Association of epilepsy and neurological impairments with homozygous recessive missense mutations found in the genes responsible for ganglioside biosynthesis (ST3GAL5) and calcium voltage-gated channels (CACNA1H) - insights through molecular dynamic simulations.

With over 2.2 million cases, the incidence rate of epilepsy in Pakistan is far higher than the rest of the world due primarily to the frequent, traditionally imposed cousin marriages. In the present study, comprehensive whole exome sequencing (WES) analyses of a three-generation family with four affected members presenting 'unexplained' childhood absence epilepsy (CAE), seizures and dementia, was performed in a quest to identify heritable, epilepsy-causal gene variants to better aid in carrier screening and genetic counselling. The WES data was generated, analyzed, and validated through Sanger's sequencing, molecular dynamic simulation (MDS) analysis, and molecular mechanics with generalized Born and surface area solvation (MM/GBSA) studies. Two homozygous recessive, missense mutations in ST3GAL5 (c.311A > G, p. His104Arg) and CACNA1H (c.6230G > A, p. Arg2077His) genes, earlier regarded as benign or of uncertain significance, have been identified as a potential etiology. Comparative MDS and free binding energy calculations revealed substantial structural perturbations in mutant forms of ST3GAL5 leading to decreased binding and reduced catalytic activity of the p.His104Arg and two other functional variants (p.Val74Glu and p.Arg288Ter) when compared with wild type. Our findings reinforce that WES analyses may uncover 'hidden', heritable variants and together with MDS and MM/GBSA may provide plausible clues to answer the unexplained causes of epilepsy for an effective management and better patient outcome. Further, revisit of epilepsy-associated mutational landscape in population context is imperative as the variants with 'benign' tags may turn out to be 'non-benign', when exist in combination with other benign.Communicated by Ramaswamy H. Sarma.

Epigenetically dysregulated NOTCH-Delta-HES signaling cascade can serve as a subtype classifier for acute lymphoblastic leukemia.

The NOTCH-Delta-HES signaling cascade is regarded as a double-edged sword owing to its dual tumor-suppressor and oncogenic roles, in different cellular environments. In the T-cells, it supports leukemogenesis by promoting differentiation while in B-cells, it controls leukemogenesis by inhibiting early differentiation/inducing growth arrest in the lead to apoptosis. The present study was undertaken to assess if this bi-faceted behavior of NOTCH family can be exploited as a diagnostic biomarker or subtype classifier of acute lymphoblastic leukemia (ALL). In this pursuit, expression of seven NOTCH cascade genes was analyzed in bone marrow (BM) biopsy and blood plasma (BP) of pediatric ALL patients using quantitative PCR (qPCR). Further, promoter DNA methylation status of the differentially expressed genes (DEGs) was assessed by methylation-specific qMSP and validated through bisulphite amplicon sequencing. Whereas hypermethylation of JAG1, DLL1, and HES-2, HES-4, and HES-5 was observed in all patients, NOTCH3 was found hypermethylated specifically in Pre-B ALL cases while DLL4 in Pre-T ALL cases. Aberrant DNA methylation strongly correlated with downregulated gene expression, which restored at complete remission stage as observed in "follow-up/post-treatment" subjects. The subtype-specific ROC curve analysis and Kaplan-Meier survival analysis predicted a clinically applicable diagnostic and prognostic potential of the panel. Moreover, the logistic regression model (Pre-B vs Pre-T ALL) was found to be the best-fitted model (McFadden's R2 = 0.28, F1 measure = 0.99). Whether analyzed in BM-aspirates or blood plasma, the NOTCH epigenetic signatures displayed comparable results (p < 0.001), advocating the potential of NOTCH-Delta-HES cascade, as a subtype classifier, in minimally invasive diagnosis of ALL.

Bioinformatics and systems biology analysis revealed PMID26394986-Compound-10 as potential repurposable drug against covid-19.

