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Monkeypox virus (MPXV) is an orthopoxvirus, causing zoonotic infections in humans with smallpox-like symptoms. The WHO reported MPXV cases in May 2022 and the outbreak caused significant morbidity threats to immunocompromised individuals and children. Currently, no clinically validated therapies are available against MPXV infections. The present study is based on immunoinformatics approaches to design mRNA-based novel vaccine models against MPXV. Three proteins were prioritized based on high antigenicity, low allergenicity, and toxicity values to predict T- and B-cell epitopes. Lead T- and B-cell epitopes were used to design vaccine constructs, linked with epitope-specific linkers and adjuvant to enhance immune responses. Additional sequences, including Kozak sequence, MITD sequence, tPA sequence, Goblin 5', 3' UTRs, and a poly(A) tail were added to design stable and highly immunogenic mRNA vaccine construct. High-quality structures were predicted by molecular modeling and 3D-structural validation of the vaccine construct. Population coverage and epitope-conservancy speculated broader protection of designed vaccine model against multiple MPXV infectious strains. MPXV-V4 was eventually prioritized based on its physicochemical and immunological parameters and docking scores. Molecular dynamics and immune simulations analyses predicted significant structural stability and binding affinity of the top-ranked vaccine model with immune receptors to elicit cellular and humoral immunogenic responses against the MPXV. The pursuance of experimental and clinical follow-up of these prioritized constructs may lay the groundwork to develop safe and effective vaccine against MPXV.Communicated by Ramaswamy H. Sarma.

Montmorillonite clay and agar are naturally occurring materials of significant importance in designing biocompatible materials tailored for applications in biotechnology and medicine. The introduction of magnetic properties has the potential to significantly boost their characteristics and expand their applications. In this study, we have successfully synthesized highly intercalated magnetic composites, incorporating magnetic iron oxide nanoparticles (MNPs), montmorillonite clay (MMT), and agar (AG), through a thermo-physicomechanical method. Three samples of MMT-AG with 2, 1.5, and 0.5% MNPs and three sample composites of MNPs-AG with 2, 1, and 0.5% MMT clay are prepared. The synthesized composites were characterized by SEM, XRD, TGA, DTA, and FTIR. SEM analysis revealed a uniform dispersion of MNPs and MMT in the composite. The XRD pattern confirmed the presence of MNPs in the composite site. The TGA and DTA results demonstrated improved thermal stability due to the MNP incorporation. FTIR spectra showed all of the constituents of agar, MNPs, and MMT clay. The swelling ratio was observed to range from 835% to 1739%. The swelling study indicated an increased hydrophobicity with the addition of MNPs to the composite. Antibacterial activities revealed a significant inhibition of Escherichia coli ( E. coli ) growth by ranging from 10 to 19 nm in the composite. The composite also exhibited a considerable antioxidant action, with IC50 values of 7.96, 46.55, and 57.58 μg/mL, and electrical properties just like conductors., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Background: Unsymmetrical thioureas 1-20 were synthesized and then characterized by various spectroscopy techniques such as UV, IR, fast atom bombardment (FAB)-MS, high-resolution FAB-MS, 1 H-NMR and 13 C-NMR. Methods: Synthetic compounds 1-20 were tested for their ability for antioxidant, lipoxygenase and xanthine oxidase activities. Results: Compounds 1 , 2 , 9 , 12 and 15 exhibited strong antioxidant potential, whereas compounds 1-3 , 9 , 12 , 15 and 19 showed good to moderate lipoxygenase activity. Ten compounds demonstrated moderate xanthine oxidase inhibition. Conclusion: Compound 15 displayed the highest potency among the series, exhibiting good antioxidant, lipoxygenase and xanthine oxidase activities. Theoretical calculations using density functional theory and molecular docking studies supported the experimental findings, indicating the potential of the synthesized compounds as potent antioxidants, lipoxygenases and xanthine oxidase agents.

Three triorganotin(IV) compounds, R 3 Sn(L), with R = CH 3 ( 1 ), n -C 4 H 9 ( 2 ) and C 6 H 5 ( 3 ), and LH = 4-[(2-chloro-4-methylphenyl)carbamoyl]butanoic acid, were prepared and confirmed by various techniques. A five-coordinate, distorted trigonal-bipyramidal geometry was elucidated for tin(IV) centres both in solution and solid states. An intercalation mode was confirmed for the compound SS-DNA interaction by UV-visible, viscometric techniques and molecular docking. MD simulation revealed stable binding of LH with SS-DNA. Anti-bacterial investigation revealed 2 to be generally the most potent, especially against Sa and Ab , i.e. having the lowest MIC values (≤0.25 μg/mL) compared to the standard anti-biotics vancomycin-HCl (MIC = 1 μg/mL) and colistin-sulphate (MIC = 0.25 μg/mL). Similarly, the anti-fungal profile shows 2 exhibits 100% inhibition against Ca and Cn fungal strains and has MIC values (≤0.25 μg/mL) comparatively lower than standard drug fluconazole (0.125 and 8 μg/mL for Ca and Cn , respectively). Compound 2 has the greatest activity with CC 50 ≤ 25 μg/mL and HC 50 > 32 μg/mL performed against HEC239 and RBC cell lines. The anti-cancer potential was assessed against the MG-U87 cell line, using cisplatin as the standard (133 µM), indicates 2 displays the greatest activity (IC 50 : 5.521 µM) at a 5 µM dose. The greatest anti-leishmanial potential was observed for 2 (87.75 at 1000 μg/mL) in comparison to amphotericin B (90.67). The biological assay correlates with the observed maximum of 89% scavenging activity exhibited by 2 . The Swiss-ADME data publicised the screened compounds generally follow the rule of 5 of drug-likeness and have good bioavailability potential.

Inhibitors of α-glucosidase have been used to treat type-2 diabetes (T2DM) by preventing the breakdown of carbohydrates into glucose and prevent enhancing glucose conversion. Structure-based virtual screening (SBVS) was used to generate novel chemical scaffold-ligand α-glucosidase inhibitors. The databases were screened against the receptor α-glucosidase using SBVS and molecular dynamics simulation (MDS) techniques in this study. Based on molecular docking studies, three and two compounds of α-glucosidase inhibitors were chosen from a commercial database (ZINC) and an In-house database for this study respectively. The mode of binding interactions of the selected compounds later predicted their α-glucosidase inhibitory potential. Finally, one out of three lead compound from ZINC and one out of two lead compound from In-house database were shortlisted based on interactions. Furthermore, MDS and post-MDS strategies were used to refine and validate the shortlisted leads along with the reference acarbose/α-glucosidase. The Hits' ability to inhibit α-glucosidase was predicted by SBVS, indicating that these compounds have good inhibitory activities. The lead inhibitor's structure may serve as templates for the design of novel inhibitors, and in vitro testing to confirm their anti-diabetic potential is necessary. These insights can help rationally design new effective anti-diabetic drugs.Communicated by Ramaswamy H. Sarma.

