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.

Investigating the Protein-Based Therapeutic Relationship between Honey Protein SHP-60 and Bevacizumab on Angiogenesis: Exploring the Synergistic Effect through In Vitro and In Silico Analysis.

Honey is a natural product produced by honeybees, which has been used not only as food but also as a medicine by humans for thousands of years. In this study, 60 kDa protein was purified from Pakistani Sidr honey named as SHP-60 (Sidr Honey Protein-60), and its antioxidant potential and the effect of Bevacizumab with purified protein on in vitro angiogenesis using human umbilical vein endothelial cells (HUVEC) were investigated. We further validated the molecular protein-protein (SHP-60 with Bevacizumab) interactions through in silico analysis. It showed very promising antioxidant activity by reducing 2,2-diphenyl-1-picrylhydrazyl free radicals with a maximum of 83% inhibition at 50 µM and an IC50 of 26.45 µM statistically significant (**p < 0.01). Angiogenesis is considered a hallmark of cancer, and without it, the tumor cannot grow or metastasize. Bevacizumab, SHP-60, and both in combination were used to treat HUVEC, and the MTT assay was used to assess cell viability. To demonstrate in vitro angiogenesis, HUVEC was grown on Geltrex, and the formation of endotubes was examined using a tube formation assay. HUVEC viability was dose-dependently decreased by Bevacizumab, SHP-60, and both together. Bevacizumab and SHP-60 both inhibited angiogenesis in vitro, and their combination displayed levels of inhibition even higher than those of Bevacizumab alone. We investigated the protein-protein molecular docking interactions and molecular dynamics simulation analysis of MRJP3 (major royal jelly protein 3) similar to SHP-60 in molecular weight with both the heavy chain (HC) and light chain (LC) of Bevacizumab. We found significant interactions between the LC and MRJP3, indicating that ASN468, GLN470, and ASN473 of MRJP3 interact with SER156, SER159, and GLU161 of LC of Bevacizumab. The integration of experimental data and computational techniques is believed to improve the reliability of the findings and aid in future drug design.

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.

Transcript-Level In Silico Analysis of Alzheimer's Disease-Related Gene Biomarkers and Their Evaluation with Bioactive Flavonoids to Explore Therapeutic Interactions.

Alzheimer's disease (AD) is a progressive brain disorder that can significantly affect the quality of life. We used a variety of in silico tools to investigate the transcript-level mutational impact of exonic missense rare variations (single nucleotide polymorphisms, SNPs) on protein function and to identify potential druggable protein cavities that correspond to potential therapeutic targets for the management of AD. According to the NIA-AA (National Institute on Aging-Alzheimer's Association) framework, we selected three AD biomarker genes (APP, NEFL, and MAPT). We systematically screened transcript-level exonic rare SNPs from these genes with a minor allele frequency of 1% in 1KGD (1000 Genomes Project Database) and gnomAD (Genome Aggregation Database). With downstream functional effect predictions, a single variation (rs182024939: K > N) of the MAPT gene with nine transcript SNPs was identified as the most pathogenic variation from the large dataset of mutations. The machine learning consensus classifier predictor categorized these transcript-level SNPs as the most deleterious variations, resulting in a large decrease in protein structural stability (ΔΔG kcal/mol). The bioactive flavonoid library was screened for drug-likeness and toxicity risk. Virtual screening of eligible flavonoids was performed using the MAPT protein. Identification of druggable protein-binding cavities showed VAL305, GLU655, and LYS657 as consensus-interacting residues present in the MAPT-docked top-ranked flavonoid compounds. The MM/PB(GB)SA analysis indicated hesperetin (-5.64 kcal/mol), eriodictyol (-5.63 kcal/mol), and sakuranetin (-5.60 kcal/mol) as the best docked flavonoids with the near-native binding pose. The findings of this study provide important insights into the potential of hesperetin as a promising flavonoid that can be utilized for further rational drug design and lead optimization to open new gateways in the field of AD therapeutics.

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.

Identification of potential therapeutic intervening targets by in-silico analysis of nsSNPs in preterm birth-related genes.