The global health pandemic known as COVID-19, which stems from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a significant concern worldwide. Several treatment methods exist for COVID-19; however, there is an urgent demand for previously established drugs and vaccines to effectively combat the disease. Since, discovering new drugs poses a significant challenge, making the repurposing of existing drugs can potentially reduce time and costs compared to developing entirely new drugs from scratch. The objective of this study is to identify hub genes and associated repurposed drugs targeting them. We analyzed differentially expressed genes (DEGs) by analyzing RNA-seq transcriptomic datasets and integrated with genes associated with COVID-19 present in different databases. We detected 173 Covid-19 associated genes for the construction of a protein-protein interaction (PPI) network which resulted in the identification of the top 10 hub genes/proteins (STAT1, IRF7, MX1, IRF9, ISG15, OAS3, OAS2, OAS1, IRF3, and IRF1). Hub genes were subjected to GO functional and KEGG pathway enrichment analyses, which indicated some key roles and signaling pathways that were strongly related to SARS-CoV-2 infections. We conducted drug repurposing analysis using CMap, TTD, and DrugBank databases with these 10 hub genes, leading to the identification of Piceatannol, CKD-712, and PMID26394986-Compound-10 as top-ranked candidate drugs. Finally, drug-gene interactions analysis through molecular docking and validated via molecular dynamic simulation for 80 ns suggests PMID26394986-Compound-10 as the only potential drug. Our research demonstrates how in silico analysis might produce repurposing candidates to help respond faster to new disease outbreaks.Communicated by Ramaswamy H. Sarma.

Scientific considerations in the regulatory approval of generic (or biosimilar) version of enoxaparin sodium - A lifesaving carbohydrate polymer.

Enoxaparin sodium (Clexane®/Klexane®/Lovenox®) is one amongst the few drugs that have assumed a central role as drug of treatment and/or prevention against thromboembolic complications during COVID-19. The increase in demand resulting in many generic (or biosimilar) versions entering the market has increased the risks of quality and safety (including immunogenicity) related issues. Under the circumstances, development of stringent regulatory approaches has received much attention as investigation of new drug delivery systems for improved therapeutic activity. As one of the measures to increase quality testing and ensure uninterrupted supply of this life-saving drug globally, determination of enoxaparin molecular weight (MW) has been added in the United States Pharmacopoeia (USP) monograph for enoxaparin sodium. In addition, the presence of a unique 1,6-anhydro-ring structure at the reducing end of about 15-25% of the poly (oligo) saccharide chains of the generic (or biosimilar) product has been set as a mandatory requirement. This article presents an overview of the scientific considerations in the quality manufacturing and testing of the generic (or biosimilar) enoxaparin for regulatory review and approval. In certain cases of strong analytical similarity (structural and functional), abandonment of in vivo testing in animals and humans represents a major advancement in the approval of generic (or biosimilar) version of innovator enoxaparin sodium (lovenox®, injections).

A novel missense compound heterozygous variant in TLR1 gene is associated with susceptibility to rheumatoid arthritis - structural perspective and functional annotations.

INTRODUCTION: Besides human leukocyte antigen (HLA-DRB1) locus, more than 100 loci across the genome have been identified and linked with the onset, expression and/or progression of rheumatoid arthritis (RA). However, there are still grey areas in our understanding of the key genetic contributors of the disease, particularly in familial cases. METHODS: In the present study, we have performed the whole exome sequencing (WES) of RA patients from two consanguineous families of Pakistan in a quest to identify novel, high-impact, RA-susceptibility genetic variants. RESULTS: Through stepwise filtering, around 17,000 variants (common in the affected members) were recognized, out of which 2651 were predicted to be deleterious. Of these, 196 had direct relevance to RA. When selected for homozygous recessive mode of inheritance, two novel pathogenic variants (c.1324T>C, p.Cys442→Arg442; c.2036T>C, p.Ile679→Thr679) in the TLR1 gene displayed the role of compound heterozygosity in modulating the phenotypic expression and penetrance of RA. The structural and functional consequences of the TLR1 missense single nucleotide mutations (Cys442→Arg442; Ile679→Thr679) were evaluated through molecular dynamic simulation (MDS) studies. Analysis showed domain's rigidification, conferring stability to mutant TLR1-TIR/TIRAP-TIR complex with concomitant increase in molecular interactions with pro-inflammatory cytokines, compared to the wild-type conformation. Gene co-expression network analysis highlighted interlinked partnering genes along with interleukin-6 production of TLR1 (corrected p-value 2.98e-4) and acetylcholine receptor activity of CHRNG (corrected p-value 6.12e-2) as highly enriched associated functions. CONCLUSION: The results, validated through case-control study subjects, suggested that the variants identified through WES and confirmed through Sanger sequencing and MDS are the novel disease variants and are likely to confer RA-susceptibility, independently and/or in a family-specific context. Key Points • Exploration of population based/ethno-specific big data is imperative to identify novel causal variants of RA. • Two new deleterious missense mutations in mutational hotspot exon 4 of TLR1 gene have been identified in Pakistani RA patients. • MD simulation data provides evidence for domain's rigidification, conferring stability to mutant TLR1-TIR/TIRAP-TIR complex, with concomitant increase in production of pro-inflammatory cytokines, thus adding to the onset/erosive outcome of RA.