One of the most widespread metabolic diseases, Type-2 Diabetes Mellitus (T2DM) is defined by high blood sugar levels brought on by decreased insulin secretion, reduced insulin action, or both. Due to its cost-effectiveness and eco-friendliness, plant-mediated green synthesis of nanomaterials has become more and more popular. The aim of the study is to synthesize AgNPs, their characterizations and further in-vitro and in-vivo studies. Several methods were used to morphologically characterise the AgNPs. The AgNPs were crystalline, spherical, and clustered, with sizes ranging from 20 to 50 nm. AgNPs were found to contain various functional groups using Fourier transform infrared spectroscopy. This study focuses on the green-synthesis of AgNPs from Fagonia cretica ( F. cretica ) leaves extract to evaluate their synthesized AgNPs for in-vitro and in-vivo anti-diabetic function. For the in-vivo tests, 20 male Balb/C albino-mice were split up into four different groups. Anti-diabetic in-vivo studies showed significant weight gain and a decrease in all biochemical markers (pancreas panel, liver function panel, renal function panel, and lipid profile) in Streptozotocin (STZ)-induced diabetic mice. In vitro anti-diabetic investigations were also conducted on AgNPs, comprising α-amylase, α-glucosidase inhibitions, and antioxidant assays. AgNPs showed antioxidant activity in both the DPPH and ABTS assays. The research showed that the isolated nanoparticles have powerful antioxidant and enzyme inhibitory properties, especially against the main enzymes involved in T2DM., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Khan, Ghufran, Shams, Jamal, Khan, Abdullah, Awan and Khan.)

In the current research work, an amide based metal carboxylate chemical ([((5-((5-(2-hydroxyethyl)-4-methylthiazol-3-ium-3-yl)methyl)-2-methylpyrimidin-4-yl)amino)bis((4-((4-methoxy-2-nitrophenyl)amino)-4-oxobutanoyl)oxy)zinc]) was identified as anti-diabetic analgesic and anti-inflammatory. The identified chemical(MT-1) was tested for acute toxicity (the MT-1 was fund safe), antidiabetic analgesic, and anti-inflammatory potentials. The in-vitro study was conducted for antidiabetic enzyme inhibition (α-amylase and α-glucosidase) and the in-vivo studies included analgesic (acetic acid-induced writing and hot plate model) and anti-inflammatory (carrageenan etc induced edema) effects. The tested compound showed 88.63% (IC 50  = 3.23 μg/ml) and 89.10%(IC 50  = 5.10 μg/ml) againstα-amylase and α-glucosidase respectively. A significant (p < 0.001) analgesic effect was noted by MT-1 in acetic acid-induced animal models with a percent effect of 86.00, 60.,06, and 55.29 at the tested doses of 20, 1,0, and 5 mg/kg respectively. In the case of the hot plate model, the MT-1 showed a significant (p < 0.001) effect with maximum percent prolongation in latency observed after 60 min.08, 22.2,9, and 11.61) against 20, 1,0, and 5 mg/kg. The analgesic effect in the hot plate model was significantly (p < 0.01) reversed by the injection of naloxone (0.125 mg/kg). The paw edema induced by carrageenan, histamine, bradykinin, arachidonic acid, and PGE2 was significantly antagonized with percent attenuation of 34.09, 33.57, 34.60, 34.14, and 48.04 respectively. Furthermore, to predict the interactions between the MT-1 compound and COX-2 molecular docking was carried out and the result was compared with the standard compound. The docking score of MT-1 was predicted as -6.30 while that of Diclofenac was predicted as -6.82. Both compounds made several hydrogen bond interactions with the active site of the COX-2 enzyme. The docking study revealed the potent inhibitory potential of the compound MT-1 against the COX-2 receptor., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Therapeutic moieties derived from medicinal plants as well as plants-based ecofriendly processes for producing selenium nanoparticles have shown great promise in the management of type 2 diabetes mellitus (T2DM). The current study was aimed to assess the anti-diabetic potentials of Fagonia cretica mediated biogenic selenium nanoparticles (FcSeNPs) using in-vitro and in-vivo approaches. The bio-synthesized FcSeNPs were characterized using various techniques including UV-VIS spectrophotometry and FTIR analysis. The in-vitro efficacy of FcSeNPs were assessed against α-glucosidase, α-amylase enzymes as well as the anti-radical studies were performed using DPPH and ABTS free radicals scavenging assays. For in-vivo studies, 20 Male Balb/C albino-mice were randomly divided into 4 groups (n = 5) including normal group, disease group (Diabetic group with no treatment), control group and treatment group (Diabetic group treated with FcSeNPs). Further, biochemistry markers including pancreas, liver, kidney and lipid profile were assessed for all treatment groups. The FcSeNPs exhibited a dose-dependent inhibition against α-amylase and α-glucosidase at 62-1000 µg mL -1 concentration with IC 50 values of 92 and 100 µg mL -1 respectively. In antioxidant experiments, the FcSeNPs demonstrated significant radicals scavenging effect against DPPH and ABTS radicals. In STZ-induced diabetic mice, a considerable decline in blood glucose level was observed after treatment with FcSeNPs. Anti-hyperglycemic effect of FcSeNPs treated animals were high (105 ± 3.22**) as compared to standard drug (128.6 ± 2.73** mg dL -1 ). Biochemical investigations revealed that all biochemical parameters for pancreas, liver function, renal function panel and lipid profile were significantly lowered in FcSeNPs treated animals. Our findings indicate a preliminary multi-target efficacy for FcSeNPs against type-2 diabetes and thus warrant further detailed studies., Competing Interests: Declaration of Competing Interest No potential conflict of interest was reported by the authors., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The core objective of forensic DNA typing is developing DNA profiles from biological evidence for personal identification. The present study was designed to check the validation of the IrisPlex system and the Prevalence of eye colour in the Pakhtoon population residing within the Malakand Division., Methods: Eye colour digital photographs and buccal swab samples of 893 individuals of different age groups were collected. Multiplexed SNaPshot single base extension chemistry was used, and the genotypic results were analysed. Snapshot data were used for eye colour prediction through the IrisPlex and FROG-kb tool., Results: The results of the present study found brown eye colour to be the most prevalent eye colour in comparison to intermediate and blue coloured. Overall, individuals with brown-coloured eyes possess CT (46.84%) and TT (53.16%) genotypes. Blue eye-coloured individuals are solely of the CC genotype, while individuals of intermediate eye colour carry CT (45.15%) and CC (53.85%) genotypes in rs12913832 SNP in the HERC2 gene. It was also revealed that brown-coloured eyes individuals were dominant among all age groups followed by intermediate and blue. Statistical analysis between particular variables and eye colour showed a significant p -value (<0.05) for rs16891982 SNP in SLC45A2 gene, rs12913832 SNP in HERC2 gene, rs1393350 SNP in SLC45A2 , districts and gender. The rest of the SNPs were non-significant with eye colour, respectively. The rs12896399 SNP and SNP rs1800407 were found significant with rs16891982 SNP. The result also demonstrated that the study group differs from the world population based on eye colour. The two eye colour prediction results were compared, and it was discovered that IrisPlex and FROG-Kb had similar higher prediction ratios for Brown and Blue eye colour., Conclusions: The results of the current study revealed brown eye colour to be the most prevalent amongst members of the local population of Pakhtoon ethnicity in the Malakand Division of northern Pakistan. A set of contemporary human DNA samples with known phenotypes are used in this research to evaluate the custom panel's prediction accuracy. With the aid of this forensic test, DNA typing can be supplemented with details about the appearance of the person from whom the sample was taken in cases involving missing persons, ancient human remains, and trace samples. This study may be helpful for future population genetics and forensics studies.

