Cloning of Acinetobacter calcoaceticus chromosomal region involved in catechin degradation

Acinetobacter calcoaceticus utilizes catechin as sole carbon source. The chromosomal region involved in catechin catabolism was cloned in Escherichia coli DH5alpha from the genomic DNA of A. calcoaceticus. A recombinant E. coli containing 9.2 kb DNA fragment of A. calcoaceticus inserted in pUC19 showed a halo zone around the colony in plate assays, indicating the catechin utilizing ability of the clone. Enzyme assays revealed the expression of the cloned DNA fragment of A. calcoaceticus. High performance thin layer chromatography confirmed protocatechuic acid and phloroglucinol carboxylic acid as cleavage products of catechin in A. calcoaceticus and the catechin degrading ability of the clones. A. calcoaceticus followed the beta-ketoadipate pathway for catechin degradation. The sub-clone (pASCI) of this insert was sequenced and analyzed. The sequence showed three major ORFs but only ORF 2 showed similarities to other aromatic oxygenases and the sequence of ORF 2 was submitted to GenBank (AF369935).     

Effect of Azadirachta indica on paracetamol-induced hepatic damage in albino rats.

Azadirachta indica, a plant used widely in Ayurveda, has been reported to have anti-inflammatory, immunomodulatory and adaptogenic properties. The present study evaluates its hepatoprotective role. Fresh juice of tender leaves of Azadirachta indica (200 mg/kg body wt. p.o.) inhibited paracetamol (2 g/kg body wt. p.o.)-induced lipid peroxidation and prevented depletion of sulfhydryl groups in liver cells. There was an increase in serum marker enzymes of hepatic damage (aspartate transaminase, alanine transaminase and alkaline phosphatase) after paracetamol administration. Azadirachta indica pretreatment stabilized the serum levels of these enzymes. Histopathological observations of liver tissues corroborated these findings.     

Strategies to target SARS-CoV-2 entry and infection using dual mechanisms of inhibition by acidification inhibitors

Many viruses utilize the host endo-lysosomal network for infection. Tracing the endocytic itinerary of SARS-CoV-2 can provide insights into viral trafficking and aid in designing new therapeutic strategies. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV-2 spike protein is internalized via the pH-dependent CLIC/GEEC (CG) endocytic pathway in human gastric-adenocarcinoma (AGS) cells expressing undetectable levels of ACE2. Ectopic expression of ACE2 (AGS-ACE2) results in RBD traffic via both CG and clathrin-mediated endocytosis. Endosomal acidification inhibitors like BafilomycinA1 and NH₄Cl, which inhibit the CG pathway, reduce the uptake of RBD and impede Spike-pseudoviral infection in both AGS and AGS-ACE2 cells. The inhibition by BafilomycinA1 was found to be distinct from Chloroquine which neither affects RBD uptake nor alters endosomal pH, yet attenuates Spike-pseudovirus entry. By screening a subset of FDA-approved inhibitors for functionality similar to BafilomycinA1, we identified Niclosamide as a SARS-CoV-2 entry inhibitor. Further validation using a clinical isolate of SARS-CoV-2 in AGS-ACE2 and Vero cells confirmed its antiviral effect. We propose that Niclosamide, and other drugs which neutralize endosomal pH as well as inhibit the endocytic uptake, could provide broader applicability in subverting infection of viruses entering host cells via a pH-dependent endocytic pathway.     

Plasma-Derived Exosomal Survivin,a Plausible Biomarker for Early Detection of Prostate Cancer

Background

Survivin is expressed in prostate cancer (PCa), and its downregulation sensitizes PCa cells to chemotherapeutic agents in vitro and in vivo. Small membrane-bound vesicles called exosomes, secreted from the endosomal membrane compartment, contain RNA and protein that they readily transport via exosome internalization into recipient cells. Recent progress has shown that tumor-derived exosomes play multiple roles in tumor growth and metastasis and may produce these functions via immune escape, tumor invasion and angiogenesis. Furthermore, exosome analysis may provide novel biomarkers to diagnose or monitor PCa treatment.