Molecular Dynamic Simulations Unravel the Underlying Impact of Missense Mutation in Autoimmunity Gene PTPN22 on Predisposition to Rheumatoid Arthritis.

Genetic mutations in various proteins have been implicated with increased risk or severity of rheumatoid arthritis (RA) in different population groups. In the present case-control study, we have investigated the risk association of single nucleotide mutations present in some of the highly reported anti-inflammatory proteins and/or cytokines, with RA susceptibility in the Pakistani subjects. The study involves 310 ethnically and demographically similar participants from whom blood samples were taken and processed for DNA extraction. Through extensive data mining, 5 hotspot mutations reported in 4 genes, that is, interleukin (IL)-4 (-590; rs2243250), IL-10 (-592; rs1800872), IL-10 (-1082; rs1800896), PTPN22 (C1858T; rs2476601), and TNFAIP3 (T380G; rs2230926), were selected for RA susceptibility analyses using genotyping assays. The results demonstrated the association of only 2 DNA variants [rs2243250 (odds ratio, OR = 2.025, 95% confidence interval, CI = 1.357-3.002, P = 0.0005 Allelic) and rs2476601 (OR = 4.25, 95% CI = 1.569-11.55, P = 0.004 Allelic)] with RA susceptibility in the local population. The former single nucleotide mutation was nonfunctional, whereas the latter, residing in the exonic region of a linkage-proven autoimmunity gene PTPN22, was involved in R620→W620 substitution. Comparative molecular dynamic simulations and free-energy calculations revealed a radical impact on the geometry/confirmation of key functional moieties in the mutant protein leading to a rather weak binding of W620 variant with the interacting receptor (SRC kinase). The interaction imbalance and binding instabilities provide convincing clues about the insufficient inhibition of T cell activation and/or ineffective clearance of autoimmune clones-a hallmark of several autoimmune disorders. In conclusion, the present research describes the association of 2 hotspot mutations in IL-4 promoter and PTPN22 gene with RA susceptibility in the Pakistani study cohort. It also details how a functional mutation in PTPN22 impacts the overall protein geometry, charge, and/or receptor interactions to contribute to RA susceptibility.

Comparative molecular docking analysis for analyzing the inhibitory effect of Anakinra and Ustekinumab against IL17F.

Interleukin 17 F is a member of IL-17 cytokine family with a 50% structural homology to IL-17A and plays a significant role either alone or in combination with IL-17A towards inflammation in Rheumatoid arthritis (RA). A growing number of drugs targeting IL-17 pathway are being tested against population specific disease markers. The major objective of this research was to investigate the anti-inflammatory effect of Anakinra (an IL-1 R1 inhibitor) and Ustekinumab (an IL-12 and IL-23 inhibitor) by targeting IL17F. The three dimensional structures of IL17F was taken from PDB while structures of drugs were taken from PubChem database. Docking was performed using MOE and Schrodinger ligand docking software and binding energies, including s-score using London-dG fitness function and glide score using glide internal energy function, between drug and targets were compared. Furthermore, Protein-Drug complex were subjected to 150 ns Molecular Dynamics (MD) Simulations using Schrodinger's Desmond Module. Docking and MD simulation results suggest anakinra as a more potent IL17F inhibitor and forming a more structurally stable complex.Communicated by Ramaswamy H. Sarma.

