Analyzing molecular signatures in preeclampsia and fetal growth restriction: Identifying key genes, pathways, and therapeutic targets for preterm birth.

Background: Intrauterine growth restriction (IUGR) and preeclampsia (PE) are intricately linked with specific maternal health conditions, exhibit shared placental abnormalities, and play pivotal roles in precipitating preterm birth (PTB) incidences. However, the molecular mechanism underlying the association between PE and IUGR has not been determined. Therefore, we aimed to analyze the data of females with PE and those with PE + IUGR to identify the key gene(s), their molecular pathways, and potential therapeutic interactions. Methods: In this study, a comprehensive relationship analysis of both PE and PE + IUGR was conducted using RNA sequence datasets. Using two datasets (GSE148241 and GSE114691), differential gene expression analysis via DESeq2 through R-programming was performed. Gene set enrichment analysis was performed using ClusterProfiler, protein‒protein interaction (PPI) networks were constructed, and cluster analyses were conducted using String and MCODE in Cytoscape. Functional enrichment analyses of the resulting subnetworks were performed using ClueGO software. The hub genes were identified under both conditions using the CytoHubba method. Finally, the most common hub protein was docked against a library of bioactive flavonoids and PTB drugs using the PyRx AutoDock tool, followed by molecular dynamic (MD) simulation analysis. Pharmacokinetic analysis was performed to determine the ADMET properties of the compounds using pkCSM. Results: We identified eight hub genes highly expressed in the case of PE, namely, PTGS2, ENG, KIT, MME, CGA, GAPDH, GPX3, and P4HA1, and the network of the PE + IUGR gene set demonstrated that nine hub genes were overexpressed, namely, PTGS2, FGF7, FGF10, IL10, SPP1, MPO, THBS1, CYBB, and PF4. PTGS2 was the most common hub gene found under both conditions (PE and PEIUGR). Moreover, the greater (-9.1 kcal/mol) molecular binding of flavoxate to PTGS2 was found to have satisfactory pharmacokinetic properties compared with those of other compounds. The flavoxate-bound PTGS2 protein complex remained stable throughout the simulation; with a ligand fit to protein, i.e., a RMSD ranging from ∼2.0 to 4.0 Å and a RMSF ranging from ∼0.5 to 2.9 Å, was observed throughout the 100 ns analysis. Conclusion: The findings of this study may be useful for treating PE and IUGR in the management of PTB.

Genetic insights into obesity: in silico identification of pathogenic SNPs in MBOAT4 gene and their structural molecular dynamics consequences.

Membrane Bound O-Acyltransferase Domain-Containing 4 (MBOAT4) protein catalyzes ghrelin acylation, leading to prominent ghrelin activity, hence characterizing its role as an anti-obesity target. We extracted 625 exonic SNPs from the ENSEMBL database and one phenotype-based missense mutation associated with obesity (A46T) from the HGMD (Human Gene Mutation Database). These were differentiated on deleterious missense SNPs of the MBOAT4 gene through MAF (minor allele frequency: <0.01) cut-off criteria in relation to some bioinformatics-based supervised machine learning tools. We found 8 rare-coding and harmful missense SNPs. The consensus classifier (PredictSNP) tool predicted that the SNP (G57S, C: rs561065025) was the most pathogenic. Several trained in silico algorithms have predicted decreased protein stability [ΔΔG (kcal/mol)] function in the presence of these rare-coding pathogenic mutations in the MBOAT4 gene. Then, a stereochemical quality check (i.e. validation and assessment) of the 3D model was performed, followed by a blind cavity docking approach, used to search for druggable cavities and molecular interactions with citrus flavonoids of the Rutaceae family, ranked with energetic estimations. Significant interactions with Phloretin 3',5'-Di-C-Glucoside were also observed at R304, W306, N307, A311, L314 and H338 with (iGEMDOCK: -95.82 kcal/mol and AutoDock: -7.80 kcal/mol). The RMSD values and other variables of MD simulation analyses on this protein further validated its significant interactions with the above flavonoids. The MBOAT4 gene and its molecular interactions could serve as an interventional future anti-obesity target. The current study's findings will benefit future prospects for large population-based studies and drug development, particularly for generating personalized medicine.Communicated by Ramaswamy H. Sarma.

