Multi-Dimensional Screening Strategy for Drug Repurposing with Statistical Framework—A New Road to Influenza Drug discovery Abstract Influenza virus is known for its intermittent outbreaks affecting billions of people worldwide. Several neuraminidase inhibitors have been used in practice to overcome this situation. However, advent of new resistant mutants has limited its clinical utilization. In the recent years drug repurposing technique has attained the limelight as it is cost effective and reduces the time consumed for drug discovery. Here, we present multi-dimensional repurposing strategy that integrates the results of ligand-, energy-, receptor cavity, and shape-based pharmacophore algorithm to effectively identify novel drug candidate for influenza. The pharmacophore hypotheses were generated by utilizing the PHASE module of Schrödinger. The generated hypotheses such as AADP, AADDD, and DDRRNH, respectively, for ligand-, e-pharmacophore and receptor cavity based approach alongside shape of oseltamivir were successfully utilized to screen the DrugBank database. Subsequently, these models were evaluated for their differentiating ability using Enrichment calculation. Receiver operating curve and enrichment factors from the analysis indicate that the models possess better capability to screen actives from decoy set of molecules. Eventually, the hits retrieved from different hypotheses were subjected to molecular docking using Glide module of Schrödinger Suite. The results of different algorithms were then combined to eliminate false positive hits and to demonstrate reliable prediction performance than existing approaches. Of note, Pearson’s correlation coefficients were calculated to examine the extent of correlation between the glide score and IC50 values. Further, the interaction profile, pharmacokinetic, and pharmacodynamics properties were analyzed for the hit compounds. The results from our analysis showed that alprostadil (DB00770) exhibits better binding affinity toward NA protein than the existing drug molecules. The biological activity of the hit was also predicted using PASS algorithm that renders the antiviral activity of the compound. Further, the results were validated using mutation analysis and molecular dynamic simulation studies. Indeed, this integrative filtering is able to exceed accuracy of other state-of-the-art methods for the drug discovery.

Profiles of Physarum Microplasmodial Phosphatase Activity Crucial to Cytoplasmic Streaming and Spherule Formation Abstract This study aimed to investigate for the first time, the profile of Physarum microplasmodial phosphatase (PPH) activity toward the phosphorylated light chain of Physarum myosin II (PLCM) at pH 7.6, the velocity of cytoplasmic streaming, and PPH expression in spherule formation during dark starvation (DS). In this study, we cloned the full-length cDNA of PPH using polymerase chain reaction, based on the N-terminal amino acid sequence of the purified enzyme. The cDNA contained an open reading frame (ORF) of 1245 bp, corresponding to 415 amino acids. We confirmed that a rapid increase in PPH activity toward PLCM and a rapid decrease in cytoplasmic streaming velocity precede spherule formation by Physarum microplasmodia. The profiles of increase in PPH activity toward PLCM, PPH expression, and PPH accumulation during DS were correlated with spherule formation in the Physarum microplasmodia. Moreover, application of the wheat germ cell-free expression system resulted in the successful production of recombinant PPH and in the expression of phosphatase activity toward PLCM. These results suggest that PPH is involved in the cessation of cytoplasmic streaming in Physarum microplasmodia during DS.

Age-Dependent Variation in Glycosylation Features of Alpha-2-Macroglobulin Abstract Alpha-2-macroglobulin (A2M) is a glycosylated broad spectrum inhibitor of numerous proteases, including those involved in blood coagulation. Glycosylation characteristics can affect protein structure and function. This study compares glycosylation characteristics of A2M in newborn umbilical cord (NUCP) and adult pooled plasmas. Peptide N-Glycosidase F treatment was used to evaluate the total N-glycan content of the molecules. Neuraminidase treatment, and affinity for Ricinus Communis Agglutinin I were used to examine terminal sialic acid and galactose content, respectively. Two-dimensional (2D) electrophoresis was used to determine charge-related isoform profiles and fluorophore-assisted carbohydrate electrophoresis (FACE) was used to characterize N-glycan profiles. Results revealed no difference in total N-glycan mass, however, a statistically significant difference was shown in the change in charge associated with sialic acid loss in the NUCP A2M population. 2D electrophoresis indicated a lower pI range for NUCP A2M isoforms. In addition, NUCP A2M displayed a trend toward higher terminal galactose quantities than adult A2M. FACE revealed an increased abundance of more branched, higher molecular weight glycans in NUCP A2M. These differences in glycan branching and charged residues may impact A2M receptor-based clearance and thus could be responsible for the increased A2M concentration seen in NUCP, and newborns.