Nanotechnology has recently appeared as an important study subject in modern material sciences. Greener synthesis of nanoparticles has gained the attention of many scientists because of its integral characteristics such as effectiveness, eco-friendly, and low cost. In the present study by following the green synthesis approach, zinc oxide nanoparticles (ZnO NPs) were formed for the very first time by using Senecio chrysanthemoides leaf extract as a reducing agent. The UV-Vis spectrophotometer was used to study the synthesized ZnO NPs, and the specific peak was found to be at 349 nm. The characteristic Fourier transform infrared (FTIR) peak was found to be at 449 cm -1 which displays the peak of ZnO molecules. The surface morphology of the ZnO NPs was determined via scanning electron microscopy (SEM). The energy-dispersive X-ray spectroscopy (EDX) study showed that the synthesized ZnO NPs are present at the weight percentage of 66.38%. The X-ray diffraction (XRD) spectrum confirmed the hexagonal phase wurtzite structure, with the average particle size of 31 nm, and demonstrated the crystalline structure of ZnO NPs. Additionally, to all these experiments, we compared the anti-inflammatory properties of biogenic ZnO NPs to a standard drug. Biosynthesized ZnO NPs have revealed an effective anti-inflammatory activity at a higher concentration (100 mL -1 ) and showed 73% inhibition in comparison with diclofenac sodium drug. Zinc oxide was shown to be compatible with diclofenac sodium, according to the results. The ZnO NPs produced using the greener synthesis process have the potential to be used in a broad range of fields and also used as a good anti-inflammatory agent., Competing Interests: The authors declare no competing financial interest., (Copyright © 2023 Sana Zahoor et al.)

Alzheimer's disease (AD) is a neurodegenerative disorder that affects 35 million people worldwide. However, no potential therapeutics currently are available for AD because of the multiple factors involved in it, such as regulatory factors with their candidate genes, factors associated with the expression levels of its corresponding genes, and many others. To date, 29 novel loci from GWAS have been reported for AD by the Psychiatric Genomics Consortium (PGC2). Nevertheless, the main challenge of the post-GWAS era, namely to detect significant variants of the target disease, has not been conducted for AD. N6-methyladenosine (m6a) is reported as the most prevalent mRNA modification that exists in eukaryotes and that influences mRNA nuclear export, translation, splicing, and the stability of mRNA. Furthermore, studies have also reported m6a's association with neurogenesis and brain development. We carried out an integrative genomic analysis of AD variants from GWAS and m6a-SNPs from m6AVAR to identify the effects of m6a-SNPs on AD and identified the significant variants using the statistically significance value ( p -value <0.05). The cis-regularity variants with their corresponding genes and their influence on gene expression in the gene expression profiles of AD patients were determined, and showed 1458 potential m6a-SNPs (based on p -value <0.05) associated with AD. eQTL analysis showed that 258 m6a-SNPs had cis-eQTL signals that overlapped with six significant differentially expressed genes based on p -value <0.05 in two datasets of AD gene expression profiles. A follow-up study to elucidate the impact of our identified m6a-SNPs in the experimental study would validate our findings for AD, which would contribute to the etiology of AD., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Lumpy skin disease is a fatal emerging disease of cattle, which has started to gain extensive attention due to its rapid incursions across the globe. The disease epidemic causes economic loss and cattle morbidity. Currently, there are no specific treatments and safe vaccines against the lumpy skin disease virus (LSDV) to halt the spread of the disease. The current study uses genome-scan vaccinomics analyses to prioritize promiscuous vaccine candidate proteins of the LSDV. These proteins were subjected to top-ranked B- and T-cell epitope prediction based on their antigenicity, allergenicity, and toxicity values. The shortlisted epitopes were connected using appropriate linkers and adjuvant sequences to design multi-epitope vaccine constructs. Three vaccine constructs were prioritized based on their immunological and physicochemical properties. The model constructs were back-translated to nucleotide sequences and codons were optimized. The Kozak sequence with a start codon along with MITD, tPA, Goblin 5', 3' UTRs, and a poly(A) tail sequences were added to design a stable and highly immunogenic mRNA vaccine. Molecular docking followed by MD simulation analysis predicted significant binding affinity and stability of LSDV-V2 construct within bovine immune receptors and predicted it to be the top-ranked candidate to stimulate the humeral and cellular immunogenic responses. Furthermore, in silico restriction cloning predicted feasible gene expression of the LSDV-V2 construct in a bacterial expression vector. It could prove worthwhile to validate the predicted vaccine models experimentally and clinically against LSDV.

Neisseria gonorrhoeae is an emerging multidrug resistance pathogen that causes sexually transmitted infections in men and women. The N. gonorrhoeae has demonstrated an emerging antimicrobial resistance against reported antibiotics, hence fetching the attention of researchers to address this problem. The present in-silico study aimed to find putative novel drug and vaccine targets against N. gonorrhoeae infection by the application of bioinformatics approaches. Core genes set of 69 N . gonorrhoeae strains was acquired from complete genome sequences. The essential and non-homologous metabolic pathway proteins of N. gonorrhoeae were identified. Moreover, different bioinformatics databases were used for the downstream analysis. The DrugBank database scanning identified 12 novel drug targets in the prioritized list. They were preferred as drug targets against this bacterium. A viable vaccine is unavailable so far against N. gonorrhoeae infection. In the current study, two outer-membrane proteins were prioritized as vaccine candidates via reverse vaccinology approach. The top lead B and T-cells overlapped epitopes were utilized to generate a chimeric vaccine construct combined with immune-modulating adjuvants, linkers, and PADRE sequences. The top ranked prioritized vaccine construct (V7) showed stable molecular interaction with human immune cell receptors as inferred during the molecular docking and MD simulation analyses. Considerable response for immune cells was interpreted by in-silico immune studies. Additional tentative validation is required to ensure the effectiveness of the prioritized vaccine construct against N. gonorrhoeae infection. The identified proteins can be used for further rational drug and vaccine designing to develop potential therapeutic entities against the multi-drug resistant N. gonorrhoeae ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Qasim, Jaan, Wara, Shehroz, Nishan, Shams, Shah and Ojha.)

(1) Background: Dyggve-Melchior-Clausen Syndrome is a skeletal dysplasia caused by a defect in the DYM gene (OMIM number 607461). Pathogenic variants in the gene have been reported to cause Dyggve-Melchior-Clausen (DMC; OMIM 223800) dysplasia and Smith-McCort (SMC; OMIM 607326) dysplasia. (2) Methods: In the present study, large consanguineous families with five affected individuals with osteochondrodysplasia phenotypes were recruited. The family members were analyzed by polymerase chain reaction for homozygosity mapping using highly polymorphic microsatellite markers. Subsequent to linkage analysis, the coding exons and exon intron border of the DYM gene were amplified. The amplified products were then sent for Sanger sequencing. The structural effect of the pathogenic variant was analyzed by different bioinformatics tools. (3) Results: Homozygosity mapping revealed a 9 Mb homozygous region on chromosome 18q21.1 harboring DYM shared by all available affected individuals. Sanger sequencing of the coding exons and exon intron borders of the DYM gene revealed a novel homozygous nonsense variant [DYM (NM&#95;017653.6):c.1205T>A, p.(Leu402Ter)] in affected individuals. All the available unaffected individuals were either heterozygous or wild type for the identified variant. The identified mutation results in loss of protein stability and weekend interactions with other proteins making them pathogenic (4) Conclusions: This is the second nonsense mutation reported in a Pakistani population causing DMC. The study presented would be helpful in prenatal screening, genetic counseling, and carrier testing of other members in the Pakistani community.