Understanding the impact of structural modifications at the NNAT gene's post-translational acetylation site: in silico approach for predicting its drug-interaction role in anorexia nervosa.

PURPOSE: Anorexia nervosa (AN) is a neuropsychological public health concern with a socially disabling routine and affects a person's healthy relationship with food. The role of the NNAT (Neuronatin) gene in AN is well established. The impact of mutation at the protein's post-translational modification (PTM) site has been exclusively associated with the worsening of the protein's biochemical dynamics. METHODS: To understand the relationship between genotype and phenotype, it is essential to investigate the appropriate molecular stability of protein required for proper biological functioning. In this regard, we investigated the PTM-acetylation site of the NNAT gene in terms of 19 other specific amino acid probabilities in place of wild type (WT) through various in silico algorithms. Based on the highest pathogenic impact computed through the consensus classifier tool, we generated 3 residue-specific (K59D, P, W) structurally modified 3D models of NNAT. These models were further tested through the AutoDock Vina tool to compute the molecular drug binding affinities and inhibition constant (Ki) of structural variants and WT 3D models. RESULTS: With trained in silico machine learning algorithms and consensus classifier; the three structural modifications (K59D, P, W), which were also the most deleterious substitution at the acetylation site of the NNAT gene, showed the highest structural destabilization and decreased molecular flexibility. The validation and quality assessment of the 3D model of these structural modifications and WT were performed. They were further docked with drugs used to manage AN, it was found that the ΔGbind (kcal/mol) values and the inhibition constants (Ki) were relatively lower in structurally modified models as compared to WT. CONCLUSION: We concluded that any future structural variation(s) at the PTM-acetylation site of the NNAT gene due to possible mutational consequences, will serve as a basis to explore its relationship with the propensity of developing AN. LEVEL OF EVIDENCE: No level of evidence-open access bioinformatics research.

In Silico Characterization of Withania coagulans Bioactive Compounds as Potential Inhibitors of Hydroxymethylglutaryl (HMG-CoA) Reductase of Mus musculus.

Hypercholesterolemia is a mediator for the etiology of cardiovascular diseases, which are characterized as the global leading cause of mortality. We aimed to investigate the inhibitory activity of Withania coagulans compounds against 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr) of Mus musculus using an extensive in silico approach. The 3D structure of the Hmgcr protein is not yet known, so we performed the homology modeling using MODELLER and SWISS-MODEL tools, followed with structural validation and assessment. The PROCHECK web server showed that the top-ranked homology model from SWISS-MODEL has 93.4% of residues in the most-favorable region, the quality factor was 98%, and the Verify3D score was 91.43%, compared to the other generated models. The druggable protein-binding cavities in a 3D model of Hmgcr were investigated with the aid of commonly prescribed statin compounds using the CB-dock approach. We compiled a 3D compound library of W. coagulans, followed by drug-likeness evaluation, and found 20 eligible compounds. The pattern of consensus residues obtained from the CB-dock procedure was then used for grid-box docking of W. coagulans compounds and statin drugs using AutoDock 4.2, respectively. The results showed that withanolide R (-10.77 kcal/mol), withanolide Q (-10.56 kcal/mol), withanolide J (-10.52 kcal/mol), atorvastatin (-8.99 kcal/mol), simvastatin (-8.66 kcal/mol), and rosuvastatin (-8.58 kcal/mol) were promising candidates that bind Hmgcr protein. The key residues involved in protein-ligand (withanolide R) interactions were Y516, C526, V529, I530, M533, I535, and V537, and the formation of a H-bond was at C526, M533, and I535 residues. M533 was the consensus residue having a tendency to form a H-bond with withanolide Q, too. Molecular dynamics simulations were used to validate the top-ranked docked complexes for the stability of the modeled protein. We also predicted the pharmacokinetic properties of binding affinity-based top-ranked compounds and concluded that they could be used as potential inhibitors of Hmgcr. However, further in vitro and in vivo studies are essential to completing the drug development process.

Antioxidant and anti-inflammatory activities of Centratherum anthelminticum (L.) Kuntze seed oil in diabetic nephropathy via modulation of Nrf-2/HO-1 and NF-κB pathway.

