Genetic structure of apical membrane antigen-1 in Plasmodium falciparum isolates from Pakistan.

Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is a major candidate for the blood-stage malaria vaccine. Genetic polymorphisms of global pfama-1suggest that the genetic diversity of the gene can disturb effective vaccine development targeting this antigen. This study was conducted to explore the genetic diversity and gene structure of pfama-1 among P. falciparum isolates collected in the Khyber Pakhtunkhwa (KP) province of Pakistan. A total of 19 full-length pfama-1 sequences were obtained from KP-Pakistan P. falciparum isolates, and genetic polymorphism and natural selection were investigated. KP-Pakistan pfama-1 exhibited genetic diversity, wherein 58 amino acid changes were identified, most of which were located in ectodomains, and domains I, II, and III. The amino acid changes commonly found in the ectodomain of global pfama-1 were also detected in KP-Pakistan pfama-1. Interestingly, 13 novel amino acid changes not reported in the global population were identified in KP-Pakistan pfama-1. KP-Pakistan pfama-1 shared similar levels of genetic diversity with global pfama-1. Evidence of natural selection and recombination events were also detected in KP-Pakistan pfama-1.

Population genetic analysis of Plasmodium vivax vir genes in Pakistan.

Plasmodium vivax variant interspersed repeats (vir) refer to the key protein used for escaping the host immune system. Knowledge in the genetic variation of vir genes can be used for the development of vaccines or diagnostic methods. Therefore, we evaluated the genetic diversity of the vir genes of P. vivax populations of several Asian countries, including Pakistan, which is a malaria-endemic country experiencing a significant rise in malaria cases in recent years. We analyzed the genetic diversity and population structure of 4 vir genes (vir 4, vir 12, vir 21, and vir 27) in the Pakistan P. vivax population and compared these features to those of the corresponding vir genes in other Asian countries. In Pakistan, vir 4 (S=198, H=9, Hd=0.889, Tajima's D value=1.12321) was the most genetically heterogenous, while the features of vir 21 (S=8, H=7, Hd=0.664, Tajima's D value =-0.63763) and vir 27 (S =25, H =11, Hd =0.682, Tajima's D value=-2.10836) were relatively conserved. Additionally, vir 4 was the most genetically diverse among Asian P. vivax populations, although within population diversity was low. Meanwhile, vir 21 and vir 27 among all Asian populations were closely related genetically. Our findings on the genetic diversity of vir genes and its relationships between populations in diverse geographical locations contribute toward a better understanding of the genetic characteristics of vir. The high level of genetic diversity of vir 4 suggests that this gene can be a useful genetic marker for understanding the P. vivax population structure. Longitudinal genetic diversity studies of vir genes in P. vivax isolates obtained from more diverse geographical areas are needed to better understand the function of vir genes and their use for the development of malaria control measures, such as vaccines.

Identification of new potent NLRP3 inhibitors by multi-level in-silico approaches.

Nod-like receptor protein 3 (NLRP-3), is an intracellular sensor that is involved in inflammasome activation, and the aberrant expression of NLRP3 is responsible for diabetes mellitus, its complications, and many other inflammatory diseases. NLRP3 is considered a promising drug target for novel drug design. Here, a pharmacophore model was generated from the most potent inhibitor, and its validation was performed by the Gunner-Henry scoring method. The validated pharmacophore was used to screen selected compounds databases. As a result, 646 compounds were mapped on the pharmacophore model. After applying Lipinski's rule of five, 391 hits were obtained. All the hits were docked into the binding pocket of target protein. Based on docking scores and interactions with binding site residues, six compounds were selected potential hits. To check the stability of these compounds, 100 ns molecular dynamic (MD) simulations were performed. The RMSD, RMSF, DCCM and hydrogen bond analysis showed that all the six compounds formed stable complex with NLRP3. The binding free energy with the MM-PBSA approach suggested that electrostatic force, and van der Waals interactions, played a significant role in the binding pattern of these compounds. Thus, the outcomes of the current study could provide insights into the identification of new potential NLRP3 inflammasome inhibitors against diabetes and its related disorders.

Salivary Oxidative Stress and Antioxidant Capacity in Smokeless Tobacco (Naswar) Users.