Commercial Low Molecular Weight Heparins - Patent Ecosystem and Technology Paradigm for Quality Characterization.

Heparin is a subject of ever-growing interest for laboratory researchers and pharmaceutical industry. One of the driving factors is its critical life-saving drug status, which during the COVID-19 pandemic has assumed a central role in disease treatment and/or prevention. Apart, heparin is one amongst few drugs enjoying a "demand constant" status. In 2020, heparin market size was valued to US$6.5 bn., and given the ongoing stability in the COVID-19 health crisis, it is expected to reach US$11.43 bn. by 2027 with yearly growth rate momentum (CAGR) of 3.9% during the forecast period (Pepi et al., Mol Cell Proteomics 20:100,025, 2021). As patent is a limited monopoly, every year, many patents on low molecular weight heparin (LMWH; a chemically or enzymatically degraded product of unfractionated heparin) are losing market exclusivity worldwide, inviting the generic/biosimilar drug manufacturers to capture market share with cheaper drug products. By tracking patent expiration, drugs in patent litigation, regulatory setbacks for innovator companies (such as those seeking data exclusivity or patent term extension), or other unexpected events affecting market demand and competition, generics can make investment decisions in manufacturing off-patent LMWH drug products of commercial significance. However, given the US Food and Drug Administration (FDA), European Medicine Agency (EMA), Drug Regulatory Authority of Pakistan (DRAP), and other regulatory authorities scientifically rigorous standards for generic/biosimilar LMWH drug products marketing approval, the market is secured and momentous for drug makers that could demonstrate through scientific and clinical dataset that the generic/biosimilar LMWH drug product is of the same quality and purity as the innovator drug product. This study presents an overview of the patent landscape of commercially available LMWHs and advanced analytical techniques for their structural and biochemical characterization for quality control and quality assurance during manufacturing and post-marketing. The study also covers FDA, EMA, Health Canada, and DRAP's current approaches to evaluating the generic/biosimilar LMWH drug products for quality, safety including immunogenicity, and efficacy.

Hepatitis C virus transmission cluster among injection drug users in Pakistan.

Hepatitis C virus (HCV) infections are public health problem across the globe, particularly in developing countries. Pakistan has the second highest prevalence of HCV infection worldwide. Limited data exist from Pakistan about persons who inject drugs (PWID) and are at significant risk of exposure to HCV infection and transmission. Serum specimens (n = 110) collected from PWID residing in four provinces were tested for molecular markers of HCV infection. Next generation sequencing (NGS) of the hypervariable region (HVR1) of HCV and Global Hepatitis Outbreak and Surveillance Technology (GHOST) were used to determine HCV genotype, genetic heterogeneity, and construct transmission networks. Among tested specimens, 47.3% were found anti-HCV positive and 34.6% were HCV RNA-positive and belonged to four genotypes, with 3a most prevalent followed by 1a, 1b and 4a. Variants sampled from five cases formed phylogenetic cluster and a transmission network. One case harbored infection with two different genotypes. High prevalence of infections and presence of various genotypes indicate frequent introduction and transmission of HCV among PWID in Pakistan. Identification of a transmission cluster across three provinces, involving 20% of all cases, suggests the existence of a countrywide transmission network among PWIDs. Understanding the structure of this network should assist in devising effective public health strategies to eliminate HCV infection in Pakistan.

Biosimilars: A Comparative Study of Regulatory, Safety and Pharmacovigilance Monograph in the Developed and Developing Economies.

