👤 Afzal Ansari

ApoB is an essential structural protein for the assembly and secretion of triglyceride-rich lipoproteins and therefore remains a potential target to lower plasma cholesterol levels in hypercholesterolemia patients. To understand the global consequences of APOB gene deficiency, we employed CRISPR-Cas9 system to generate apoB-deficient human hepatoma Huh-7 cells (Ako cells). ApoB was not detectable in the cells and media of the Ako cells. ApoB deficiency had no effect on microsomal triglyceride transfer protein expression and activity. These cells supported apoB48 secretion when transfected with plasmids for the expression of apoB48 suggesting that these cells retain all the lipoprotein assembly and secretion machinery except for apoB expression. APOB gene deficiency had no significant effect on cellular lipid levels, cell growth, and ER stress markers. Proteome analysis of secreted proteins revealed that the most upregulated protein was the vitamin D binding protein, while the most downregulated protein was apoB in Ako cells compared to control cells. This analysis also identified coagulation as an upregulated pathway. Total RNA transcriptome analysis identified DNA replication and complement and coagulation pathways as the most upregulated pathways in Ako cells. Further detailed studies are needed to establish how apoB regulates these pathways. These Ako cells may be useful in studying structure-function analysis of apoB peptides and to address the cellular consequences of disruptions in lipoprotein assembly and secretion. Show less

High apolipoprotein B-containing (apoB-containing) low-density lipoproteins (LDLs) and low apoA1-containing high-density lipoproteins (HDLs) are associated with atherosclerotic cardiovascular diseases. In search of a molecular regulator that could simultaneously and reciprocally control both LDL and HDL levels, we screened a microRNA (miR) library using human hepatoma Huh-7 cells. We identified miR-541-3p that both significantly decreases apoB and increases apoA1 expression by inducing mRNA degradation of 2 different transcription factors, Znf101 and Casz1. We found that Znf101 enhances apoB expression, while Casz1 represses apoA1 expression. The hepatic knockdown of Casz1 in mice increased plasma apoA1, HDL, and cholesterol efflux capacity. The hepatic knockdown of Zfp961, an ortholog of Znf101, reduced lipogenesis and production of triglyceride-rich lipoproteins and atherosclerosis, without causing hepatic lipid accumulation. This study identifies hepatic Znf101/Zfp961 and Casz1 as potential therapeutic targets to alter plasma lipoproteins and reduce atherosclerosis without causing liver steatosis. Show less

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cognitive decline, driven by the accumulation of amyloid-beta plaques and neurofibrillary tangles. It involves the dysfunction of key enzymes such as Acetylcholinesterase (AChE) and β-secretase (BACE1), making them critical targets for therapeutic intervention. In this study we investigated an in-house library of 820 secondary metabolites obtained from Ayurvedic plants against AChE and BACE1 with the aim to discover novel leads for AD. Virtual screening resulted in 15 ligands, mostly belonging to the ursane-type or dammarene-type triterpene saponins of Centella asiatica, reestablishing the potency of this plant in drug discovery against AD. The binding affinities were further verified by molecular dynamics (MD) simulation trajectories, including root mean square fluctuations (RMSF), root mean square deviation (RMSD), hydrogen bonding analysis, Coulomb interaction calculation, Lennard-Jones interactions, and the total interaction energy. Moreover, extensive Principal Component Analysis (PCA) and Gibbs free energy landscape were performed. Our results demonstrated three compounds, namely (S)-eriodictyol 7-O-(6-β-O-trans-p-coumaroyl)-β-d-glucopyranoside, sitoindoside-X and 1,5-di-o-caffeoyl quinic acid as more effective in treating AD due to their comparable drug-like properties. Drug-likeness, structural chemistry, pharmacophore, and ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) analysis support their potential for future drug development. To establish the effectiveness of these lead compounds against AD, additional experimental testing should be performed. Show less

The widespread detection of antibiotics in aquatic environments, particularly in effluent-receiving surface waters, poses significant ecological and public health concerns due to their role in promoting antimicrobial resistance. Accurate trace-level antibiotic measurement is essential for environmental risk assessment and for improving wastewater treatment strategies. This study presents the development, optimization, and validation of two complementary liquid chromatography-mass spectrometry (LC-MS) workflows for the simultaneous quantification of nine antibiotics across five therapeutic classes in creek water impacted by a Common Effluent Treatment Plant (CETP). The performance of a triple quadrupole LC-MS/MS system (LC-QqQ-MS) was compared to that of a high-resolution Orbitrap mass spectrometer (LC-Orbitrap-HRMS). Both instruments demonstrated excellent linearity ( Show less