Prematurity is the foremost cause of death in children under 5 years of age. Genetics contributes to 25-40% of all preterm births (PTB) yet we still need to identify specific targets for intervention based on genetic pathways. This study involved the effect of region-specific non-synonymous variations and their transcript level mutational impact on protein functioning and stability by various in-silico tools. This investigation identifies potential therapeutic targets to manage the challenge of PTB, corresponding protein cavities and explores their binding interactions with intervening compounds. We searched 20 genes coding 55 PTB proteins from NCBI. Single Nucleotide Polymorphisms (SNPs) of concerned genes were extracted from ENSEMBL, and filtration of exonic variants (non-synonymous) was performed. Several in-silico downstream protein functional effect prediction tools were used to identify damaging variants. Rare coding variants were selected with an allele frequency of ≤1% in 1KGD, further supported by South Asian ALFA frequencies and GTEx gene/tissue expression database. CNN1, COL24A1, IQGAP2 and SLIT2 were identified with 7 rare pathogenic variants found in 17 transcript sequences. The functional impact analyses of rs532147352 (R>H) of CNN1 computed through PhD-SNP, PROVEAN, SNP&GO, PMut and MutPred2 algorithms showed impending deleterious effects, and the presence of this pathogenic mutation in CNN1 resulted in large decrease in protein structural stability (ΔΔG (kcal/mol). After structural protein identification, homology modelling of CNN1, which has been previously reported as a biomarker for the prediction of PTB, was performed, followed by the stereochemical quality checks of the 3D model. Blind docking approach were used to search the binding cavities and molecular interactions with progesterone, ranked with energetic estimations. Molecular interactions of CNN1 with progesterone were investigated through LigPlot 2D. Further, molecular docking experimentation of CNN1 showed the significant interactions at S102, L105, A106, K123, Y124 with five selected PTB-drugs, Allylestrenol (-7.56 kcal/mol), Hydroxyprogesterone caproate (-8.19 kcal/mol), Retosiban (-9.43 kcal/mol), Ritodrine (-7.39 kcal/mol) and Terbutaline (-6.87 kcal/mol). Calponin-1 gene and its molecular interaction analysis could serve as an intervention target for the prevention of PTB.

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.

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.

Antioxidant and haematinic effects of methanolic and aqueous methanolic roots extracts of Rauwolfia serpentina Benth in type 2 diabetic mice.

Present investigation was carried out to evaluate the antioxidant and haematinic effects of methanolic (MREt) and aqueous methanolic (AqMREt) root extracts of R. serpentina in mice model of type 2 diabetes (T2D). Experimental mice were divided into nine groups (six per group) as: fructose-induced (T2D) diabetic group (distilled water 1ml/kg), negative control (0.05% DMSO 1ml/kg), positive control (pioglitazone 15mg/kg) and six test groups (MREt 10, 30 & 60mg/kg & AqMREt 50, 100 & 150mg/kg). Whereas tenth group was served as normal control (1ml/kg distilled water). All test doses of MREt & AqMREt significantly (p<0.05) decreases the percent inhibition of catalase (CAT) and superoxide dismutase (SOD) when compared with diabetic controls. Treatment with both extracts also improved the total hemoglobin (Hb), red blood cell (RBC), white blood cell (WBC) counts, packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) in test groups. Fourier transform infrared (FTIR) spectral analysis revealed the presence of phenols moiety in both extracts. Findings suggested that AqMREt possesses more antioxidant and haematinic potential while the MREt of R. serpentina moderately possesses the same activities, which might be due to the high content of phenols present in AqMREt.

Risk association of fasting insulin resistance index in non-diabetic heart failure patients: A case-control study at Karachi, Pakistan.

Present work investigates the risk association of insulin resistance (IResistance) in Non-Diabetic Heart Failure (NDHF) patients. Eighty (n=80) NDHF patients and same numbers of healthy controls were included to investigate with anthropometric measures, fasting blood glucose level (FBGL), serum insulin (SI), FIRI and ß-cells quantification was computed through HOMA-IR. Mean rank assessment of NDHF patients showed higher significant (p<0.0001) set of values in FBGL, SI, FIRI and HOMA-IR, when compared with controls. High (p<0.0001 & p<0.05) risk in NDHF patients was associated in SI status (OR=8.93-95% CI: 4.1-19.42) and also in HOMA-IR (OR=6.6-95% CI: 3.30-13.19), when compared for Pearson value based probability through Chi Square (χ2 Test) values estimates of probability, respectively. Area under the curve (AUC) of targeted NDHF patients showed higher set of estimation (FBGL-AUC =0.667, SI-AUC =0.763, FIRI-AUC=0.780 and HOMA-IR-AUC=0.776). Association of determinants through Pearson's (r) correlation was found significantly (p<0.0001) linked with HOMA-IR and FIRI. Regression coefficient shows that for every additional unit score in FBGL and SI can expect HOMA-IR to increase by an average of 0.883 (for FBGL) and 0.0368 (for SI), respectively. Findings concluded the association of IResistance with greater risk estimation in NDHF patients.

