Enhancing the evaluation of PI3K inhibitors through 3D melanoma models

Targeted therapies for mutant BRAF metastatic melanoma are effective but not curative due to acquisition of resistance. PI3K signaling is a common mediator of therapy resistance in melanoma; thus, the need for effective PI3K inhibitors is critical. However, testing PI3K inhibitors in adherent cultures is not always reflective of their potential in vivo. To emphasize this, we compared PI3K inhibitors of different specificity in two‐ and three‐dimensional (2D, 3D) melanoma models and show that drug response predictions gain from evaluation using 3D models. Our results in 3D demonstrate the anti‐invasive potential of PI3K inhibitors and that drugs such as PX‐866 have beneficial activity in physiological models alone and when combined with BRAF inhibition. These assays finally help highlight pathway effectors that could be involved in drug response in different environments (e.g. p4E‐BP1). Our findings show the advantages of 3D melanoma models to enhance our understanding of PI3K inhibitors.     

Comparison of laboratory and field testing performance evaluations of siloxane-polyurethane fouling-release marine coatings

A series of eight novel siloxane-polyurethane fouling-release (FR) coatings were assessed for their FR performance in both the laboratory and in the field. Laboratory analysis included adhesion assessments of bacteria, microalgae, macroalgal spores, adult barnacles and pseudobarnacles using high-throughput screening techniques, while field evaluations were conducted in accordance with standardized testing methods at three different ocean testing sites over the course of six-months exposure. The data collected were subjected to statistical analysis in order to identify potential correlations. In general, there was good agreement between the laboratory screening assays and the field assessments, with both regimes clearly distinguishing the siloxane-polyurethane compositions comprising monofunctional poly(dimethyl siloxane) (PDMS) (m-PDMS) as possessing superior, broad-spectrum FR properties compared to those prepared with difunctional PDMS (d-PDMS). Of the seven laboratory screening techniques, the Cellulophaga lytica biofilm retraction and reattached barnacle (Amphibalanus amphitrite) adhesion assays were shown to be the most predictive of broad-spectrum field performance.     

In Silico Derived Peptides for Inhibiting the Toxin–Antitoxin Systems of Mycobacterium tuberculosis: Basis for Developing Peptide-Based Therapeutics

Toxin–antitoxin (TA) systems of Mycobacterium tuberculosis (Mtb) is a prerequisite for the bacterium to survive in extreme conditions. Antimicrobial peptides inhibiting the formation of these complexes provide a novel strategy for TB drug discovery process. Absence of TA genes in human, makes these systems as an attractive target for drug development. In this study using Peptiderive server, we have derived a number of potential inhibitory peptides for nine TA complexes—VapBC3, VapBC5, VapBC11, VapBC15, VapBC26, VapBC30, RelBE2, RelJK, MazEF4 of Mtb. We have studied about the common interacting toxin residues with the antitoxin and with the derived peptide. Further, using Cluspro server, we compared the binding efficacy of the in silico derived peptides with the published potential peptides for the toxins VapC26, VapC30 and MazF. Thus, these in silico derived peptides would serve as basis for developing peptide based therapeutics for TA complexes of Mtb.

     

Exploratory algorithm to devise multi-epitope subunit vaccine by investigating Leishmania donovani membrane proteins

Visceral leishmaniasis (VL) is a deadly parasitic infection which affects poorest to poor population living in the endemic countries. Increasing resistant to existing drugs, disease burden and a significant number of deaths, necessitates the need for an effective vaccine to prevent the VL infection. This study employed a combinatorial approach to develop a multi-epitope subunit vaccine by exploiting Leishmania donovani membrane proteins. Cytotoxic T- and helper T-lymphocyte binding epitopes along with suitable adjuvant and linkers were joined together in a sequential manner to design the subunit vaccine. The occurrence of B-cell and IFN-γ inducing epitopes approves the ability of subunit vaccine to develop humoral and cell-mediated immune response. Physiochemical parameters of vaccine protein were also assessed followed by homology modeling, model refinement and validation. Moreover, disulfide engineering was performed for the increasing stability of the designed vaccine and molecular dynamics simulation was performed for the comparative stability purposes and to conform the geometric conformations. Further, molecular docking and molecular dynamics simulation study of a mutated and non-mutated subunit vaccine against TLR-4 immune receptor were performed and respective complex stability was determined. In silico cloning ensures the expression of designed vaccine in pET28a(+) expression vector. This study offers a cost-effective and time-saving way to design a novel immunogenic vaccine that could be used to prevent VL infection.