The discovery of novel therapeutic agents with potent anticancer activity remains a critical challenge in drug development. Natural products, particularly bioactive phytoconstituents derived from plants, have emerged as promising sources for anticancer drug discovery. In this study, we used virtual screening techniques to explore the potential of bioactive phytoconstituents as inhibitors of fibroblast growth factor receptor 1 (FGFR1), a key signaling protein implicated in cancer progression. We used virtual screening techniques to analyze phytoconstituents extracted from the IMPPAT 2.0 database. Our primary objective was to discover promising inhibitors of FGFR1. To ensure the selection of promising candidates, we initially filtered the molecules based on their physicochemical properties. Subsequently, we performed binding affinity calculations, PAINS, ADMET, and PASS filters to identify nontoxic and highly effective hits. Through this screening process, one phytocompound, namely Mundulone, emerged as a potential lead. This compound demonstrated an appreciable affinity for FGFR1 and exhibited specific interactions with the ATP-binding site residues. To gain further insights into the conformational dynamics of Mundulone and the reference FGFR1 inhibitor, Lenvatinib, we conducted time-evolution analyses employing 200 ns molecular dynamics simulations (MDS) and essential dynamics. These analyses provided valuable information regarding the dynamic behavior and stability of the compounds in complexes with FGFR1. Overall, the findings indicate that Mundulone exhibits promising binding affinity, specific interactions, and favorable drug profiles, making it a promising lead candidate. Further experimental analysis will be necessary to confirm its effectiveness and safety profiles for therapeutic advancement in the cancer field. Show less

This study was aimed at assessing the diagnostic utility of whole genome sequence analysis in a well-characterised research cohort of individuals referred with a clinical suspicion of Cornelia de Lange syndrome (CdLS) in whom prior genetic testing had not identified a causative variant. Short-read whole genome sequencing was performed on 195 individuals from 105 families, 108 of whom were affected. 100/108 of the affected individuals had prior relevant genetic testing, with no pathogenic variant being identified. The study group comprised 42 trios in which both parental samples were available for testing (42 affected individuals and 126 unaffected parents), 61 singletons (unrelated affected individuals), and two families with more than one affected individual. The results showed that 32 unrelated probands from 105 families (30.5%) had likely causative coding region-disrupting variants. Four loci were identified in > 1 proband: Show less

Plasma lipids are mainly carried in apolipoprotein B (apoB) containing lipoproteins. High levels of these lipoproteins are associated with several metabolic diseases and lowering their plasma levels is associated with reduced incidence of atherosclerotic cardiovascular disease. MicroRNAs (miRs) are small non-coding RNAs that reduce the protein expression of their target mRNAs and are potential therapeutic agents. Here, we identified a novel miR-615-3p that interacts with human 3'-UTR of apoB mRNA, induces post-transcriptional mRNA degradation, and reduces cellular and secreted apoB100 in human hepatoma Huh-7 cells. Reducing cellular miR-615-3p levels by CRISPR-sgRNA increased cellular and secreted apoB100 indicating endogenous miR regulates apoB expression. Overexpression of miR-615-3p along with or without palmitic acid treatment decreased cellular and media apoB and increased cellular triglyceride levels without inducing endoplasmic reticulum stress. These studies have identified miR-615-3p as a negative regulator of apoB expression in human liver-derived cells. It is likely that there are more miRs that regulate apoB-containing lipoprotein assembly and secretion. Discovery of additional miRs may uncover novel mechanisms that control lipoprotein assembly and secretion. Show less

Alzheimer's disease (AD) is a chronic neurological condition that has become a leading cause of cognitive decline in elder individuals. Hardly any effective medication has been developed to halt the progression of AD due to the disease's complexity. Several theories have been put forward to clarify the mechanisms underlying AD etiology. The identification of amyloid plaques as a hallmark of AD has sparked the development of numerous drugs targeting the players involved in the amyloidogenic pathway, such as the β-site of amyloid precursor protein cleavage enzyme 1 (BACE1) blockers. Over the last ten years, preclinical and early experimental research has led several pharmaceutical companies to prioritize producing BACE1 inhibitors. Despite all these efforts, earlier discovered inhibitors were discontinued in consideration of another second-generation small molecules and recent BACE1 antagonists failed in the final stages of clinical trials because of the complications associated either with toxicity or effectiveness. In addition to discussing the difficulties associated with development of BACE1 inhibitors, this review aims to provide an overview of BACE1 and offer perspectives on the causes behind the failure of five recent BACE1 inhibitors, that would be beneficial for choosing effective treatment approaches in the future. Show less