(1) Background: Liver fibrosis is currently one of the top ten causes of death worldwide. Stem cells transplantation using mesenchymal stem cells (MSCs) is an alternative therapy which is used in the place of organ transplant, due to the incapacity of stem cells to endure oxidative stress in the damage site, thus affecting the healing process. The present study aimed to enhance the therapeutic potential of MSCs using combined therapy, along with the novel synthetic compounds of benzimidazol derivatives. (2) Methods: Eighteen compound series (benzimidazol derivatives) were screened against liver fibrosis using an in vitro CCl 4 -induced injury model on cultured hepatocytes. IC50 values were calculated on the bases of LDH assay and cell viability assay. (3) Results: Among the eighteen compounds, compounds ( 10 ), ( 14 ) and ( 18 ) were selected on the basis of IC50 value, and compound ( 10 ) was the most potent and had the lowest IC50 value in the LDH assay (8.399 ± 0.23 uM) and cell viability assay (4.73 ± 0.37 uM). Next, these compounds were combined with MSCs using an in vitro hepatocytes injury culture and in vivo rat fibrotic model. The effect of the MSCs + compounds treatment on injured hepatocytes was evaluated using LDH assay, cell viability assay, GSH assay and real-time PCR analysis and immuno-staining for caspase-3. Significant reductions in LDH level, caspase-3 and apoptotic marker genes were noted in MSCs + compounds-treated injured hepatocytes. In vivo data also showed the increased homing of the MSCs, along with compounds after transplantation. Real-time PCR analysis and TUNEL assay results also support our study. (4) Conclusions: It was concluded that compounds ( 10 ), ( 14 ) and ( 18 ) can be used in combination with MSCs to reduce liver fibrosis.

Hepatitis is one of the common liver diseases, imposing a heavy health burden worldwide. Acute hepatitis may develop into chronic hepatitis, progressing to cirrhosis and hepatocellular carcinoma. In the present study, the expression of miRNAs was quantified by real-time PCR, such as miRNA-182, 122, 21, 150, 199, and 222. Along with the control group, HCV was divided into chronic, cirrhosis, and HCC groups. The treated group was also included after the successful treatment of HCV. Biochemical parameters, such as ALT, AST, ALP, bilirubin, viral load, and AFP (HCC), were also evaluated in all of the study groups. We compared the control and diseased groups; these parameters showed significant results ( p = 0.000). The viral load was high in HCV but was not detected after treatment. miRNA-182 and miRNA-21 were overexpressed with disease progression, while the expression of miRNA-122 and miRNA-199 was increased compared with the control, but decreased in the cirrhosis stage compared with chronic and HCC. The expression of miRNA-150 was increased in all of the diseased groups compared with the control, but decreased compared with the chronic group. We compared the chronic and treated groups and then all of these miRNAs were down-regulated after treatment. These microRNAs could be used as potential biomarkers for diagnosing different stages of HCV.

Single-Nucleotide Polymorphisms (SNPs) are common genetic variations implicated in human diseases. The non-synonymous SNPs (nsSNPs) affect the proteins' structures and their molecular interactions with other interacting proteins during the accomplishment of biochemical processes. This ultimately causes proteins functional perturbation and disease phenotypes. The Insulin receptor substrate-2 (IRS-2) protein promotes glucose absorption and participates in the biological regulation of glucose metabolism and energy production. Several IRS-2 SNPs are reported in association with type 2 diabetes and obesity in human populations. However, there are no comprehensive reports about the protein structural consequences of these nsSNPs. Keeping in view the pathophysiological consequences of the IRS-2 nsSNPs, we designed the current study to understand their possible structural impact on coding protein. The prioritized list of the deleterious IRS-2 nsSNPs was acquired from multiple bioinformatics resources, including VEP (SIFT, PolyPhen, and Condel), PROVEAN, SNPs&GO, PMut, and SNAP2. The protein structure stability assessment of these nsSNPs was performed by MuPro and I-Mutant-3.0 servers via structural modeling approaches. The atomic-level structural and molecular dynamics (MD) impact of these nsSNPs were examined using GROMACS 2019.2 software package. The analyses initially predicted 8 high-risk nsSNPs located in the highly conserved regions of IRS-2. The MD simulation analysis eventually prioritized the N232Y, R218C, and R104H nsSNPs that predicted to significantly compromise the structure stability and may affect the biological function of IRS-2. These nsSNPs are predicted as high-risk candidates for diabetes and obesity. The validation of protein structural impact of these shortlisted nsSNPs may provide biochemical insight into the IRS-2-mediated type-2 diabetes., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Twenty-one analogs were synthesized based on benzimidazole, incorporating a substituted benzaldehyde moiety ( 1 - 21 ). These were then screened for their acetylcholinesterase and butyrylcholinesterase inhibition profiles. All the derivatives except 13 , 14 , and 20 showed various inhibitory potentials, ranging from IC 50 values of 0.050 ± 0.001 µM to 25.30 ± 0.40 µM against acetylcholinesterase, and 0.080 ± 0.001 µM to 25.80 ± 0.40 µM against butyrylcholinesterase, when compared with the standard drug donepezil (0.016 ± 0.12 µM and 0.30 ± 0.010 µM, against acetylcholinesterase and butyrylcholinesterase, respectively). Compound 3 in both cases was found to be the most potent compound due to the presence of chloro groups at the 3 and 4 positions of the phenyl ring. A structure-activity relationship study was performed for all the analogs except 13 , 14 , and 20 , further, molecular dynamics simulations were performed for the top two compounds as well as the reference compound in a complex with acetylcholinesterase and butyrylcholinesterase. The molecular dynamics simulation analysis revealed that compound 3 formed the most stable complex with both acetylcholinesterase and butyrylcholinesterase, followed by compound 10 . As compared to the standard inhibitor donepezil both compounds revealed greater stabilities and higher binding affinities for both acetylcholinesterase and butyrylcholinesterase.

Background: Plasmodium vivax apical membrane antigen-1 (pvama-1) is an important vaccine candidate against Malaria. The genetic composition assessment of pvama-1 from wide-range geography is vital to plan the antigen based vaccine designing against Malaria., Methods: The blood samples were collected from 84 P. vivax positive malaria patients from different districts of Khyber Pakhtunkhwa (KP) province of Pakistan. The highly polymorphic and immunogenic domain-I (DI) region of pvama-1 was PCR amplified and DNA sequenced. The QC based sequences raw data filtration was done using DNASTAR package. The downstream population genetic analyses were performed using MEGA4, DnaSP, Arlequin v3.5 and Network.5 resources., Results: The analyses unveiled total 57 haplotypes of pvama-1 (DI) in KP samples with majorly prevalent H-14 and H-5 haplotypes. Pairwise comparative population genetics analyses identified limited to moderate genetic distinctions among the samples collected from different districts of KP, Pakistan. In context of worldwide available data, the KP samples depicted major genetic differentiation against the Korean samples with Fst = 0.40915 (P-value = 0.0001), while least distinction was observed against Indian and Iranian samples. The statistically significant negative values of Fu and Li's D* and F* tests indicate the evidence of population expansion and directional positive selection signature. The slow LD decay across the nucleotide distance in KP isolates indicates low nucleotide diversity. In context of reference pvama-1 sequence, the KP samples were identified to have 09 novel non-synonymous single nucleotide polymorphisms (nsSNPs), including several trimorphic and tetramorphic substitutions. Few of these nsSNPs are mapped within the B-cell predicted epitopic motifs of the pvama-1, and possibly modulate the immune response mechanism., Conclusion: Low genetic differentiation was observed across the pvama-1 DI among the P. vivax isolates acquired from widespread regions of KP province of Pakistan. The information may implicate in future vaccine designing strategies based on antigenic features of pvama-1., (© 2022. The Author(s).)