Tumour Initiation: a Discussion on Evidence for a “Load-Trigger” Mechanism Abstract Appropriate mechanical forces on cells are vital for normal cell behaviour and this review discusses the possibility that tumour initiation depends partly on the disruption of the normal physical architecture of the extracellular matrix (ECM) around a cell. The alterations that occur thence promote oncogene expression. Some questions, that are not answered with certainty by current consensus mechanisms of tumourigenesis, are elegantly explained by the triggering of tumours being a property of the physical characteristics of the ECM, which is operative following loading of the tumour initiation process with a relevant gene variant. Clinical observations are consistent with this alternative hypothesis which is derived from studies that have, together, accumulated an extensive variety of data incorporating biochemical, genetic and clinical findings. Thus, this review provides support for the view that the ECM may have an executive function in induction of a tumour. Overall, reported observations suggest that either restoring an ECM associated with homeostasis or targeting the related signal transduction mechanisms may possibly be utilised to modify or control the early progression of cancers. The review provides a coherent template for discussing the notion, in the context of contemporary knowledge, that tumourigenesis is an alliance of biochemistry, genetics and biophysics, in which the physical architecture of the ECM may be a fundamental component. For more definitive clarification of the concept there needs to be a phalanx of experiments conceived around direct questions that are raised by this paper.

Grape Seed Procyanidins Attenuates Cisplatin-induced Human Embryonic Renal Cell Cytotoxicity by Modulating Heme Oxygenase-1 in Vitro Abstract Cisplatin is a widely used anti-cancer drug. However, cisplatin is limited in clinical treatment because of its severe nephrotoxicity. This study reported whether O-GSP can antagonize the cisplatin-induced cytotoxicity in HEK293 cells through inducing HO-1 protein expression. We previously demonstrated O-GSP can increase the survival rate of HEK293 and have protective effect on HEK293 cells. Herein, We found that O-GSP can antagonize cisplatin nephrotoxicity through regulating the expression of HO-1. O-GSP promotes the translocation of Nrf2 in the nucleus, and activates the ERKN JNK pathway and p38 MAPK pathway. Interestingly, p38 MAPK plays a major role in HO-1 expression induced by O-GSP. And O-GSP can modulate the decrease of Nrf2 and HO-1 expression induced by cisplatin, and improve the cisplatin-induced activity and apoptosis rate of cells by stimulating the expression of HO-1. However, the protective effects of O-GSP are inhibited by ZnPP IX. Collectively, the results indicated that O-GSP induced the expression of HO-1 through p38MAPK and Nrf2 pathway in HEK293 cells.

What is the Mechanism of Nitric Oxide Conversion into Nitrosonium Ions Ensuring S-Nitrosating Processes in Living Organisms Abstract Here, I present the data testifying that the conversion of free radical NO molecules to nitrosonium ions (NO+), which are necessary for the realization of one of NO biological effects (S-nitrosation), may occur in living organisms after binding NO molecules to loosely bound iron (Fe2+ ions) with the subsequent mutual one-electron oxidation–reduction of NO molecules (their disproportionation). Inclusion of thiol-containing substances as iron ligands into this process prevents hydrolysis of NO+ ions bound to iron thus providing the formation of stable dinitrosyl iron complexes (DNIC) with thiol ligands. Such complexes act in living organisms as donors of NO and NO+, providing stabilization and transfer of these agents via the autocrine and paracrine pathways. Without loosely bound iron (labile iron pool) and thiols participating in the DNIC formation, NO functioning as one of universal regulators of diverse metabolic processes would be impossible.

Carnosine Prevents Different Structural Damages Induced by Methylglyoxal in Lens Crystallins Abstract The elevated lenticular level of methylglyoxal (MGO) in diabetic patients is an important risk factor for the development of age-related (senile) cataract disorders. Carnosine (β-alanyl L-histidine), a natural antioxidant dipeptide product, has been indicated to prevent the development of cataract diseases. Therefore, it is possible that the anti-cataract activity of this naturally occurring compound is mediated by its anti-glycoxidation effect. In this study, the eye lens proteins were treated with MGO in the presence of carnosine. Then, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and several spectroscopic evaluations were used with the aim to investigate the possible inhibitory activity of carnosine against the MGO-induced structural damages of eye lens proteins. Our results indicated that MGO causes a substantial structural alteration in the lens proteins. The results of fluorescence assessments also suggested that MGO-induced structural changes were accompanied with the formation of additional chromophores in the protein structure. Also, in the presence of carnosine, a notable reduction in the protein structural damages was observed. Our results may highlight an important protective role of carnosine against MGO-induced structural insults in the lens proteins. This study suggests carnosine supplementation as a possible preventive strategy against development of the diabetic cataracts in human.