Methods

Exosomes were purified from the plasma and serum from 39 PCa patients, 20 BPH patients, 8 prostate cancer recurrent and 16 healthy controls using ultracentrifugation and their quantities and qualities were quantified and visualized from both the plasma and the purified exosomes using ELISA and Western blotting, respectively.

Results

Survivin was significantly increased in the tumor-derived samples, compared to those from BPH and controls with virtually no difference in the quantity of Survivin detected in exosomes collected from newly diagnosed patients exhibiting low (six) or high (nine) Gleason scores. Exosome Survivin levels were also higher in patients that had relapsed on chemotherapy compared to controls.

Conclusions

These studies demonstrate that Survivin exists in plasma exosomes from both normal, BPH and PCa subjects. The relative amounts of exosomal Survivin in PCa plasma was significantly higher than in those with pre-inflammatory BPH and control plasma. This differential expression of exosomal Survivin was seen with both newly diagnosed and advanced PCa subjects with high or low-grade cancers. Analysis of plasma exosomal Survivin levels may offer a convenient tool for diagnosing or monitoring PCa and may, as it is elevated in low as well as high Gleason scored samples, be used for early detection.     

Assessing Patterns of Human-Wildlife Conflicts and Compensation around a Central Indian Protected Area

Mitigating crop and livestock loss to wildlife and improving compensation distribution are important for conservation efforts in landscapes where people and wildlife co-occur outside protected areas. The lack of rigorously collected spatial data poses a challenge to management efforts to minimize loss and mitigate conflicts. We surveyed 735 households from 347 villages in a 5154 km² area surrounding Kanha Tiger Reserve in India. We modeled self-reported household crop and livestock loss as a function of agricultural, demographic and environmental factors, and mitigation measures. We also modeled self-reported compensation received by households as a function of demographic factors, conflict type, reporting to authorities, and wildlife species involved. Seventy-three percent of households reported crop loss and 33% livestock loss in the previous year, but less than 8% reported human injury or death. Crop loss was associated with greater number of cropping months per year and proximity to the park. Livestock loss was associated with grazing animals inside the park and proximity to the park. Among mitigation measures only use of protective physical structures were associated with reduced livestock loss. Compensation distribution was more likely for tiger related incidents, and households reporting loss and located in the buffer. Average estimated probability of crop loss was 0.93 and livestock loss was 0.60 for surveyed households. Estimated crop and livestock loss and compensation distribution were higher for households located inside the buffer. Our approach modeled conflict data to aid managers in identifying potential conflict hotspots, influential factors, and spatially maps risk probability of crop and livestock loss. This approach could help focus allocation of conservation efforts and funds directed at conflict prevention and mitigation where high densities of people and wildlife co-occur.     

Chlorophyllase in <Emphasis Type="Italic">Piper betle</Emphasis> L. has a role in chlorophyll homeostasis and senescence dependent chlorophyll breakdown

Total chlorophyll content and chlorophyllase (chlorophyll-chlorophyllido hydrolase EC 3.1.1.14) activity in fresh leaves of Piper betle L. landrace KS was, respectively, twofold higher and eight fold lower than KV, showing negative correlation between chlorophyll and chlorophyllase activity. Specific chlorophyllase activity was nearly eightfold more in KV than KS. ORF of 918 nt was found in cloned putative chlorophyllase cDNAs from KV and KS. The gene was present as single copy in both the landraces. The encoded polypeptide of 306 amino acids differed only at two positions between the KV and KS; 203 (cysteine to tyrosine) and 301 (glutamine to glycine). Difference in chlorophyllase gene expression between KV and KS was evident in fresh and excised leaves. Up regulation of chlorophyllase gene by ABA and down regulation by BAP was observed in both the landraces; however, there was quantitative difference between KV and KS. Data suggests that chlorophyllase in P. betle is involved in chlorophyll homeostasis and chlorophyll loss during post harvest senescence.