Introduction: The genomic miscellany of malaria parasites can help inform the intensity of malaria transmission and identify potential deficiencies in malaria control programs. This study was aimed at investigating the genomic miscellany, allele frequencies, and MOI of P. falciparum infection., Methods: A total of 85 P. falciparum confirmed isolates out of 100 were included in this study that were collected from P. falciparum patients aged 4 months to 60 years in nine districts of Khyber Pakhtunkhwa Province. Parasite DNA was extracted from 200µL whole blood samples using the Qiagen DNA extraction kit following the manufacturer's instructions. The polymorphic regions of msp-1, msp-2 and glurp loci were genotyped using nested PCR followed by gel electrophoresis for amplified fragments identification and subsequent data analysis., Results: Out of 85 P. falciparum infections detected, 30 were msp-1 and 32 were msp-2 alleles specific. Successful amplification occurred in 88.23% (75/85) isolates for msp-1, 78.9% (67/85) for msp-2 and 70% (60/85) for glurp gene. In msp-1, the K1 allelic family was predominantly prevalent as 66.66% (50/75), followed by RO33 and MAD20. The frequency of samples with single infection having only K1, MAD20 and RO33 were 21.34% (16/75), 8% (6/75), and 10.67% (8/75), respectively. In msp-2, both the FC27 and 3D7 allelic families revealed almost the same frequencies as 70.14% (47/67) and 67.16% (45/67), respectively. Nine glurp RII region alleles were identified in 60 isolates. The overall mean multiplicity of infection for msp genes was 1.6 with 1.8 for msp-1 and 1.4 for msp-2, while for glurp the MOI was 1.03. There was no significant association between multiplicity of infection and age groups (Spearman's rank coefficient = 0.050; P = 0.6) while MOI and parasite density correlated for only msp-2 allelic marker., Conclusions: The study showed high genetic diversity and allelic frequency with multiple clones of msp-1, msp-2 and glurp in P. falciparum isolates in Khyber Pakhtunkhwa, Pakistan. In the present study the genotype data may provide valuable information essential for monitoring the impact of malaria eradication efforts in this region., Competing Interests: The authors have declared that no competing interests exist.

Pyrazinoic acid-resistant tuberculosis is a severe chronic disorder. First-line drugs specifically target the ribosomal protein subunit-1 (RpsA) and stop trans-translation in the wild-type bacterium, causing bacterial cell death. In mutant bacterial strain, the deletion of ala438 does not let the pyrazinoic acid to bind to the active site of RpsA and ensures that the bacterium survives. Hence, such tuberculosis cases require an immediate and successful regime. The current study was designed to identify inhibitors that could bind to the mutant state of the RpsA protein. Initially, a pharmacophore model was generated based on the recently published most potent inhibitor for the mutant state of RpsA, i.e., zrl 15. The validated pharmacophore model was further used for virtual screening of two chemical libraries, i.e., ZINC and ChemBridge. After applying the Lipinski rule of five (Ro5), a total of 260 and 749 hits from the ChemBridge and ZINC libraries, respectively, were identified using pharmacophore mapping. These hits were then docked into the active site of the mutant state of the RpsA protein, and later, the top 150 compounds from each library were chosen based on the docking score. A total of 21 compounds were shortlisted from each library based on the best protein-ligand interactions. Finally, a total of 05 compounds were subjected to molecular dynamics study to examine the dynamic behavior of each compound in the active site of the mutant state of the RpsA protein. The results revealed that all compounds had good chemical properties such as absorption, distribution, metabolism, excretion, and toxicity (ADMET), and there was no Pan Assay Interference (PAINS) or deviation from Ro5, indicating that these compounds could be useful antagonists for the mutant state of the RpsA protein., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Plant species represent the hierarchical expression of vegetation as it is affected by various environmental gradients. We explored the plant species composition, distribution pattern, communities formation and their respective indicators under the influence of various environmental factors in the Dhirkot region, Azad Jammu and Kashmir. It was hypothesized that different environmental factors were responsible for the formation of various plant communities each with a distinct indicator. Quantitative ecological techniques were used for the sampling of vegetation. A total of 114 quadrats were established in 13 selected sampling sites. Phytosociological attributes were calculated for each plant species at each quadrat. Soil samples were collected and analyzed using different standard protocols. All the collected data were analyzed using Cluster Analysis, Indicator Species Analysis and Canonical Correspondence Analysis of PCORD and CANOCO software, respectively. A total of 145 plant species were recorded belong to 62 different families. Asteraceae and Lamiaceae were the dominant families, represented by 12 species each (8.27%). Cluster Analysis classify all the stations and plants into four major plant communities as 1) Olea-Desmodium-Prunilla community. 2) Abies-Zanthoxylum-Pteracanthus community 3) Cedrus-Elaeagnus-Hypericum community 4) Alnus-Myrsine-Ranunculus community. Soil pH, electrical conductivity, soil saturation, organic matter and altitude were the significant environmental factors that play its essential role in the plant species distribution, composition, formation of major plant communities and their respective indicators in the region. It is recommended that the identified indicator and rare plant species of the investigated area can further be grown for conservation and management purposes in in-situ environment., Competing Interests: The authors have declared that no competing interests exist.

Background: Plasmodium falciparum is an obligate intracellular parasite of humans that causes malaria. Falciparum malaria is a major public health threat to human life responsible for high mortality. Currently, the risk of multi-drug resistance of P. falciparum is rapidly increasing. There is a need to address new anti-malarial therapeutics strategies to combat the drug-resistance threat., Methods: The P. falciparum essential proteins were retrieved from the recently published studies. These proteins were initially scanned against human host and its gut microbiome proteome sets by comparative proteomics analyses. The human host non-homologs essential proteins of P. falciparum were additionally analysed for druggability potential via in silico methods to possibly identify novel therapeutic targets. Finally, the PfAp4AH target was prioritized for pharmacophore modelling based virtual screening and molecular docking analyses to identify potent inhibitors from drug-like compounds databases., Results: The analyses identified six P. falciparum essential and human host non-homolog proteins that follow the key druggability features. These druggable targets have not been catalogued so far in the Drugbank repository. These prioritized proteins seem novel and promising drug targets against P. falciparum due to their key protein-protein interactions features in pathogen-specific biological pathways and to hold appropriate drug-like molecule binding pockets. The pharmacophore features based virtual screening of Pharmit resource predicted a lead compound i.e. MolPort-045-917-542 as a promising inhibitor of PfAp4AH among prioritized targets., Conclusion: The prioritized protein targets may worthy to test in malarial drug discovery programme to overcome the anti-malarial resistance issues. The in-vitro and in-vivo studies might be promising for additional validation of these prioritized lists of drug targets against malaria., (© 2021. The Author(s).)

Liver dysfunction is a major health problem worldwide. Stem cells therapy has opened up new avenues for researches to treat liver diseases due to their multi lineage differentiation. As mesenchymal stem cells (MSCs) can be differentiated into hepatic lineages in the presence of different exogenous factors, the current study aimed to investigate the impact of carbon tetrachloride (CCl 4 ) induced liver injured mice serum on MSCs differentiation toward hepatocytes in vitro. Male Balb/c mice were treated for liver injury with CCl 4 as determined through biochemical tests spectrophotometrically and different growth factors (EGF, HGF) quantification through Sandwich ELISA in both normal and CCl 4 -induced liver injured mice serum. Mice bone marrow derived-MSCs at second passage were treated with normal and CCl 4 -induced liver injured mice serum. After 7 days, serum treated MSCs were investigated for hepatocytes like characteristics through RT-PCR. Serum biochemical tests (Bilirubin, ALT and ALP) and sandwich ELISA results of EGF and HGF showed marked increase in CCl 4 treated mice serum as compared to normal mice serum . Periodic acid Schiff's staining and urea assay kit confirmed high level of glycogen storage and urea production in cells treated with CCl 4 -induced liver injured mice serum. RT-PCR results of CCl 4 -induced liver injured mice serum treated cells also showed expression of hepatic markers (Albumin, Cyto-8, Cyto-18, and Cyto-19). This study confirmed that CCl 4 -induced liver injured serum treatment can differentiate MSCs into hepatocyte-like cells in vitro.