Epitomizing one of the rapidly maturing segments of pharmaceutical industry, biologics gestalt has severely implicated treatment algorithms of many life-threatening diseases especially in oncology, immunology, diabetes, and irresistible infections through integration of biologics in the clinical practice guidelines. As of 2021, the impact is expected to gain resilience as more patents on new biological drugs (such as Erbitux, Avastin, Orencis) are going off. Growing acceptance, trusting on stringent risk-benefits assessment, cost-effectiveness, and potential for return on investment, drive the global market of biosimilars is expected to remain steadfast in the following years; hence knowing about regulatory requirements for approval, opportunities, and barriers to biosimilars uptake in the biggest markets of USA, European Union, Canada, and Asia-Pacific (India and Pakistan) is warranted for development of effective biosimilars marketing strategies. This article reviews the biosimilars development from the beginning (historic) to the end (development & marketing approval perspectives) and then tries to present a clear picture on areas that are still uncertain concerning the biosimilars landscape especially the biologics effect on immunogenicity, the provocative issue of interchangeability, and extrapolation of indications.

A patent-based consideration of latest platforms in the art of directed evolution: a decade long untold story.

Directed (or in vitro) evolution of proteins and metabolic pathways requires tools for creating genetic diversity and identifying protein variants with new or improved functional properties. Besides simplicity, reliability, speed, versatility, universal applicability and economy of the technique, the new science of synthetic biology requires improved means for construction of smart and high-quality mutant libraries to better navigate the sequence diversity. In vitro CRISPR/Cas9-mediated mutagenic (ICM) system and machine-learning (ML)-assisted approaches to directed evolution are now in the field to achieve the goal. This review describes the gene diversification strategies, screening and selection methods, in silico (computer-aided), Cas9-mediated and ML-based approaches to mutagenesis, developed especially in the last decade, and their patent position. The objective behind is to emphasize researchers the need for noting which mutagenesis, screening or selection method is patented and then selecting a suitable restriction-free approach to sequence diversity. Techniques and evolved products subject to patent rights need commercial license if their use is for purposes other than private or experimental research.

De-novo fabrication of sunlight irradiated silver nanoparticles and their efficacy against E. coli and S. epidermidis.

Silver nanoparticles (AgNPs) gained significant attention due to their activity against microbial pathogens, cancer cells, and viral particles etc. Traditional fabrication methods require hazardous chemicals as reducing agents and their usage and disposal pose a significant hazard to environmental ecosystem. Here, a de novo, robust, cost effective and an eco-friendly method is reported to fabricate AgNPs irradiated with sunlight (SL) while using Salvadora persica root extract (SPE) as reducing agent. Sunlight (SL) irradiated S. persica silver nanoparticles (SpNPs) i.e., SL-SpNPs were characterized using multiple techniques and their antibacterial efficacy was evaluated. The SL-SpNPs were synthesized in 10 min. Field emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) analysis revealed their spherical morphology with a size range of 4.5-39.7 nm, while surface plasmon resonance (SPR) peaked at 425 nm. Fourier transform infrared spectroscopy (FTIR) analysis suggested that the reduction of SL-SpNPs was due to the presence of phytochemicals in the SPE. Furthermore, X-ray powder diffraction (P-XRD) pattern depicted the crystal structure of SL-SpNPs, hence proving the presence of AgNPs. Further the antibacterial studies were carried out against Escherichia coli (ATCC 11229) and Staphylococcus epidermidis (ATCC 12228) using Kirby Bauer method. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for E. coli were determined to be 1.5 µg/mL and 3.0 µg/mL respectively while MIC and MBC values for S. epidermidis were found to be 12.5 µg/mL and 25 µg/mL respectively. The solar irradiation-based fabrication method and resulting SL-SpNPs can find their utility in many biomedical and environmental applications.

Forty years of directed evolution and its continuously evolving technology toolbox: A review of the patent landscape.

