitivity to drugs and displayed impaired efflux of drugs. Interestingly, first category mutants, when replaced with leucine, resulted in more dramatic loss of drug resistance and efflux. Notwithstanding the location in the core motif, the second category included residues which are part of the motif, such as P260, and those which were not part of the motif, such as L245, W248, P256, and F262, whose substitution with alanine resulted in a severe loss of drug resistance and efflux. The third category included G263, which is a part of motif C, but unlike other conserved glycines, its replacement with alanine or leucine showed no change in the phenotype. The replacement of the remaining 11 residues of the fourth category did not result in any change. The putative helical wheel projection showed clustering of functionally critical residues to one side and thus suggests an asymmetric nature of TMS 5.     

Microbial diversity and function in crystalline basement beneath the Deccan Traps explored in a 3 km borehole at Koyna,western India

Deep terrestrial subsurface represents a huge repository of global prokaryotic biomass. Given its vastness and importance, microbial life within the deep subsurface continental crust remains under-represented in global studies. We characterize the microbial communities of deep, extreme and oligotrophic realm hosted by crystalline Archaean granitic rocks underneath the Deccan Traps, through sampling via 3000 m deep scientific borehole at Koyna, India through metagenomics, amplicon sequencing and cultivation-based analyses. Gene sequences 16S rRNA (7.37 × 10⁶) show considerable bacterial diversity and the existence of a core microbiome (5724 operational taxonomic units conserved out of a total 118,064 OTUs) across the depths. Relative abundance of different taxa of core microbiome varies with depth in response to prevailing lithology and geochemistry. Co-occurrence network analysis and cultivation attempt to elucidate close interactions among autotrophic and organotrophic bacteria. Shotgun metagenomics reveals a major role of autotrophic carbon fixation via the Wood–Ljungdahl pathway and genes responsible for energy and carbon metabolism. Deeper analysis suggests the existence of an ‘acetate switch’, coordinating biosynthesis and cellular homeostasis. We conclude that the microbial life in the nutrient- and energy-limited deep granitic crust is constrained by the depth and managed by a few core members via a close interplay between autotrophy and organotrophy.