Objective: To determine the frequency of hyperemesis gravidarum (HG) and associated factors among pregnant women., Methods: The hospital-based cross-sectional study was conducted from October 2016 to March 2017 at Lady Reading Hospital (LRH), Peshawar, District Headquarter Hospital (DHQ), Mardan, and District Headquarter Hospital, Nowshera, Khyber Pakhtunkhwa, Pakistan, and comprised data of 146 pregnant women with hyperemesis gravidarum. Data was compiled using pre-designed proforma. Frequency data of HG was also collected from the two hospitals of Peshawar and Mardan presenting in 2015 and 2016. Blood samples of all patients were analysed for serum electrolytes and complete blood count. Data was analyzed using Microsoft Excel 2010.., Results: Mean frequency of HG in LRH Peshawar and DHQ Mardan during 2015 and 2016 was 14.5% and 8.34% respectively. Of the 146 women, 103(70.5%) belonged to Nowshera, 24(16.4%) to Peshawar and 19(13%) to Mardan. The overall mean age was 27±4.9 years, and maximum number of patients 67(45.89%) were aged 26-30 years. Major risk factor was urinary tract infection in Nowshera 30(29%) and Mardan 5(26.3%), while no major factor was identified in Peshawar. Patients in the first trimester were 59(57.28%) in Nowshera, 19(100%) in Mardan and 19(83.3%) in Peshawar, and primigravidas were 19(18.4%), 6(25%) and 8(42%) respectively. Overall, 119(81.5%) patients had no history of abortion., Conclusions: The prevalence of hyperemesis gravidarum was high in Nowshera, Mardan and Peshawar, predominantly during the first trimester of pregnancy.

Bardet-Biedl syndrome (BBS) is characterized by six major features: postaxial polydactyly, obesity, learning disabilities, renal anomalies, retinitis pigmentosa and hypogonadism and is inherited in an autosomal recessive manner. BBS is caused by disease causing sequence variants in the 22 BBS genes identified to date. In the present study, a single consanguineous Pakistani Family with BBS was clinically and genetically characterized. After establishing linkage to a BBS gene on chromosome 4q27, Sanger sequencing was performed in all available affected and unaffected members. Sequence analysis of the BBS7 gene revealed novel substitution mutation (c.719G>T; p. Gly240Val). Our findings further extend the body of evidence implicating BBS7 in causing BBS and expand the mutation spectrum.

The present study aimed to gain insight into the genetic origin of the Shina population from Gilgit-Baltistan, Pakistan. We partially performed the mitochondrial DNA (mtDNA) control region of healthy unrelated individuals of Shina tribe residing in the remote area of Gilgit-Baltistan to investigate their maternal lineages. The present study is the first report about Shina's genetic structure, origin, and relationship with the surrounding north-western Pakistani population. The mtDNA sequences of the Shina samples were compared with the revised Cambridge Reference Sequence (rCRS) and the HVR-1 D-loop region was covered. The comparison with rCRS identified overall 38 haplotypes and 08 haplogroups for Shina samples. Among these haplotypes, 18 were shared by more than one individual of the Shina tribe. The obtained mtDNA sequences of Shina were compared with surrounding north-western Pakistani population groups, i.e. Kho, Kashmiri, and Pathan. The genetic diversity (i.e. 1.0424) and power of discrimination (i.e. 0.9266) of the Shina was found equivalent to surrounding north-western groups. The haplogroups frequencies, phylogenetic tree and network analysis identified the west Eurasian ancestral origin of Shina group with nearby maternal ancestral relationships with the Kashmiri population. However, no close genetic relationship of Shina was depicted with nearby residing Kho population group., Competing Interests: No potential conflict of interest was reported by the authors., (© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Background: Barbituric acid derivatives are a versatile group of compounds which are identified as potential pharmacophores for the treatment of anxiety, epilepsy and other psychiatric disorders. They are also used as anesthetics and have sound effects on the motor and sensory functions. Barbiturates are malonylurea derivatives with a variety of substituents at C-5 position showing resemblance with nitrogen and sulfur containing compounds like thiouracil which exhibited potent anticancer and antiviral activities. Recently, barbituric acid derivatives have also received great interest for applications in nanoscience., Objective: Synthesis of 5-arylidene-N,N-diethylthiobarbiturates, biological evaluation as potential α-glucosidase inhibitors and molecular modeling., Methods: In the present study, N,N-Diethylthiobarbituric acid derivatives were synthesized by refluxing of N,N-diethylthiobarbituric acid and different aromatic aldehydes in distilled water. In a typical reaction; a mixture of N,N-diethylthiobarbituric acid 0.20 g (1 mmol) and 5-bromo-2- hydroxybenzaldehyde 0.199 g (1 mmol) mixed in 10 mL distilled water and reflux for 30 minutes. After completion of the reaction, the corresponding product 1 was filtered and dried and yield calculated. It was crystallized from ethanol. The structures of synthesized compounds 1-25 were carried out by using 1H, 13C NMR, EI spectroscopy and CHN analysis used for the determination of their structures. The α-glucosidase inhibition assay was performed as given by Chapdelaine et al., with slight modifications and optimization., Results: Our newly synthesized compounds showed a varying degree of α-glucosidase inhibition and at least four of them were found as potent inhibitors. Compounds 6, 5, 17, 11 exhibited IC50 values (Mean±SEM) of 0.0006 ± 0.0002, 18.91 ± 0.005, 19.18 ± 0.002, 36.91 ± 0.003 µM, respectively, as compared to standard acarbose (IC50, 38.25 ± 0.12 µM)., Conclusion: Our present study has shown that compounds 6, 5, 17, 11 exhibited IC50 values of 0.0006 ± 0.0002, 18.91 ± 0.005, 19.18 ± 0.002, 36.91 ± 0.003 µM, respectively. The studies were supported by in silico data analysis., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The Burkholderia pseudomallei is a unique bio-threat and causative agent of melioidosis. The B. pseudomallei Bp1651 strain has been isolated from a chronic cystic fibrosis patient. The genome-level DNA sequences information of this strain has recently been published. Unfortunately, there is no commercial vaccine available till date to combat B. pseudomallei infection. The genome-wide prioritization approaches are widely used for the identification of potential therapeutic candidates against pathogens. In the present study, we utilized the recently available annotated genomic information of B. pseudomallei Bp1651 through subtractive genomics and reverse-vaccinology strategies to identify its potential vaccine targets. The analyses identified more than 60 pathogen-specific, human host non-homologous proteins that may prioritize in future studies to investigate therapeutic targets for B. pseudomallei Bp1651. The potential B and T-cells antigenic determinant peptides from these pathogen-specific proteins were cataloged using antigenicity and epitope prediction tools. The analyses unveiled a promising antigenic peptide "FQWEFSLSV" from protein-export membrane protein (SecF) of Bp1651 strain, which was predicted to interact with multiple class I and class II MHC alleles with IC 50 value < 100 nM. The molecular docking analysis verified favorable molecular interaction of this lead antigenic peptide with the ligand-binding pocket residues of HLA A*02:06 human host immune cell surface receptor. This peptide is predicted to be a suitable epitope capable to elicit the cell-mediated immune response against the B. pseudomallei pathogen. The putative epitopes and proteins identified in this study may be promising vaccine targets against Bp1651 as well as other pathogenic strains of B. pseudomallei., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The sphingosine-1-phosphate receptors (S1PRs) are a well-studied class of transmembrane G protein-coupled sphingolipid receptors that mediate multiple cellular processes. However, S1PRs have not been previously reported to be involved in the genetic etiology of human traits. S1PR2 lies within the autosomal-recessive nonsyndromic hearing impairment (ARNSHI) locus DFNB68 on 19p13.2. From exome sequence data we identified two pathogenic S1PR2 variants, c.323G>C (p.Arg108Pro) and c.419A>G (p.Tyr140Cys). Each of these variants co-segregates with congenital profound hearing impairment in consanguineous Pakistani families with maximum LOD scores of 6.4 for family DEM4154 and 3.3 for family PKDF1400. Neither S1PR2 missense variant was reported among ∼120,000 chromosomes in the Exome Aggregation Consortium database, in 76 unrelated Pakistani exomes, or in 720 Pakistani control chromosomes. Both DNA variants affect highly conserved residues of S1PR2 and are predicted to be damaging by multiple bioinformatics tools. Molecular modeling predicts that these variants affect binding of sphingosine-1-phosphate (p.Arg108Pro) and G protein docking (p.Tyr140Cys). In the previously reported S1pr2(-/-) mice, stria vascularis abnormalities, organ of Corti degeneration, and profound hearing loss were observed. Additionally, hair cell defects were seen in both knockout mice and morphant zebrafish. Family PKDF1400 presents with ARNSHI, which is consistent with the lack of gross malformations in S1pr2(-/-) mice, whereas family DEM4154 has lower limb malformations in addition to hearing loss. Our findings suggest the possibility of developing therapies against hair cell damage (e.g., from ototoxic drugs) through targeted stimulation of S1PR2., (Copyright © 2016 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Although pluripotent stem cell (PSC) therapy has advantages for clinical applications because of the self-renewal and multi-lineage differentiation abilities of PSCs, it also has disadvantages in terms of the potential for PSCs to undergo malignant transformation or unexpected differentiation. The prevention of teratoma formation is the largest hurdle of all. Despite intensive studies that have investigated ways to block teratomas, such methods have yet to be further developed for clinical use. Here, a new approach has focused on exerting anti-tumorigenic effects using a novel mica fine particle (MFP) designated STB-HO. Treatment with STB-HO regulated pluripotency- and apoptosis-related genes in differentiating human embryonic stem (hES) cells, while there is no effects in undifferentiated hES cells. In particular, STB-HO blocked the anti-apoptotic gene BIRC5 and activated p53, p21 and the pro-apoptotic proteins Bim, Puma and p-Bad during early spontaneous differentiation. Moreover, STB-HO-pretreated differentiating hES cells did not give rise to teratomas following in vivo stem cell transplantation. Our in vitro and in vivo results suggest a method for teratoma prevention in the context of PSC-derived cell transplantation. This novel MFP could break through the limitations of PSC therapy.