Developing highly potent covalent inhibitors of Fibroblast growth factor receptors 1 (FGFR1) has always been a challenging task. In the current study, various computational techniques, such as 3D-QSAR, covalent docking, fingerprinting analysis, MD simulation followed by MMGB/PBSA, and per-residue energy decomposition analysis were used to explore the binding mechanism of pyrazolo[3,4-d]pyridazinone derivatives to FGFR1. The high q2 and r2 values for the CoMFA and CoMSIA models, suggest that the constructed 3D-QSAR models could reliably predict the bioactivities of FGFR1 inhibitors. The structural requirements revealed by the model's contour maps were strategically used to computationally create an in-house library of more than 100 new FGFR1 inhibitors using the R-group exploration technique implemented in the Spark Show less

Despite significant advances in developing selective JAK2 inhibitors, JAK2 kinase inhibitor (TKI) therapy is ineffective in suppressing the disease. Reactivation of compensatory MEK-ERK and PI3K survival pathways sustained by inflammatory cytokine signaling causes treatment failure. Concomitant inhibition of MAPK pathway and JAK2 signaling showed improved in vivo efficacy compared to JAK2 inhibition alone but lacked clonal selectivity. We hypothesized that cytokine signaling in JAK2 Show less

Cecal microbiota plays an essential role in chicken health. However, its contribution to fat metabolism, particularly in abdominal fat deposition, which is a severe problem in the poultry industry, is still unclear. Here, chickens at 1, 4, and 12 months of age with significantly (p < 0.05) higher and lower abdominal fat deposition were selected to elucidate fat metabolism. A significantly (p < 0.05) higher mRNA expression of fat anabolism genes (ACSL1, FADS1, CYP2C45, ACC, and FAS), a significantly (p < 0.05) lower mRNA expression of fat catabolism genes (CPT-1 and PPARα) and fat transport gene APOAI in liver/abdominal fat of high abdominal fat deposition chickens indicated that an unbalanced fat metabolism leads to excessive abdominal fat deposition. Parabacteroides, Parasutterella, Oscillibacter, and Anaerofustis were found significantly (p < 0.05) higher in high abdominal fat deposition chickens, while Sphaerochaeta was higher in low abdominal fat deposition chickens. Further, Spearman correlation analysis indicated that the relative abundance of cecal Parabacteroides, Parasutterella, Oscillibacter, and Anaerofustis was positively correlated with abdominal fat deposition, yet cecal Sphaerochaeta was negatively correlated with fat deposition. Interestingly, transferring fecal microbiota from adult chickens with low abdominal fat deposition into one-day-old chicks significantly (p < 0.05) decreased Parabacteroides and fat anabolism genes, while markedly increased Sphaerochaeta (p < 0.05) and fat catabolism genes (p < 0.05). Our findings might help to assess the potential mechanism of cecal microbiota regulating fat deposition in chicken production. Show less

Multiple sclerosis (MS), an immune-mediated and neurodegenerative disorder of the central nervous system (CNS), is characterized by infiltrating myelin-reactive T lymphocytes and demyelinating lesions. Experimental autoimmune encephalomyelitis (EAE) is a well-established animal model used to study MS. To explore the impact of chemokine receptor CCR1 blockade in EAE and the underlying mechanisms, we used CCR1 antagonist J-113863 in PLP Show less

Sinusoidal obstruction syndrome (SOS) is a potentially life-threatening complication after hematopoietic stem cell transplantation (HSCT) or antineoplastic treatment without HSCT. Genetic variants were investigated for their association with SOS, but the evidence is inconclusive. We performed a systematic literature review to identify genes, gene variants, and methods of association analyses of genetic markers with SOS. We identified 23 studies after HSCT and 4 studies after antineoplastic treatment without HSCT. One study (4%) performed whole-exome sequencing (WES) and replicated the analysis in an independent cohort, 26 used a candidate-gene approach. Three studies included >200 participants (11%), and six were of high quality (22%). Variants in 34 genes were tested in candidate gene studies after HSCT. Variants in Show less