Generating functional protein variants with novel or improved characteristics has been a goal of the biotechnology industry and life sciences, for decades. Rational design and directed evolution are two major pathways to achieve the desired ends. While rational protein design approach has made substantial progress, the idea of using a method based on cycles of mutagenesis and natural selection to develop novel binding proteins, enzymes and structures has attracted great attention. Laboratory evolution of proteins/enzymes requires new tools and analytical approaches to create genetic diversity and identifying variants with desired traits. In this pursuit, construction of sufficiently large libraries of target molecules to search for improved variants and the need for new protocols to alter the properties of target molecules has been a continuing challenge in the directed evolution experiments. This review will discuss the in vivo and in vitro gene diversification tools, library screening or selection approaches, and artificial intelligence/machine-learning-based strategies to mutagenesis developed in the last 40 years to accelerate the natural process of evolution in creating new functional protein variants, optimization of microbial strains, and transformation of enzymes into industrial machines. Analyzing patent position over these techniques and mechanisms also constitutes an integral and distinctive part of this review. The aim is to provide an up-to-date resource/technology toolbox for research-based and pharmaceutical companies to discover the boundaries of competitor's intellectual property (IP) portfolio, their freedom-to-operate in the relevant IP landscape, and the need for patent due diligence analysis to rule out whether use of a particular patented mutagenesis method, library screening/selection technique falls outside the safe harbor of experimental use exemption. While so doing, we have referred to some recent cases that emphasize the significance of selecting a suitable gene diversification strategy in directed evolution experiments.

Circulating miR-146a expression as a non-invasive predictive biomarker for acute lymphoblastic leukemia.

Dysregulation of non-coding microRNAs during the course of tumor development, invasion and/or progression to the distant organs, makes them a promising candidate marker for the diagnosis of cancer and associated malignancies. This exploratory study aims at evaluating the usefulness of plasma concentration of circulating mir-146a as a non-invasive biomarker for acute lymphoblastic leukemia (ALL). Total RNA including miRNA was isolated from 110 plasma samples of patients (n = 66), healthy controls (n = 24) and follow up (n = 20) cases and reverse transcribed. Relative concentrations were assessed using real-time quantitative PCR and fold-change was calculated by 2-ΔΔCt method. Finally, relative concentrations were correlated to clinicopathological factors. Patients (n = 66) were analyzed to determine fold expression of miR-146a in plasma samples of ALL. Before chemotherapy, pediatric (n = 42) and adult (n = 24) showed overexpression of miR-146a compared with healthy controls (P < 0.0001). There was no effect of age and gender on mir-146a expression in plasma. mirR-146a expression was independent of clinical and hematological features. Moreover, miR-146a levels in plasma of paired samples (n = 20) after treatment showed significant decrease in expression (P < 0.001). Expression of plasma miR-146a may be utilized as non-invasive marker to diagnose and predict prognosis in pediatric and adult patients with ALL. Moreover predicted targets may be utilized for ALL therapy in future.

Whole-genome sequencing as a first-tier diagnostic framework for rare genetic diseases.

Rare diseases affect nearly 300 million people globally with most patients aged five or less. Traditional diagnostic approaches have provided much of the diagnosis; however, there are limitations. For instance, simply inadequate and untimely diagnosis adversely affects both the patient and their families. This review advocates the use of whole genome sequencing in clinical settings for diagnosis of rare genetic diseases by showcasing five case studies. These examples specifically describe the utilization of whole genome sequencing, which helped in providing relief to patients via correct diagnosis followed by use of precision medicine.

Dysregulated Alanine as a Potential Predictive Marker of Glioma-An Insight from Untargeted HRMAS-NMR and Machine Learning Data.

Metabolic alterations play a crucial role in glioma development and progression and can be detected even before the appearance of the fatal phenotype. We have compared the circulating metabolic fingerprints of glioma patients versus healthy controls, for the first time, in a quest to identify a panel of small, dysregulated metabolites with potential to serve as a predictive and/or diagnostic marker in the clinical settings. High-resolution magic angle spinning nuclear magnetic resonance spectroscopy (HRMAS-NMR) was used for untargeted metabolomics and data acquisition followed by a machine learning (ML) approach for the analyses of large metabolic datasets. Cross-validation of ML predicted NMR spectral features was done by statistical methods (Wilcoxon-test) using JMP-pro16 software. Alanine was identified as the most critical metabolite with potential to detect glioma with precision of 1.0, recall of 0.96, and F1 measure of 0.98. The top 10 metabolites identified for glioma detection included alanine, glutamine, valine, methionine, N-acetylaspartate (NAA), γ-aminobutyric acid (GABA), serine, α-glucose, lactate, and arginine. We achieved 100% accuracy for the detection of glioma using ML algorithms, extra tree classifier, and random forest, and 98% accuracy with logistic regression. Classification of glioma in low and high grades was done with 86% accuracy using logistic regression model, and with 83% and 79% accuracy using extra tree classifier and random forest, respectively. The predictive accuracy of our ML model is superior to any of the previously reported algorithms, used in tissue- or liquid biopsy-based metabolic studies. The identified top metabolites can be targeted to develop early diagnostic methods as well as to plan personalized treatment strategies.