Stem cells have opened a new avenue to treat liver fibrosis. We investigated in vitro and in vivo the effect of cytokine (HGF and FGF4) pretreated MSCs in reduction of CCl4 liver injury. Mouse MSCs were pretreated with cytokines to improve their ability to reduce CCl4 injury. In vitro we gave CCl4 injury to mouse hepatocytes and cocultured it with untreated and cytokines pretreated MSCs. For in vivo study we labeled MSCs with PKH-26 and transplanted them into CCl4 injured mice by direct injection into liver. In vitro data showed that cytokines pretreated MSCs significantly reduce LDH level and apoptotic markers in CCl4 injured hepatocytes cocultured model. Furthermore the cytokines pretreated MSCs also improved cell viability and enhanced hepatic and antiapoptotic markers in injured hepatocytes cocultured model as compared to untreated MSCs. In vivo data in cytokines pretreated group demonstrated greater homing of MSCs in liver, restored glycogen storage, and significant reduction in collagen, alkaline phosphatase, and bilirubin levels. TUNEL assay and real time PCR also supported our hypothesis. Therefore, cytokines pretreated MSCs were shown to have a better therapeutic potential on reduction of liver injury. These results demonstrated the potential utility of this novel idea of cytokines pretreated MSCs for the treatment of liver fibrosis.

Urease is an important enzyme both in agriculture and medicine research. Strategies based on urease inhibition is critically considered as the first line treatment of infections caused by urease producing bacteria. Since, urease possess agro-chemical and medicinal importance, thus, it is necessary to search for the novel compounds capable of inhibiting this enzyme. Several computational methods were employed to design novel and potent urease inhibitors in this work. First docking simulations of known compounds consists of a set of arylidine barbiturates (termed as reference) were performed on the Bacillus pasteurii (BP) urease. Subsequently, two fold strategies were used to design new compounds against urease. Stage 1 comprised of the energy minimization of enzyme-ligand complexes of reference compounds and the accurate prediction of the molecular mechanics generalized born (MMGB) interaction energies. In the second stage, new urease inhibitors were then designed by the substitution of different groups consecutively in the aryl ring of the thiobarbiturates and N, N-diethyl thiobarbiturates of the reference ligands.. The enzyme-ligand complexes with lowest interaction energies or energies close to the calculated interaction energies of the reference molecules, were selected for the consequent chemical manipulation. This was followed by the substitution of different groups on the 2 and 5 positions of the aryl ring. As a result, several new and potent diethyl thiobarbiturates were predicted as urease inhibitors. This approach reflects a logical progression for early stage drug discovery that can be exploited to successfully identify potential drug candidates.

Urease belongs to a family of highly conserved urea-hydrolyzing enzymes. A common feature of these enzymes is the presence of two Lewis acid nickel ions and reactive cysteine residue in the active sites. In the current study we examined a series of biscoumarins 1-10 for their mechanisms of inhibition with the nickel containing active sites of Jack bean and Bacillus pasteurii ureases. All these compounds competitively inhibited Jack bean urease through interaction with the nickel metallocentre, as deduced from Michaelis-Menten kinetics, UV-visible absorbance spectroscopic, and molecular docking simulation studies. Some of the compounds behaved differently in case of Bacillus pasteurii urease. We conducted the enzyme kinetics, UV-visible spectroscopy, and molecular docking results in terms of the known protein structure of the enzyme. We also evaluated possible molecular interpretations for the site of biscoumarins binding and found that phenyl ring is the major active pharmacophore. The excellent in vitro potency and selectivity profile of the several compounds described combined with their nontoxicity against the human cells and plants suggest that these compounds may represent a viable lead series for the treatment of urease associated problems.