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.

Changing trends of anaemia prevalence among female medical students in a metropolitan setting: Assessment through self-grading and haematological parameters.

OBJECTIVE: To determine the trend of anaemia prevalence among female medical students (FMS) through self-grading along haematological parameters and its association with their nutritional indicators. METHODS: This cross-sectional study was conducted at a public sector health university of Karachi among FMS from April to September 2016. After written informed consent, 216 FMS were assessed for height and weight, for nutrition habits by calculating minimum dietary diversity for women (MDD-W) and anaemia status by self-administered validated self-grading anaemia assessment questionnaire (SGAAQ). The haematological parameters were examined in venous blood sample on Sysmex (XN-3000). The data was analysed using IBM SPSS software version 24. Association between anaemia and nutritional indicators was determined by Chi-square and considered significant when p < 0.05. RESULTS: Anaemia prevalence was 31% with highest frequency among obese (56%) and 29% FMS achieved MDD-W. The mean SGAAQ score, Hb (g/dl), MCV (fl), MCH (pg) and Ret-He (pg) differed significantly (p = <0.001) between anaemic and non-anaemic students. The mean Hb (g/dl) level was significantly higher for FMS who scored MDD-W >5 (p= 0.04). CONCLUSION: Malnutrition and anaemia co-exist despite appropriate awareness of anaemia among FMS. It was associated with self-assessment of anaemia and BMI groups but not with dietary diversity in the present study.

Persea americana seeds improve glycosylation and dyslipidemia in fructose-fed streptozotocin-injected type 2 diabetic male rats.

This work explored the in-vitro phytochemical contents and antidiabetic activity of crude seeds powder of Persea americana (CSSPa) and their in-vivo biochemical effects on glycated hemoglobin, lipid profile and other parameters in type 2 diabetic rats (fructose-STZ model). There were 2 groups of over night fasted rats, control (normal diet) and diabetic (35% Fructose for 6 weeks followed with injection (i.p.) of streptozotocin (STZ) (40mg/kg bw). Diabetic group was further divided into diabetic control, positive control (pioglitazone 15mg) and test (CSSPa 500mg) groups. After the appropriate treatments in each group for 2 weeks fasting glucose level (FGL), serum lipids, insulin, alanine aminotransferase (ALT), creatine Kinase (CK) & uric acid were determined. CSPPa showed presence of alkaloids, flavonoids, phenols etc and potent antidiabetic activity with IC50 13.23±0.76µM. CSPPa treatment showed a significant (p<0.01) decline in lipid profile, while HDL showed significant increase (p<0.01) in test group as compared with positive and diabetic control groups. The serum ALT, CK, uric acid, bilirubin & fasting glucose (fbg) showed significant improvements in test group (p<0.01). Coronary risk index (CRI), Fasting insulin resistance index (FIRI), Percent glycemic change (PGC) and HbA1c values also significantly (p<0.01) improved.

Raisins: A kitchen cabinet item can restores the liver function and structure.

The aqueous methanol extract of raisins (Vitis vinifera) was investigated in carbon tetrachloride (CCl4)-induced hepatotoxic rats model. Where it was found to revert the alteration induced by CCl4 in liver structure and function by improving the body weights, liver index, liver and bile duct specific enzymes, liver conjugative and synthetic markers, reduced glutathione and the total bilirubin/ albumin ratio while increasing the percent inhibition of lipid peroxidation in test groups treated with extract in doses of 400 and 800 mg/kg body weight as compared to negative control group only treated with CCl4 3mL/kg that showed entirely opposite picture of all these parameters. Silymarin 100 mg/kg was used as reference hepatoprotective medicine in present study. In addition, histopathological studies of liver tissues of test groups displayed the restoration of liver anatomy. Therefore, raisins' extract proved to have liver protective, regenerative and antioxidant properties. These might reside in total phenolic content particularly in gallic acid and rutin in extract estimated and detected by spectrophotometric and high performance liquid chromatographic methods.