A panel of epigenetically dysregulated Wnt signaling pathway genes for non-invasive diagnosis of pediatric acute lymphoblastic leukemia.

BACKGROUND: Genetic and epigenetic dysregulation of Wnt signaling pathway is widely linked up with abnormal proliferation and/or epithelial-to-mesenchymal transition, in different cancer cell types. OBJECTIVE: In the present research, we have tested whether promoter DNA methylation of a set of Wnt/non-Wnt genes such as [cadherin-2 (CDH2)], "present in circulation", could serve as "bone-marrow biopsy surrogate" and help in diagnosing the status, sub-type or treatment outcome in pediatric acute lymphoblastic leukemia (ALL) patients. METHODS: Promoter DNA methylation was quantified in the bisulfite modified blood from the pediatric ALL patients (n= 86) in comparison with age-matched cancer-free subjects (n= 28), using real-time methylation specific PCR followed by rigorous statistical validations. RESULTS: The observed methylation index, sensitivity and specificity of selected molecular markers (viz., SALL1, WNT5α, LRP1b, CDH2) in patients' liquid-biopsies was clinically significant showing high positive correlation in the pre-B ALL cases (p-value < 0.001). A substantial drop in promoter methylation signal of the follow-up/post-treatment patients was also noted (p-value < 0.001), which suggested an impending role of minimally invasive liquid-biopsy approach in the diagnosis and/or therapeutic monitoring of pediatric leukemia. CONCLUSIONS: Whilst the reported metadata provides useful insight into the plausible involvement of epigenetic glitches in leukemogensis, our findings strengthen the remarkable functional consequences of dysregulated Wnt signaling genes in the hematological malignancies besides offering a novel panel of epigenetic marks.

Salvadora persica mediated synthesis of silver nanoparticles and their antimicrobial efficacy.

Silver nanoparticles (AgNPs) exhibit strong antimicrobial properties against many pathogens. Traditionally employed chemical methods for AgNPs synthesis are toxic for the environment. Here, we report a quicker, simpler, and environmentally benign process to synthesize AgNPs by using an aqueous 'root extract' of Salvadora persica (Sp) plant as a reducing agent. The synthesized Salvadora persica nano particles (SpNPs) showed significantly higher antimicrobial efficacy compared to earlier reported studies. We characterized SpNPs using UV-Vis spectroscopy, Fourier Transform Infrared Spectroscopy (FTIR), Transmission Electron Microscopy (TEM), Field Emission Scanning Electron Microscopy (FE-SEM), Dynamic Light Scattering (DLS) and X-ray powder diffraction (P-XRD). UV-Vis spectrum showed the highest absorbance at 420 nm. FTIR analysis depicts presence of bond stretching including OH- (3300 cm-1), C=N- (2100 cm-1) and NH- (1630 cm-1) which are attributed in the involvement of phenolics, proteins or nitrogenous compounds in reduction and stabilization of AgNPs. TEM, FE-SEM and DLS analysis revealed the spherical and rod nature of SpNPs and an average size of particles as 37.5 nm. XRD analysis showed the presence of the cubic structure of Ag which confirmed the synthesis of silver nanoparticles. To demonstrate antimicrobial efficacy, we evaluated SpNPs antimicrobial activity against two bacterial pathogens (Escherichia coli (ATCC 11229) and Staphylococcus epidermidis (ATCC 12228)). SpNPs showed a significantly high inhibition for both pathogens and minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were found to be 0.39 µg/mL and 0.78 µg/mL for E. coli while 0.19 µg/mL and 0.39 µg/mL for S. epidermidis respectively. Further, Syto 16 staining of bacterial cells provided a supplemental confirmation of the antimicrobial efficacy as the bacterial cells treated with SpNPs stop to fluoresce compared to the untreated bacterial cells. Our highly potent SpNPs will likely have a great potential for many antimicrobial applications including wound healing, water purification, air filtering and other biomedical applications.

