A Homogeneous,High-Throughput Assay for Phosphatidylinositol 5-Phosphate 4-Kinase with a Novel,Rapid Substrate Preparation

Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phosphoinositide kinases often use lipid substrates prepared from lyophilized lipid preparations by sonication, which result in variability in the liposome size from preparation to preparation. Herein, we report a homogeneous 1536-well luciferase-coupled bioluminescence assay for PI5P4Kα. The substrate preparation is novel and allows the rapid production of a DMSO-containing substrate solution without the need for lengthy liposome preparation protocols, thus enabling the scale-up of this traditionally difficult type of assay. The Z’-factor value was greater than 0.7 for the PI5P4Kα assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4Kα by assessing the degree of phospho transfer of γ-³²P-ATP to PI5P; its inhibitory activity against PI5P4Kα was confirmed in the present miniaturized assay. From a pilot screen of a library of bioactive compounds, another tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538), was identified as an ATP-competitive inhibitor of PI5P4Kα with an IC₅₀ of 1 µM, affirming the suitability of the assay for inhibitor discovery campaigns. This homogeneous assay may apply to other lipid kinases and should help in the identification of leads for this class of enzymes by enabling high-throughput screening efforts.     

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.

     

Characterization of the full‐length human Grb7 protein and a phosphorylation representative mutant

It is well known the dimerization state of receptor tyrosine kinases (RTKs), in conjunction with binding partners such as the growth factor receptor bound protein 7 (Grb7) protein, plays an important role in cell signaling regulation. Previously, we proposed, downstream of RTKs, that the phosphorylation state of Grb7SH2 domain tyrosine residues could control Grb7 dimerization, and dimerization may be an important regulatory step in Grb7 binding to RTKs. In this manner, additional dimerization‐dependent regulation could occur downstream of the membrane‐bound kinase in RTK‐mediated signaling pathways. Extrapolation to the full‐length (FL) Grb7 protein, and the ability to test this hypothesis further, has been hampered by the availability of large quantities of pure and stable FL protein. Here, we report the biophysical characterization of the FL Grb7 protein and also a mutant representing a tyrosine‐phosphorylated Grb7 protein form. Through size exclusion chromatography and analytical ultracentrifugation, we show the phosphorylated‐tyrosine‐mimic Y492E‐FL‐Grb7 protein (Y492E‐FL‐Grb7) is essentially monomeric at expected physiological concentrations. It has been shown previously the wild‐type FL Grb7(WT‐FLGrb7) protein is dimeric with a dissociation constant (Kd) of approximately 11μM. Our studies here measure a FL protein dimerization K_d of WT‐FL‐Grb7 within one order of magnitude at approximately 1μM. The approximate size and shape of the WT‐FL‐Grb7 in comparison the tyrosine‐phosphorylation mimic Y492E‐FL‐Grb7 protein was determined by dynamic light scattering methods. In vitro phosphorylation of the Grb7SH2 domain indicates only one of the available tyrosine residues is phosphorylated, suggesting the same phosphorylation pattern could be relevant in the FL protein. The biophysical characterization studies in total are interpreted with a view towards understanding the functionally active Grb7 protein conformation.     

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.