BACKGROUND: Type 2 diabetes mellitus (T2DM) approximately constitutes 90% of the reported cases. 30-40% of diabetics eventually develop diabetic nephropathy (DN); accounting for one of the major causes of morbidity and mortality. Increased glucose autoxidation and non-enzymatic glycation of proteins in diabetic kidneys lead to the excessive generation of reactive oxygen species (ROS) that results in lipid peroxidation and activation of inflammatory mediators which overwhelms the scavenging capacity of the antioxidant defense system (Nrf2/Keap1/HO-1). Centratherum anthelminticum commonly called as kali zeeri (bitter cumin) and its seeds are well known for culinary purposes in Asia (Pakistan). It has reported anti-inflammatory, antioxidant, and anti-diabetic activities. The present study has attempted to explore the in-vivo anti-inflammatory, antioxidant and antihyperglycemic potential of the C. anthelminticum seed's fixed oil (FO) and its fractions in high fat-high fructose-streptozotocin (HF-HFr-STZ) induced T2DM rat model. METHODS: The T2DM rat model was developed by giving a high-fat and high-fructose diet followed by a single intraperitoneal injection of streptozotocin (STZ 60 mg/kg) on 28th day of the trial. After 72 hours of this injection, rats showing fasting blood glucose (FBG) levels≥230 mg/dL were recruited into six groups. These groups were orally administered distilled water (1 mL/kg), Gliclazide (200 mg/kg), Centratherum anthelminticum seed (FO) and its hexane (HF), chloroform (CF) and ethanol (EF) soluble fractions (200 mg/kg each), respectively for 4 weeks (i.e. 28 days). Blood, serum, and kidney tissue samples of euthanized animals were used for biochemical, pro-inflammatory, and antioxidant markers (ELISA, qRT-PCR, and spectrophotometric assays) and histology, respectively. RESULTS: C. anthelminticum FO and its fractions reduced the lipid peroxidation, and improved the antioxidant parameters: enzymatic (SOD, CAT, and GPx), non-enzymatic (GSH), and mRNA expression of anti-inflammatory markers (Nrf-2, keap1, and HO-1). mRNA expression of inflammatory and apoptotic markers (TNF-α, IL-1ß, COX-1, NF-κB, Bax, and Bcl-2) were attenuated along with improved kidney architecture. CONCLUSION: C. anthelminticum can mitigate inflammation and oxidative stress in early DN. The anti-nephropathic effect can be attributed to its ability to down-regulate NF-κB and by bringing the Nrf-2 expression levels to near normal.

Protective role of Avena fatua against drug-induced liver toxicity.

The study was conducted to examine the protective potential of ethanol seed extract (ESEt) of Avena fatua (wild oats) against antituberculosis drug (ATD)-induced hepatotoxicity in rats. Four groups of rats (n=6) were used. Of which, three groups were given ATD (Rimstar 900mg/15kg) and divided them into hepatotoxic control (distilled water 1mL/kg), positive control (silymarin 200mg/kg) and test group (ESEt 800mg/kg). The fourth was the normal control group treated only with distilled water (1mL/kg). All treatments were orally administered in their respective groups for 26 days. On the 27thday, rats were decapitated. Body and liver weights were measured whereas serum and liver samples were collected for biochemical and histopathological assessments. The rats treated with silymarin and ESEt showed a significant decrease (p<0.05, 0.01& 0.0001) in liver enzymes including alanine & aspartate transaminases, gamma glutamyltranspeptidase and alkaline phosphatase. ESEt also improved total bilirubin (particularly indirect bilirubin), total protein, albumin and low density lipoprotein cholesterol levels in test group. The hepatoprotective ability of extract was also evident by histological study of liver tissues of the test group that showed normal architecture as compared to liver of ATD treated hepatotoxic control group displayed heterogeneous hepatocytes, inflamed central vein, fatty deposits, enlarged sinusoid, Kupffer's cells infiltration, hypertrophy and fibrosis. In conclusion, ESEt of A.fatua is hepatoprotective in nature which may be due to the presence of total phenols and flavonoids already reported from the seeds of this plant.

In silico identification of the rare-coding pathogenic mutations and structural modeling of human NNAT gene associated with anorexia nervosa.