Impact of IL-17F 7488T/C Functional Polymorphism on Progressive Rheumatoid Arthritis: Novel Insight from the Molecular Dynamic Simulations.

Resorption of bones and cartilage coupled with structural changes in the inflamed joints are the major hallmark of rheumatoid arthritis (RA). Genetic polymorphisms in pro-inflammatory interleukins (ILs) appear to play an important role in the susceptibility towards progressive RA. We therefore aimed to investigate the association of single nucleotide polymorphisms (SNP), present in the hotspot coding/promoter regions of IL-6, -17 and -18, with RA susceptibility or severity in a larger study cohort from Pakistan together with finding clues as to how a functional SNP impacts the predisposition towards RA. TaqMan SNP genotyping approach was first used to assess IL-6 (rs1800795), IL-17 F (rs763780), IL-17A (rs2275913), and IL-18 (rs1946518) polymorphisms in 310 subjects (150 RA and 160 control). Molecular dynamic simulations (MDS) of wild- and mutant-type IL-17A with corresponding receptor were thereafter performed using AMBER-16; Chimera 1.13 was used for analyses. Our results showed the association of two SNPs, namely IL-6 - 174 G/C [allelic (OR = 0.960, 95% CI = 0.929-0.992, p = .009)] and IL-17 F 7488 T/C [allelic (OR = 0.907, 95%CI = 0.861-0.954, p = .000)] with increased RA risk in Pakistani subjects. When mapped, IL-17 F 7488 T/C was found involved in His161→Arg161 change near the C-terminus of IL-17 F. Comparative MDS revealed enhanced stability of the mutant hence advocating a potential role of IL-17F functional SNP in RA susceptibility and/or severity. This study provides a novel structural insight for SNP-derived functional mutation and its overall impact on binding with heterotrimeric receptor complex of IL-17 receptor thereby opening new avenues for understanding the biochemical basis of the disease.

SUMO-fusion and autoinduction-based combinatorial approach for enhanced production of bioactive human interleukin-24 in Escherichia coli.

High-level production of recombinant human interleukin-24 (IL-24), a multifunctional immunomodulatory cytokine, has been challenging due primarily to its aggregation as inclusion bodies in the bacterial host while persistent poor-expression in the insect/mammalian expression systems. The present study presents a robust, vector-host combination (pE-SUMO-IL24), auto-inducible medium (YNG/M9NG), and a simple purification scheme for soluble, bioactive, and cost-effective production of native-like IL-24 (nIL-24) in Escherichia coli. The final protein yield, following a three-step purification scheme (IMAC, SEC, dialysis), was 98 mg/L in shake-flask culture (with scale-up potential), which was several folds higher than reported earlier. In vitro cytotoxicity assays with HeLa and HCT116 cancer cell lines (performed using different concentrations of nIL-24) and the fluorescence activated cell sorting analysis (FACS) revealed a dose- and concentration-dependent increase in the population of pro-apoptotic cells with concomitant, statistically significant drop in the number of cells existent at Go/G1-, S-, and G2/M-phases (P < 0.002). The bioactive nIL-24, developed through this study, holds promise for use in further functional characterizations/applications. KEY POINTS: • Yeast SUMO fusion partner at N-terminus for improved solubility of an otherwise insoluble IL-24 in E. coli. • Enhanced cell densities with concomitant several-fold increase in protein yield by lactose-inducible media. • Improved inhibition of cervical and colorectal carcinomas by native-like nIL-24 compared with Met-containing IL. • Heterologous nIL-24 may enable better understanding of the functional intricacies linked up with its unique cancer-specific features. Graphical abstract.