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.     

Effect of Ag Ion Implantation on SPR of Cu-C60 Nanocomposite Thin Film

Plasmonics - Cu-C60 nanocomposite thin films are synthesized by co-deposition restive heating method on glass, silicon, and TEM grid substrates. Rutherford backscattering spectroscopy (RBS)...     

Stochastic Simulations Suggest that HIV-1 Survives Close to Its Error Threshold

The use of mutagenic drugs to drive HIV-1 past its error threshold presents a novel intervention strategy, as suggested by the quasispecies theory, that may be less susceptible to failure via viral mutation-induced emergence of drug resistance than current strategies. The error threshold of HIV-1, , however, is not known. Application of the quasispecies theory to determine poses significant challenges: Whereas the quasispecies theory considers the asexual reproduction of an infinitely large population of haploid individuals, HIV-1 is diploid, undergoes recombination, and is estimated to have a small effective population size in vivo. We performed population genetics-based stochastic simulations of the within-host evolution of HIV-1 and estimated the structure of the HIV-1 quasispecies and . We found that with small mutation rates, the quasispecies was dominated by genomes with few mutations. Upon increasing the mutation rate, a sharp error catastrophe occurred where the quasispecies became delocalized in sequence space. Using parameter values that quantitatively captured data of viral diversification in HIV-1 patients, we estimated to be substitutions/site/replication, ∼2–6 fold higher than the natural mutation rate of HIV-1, suggesting that HIV-1 survives close to its error threshold and may be readily susceptible to mutagenic drugs. The latter estimate was weakly dependent on the within-host effective population size of HIV-1. With large population sizes and in the absence of recombination, our simulations converged to the quasispecies theory, bridging the gap between quasispecies theory and population genetics-based approaches to describing HIV-1 evolution. Further, increased with the recombination rate, rendering HIV-1 less susceptible to error catastrophe, thus elucidating an added benefit of recombination to HIV-1. Our estimate of may serve as a quantitative guideline for the use of mutagenic drugs against HIV-1.     

Single-nucleotide variations associated with <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> KwaZulu-Natal strains

The occurrence of drug resistance in Mycobacterium tuberculosis, the aetiological agent of tuberculosis (TB), is hampering the management and control of TB in the world. Here we present a computational analysis of recently sequenced drug-sensitive (DS), multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of M. tuberculosis. Single-nucleotide variations (SNVs) were identified in a pair-wise manner using the anchor-based whole genome comparison (ABWGC) tool and its modified version. For this analysis, four fully sequenced genomes of different strains of M. tuberculosis were taken along with three KwaZulu-Natal (KZN) strains isolated from South Africa including one XDR and one MDR strain. KZN strains were compared with other fully sequenced strains and also among each other. The variations were analysed with respect to their biological influence as a result of either altered structure or synthesis. The results suggest that the DR phenotype may be due to changes in a number of genes. The database on KZN strains can be accessed through the website .     

SSR analysis of 38 genotypes of soybean (Glycine Max (L.) Merr.) genetic diversity in India

Sixteen polymorphic Simple sequence repeat (SSR) markers were used to determine the genetic diversity and varietal identification among 38 soybean (Glycine max (L.) Merr.) genotypes which are at present under seed multiplication chain in India. A total of 51 alleles with an average of 2.22 alleles per locus were detected. The polymorphic information content (PIC) among genotypes varied from 0.049 (Sat_243 and Satt337) to 0.526 (Satt431) with an average of 0.199. The pair wise genetic similarity between soybean varieties varied from 0.56 to 0.97 with an average of 0.761. These 16 SSR markers successfully distinguished 12 of the 38 soybean genotypes. These results suggest that used SSR markers are efficient for measuring genetic diversity and relatedness as well as identifying varieties of soybeans. Diverse genetic materials may be used for genetic improvements of soybean genotypes.     

Anticancer and anti-inflammatory activities of 1,8-naphthyridine-3-carboxamide derivatives

Several 1,8-naphthyridine-3-carboxamide derivatives (8-23) were synthesized and tested for in vitro cytotoxicity against eight cancer cell lines and a normal cell line. Compound 12 exhibited high cytotoxicity (IC(50)=1.37microM) in HBL-100 (breast) cell line while compounds 17 (IC(50)=3.7microM) and 22 (IC(50)=3.0microM) have shown high cytotoxicity in KB (oral) and SW-620 (colon) cell lines, respectively. The synthesized 1,8-naphthyridine-3-carboxamides were also evaluated for anti-inflammatory and myeloprotective activities, indicated by modulation in cytokine and chemokine levels secreted by dendritic cells.     