Confocal Microscopy Confirmed that in Phosphatidylcholine Giant Unilamellar Vesicles with very High Cholesterol Content Pure Cholesterol Bilayer Domains Form Abstract The cholesterol (Chol) content in the fiber cell plasma membranes of the eye lens is extremely high, exceeding the solubility threshold in the lenses of old humans. This high Chol content forms pure Chol bilayer domains (CBDs) and Chol crystals in model membranes and membranes formed from the total lipid extracts from human lenses. CBDs have been detected using electron paramagnetic resonance (EPR) spin-labeling approaches. Here, we confirm the presence of CBDs in giant unilamellar vesicles prepared using the electroformation method from Chol/1-palmitoyl-2-oleoylphosphocholine and Chol/distearoylphosphatidylcholine mixtures. Confocal microscopy experiments using phospholipid (PL) analog (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine-5,5′-disulfonic acid) and cholesterol analog fluorescent probes (23-(dipyrrometheneboron difluoride)-24-norcholesterol) were performed, allowing us to make three major conclusions: (1) In all membranes with a Chol/PL mixing ratio (expressed as a molar ratio) >2, pure CBDs were formed within the bulk PL bilayer saturated with Chol. (2) CBDs were present as the pure Chol bilayer and not as separate patches of Chol monolayers in each leaflet of the PL bilayer. (3) CBDs, presented as single large domains, were always located at the top of giant unilamellar vesicles, independent of the change in sample orientation (right-side-up/upside-down). Results obtained with confocal microscopy and fluorescent Chol and PL analogs, combined with those obtained using EPR and spin-labeled Chol and PL analogs, contribute to the understanding of the organization of lipids in the fiber cell plasma membranes of the human eye lens.

Licochalcone H Synthesized by Modifying Structure of Licochalcone C Extracted from Glycyrrhiza inflata Induces Apoptosis of Esophageal Squamous Cell Carcinoma Cells Abstract Esophageal cancer is one of the malignant cancers with a low 5-year survival rate. Licochalcone (LC) H, a chemically synthesized substance, is a regioisomer of LCC extracted from licorice. The purpose of this study was to determine whether LCH might have anticancer effect on human esophageal squamous cell carcinoma (ESCC) cell lines via apoptosis signaling pathway. After 48 h of treatment, IC50 of LCH in KYSE 30, KYSE 70, KYSE 410, KYSE 450, and KYSE 510 cells were 15, 14, 18, 15, and 16 μM, respectively. This study demonstrated that LCH potently suppressed proliferation of ESCC cells in a concentration- and time-dependent manner. LCH triggered G2/M-phase arrest by modulating expression levels of cdc2, cyclin B1, p21, and p27. LCH also induced apoptosis of ESCC cells through reactive oxygen species-mediated endoplasmic reticulum (ER) stress via JNK/p38 activation pathways. The anticancer effect of LCH was associated with ER stress and mitochondrial dysfunction. It also affected protein levels of Mcl-1, tBid, Bax, Bcl-2, cytochrome c, Apaf-1, PARP, cleaved-PARP, and ER stress-related proteins (GRP78 and CHOP). Our findings provide the first demonstration that LCH has anticancer effect on ESCC. Thus, LCH might have potential for preventing and/or treating human ESCC.

Knockout of the Placenta Specific 8 Gene Affects the Proliferation and Migration of Human Embryonic Kidney 293T Cell Abstract Candidate oncogene placenta specific 8 (PLAC8) has been identified to participate in different cellular process and human diseases. However, the effects of PLAC8 on cell proliferation and migration in human kidney cancer (KC) remained unclear. In current study, physiological effects of PLAC8 in immortalized human embryonic kidney cell line (HEK293T) were investigated in vitro. Two PLAC8 knockout (KO) cell lines were established via CRISPR/Cas9-mediated methods combined with fluorescence activated single cell sorting. To classify the characteristic of PLAC8 during cell proliferation and migration in HEK293T, cellular proliferative activity was analyzed by cell counting and colony formation assay. Cell cycle distribution was analyzed by flow cytometry. Cellular motile activity was analyzed by wound-healing and migration assay. Further underlying molecular mechanism was explored via western blot. With the KO cell lines, it was found that PLAC8 KO could decrease cell proliferation. Moreover, the inhibitory effects of PLAC8 KO on cell proliferation were associated with a G2/M arrest in cell cycle progression concomitant with a remarkable inhibition of Cyclin B1 and elevation of Cyclin A. The alteration of cell cycle proteins and E-cadherin might further associate with the enhancement of cell motility. Our study revealed a novel role for PLAC8 in cell proliferation and migration of HEK293T cells, which might shed light on further study of PLAC8 on human KC.