PURPOSE: Increased susceptibility towards anorexia nervosa (AN) was reported with reduced levels of neuronatin (NNAT) gene. We sought to investigate the most pathogenic rare-coding missense mutations, non-synonymous single-nucleotide polymorphisms (nsSNPs) of NNAT and their potential damaging impact on protein function through transcript level sequence and structure based in silico approaches. METHODS: Gene sequence, single nucleotide polymorphisms (SNPs) of NNAT was retrieved from public databases and the putative post-translational modification (PTM) sites were analyzed. Distinctive in silico algorithms were recruited for transcript level SNPs analyses and to characterized high-risk rare-coding nsSNPs along with their impact on protein stability function. Ab initio 3D-modeling of wild-type, alternate model prediction for most deleterious nsSNP, validation and recognition of druggable binding pockets were also performed. AN 3D therapeutic compounds that followed rule of drug-likeness were docked with most pathogenic variant of NNAT to estimate the drugs' binding free energies. RESULTS: Conclusively, 10 transcript (201-205)-based nsSNPs from 3 rare-coding missense variants, i.e., rs539681368, rs542858994, rs560845323 out of 840 exonic SNPs were identified. Transcript-based functional impact analyses predicted rs539681368 (C30Y) from NNAT-204 as the high-risk rare-coding pathogenic nsSNP, deviating protein functions. The 3D-modeling analysis of AN drugs' binding energies indicated lowest binding free energy (ΔG) and significant inhibition constant (Ki) with mutant models C30Y. CONCLUSIONS: Mutant model (C30Y) exhibiting significant drug binding affinity and the commonest interaction observed at the acetylation site K59. Thus, based on these findings, we concluded that the identified nsSNP may serve as potential targets for various studies, diagnosis and therapeutic interventions. LEVEL OF EVIDENCE: No level of evidence-open access bioinformatics research.

Ameliorative effect of Spatoglossum asperum and its solvent fractions against acetaminophen-induced liver dysfunction in rats.

Acetaminophen (APAP) is a widely consumed drug for pain management and treatment of pyrexia. However, beyond its recommended dose, it becomes harmful for health and may induce acute liver dysfunction. Current work is designed to ameliorate the APAP induced liver toxicity which was induced in rats by giving intra-peritoneal injection of APAP (800mg/kg) dissolved in 40% polyethylene glycol at day 1 and day 14. APAP dosed/intoxicated rats orally administered either with ethanol extract of Spatoglossum asperum (200mg/kg) and its fractions including n-hexane, chloroform and methanol soluble in a dose of 150mg/kg each daily for 14 days in their respective groups. APAP dosed rats showed remarkable elevation in hepatic biomarkers viz., alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenases, total bilirubin, pro-inflammatory cytokines interleukine-6 and apoptotic protein (caspase-3). In addition, hepatic oxidative stress (lipid per oxidation and indirect nitric oxide) and antioxidant biomarkers (glutathione peroxidase, catalase and reduced glutathione) were also altered. Whereas APAP dosed rats treated with ethanol extract of S. asperum and its fractions showed amelioration in concentration of hepatic enzymes, pro-inflammatory cytokines, apoptotic protein and reduction in hepatic oxidative stress by decreasing the lipid peroxidation, indirect nitric oxide and uplifting the activities of antioxidant enzymes and protein.

Therapeutic role of Rauwolfia serpentina in minimizing the risk of glycosylation and associated biomarkers in experimentally induced type 1 diabetic mice.

Present work investigates the effects of hydro-methanolic roots extract (HyMREt) of Rauwolfia serpentina in type 1 diabetic mice. Mice were divided into normal, diabetic, negative and positive controls (I-IV) and three test (HyMREt doses) groups (V-VII - 50, 100, &150mg/kg). Allocated treatment of each group was given orally for 14 days in overnight fasted state. Percent change in fasting blood glucose (FBG), body weights, body tissue weights, hepatic glycogen, total lipids, glycosylated hemoglobin (HbA1c), complete blood profile and antioxidant enzymes including catalase (CAT) and superoxide dismutase (SOD) were estimated. HyMREt doses produced meaningful (p<0.0001) reduction (-39 to -53%) in FBG. Hemoglobin (Hb) levels were raised, HbA1c were considerably decreased (4.5-3.77%) and glycosylation (HbA1c to Hb) ratio was expressively (p<0.0001) improved in test groups. Dose-wise improvement (p< 0.05) in total glycogen and decrement (p<0.05) in lipids were observed in livers of test groups. HyMREt significantly decreased (p<0.05) percent inhibition of SOD and CAT. HyMREt doses progressively (p<0.05) improved RBC and other hematological parameters while decrement was only noticed in leucocyte counts. Administration of test doses of HyMREt were significantly reduced the glycosylation, oxidative stress and anemia caused by alloxan intoxication in mice.