Autism spectrum disorder (ASD) is a severe neurodevelopmental disorder characterized by deficits in social interaction, communication, and repetitive behaviors. A key role for immune dysfunction has been suggested in ASD. Recent studies have indicated that inflammatory mediators and Notch-1 signaling may contribute to the development of ASD. Methylmercury chloride (MeHgCl) is an environmental pollutant that primarily affects the central nervous system, causing neurological alterations. Its effects on immunological responses have not been fully investigated in ASD. In this study, we examined the influence of MeHgCl exposure on inflammatory mediators and Notch-1 signaling in BTBR T Show less

Several studies have suggested that chemokine receptors are important mediators of inflammatory response in rheumatoid arthritis (RA). B cells are also known to play an important role in RA pathology. C-X-C chemokine receptor type 3 (CXCR3) is considered a potential therapeutic target in different inflammatory diseases; however, the mechanism remains unclear. Here, we evaluated the potentially protective effect of AMG487, a selective CXCR3 antagonist, in collagen-induced arthritis (CIA) mouse model. CIA mice were treated with AMG487 (5 mg/kg) every 48 h, from day 21 until day 41. We then investigated the effect of AMG487 on NF-κB p65-, NOS2-, MCP-1-, TNF-α-, IFN-γ, IL-4-, and IL-27-producing CD19 Show less

Premature Coronary Artery Disease (PCAD) occurs almost a decade earlier in the South Asian population as compared to the West. Inclusion of genetic information can prove to be a robust measure to improve early risk prediction of PCAD. Aim was to estimate the genotypic distribution and risk allele frequencies of 13 Coronary Artery Disease (CAD) risk Single Nucleotide Polymorphisms in loci identified by the CARDIoGRAMplusC4D consortium namely MIA3 rs17465637; 9p21 rs10757274; CXCL12 rs1746048; APOA5 rs662799; APOB rs1042031; LPA rs3798220; LPA 10455872; MRAS rs9818870; LPL rs328; SORT1 rs646776; PCSK9 rs11591147; APOE rs429358; APOE rs7412 in Pakistani PCAD patients and controls. Moreover, the differential serum cytokine levels (IL-18, IL-10, IL-6, TNF-alpha, IL-18:IL-10 & TNF-alpha:IL-10 ratios) with respect to the genotypic distribution of these selected SNPs were determined. The case-control study was carried out in National University of Sciences and Technology, Islamabad in collaboration with the Cardiovascular Genetics Institute, University College London, UK. Subjects (n=340) with >70% stenosis in at least a single major coronary artery on angiography were taken as PCAD cases along with 310 angiographically verified controls. ELISA was performed for measuring the concentrations of serum IL18, TNFA, IL6 and IL10. Genotyping was done using TAQMAN and KASPar assays. The risk allele frequencies (RAF) of APOE rs7412, CXCL12 rs1746048, 9p21 rs10757274, MIA3 rs17465637 and SORT1 rs646776 were significantly higher in the PCAD cases as compared to the controls. APOE rs429358 had the greatest influence among the selected GWAS/CARDIoGRAMplusC4D consortium CAD risk SNPs by significantly altering the serum levels of TNF-alpha, IL-10 and TNF-alpha:IL-10 ratio. It was followed by APOE rs7412 and CXCL12 rs1746048 which significantly altered the serum levels of IL-18; TNF-alpha and IL-18; IL-18:IL-10 ratio respectively. The cytokine imbalance denoted by IL-18:IL-10 was significantly higher in the risk allele carriers MIA3 rs17465637 and CXCL12 rs1746048 while TNF-alpha:IL-10 ratio was significantly raised in the risk allele carriers of APOE rs429358; MRAS rs9818870 and LPL rs328. The association of the selected SNPs with differential serum cytokine levels especially the cytokine imbalance points towards their potential causal role in the immune inflammatory pathogenic pathway of PCAD. Show less

MicroRNAs (miRNAs) are well-known key regulators of gene expression primarily at the post-transcriptional level. Plant-derived miRNAs may pass through the gastrointestinal tract, entering into the body fluid and regulate the expression of endogenous mRNAs. Show less