Beyond sequencing: re-visiting annotations for PJL as a test case.

OBJECTIVES: Current developments in sequencing techniques have enabled rapid and high-throughput generation of sequence data. However, there is a growing gap between the generation of raw sequencing data and the extraction of meaningful biological information. Variant annotation is a crucial step in the analysis of genome sequencing data. Incorrect or incomplete annotations can cause researchers to dilute interesting variants in a pool of false positives. We require consistent, accurate and reliable annotation of variants for making diagnostic and treatment decisions. Current annotation depends on the set of transcripts, and software used can be managed, with sufficient care, in the research context. Careful thought needs to be given to the choice of transcript sets and software packages for variant annotation in sequencing studies. In this project, the main objective is to analyze the genetic variants observed in Pakistani population data within the 1000 genomes project (1KGP). RESULTS: We characterized only SNVs and InDels types of genetic variations, in total ~ 1.4 million variants. Besides this, we also annotated the genetic variants with multiple annotations tools, ANNOVAR and SnpEff and compared the differential results. Our population-specific catalogue will enhance future studies on the functional impact at protein level.

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.

Risk assessment of insulin resistance with anthropometric measures in non-diabetic heart failure patients: A gender based case-control investigation at tertiary care hospitals of Karachi, Pakistan.

To assess the risk of anthropometric measures and serum markers of insulin resistance in non-diabetic heart failure (NDHF) patients and the difference among male and female subjects. 53 males, 27 females NDHF patients were enrolled and 80 healthy subjects were matched as control. Anthropometric measures, fasting blood glucose level (FBGL) and serum insulin (SI) were measured. Insulin's function (ß-cells quantification) was computed through Homeostasis Model Assessment of Insulin Resistance (HOMA-IR). Significant (p<0.05) difference in height was observed in NDHF male and female patients, whereas, weight was only significant in females. Higher mean values of FBGL, SI, and HOMA-IR in NDHF patients were observed. Highly significant (p<0.0001 & p<0.05) difference was also found in similar gender based case-control markers like FBGL, SI, and HOMA-IR. Phi coefficient for risk associations showed weak-positive correlation in both genders in FBGL, SI, and HOMA-IR. Anthropometric measures indicated lesser risk in both gender, especially in females' decreased risk in body height and waist circumference was observed. Risk measurement through odds ratios (OR) of FBGL and HOMA-IR in female subjects indicated significantly (p<0.001) double risk, whereas, in SI, three fold risks were measured in females as compared to male. Odds of exposure in cases were significantly (p<0.001) greater than in controls verified with significant Pearson and Fisher Exact Probability Test (FEPT) values with two-tailed estimates of probability in χ2 (Chi -Square) estimation test. Findings suggest increased risk of IR in female NDHF patients. Increased in FBGL IR and HOMA-IR and BMI were also found as distinguishing findings in NDHF cases.

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.

Ethanolic seeds extract of Centratherum anthelminticum reduces oxidative stress in type 2 diabetes.

This work was accomplished to assess the in-vitro antiglycation and antioxidant activities of ethanolic seeds extract of Centratherum anthelminticum (CSEt), followed by its in-vivo examination in type 2 diabetes. Overnight fasted rabbits were divided into control and diabetic groups. Rabbits in diabetic group were fed with 35% fructose solution to develop hyperglycemia that was well-monitored by glucometer. These were divided into diabetic control (distilled water 1ml/kg), positive control group (pioglitazone 15mg/kg) and two test groups (CSEt 400 and 600mg treated). All treatments were given orally. After 14 days, rabbits were sacrificed and blood samples were used to estimate glycated haemoglobin (HbA1c) while total bilirubin (direct and indirect), uric acid, alanine aminotransferase (ALT) and creatine kinase (CK) were done in sera. In addition, antioxidant parameters viz., catalase (CAT), superoxide dismutase (SOD), reduced glutathione (GSH) and lipid peroxidation (LPO) were in liver tissues. The in-vitro studies showed good antiglycation and antioxidant potential of CSEt. Similarly, in-vivo investigation showed significant reduction in glycemia and body weights in type 2 diabetic test groups. Plus values of HbA1c, ALT, CK, uric acid and bilirubin were almost back to normal along with improvement found in efficiency of antioxidant parameters.