In silico investigation on alkaloids of Rauwolfia serpentina as potential inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA reductase.

Present work aimed to investigate the in silico activity of the alkaloids of roots of Rauwolfia serpentina as inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). For this purpose, the three-dimensional (3D) structure of the protein HMGCR (PDB ID: 1HW9) was downloaded from Protein Data Bank (PDB) database, as a target enzyme. The structures of twelve alkaloids from the roots of R. serpentina were selected as ligands and docked with the selected HMGCR enzyme using Molegro Virtual Docker (MVD) software. The software 'MVD' computes the binding (atom) energies of selected protein (enzyme) and each ligand at minimum energetic conformation state by using the PLP (Piecewise Linear Potential) scoring mechanism. Docking results of twelve tested alkaloids showed that five alkaloids including compound 1 (ajmalicine), 2 (reserpine), 3 (indobinine), 4 (yohimbine), and 5 (indobine) have displayed the highest MolDock scores and best fit within the prominent active site residues (positioned between 684 and 692 of cis-loop) of HMGCR. According to the lowest MolDock energies obtained through non-covalent interactions of alkaloids with HMGCR, these are characterized to be the potential inhibitors of HMGCR. Therefore, the alkaloids from R. serpentina can effectively suppress the cholesterol biosynthesis pathway through inhibition of HMGCR and can serve as potential lead compounds for the development of new drugs for the treatment of hyperlipidaemia.

Vitamin D and bone metabolism in breast cancer patients in Karachi, Pakistan.

Breast cancer is one of the common types of malignancy worldwide and in Pakistan. The heterogeneous disease itself and its complex treatment leads to various bone-affecting complications that make breast cancer patients more vulnerable to bone fractures. Vitamin D deficiency among these women worsens the condition and promotes breast cancer growth. Thus, the purpose of the study was to assess serum levels of 25-hydroxyvitamin D (25OHD) and bone markers in women suffering from breast cancer. Serum levels of 25OHD, alkaline phosphatase (ALP), bone specific ALP, calcium (Ca), phosphorus (P), magnesium (Mg), albumin (Alb) and beta carboxyl terminal collagen crosslink (ß-CTx) were analyzed in 201 histological diagnosed patient volunteers from breast cancer clinic. Vitamin D insufficiency was present among the total study population and deficiency was particularly observed among women with metastases. These patients had significantly increased serum levels of ß-CTx and bone specific ALP when compared with the non-metastatic group. No significant difference was observed in other biochemical parameters. A weak correlation between serum levels of 25OHD and ß-CTx was observed. Therefore, monitoring of serum levels of 25OHD and bone markers at the time of diagnosis and during the course of treatment will endeavor a better overall health status.

Presence of gallic acid and rutin improve the hepatoprotective strength of Withania coagulans.

Carbon tetrachloride (CCl4) is one of the chemicals used in industry reported to accelerate the risk of liver diseases in workers especially in developing countries, if it is not handled carefully. Therefore, the present study conducted to evaluate the liver protective and oxidative stress reducing activities of methanolic (MFEt) and aqueous methanolic fruits (AqMFEt) extracts of Withania coagulans against CCl4-induced liver damage in rats. These fruits extracts in oral doses of 800 mg/kg were found effective in their respective test groups in decreasing weight loss, maintaining hepatic membrane integrity, biosynthetic and conjugative abilities by improving liver and bile duct specific enzymes (alanine and aspartate transferases, alkaline phosphatase, γ-glutamyltranstransferase), total protein and bilirubin profiles, uric acid levels plus uplifting the efficacy of hepatic antioxidant enzymes and protein by minimizing lipid peroxidation. All these beneficial effects confirmed by observing normal anatomical features of liver tissues in test groups. Total phenolic compounds were found high in AqMFEt. Interestingly, for the first time, gallic acid and rutin are identified and quantified in these extracts and thought to improve hepatoprotective potential of W. coagulans.