Communicated by Ramaswamy H. Sarma     

Habitat occupancy of sloth bear Melursus ursinus in Chitwan National Park,Nepal

Mammals have experienced a massive decline in their populations and geographic ranges worldwide. The sloth bear, Melursus ursinus (Shaw, 1791), is one of many species facing conservation threats. Despite being endangered in Nepal, decades of inattention to the situation have hindered their conservation and management. We assessed the distribution and patterns of habitat use by sloth bears in Chitwan National Park (CNP), Nepal. We conducted sign surveys from March to June, 2020, in 4 × 4 km grids (n = 45). We collected detection/non‐detection data along a 4‐km trail that was divided into 20 continuous segments of 200 m each. We obtained environmental, ecological, and anthropogenic covariates to understand determinants of sloth bear habitat occupancy. The data were analyzed using the single‐species single‐season occupancy method, with a spatially correlated detection. Using repeated observations, these models accounted for the imperfect detectability of the species to provide robust estimates of habitat occupancy. The model‐averaged occupancy estimate for the sloth bear was 69% and the detection probability was 0.25. The probability of habitat occupancy by sloth bears increased with the presence of termites and fruits and in rugged, dry, open, undisturbed habitats. Our results indicate that the sloth bear is elusive, functionally unique, and widespread in CNP. Future conservation interventions and action plans aimed at sloth bear management must adequately consider their habitat requirements.     

Is the mechanism of COVID-19 coagulopathy still a rabbit’s hole?

The pathophysiology of COVID-19 is an enigma with its severity often determined by the extent of coagulopathy. Several regulatory pathways targeted by the SARS-CoV-2 include the renin-angiotensin system, von Willebrand Factor, and most importantly, the complement pathway. This article discusses these pathways to help design potential future therapies.     

Immunodeficient mice as hosts for hemoparasitic infections

Thiruchandurai Rajan, Julie Moore and Leonard Shultz here review the evolution of technology in murine xeno-lymphohemopoietic chimeras, produced by engraftment with xenogeneic (fetal or adult) progenitor cells or mature lymphohemopoietic tissues into immunodeficient mice, and their use as hosts for hemoprotozoan parasites. Particular attention is paid to the development of chimeras that house xenogeneic peripheral red blood cells (xeno-RBC). These chimeras are potentially invaluable models for hemoprotozoan parasites, such as Babesia and Plasmodium. There are, however, daunting limitations that have to be overcome before these models can become universally acceptable systems for the study of these parasitic agents.     

Cloning,characterization, expression analysis and inhibition studies of a novel gene encoding Bowman–Birk type protease inhibitor from rice bean

This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman–Birk protease inhibitor (RbTI) from rice bean (Vigna umbellata). A full-length protease inhibitor gene with complete open reading frame of 327 bp encoding 109 amino acids was cloned from rice bean seeds using degenerate primer set. BlastP search revealed that the RbTI encoded amino acid of approx 13.0 kDa and shared 99% homology each with BBI from Phaseolus parvulus, Vigna trilobata and Vigna vexilata. Phylogenetic tree also showed close relationship of RbTI with BBI from other members of Leguminaceae family. RbTI gene was further confirmed as intronless (GenBank accession no. KJ159908). The secondary and 3D-structural models for the RbTI were predicted with homology modeling. qRT-PCR studies revealed the highest RbTI expression in the seeds nearing maturity, whereas the low expression of the gene was noticed in young leaves. The isolated RbTI was successfully expressed in Escherichiacoli and the highest expression was recorded after 5.5 h of induction. Study on the inhibitory activity of expressed protein against the gut proteases of Hessian fly larvae revealed 87% inhibition. The novel RbTI gene will further broaden the pool of plant defense genes and could be an ideal choice for developing transgenic crops resistant to insect pests with high economic value. In addition, it has the potential to be used as a probe for selection of insect- and pathogen-resistant genotypes.     

The hinge region of human thyroid-stimulating hormone (TSH) receptor operates as a tunable switch between hormone binding and receptor activation

The mechanism by which the hinge regions of glycoprotein hormone receptors couple hormone binding to activation of downstream effecters is not clearly understood. In the present study, agonistic (311.62) and antagonistic (311.87) monoclonal antibodies (MAbs) directed against the TSH receptor extracellular domain were used to elucidate role of the hinge region in receptor activation. MAb 311.62 which identifies the LRR/Cb-2 junction (aa 265-275), increased the affinity of TSHR for the hormone while concomitantly decreasing its efficacy, whereas MAb 311.87 recognizing LRR 7-9 (aa 201-259) acted as a non-competitive inhibitor of Thyroid stimulating hormone (TSH) binding. Binding of MAbs was sensitive to the conformational changes caused by the activating and inactivating mutations and exhibited differential effects on hormone binding and response of these mutants. By studying the effects of these MAbs on truncation and chimeric mutants of thyroid stimulating hormone receptor (TSHR), this study confirms the tethered inverse agonistic role played by the hinge region and maps the interactions between TSHR hinge region and exoloops responsible for maintenance of the receptor in its basal state. Mechanistic studies on the antibody-receptor interactions suggest that MAb 311.87 is an allosteric insurmountable antagonist and inhibits initiation of the hormone induced conformational changes in the hinge region, whereas MAb 311.62 acts as a partial agonist that recognizes a conformational epitope critical for coupling of hormone binding to receptor activation. The hinge region, probably in close proximity with the α-subunit in the hormone-receptor complex, acts as a tunable switch between hormone binding and receptor activation.