Transformation of pWWO in Rhizobium leguminosarum DPT to Engineer Toluene Degrading Ability for Rhizoremediation

Rhizoremediation of organic xenobiotics is based on interactions between plants and their associated micro-organisms. The present work was designed to engineer a bacterial system having toluene degradation ability along with plant growth promoting characteristics for effective rhizoremediation. pWWO harboring the genes responsible for toluene breakdown was isolated from Pseudomonas putida MTCC 979 and successfully transformed in Rhizobium DPT. This resulted in a bacterial strain (DPT^T) which had the ability to degrade toluene as well as enhance growth of host plant. The frequency of transformation was recorded 5.7 × 10⁻⁶. DPT produced IAA, siderophore, chitinase, HCN, ACC deaminase, solubilized inorganic phosphate, fixed atmospheric nitrogen and inhibited the growth of Fusarium oxysporum and Macrophomina phaseolina in vitro. During pot assay, 50 ppm toluene in soil was found to inhibit the germination of Cajanus cajan seeds. However when the seeds bacterized with toluene degrading P. putida or R. leguminosarum DPT were sown in pots, again no germination was observed. Non-bacterized as well as bacterized seeds germinated successfully in toluene free soil as control. The results forced for an alternative mode of application of bacteria for rhizoremediation purpose. Hence bacterial suspension was mixed with soil having 50 ppm of toluene. Germination index in DPT treated soil was 100% while in P. putida it was 50%. Untreated soil with toluene restricted the seeds to germinate.     

Anchor-based whole genome phylogeny (ABWGP): a tool for inferring evolutionary relationship among closely related microorganisms [corrected

Phenotypic behavior of a group of organisms can be studied using a range of molecular evolutionary tools that help to determine evolutionary relationships. Traditionally a gene or a set of gene sequences was used for generating phylogenetic trees. Incomplete evolutionary information in few selected genes causes problems in phylogenetic tree construction. Whole genomes are used as remedy. Now, the task is to identify the suitable parameters to extract the hidden information from whole genome sequences that truly represent evolutionary information. In this study we explored a random anchor (a stretch of 100 nucleotides) based approach (ABWGP) for finding distance between any two genomes, and used the distance estimates to compute evolutionary trees. A number of strains and species of Mycobacteria were used for this study. Anchor-derived parameters, such as cumulative normalized score, anchor order and indels were computed in a pair-wise manner, and the scores were used to compute distance/phylogenetic trees. The strength of branching was determined by bootstrap analysis. The terminal branches are clearly discernable using the distance estimates described here. In general, different measures gave similar trees except the trees based on indels. Overall the tree topology reflected the known biology of the organisms. This was also true for different strains of Escherichia coli. A new whole genome-based approach has been described here for studying evolutionary relationships among bacterial strains and species.     

Investigations on the Thermal Stability of Fullerene-Based (Ag-C<Subscript>70</Subscript>) Nanocomposite Thin Films

Fullerene-based bi-functional nanocomposite thin film (Ag nanoparticles embedded in fullerene C₇₀ matrix) is synthesized by thermal co-deposition method. Thermal stability of Ag-C₇₀ nanocomposite is investigated by annealing the nanocomposite thin film at different temperatures from 80 to 350 °C for 30 min. Optical and structural properties of nanocomposite thin film with respect to high temperature are studied by UV-visible spectroscopy and x-ray diffraction, respectively. Transmission electron microscopy is performed to observe the temperature-dependent size evolution of Ag nanoparticles in fullerene C₇₀ matrix. A large growth of Ag nanoparticles is observed with temperature especially above 200 °C due to enhanced diffusion of Ag in fullerene C₇₀ at higher temperature and Ostwald ripening. The properties of metal-fullerene nanocomposite is not significantly affected up to a temperature of 150 °C. With a further increase in temperature, a major blue shift of ~?33 nm in SPR wavelength is seen at a temperature of 300 °C due to the thermal induced structural transformation of fullerene C₇₀ matrix into amorphous carbon. A very large-sized Ag nanoparticle with a wide size distribution varying from 27.8 ± 0.6 to 330.0 ± 4.5 nm is seen at 350 °C and due to which, a red shift of ~?16 nm is obtained at this temperature. This study throws light on the thermal stability of the devices based on metal-fullerene bi-functional nanocomposite.