Background: Mesenchymal stem cell (MSC) transplantation has emerged as a promising therapy for liver fibrosis. Issues concerning poor MSC survival and engraftment in the fibrotic liver still persist and warrant development of a strategy to increase MSC potency for liver repair. The present study was designed to examine a synergistic role for Interleukin-6 (IL-6) and MSCs therapy in the recovery of carbon tetrachloride (CCl(4)) induced injured hepatocytes in vitro and in vivo., Methods: Injury was induced through 3 mM and 5 mM CCl(4) treatment of cultured hepatocytes while fibrotic mouse model was established by injecting 0.5 ml/kg CCl(4) followed by treatment with IL-6 and MSCs. Effect of MSCs and IL-6 treatment on injured hepatocytes was determined by lactate dehydrogenase release, RT-PCR for (Bax, Bcl-xl, Caspase3, Cytokeratin 8, NFκB, TNF-α) and annexin V apoptotic detection. Analysis of MSC and IL-6 treatment on liver fibrosis was measured by histopathology, PAS, TUNEL and Sirius red staining, RT-PCR, and liver function tests for Bilirubin and Alkaline Phosphatase (ALP)., Results: A significant reduction in LDH release and apoptosis was observed in hepatocytes treated with a combination of MSCs and IL-6 concomitant with upregulation of anti-apoptotic gene Bcl-xl expression and down regulation of bax, caspase3, NFκB and TNF-α. Adoptive transfer of MSCs in fibrotic liver pretreated with IL-6 resulted increased MSCs homing and reduced fibrosis and apoptosis. Hepatic functional assessment demonstrated reduced serum levels of Bilirubin and ALP., Conclusion: Pretreatment of fibrotic liver with IL-6 improves hepatic microenvironment and primes it for MSC transplantation leading to enhanced reduction of liver injury after fibrosis. Synergistic effect of IL-6 and MSCs seems a favored therapeutic option in attenuation of liver apoptosis and fibrosis accompanied by improved liver function.

Background: Liver fibrosis is a major health problem worldwide and poses a serious obstacle for cell based therapies. Mesenchymal stem cells (MSCs) are multipotent and important candidate cells for future clinical applications however success of MSC therapy depends upon their homing and survival in recipient organs. This study was designed to improve the repair potential of MSCs by transplanting them in sodium nitroprusside (SNP) pretreated mice with CCl(4) induced liver fibrosis., Methods: SNP 100 mM, a nitric oxide (NO) donor, was administered twice a week for 4 weeks to CCl(4)-injured mice. MSCs were isolated from C57BL/6 wild type mice and transplanted in the left lateral lobe of the liver in experimental animals. After 4 weeks, animals were sacrificed and liver improvement was analyzed. Analysis of fibrosis by qRT-PCR and sirius red staining, homing, bilirubin and alkaline phosphatase (ALP) serum levels between different treatment groups were compared to control., Results: Liver histology demonstrated enhanced MSCs homing in SNP-MSCs group compared to MSCs group. The gene expression of fibrotic markers; αSMA, collagen 1α1, TIMP, NFκB and iNOS was down regulated while cytokeratin 18, albumin and eNOS was up-regulated in SNP-MSCs group. Combine treatment sequentially reduced fibrosis in SNP-MSCs treated liver compared to the other treatment groups. These results were also comparable with reduced serum levels of bilirubin and ALP observed in SNP-MSCs treated group., Conclusion: This study demonstrated that NO effectively augments MSC ability to repair liver fibrosis induced by CCl(4) in mice and therefore is a better treatment regimen to reduce liver fibrosis.

Background: Countrywide 5.9 million, 0-11 Month old children are immunized annually by EPI (Expended Program on Immunization) against 8 vaccine preventable diseases including measles and so on. Unfortunately the basic immunity centers are not uniform throughout the country. Each center provides services to about 27000 people which is inadequate. The purpose of this study was to explore the development of EPI Pakistan in terms of immunization of measles., Methods: Nucleotide sequences were analyzed by neighbor joining method (bootstrap test) using Bio- edit and MEGA-5 software to find evolutionary relationship between wild type measles strain and vaccine strain (Edmonston strain) used in Pakistan. For statistical analysis of data SPSS 16 was used., Results: Currently 1.3 vaccinators are working at each U C (union council) which according to national EPI policy should be at least 2. About 56% and 44% children of age 0-11 months did not received second dose of measles in the last two years respectively. Out of these 4231 cases which were reported last year, 1370 have received their first dose of measles vaccine., Conclusion: Seroconversion and seroprevalence study of the vaccine and field strain of measles virus is needed to confirm whether its failure is due to service unavailability or vaccine in-affectivity.

Background: Hepatitis B virus (HBV) infection is a serious health problem in the developing countries including Pakistan. Various risk factors are responsible for the spread of this infectious disease. Prevalence of HBV infection in apparently suspected individual of Punjab province of Pakistan was analyzed during January 2008 to December 2010. Current study was aimed to investigate the epidemiology and risk factors of HBV infection., Methodology: Four thousand eight hundred and ninety patients suffering from chronic liver disease were screened for the presence of HBV DNA using qualitative Real Time PCR methodology to confirm their status of infection. A predesigned standard questionnaire was filled for all the patients that included information about the possible risk factors., Results: A total of 4890 ELISA positive patients were screened for Hepatitis B virus infection. Of these 3143 were positive for HBV, includes 68.15% males and 31.85% females. Male were observed to be more frequently infected as compared to the female with a positivity ratio of 2.14: 1. The rate of infection increases with the passage of time in the course of three years. Highest frequency of infection was found in the age of 21-30 was 34.93% followed by 23.83% in 31-40. Only (13.39%) were belonging to the age group 11-20 year. The rate of infection declines with increasing age as shown by age groups 41-50 (16.13%) and 51-60 (7.09%). While children aged 0-10 and very old >60 age groups were very less frequently 1.49% and 1.65% infected respectively. Important risk factors contributing to HBV spread include barber risk (23.60%), blood transfusion (4.04%), History of injection 26.19%, Reuse of syringes 26.60%, dental risk (11.20%) and surgical procedure (4.26%). Among the entire respondents trend sharing personal items was very common. History of injection, barber risk, surgery and dental procedure and reuse of syringes appear as major risk factors for the transmission., Conclusion: Male were more frequently exposed to the risk factors as compared to female. Similarly the younger age group had high rate of infection as compared to the children's and the older age groups. Reuse of syringes', barber risk and History of injection were main risk identified during the present study. To lower HBV transmission rate Government should take aggressive steps towards massive awareness and vaccination programs to decrease the burden of HBV from the Punjab province of Pakistan.

Liver failure represents a serious challenge for cell based therapies. Mesenchymal stem cells (MSCs) possess potential for regeneration of fibrotic liver; however, there is a dire need to improve their hepatic differentiation. This study examines a pretreatment strategy to augment the differentiation potential of MSCs towards hepatic lineage. MSCs were isolated from C57BL/6 wild type mice and were characterized by flow cytometry for CD44 (92.4%), CD90 (96.6%), CD105 (94.7%), CD45 (0.8%) and CD34 (1.4%) markers. To improve the differentiation potential of MSCs towards hepatic lineage, cells were pretreated with injured liver tissue in an in-vitro model, which resulted in high expression of albumin, cytokeratin 8, 18, TAT and HNF1α as compared to untreated MSCs. The efficacy of pretreated MSCs was evaluated by preparing in-vivo mouse model with liver fibrosis by intraperitoneal administration of CCl(4). Pretreated MSCs were transplanted in the left lateral lobe of mice with liver fibrosis and showed enhanced localization and differentiation abilities after 1 month. The expression for cytokeratin 8, 18, albumin and Bcl-xl was up-regulated and that of HGF, Bax and Caspase- 3 was down-regulated in animals transplanted with pretreated MSCs. Sirus red staining also confirmed a significant reduction in the fibrotic area in liver tissue transplanted with pretreated MSCs as compared to untreated MSCs and was concomitant with improved serum levels of bilirubin and alkaline phosphatase (ALP). Therefore, it was concluded that pretreatment with injured liver tissue augment homing and hepatic differentiation abilities of MSCs and provides an improved procedure for the treatment of liver fibrosis., (Copyright © 2010 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)