Evaluation and analysis of novel germline variants in ethanol metabolism pathway genes predisposition to liver disease.

The pathogenetic events of liver disease are seemingly determined by factors linked to ethanol metabolism. The variations in genes encoding enzymes of the ethanol metabolic pathway can influence exposure to alcohol and thus may act as risk factors for the development of liver disease. The present study aimed to understand the genetic aspect of germline variations in ethanol metabolic pathway genes in two major categories of liver disease i.e. ALD and NAFLD. Targeted Re-sequencing was performed in the two disease categories along with healthy control followed by an assessment and evaluation of the variants in a case vs control manner. The pathogenicity prediction was evaluated using SIFT, PolyPhen, PROVEN, LRT, CADD, FATHMM, EIGEN, REVEL and VarSome, while MD simulation of a novel significant variant was performed using the GROMACS 5.1.4 package. The annotation of targeted re-sequencing results revealed 2172 variants in different locations of the genes. Upon recurrent assessment predominantly focusing on exonic missense variants from these genes of the alcohol metabolism pathway, the ALDH1L2 [c.337C > G, p.Pro113Ala, (rs199841702)] variant was found highly significant with comprehensive results. The amino acid substitution tool that predicted protein stability due to a point mutation showed a decrease in stability. The genotyping distribution of the identified novel variant in the population revealed that heterozygosity is significantly distributed in ALD patients. However, the predominant association between the inherited variant and the cause of developing disease needs further robust study.

Accelerated identification of serine racemase inhibitor from Centella asiatica.

Serine racemase (SR) converts the free form of L-serine into D-serine (DS) in the mammalian brain. The DS functions as a co-agonist of N-methyl D-aspartate (NMDA) receptor. The over- activation of NMDA receptor leads to many neurological disorders like stroke, amyotrophic lateral sclerosis, Alzheimer's disease and an effective inhibitor of SR could be a corrective method for the receptor over-activation. We report for the first time here a rapid way of purifying and identifying an inhibitor from medicinal plants known to have the neuro-protective effect. We have purified SR inhibitor from the methanolic extract of Centella asiatica by affinity method. High resolution mass spectrometry and infrared spectroscopy were used to identify the ligand to be madecassoside. We have shown the madecassoside binding in silico and its inhibition of recombinant human serine racemase in vitro and ex vivo.

Bioinformatics Analysis and Functional Prediction of Transmembrane Proteins in Entamoeba histolytica.

Entamoeba histolytica is an invasive, pathogenic parasite causing amoebiasis. Given that proteins involved in transmembrane (TM) transport are crucial for the adherence, invasion, and nutrition of the parasite, we conducted a genome-wide bioinformatics analysis of encoding proteins to functionally classify and characterize all the TM proteins in E. histolytica. In the present study, 692 TM proteins have been identified, of which 546 are TM transporters. For the first time, we report a set of 141 uncharacterized proteins predicted as TM transporters. The percentage of TM proteins was found to be lower in comparison to the free-living eukaryotes, due to the extracellular nature and functional diversification of the TM proteins. The number of multi-pass proteins is larger than the single-pass proteins; though both have their own significance in parasitism, multi-pass proteins are more extensively required as these are involved in acquiring nutrition and for ion transport, while single-pass proteins are only required at the time of inciting infection. Overall, this intestinal parasite implements multiple mechanisms for establishing infection, obtaining nutrition, and adapting itself to the new host environment. A classification of the repertoire of TM transporters in the present study augments several hints on potential methods of targeting the parasite for therapeutic benefits.

N-acetyl ornithine deacetylase is a moonlighting protein and is involved in the adaptation of Entamoeba histolytica to nitrosative stress.

Adaptation of the Entamoeba histolytica parasite to toxic levels of nitric oxide (NO) that are produced by phagocytes may be essential for the establishment of chronic amebiasis and the parasite's survival in its host. In order to obtain insight into the mechanism of E. histolytica's adaptation to NO, E. histolytica trophozoites were progressively adapted to increasing concentrations of the NO donor drug, S-nitrosoglutathione (GSNO) up to a concentration of 110 µM. The transcriptome of NO adapted trophozoites (NAT) was investigated by RNA sequencing (RNA-seq). N-acetyl ornithine deacetylase (NAOD) was among the 208 genes that were upregulated in NAT. NAOD catalyzes the deacetylation of N-acetyl-L-ornithine to yield ornithine and acetate. Here, we report that NAOD contributes to the better adaptation of the parasite to nitrosative stress (NS) and that this function does not depend on NAOD catalytic activity. We also demonstrated that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is detrimental to E. histolytica exposed to NS and that this detrimental effect is neutralized by NAOD or by a catalytically inactive NAOD (mNAOD). These results establish NAOD as a moonlighting protein, and highlight the unexpected role of this metabolic enzyme in the adaptation of the parasite to NS.