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.     

Rapid and simple DNA extraction method for the detection of enterotoxigenic Staphylococcus aureus directly from food samples: comparison of PCR and LAMP methods

Aims: The study describes the development of simple and rapid DNA extraction method in combination with loop‐mediated isothermal amplification (LAMP) to detect enterotoxigenic Staphylococcus aureus in food samples. Methods and Results: In this study, isolation of genomic DNA of enterotoxigenic Staph. aureus from spiked milk, milk burfi, khoa, sugarcane juice and boiled rice was carried out by boiling the isolated sample pellets for 10 min with 1% Triton X‐100. The isolated DNA was evaluated by polymerase chain reaction (PCR) and LAMP method. The LAMP was found to be 100 times more sensitive than PCR. The LAMP assay was very specific for Staph. aureus, and the presence of other contaminating bacterial DNAs and food matrix did not interfere or inhibit the LAMP assay. Conclusions: The template DNA extraction method developed in this study for food samples is simple, rapid and cost‐effective. LAMP was found to be less sensitive to matrix effect of food, compared to PCR. Significance and Impact of the Study: The method is suitable for direct detection of Staph. aureus without any enrichment in contaminated food samples and hence finds its application in food safety analysis, in permutation with LAMP.     

Judging a tumor cell by its cover: a matter of mitochondrial contact sites

Glioblastoma is a highly aggressive brain tumor constituted by glioma stem cell and differentiated cell populations with distinct susceptibility to cytotoxic T lymphocytes crucial for tumor immune surveillance. In this issue of The EMBO Journal, Bassoy et al ( 2017 ) show that the surface expression of certain glycans that favor recognition and killing by T cells depends on mitochondrial contacts with the endoplasmic reticulum, extending the function of this interface even to immune recognition.     

The walnut (Juglans regia) genome sequence reveals diversity in genes coding for the biosynthesis of non‐structural polyphenols

The Persian walnut (Juglans regia L.), a diploid species native to the mountainous regions of Central Asia, is the major walnut species cultivated for nut production and is one of the most widespread tree nut species in the world. The high nutritional value of J. regia nuts is associated with a rich array of polyphenolic compounds, whose complete biosynthetic pathways are still unknown. A J. regia genome sequence was obtained from the cultivar ‘Chandler’ to discover target genes and additional unknown genes. The 667‐Mbp genome was assembled using two different methods (SOAPdenovo2 and MaSuRCA), with an N50 scaffold size of 464 955 bp (based on a genome size of 606 Mbp), 221 640 contigs and a GC content of 37%. Annotation with MAKER‐P and other genomic resources yielded 32 498 gene models. Previous studies in walnut relying on tissue‐specific methods have only identified a single polyphenol oxidase (PPO) gene (JrPPO1). Enabled by the J. regia genome sequence, a second homolog of PPO (JrPPO2) was discovered. In addition, about 130 genes in the large gallate 1‐β‐glucosyltransferase (GGT) superfamily were detected. Specifically, two genes, JrGGT1 and JrGGT2, were significantly homologous to the GGT from Quercus robur (QrGGT), which is involved in the synthesis of 1‐O‐galloyl‐β‐d ‐glucose, a precursor for the synthesis of hydrolysable tannins. The reference genome for J. regia provides meaningful insight into the complex pathways required for the synthesis of polyphenols. The walnut genome sequence provides important tools and methods to accelerate breeding and to facilitate the genetic dissection of complex traits.     

Positional Preferences of Polypurine/Polypyrimidine Tracts in Saccharomyces cerevisiae Genome: Implications for cis Regulation of Gene Expression

The complete genome of the baker's yeast S. cerevisiae was analyzed for the presence of polypurine/polypyrimidine (poly[pu/py]) repeats and their occurrences were classified on the basis of their location within and outside open reading frames (ORFs). The analysis reveals that such sequence motifs are present abundantly both in coding as well as noncoding regions. Clear positional preferences are seen when these tracts occur in noncoding regions. These motifs appear to occur predominantly at a unit nucleosomal length both upstream and downstream of ORFs. Moreover, there is a biased distribution of polypurines in the coding strands when these motifs occur within open reading frames. The significance of the biased distribution is discussed with reference to the occurrence of these motifs in other known mRNA sequences and expressed sequence tags. A model for cis regulation of gene expression is proposed based on the ability of these motifs to form an intermolecular triple helix structure when present within the coding region and/or to modulate nucleosome positioning via enhanced histone affinity when present outside coding regions. Received: 14 November 1996 / Accepted: 7 May 1997     

Modulation of complex coordinated molecular signaling by 5HT and a cocktail of inhibitors leads to ovarian maturation of Penaeus monodon in captivity

In aquaculture practices, prawn cultivation holds the major share and Penaeus monodon is the main species cultured. The decline in production of P. monodon is mainly due to the limited availability of domesticated broodstock, which is attributed to its reproductive cycle, controlled by complex coordinated signaling mechanisms. Unilateral eyestalk ablation of domesticated females held in captivity is done to induce ovary development, which has certain disadvantages, including a high rate of mortality. Thus, developing alternative techniques for eyestalk ablation in captive broodstock is necessary to induce maturation of ovary. This study exemplifies the role of 5HT along with a cocktail of inhibitors (U0126, Rp‐cAMP, and LY294002) in inducing ovarian maturation. In this study, inhibition of pERK by U0126 inhibited vitellogenesis‐inhibiting hormone (VIH), which in turn led to the overexpression of vitellogenin. 5HT induces steroidogenesis (estradiol‐17β) through induction of the gonadotropin‐releasing hormone by activating calcium–calmodulin signaling. Steroidogenesis is also aided by synthesis of StAR protein. Estradiol‐17β stimulates the formation of the maturation‐promoting factor (MPF) complex by cdc25 activation and Myt1 inactivation. LY294002 aids in keeping cdc25 activated by inhibiting calcium–calmodulin induced phosphorylation of Akt which is a negative regulator of mitogen‐activated protein kinases. VIH induced activation of Myt1, through protein kinase A (PKA), was inhibited by Rp‐cAMP which inhibits adenylate cyclase, thus stabilizing the activated MPF complex. To conclude, the coordinated effect of inhibitors and 5HT accelerates the development of ovary from previtellogenic to matured oocytes, yielding high quality and quantity larvae compared with eyestalk‐ablated P. monodon.     

Design,synthesis, docking,Hirshfeld surface analysis and DFT calculations of 2-methylxanthen-9-with the FtsZ protein from Staphylococcus aureus

It is of interest to document the design, synthesis, docking, Hirshfeld surface analysis and DFT calculations of 2-methylxanthen-9-with the FtsZ protein (PDB ID: 3VOB) from Staphylococcus aureus for antimicrobial applications. We report the quantitative structure function data in this context.