Diabetic Nephropathy (DN), a serious complication of Type 2 Diabetic Mellitus (T2DM), is progressive and susceptibility to DN varies among T2DM patients. ApoA5-1131T>C polymorphism revealed that is strongly associated with triglyceride levels and proposed as a predisposing factor for DN. The purpose of this study was to investigate the association -1131T>C ApoA5 gene polymorphism with serum lipids levels in Type 2 diabetic (DM) patients with or without DN in north of Iran (Mazandaran province). This study comprised patients with established T2DM (n=161) and controls (n=58). Genotyping of APOA5 -1131T>C polymorphisms was performed by PCR-RFLP. Diabetic patients were divided into two groups: with nephropathy (DN+, n = 90) and without nephropathy (DN-, n = 71). Lipids and lipoproteins were assessed by enzymatic methods. The genotype frequencies were 63.8 % TT, 31 % TC, 5.2 % CC in controls, 33.8% TT, 52.1 % TC, 14.1 % CC in DN- and 44.4 % TT, 36.7 % TC, 18.9 % CC in DN+ patients. The TC genotype and the CC genotype were overexpressed among DN+ and DN-population in comparison to the control group. The highest and the lowest TG levels in both diabetic patients and controls belonged to CC+TC and TT genotypes, respectively. Furthermore in both patients TG increased with this order: TT< TCC polymorphisms influence lipid levels in type 2 diabetic patients. Show less

Hepatic resection is the most curative treatment option for early-stage hepatocellular carcinoma, but is associated with a high recurrence rate, which exceeds 50% at 5 years after surgery. Understanding the genetic basis of hepatocellular carcinoma at surgically curable stages may enable the identification of new molecular biomarkers that accurately identify patients in need of additional early therapeutic interventions. Whole exome sequencing and copy number analysis was performed on 231 hepatocellular carcinomas (72% with hepatitis B viral infection) that were classified as early-stage hepatocellular carcinomas, candidates for surgical resection. Recurrent mutations were validated by Sanger sequencing. Unsupervised genomic analyses identified an association between specific genetic aberrations and postoperative clinical outcomes. Recurrent somatic mutations were identified in nine genes, including TP53, CTNNB1, AXIN1, RPS6KA3, and RB1. Recurrent homozygous deletions in FAM123A, RB1, and CDKN2A, and high-copy amplifications in MYC, RSPO2, CCND1, and FGF19 were detected. Pathway analyses of these genes revealed aberrations in the p53, Wnt, PIK3/Ras, cell cycle, and chromatin remodeling pathways. RB1 mutations were significantly associated with cancer-specific and recurrence-free survival after resection (multivariate P = 0.038 and P = 0.012, respectively). FGF19 amplifications, known to activate Wnt signaling, were mutually exclusive with CTNNB1 and AXIN1 mutations, and significantly associated with cirrhosis (P = 0.017). RB1 mutations can be used as a prognostic molecular biomarker for resectable hepatocellular carcinoma. Further study is required to investigate the potential role of FGF19 amplification in driving hepatocarcinogenesis in patients with liver cirrhosis and to investigate the potential of anti-FGF19 treatment in these patients. Show less

The peptidyl-prolyl isomerase (PPIase) cyclophilin A (Cpr1p) is conserved from eubacteria to mammals, yet its biological function has resisted elucidation. Unable to identify a phenotype that is suggestive of Cpr1p's function in a cpr1Delta Saccharomyces cerevisiae strain, we screened for CPR1-dependent strains. In all cases, dependence was conferred by mutations in ZPR1, a gene encoding an essential zinc finger protein. CPR1 dependence was suppressed by overexpression of EF1alpha (a translation factor that binds Zpr1p), Cpr6p (another cyclophilin), or Fpr1p (a structurally unrelated PPIase). Suppression by a panel of cyclophilin A mutants correlated with PPIase activity, confirming the relevance of this activity in CPR1-dependent strains. In CPR1(+) cells, wild-type Zpr1p was distributed equally between the nucleus and cytoplasm. In contrast, proteins encoded by CPR1-dependent alleles of ZPR1 accumulated in the nucleus, as did wild-type Zpr1p in cpr1Delta cells. Transport kinetic studies indicated that nuclear export of Zpr1p was defective in cpr1Delta cells, and rescue of this defect correlated with PPIase activity. Our results demonstrate a functional interaction between Cpr1p, Zpr1p, and EF1alpha, a role for Cpr1p in Zpr1p nuclear export, and a biological function for Cpr1p PPIase activity. Show less