Background: Naswar, a smokeless tobacco product, commonly consumed in Pakistan, is associated with a 10-fold increase in the risk of oral cancer. However, little is known about Naswar's underlying toxicity mechanisms. Objective: The current study aimed to investigate the effects of Naswar use on oral health and salivary parameters. Methods: A case-control study was conducted among Naswar users (n=42) and age-matched healthy controls (n=42) in Pakistan in 2019. Participant data were collected using questionnaires. Decayed, missing, and filled teeth (DMFT) scores were computed during clinical examinations. Unstimulated whole saliva was collected to assess salivary flow rate, pH, and salivary total oxidative stress (TOS)/total antioxidant capacity (TAC) using commercially available kits. Participants' oral health parameters were compared between cases and controls using ANOVA. Results: No significant differences were observed between the two groups in terms of age, oral health, and hygiene practices and mean DMFT score. Mean salivary pH and the salivary flow rate was significantly (p<0.001) higher in Naswar users (7.7 and 0.71 mL/minute, respectively) than in non-users (6.95 and 0.52 mL/minute, respectively). Although TOS and TAC were not significantly different between the groups, Naswar users generally had a higher TOS (51.6±42 µmol/L) and lower TAC (0.55±0.18 mmol/L) than non-users (TOS 45.5±38.2 µmol/L and TAC 0.57±0.17 mmol/L). Correlational analysis also revealed a significant positive correlation between DMFT score and Naswar use duration (r=0.796, p<0.001) and the number of dips/units consumed each day (r=0.515; p<0.001). Conclusion: Habitual Naswar use is associated with increased salivary flow rate, pH, and TOS, and reduced TAC levels in Pakistani adults compared to non-users. The pro-oxidant changes may contribute toward deleterious effects of Naswar use including oral cancer.

Combinatorial Therapeutic Potential of Stem Cells and Benzimidazol Derivatives for the Reduction of Liver Fibrosis.

(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 CCl4-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.

Structural Consequences of IRS-2 nsSNPs and Implication for Insulin Receptor Substrate-2 Protein Stability.

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.

Molecular Mimicry Analyses Unveiled the Human Herpes Simplex and Poxvirus Epitopes as Possible Candidates to Incite Autoimmunity.

Clinical epidemiological studies have reported that viral infections cause autoimmune pathology in humans. Host-pathogen protein sequences and structure-based molecular mimicry cause autoreactive T cells to cross-activate. The aim of the current study was to implement immunoinformatics approaches to infer sequence- and structure-based molecular mimicry between viral and human proteomic datasets. The protein sequences of all the so far known human-infecting viruses were obtained from the VIPR database, and complete human proteome data were retrieved from the NCBI repository. Based on a predefined, stringent threshold of comparative sequence analyses, 24 viral proteins were identified with significant sequence similarity to human proteins. PathDIP identified the enrichment of these homologous proteins in nine metabolic pathways with a p-value < 0.0001. Several viral and human mimic epitopes from these homologous proteins were predicted as strong binders of human HLA alleles, with IC50 < 50 nM. Downstream molecular docking analyses identified that lead virus-human homologous epitopes feasibly interact with HLA and TLR4 types of immune receptors. The vast majority of these top-hit homolog epitopic peptides belong to the herpes simplex and poxvirus families. These lead epitope biological sequences and 3D structural-based molecular mimicry may be promising for interpreting herpes simplex virus and poxvirus infection-mediated autoimmune disorders in humans.

Population genetic analyses inferred a limited genetic diversity across the pvama-1 DI domain among Plasmodium vivax isolates from Khyber Pakhtunkhwa regions of Pakistan.

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.

An insight in Salmonella typhi associated autoimmunity candidates' prediction by molecular mimicry.

Autoimmune diseases develop when the immune system targets healthy cells and tissues of an individual. In developing countries, S. typhi (a gram-negative pathogenic bacteria) remains a major public health issue. This study aimed to employ bioinformatics analyses to determine the 3D structural-based molecular mimicry and sequence of S. typhi and human host proteins. In addition, to classify possible antigenic microbial peptides homologous to human peptides and comprehend the molecular basis of S. typhi-related autoimmune disorders. Protein sequences were obtained from the NCBI database, and redundancy was removed using the CD-HIT tool. The BLASTp comparative sequence analysis was followed for molecular mimicry identification of human and S. typhi protein sequences. The PathDIP database was utilized to simulate essential physical relationships between proteins and curated pathways for metabolic processes. Subsequently, the IEDB database was used to find cross-reactive MHC class-II binding epitopes that could trigger an autoimmune reaction. SPARKS-X computational biology resource was also used to determine the structural homology between human and S. typhi peptides. The BLASTp study showed that S. typhi and the human host have several proteins holding considerable sequence similarities based on a set threshold of e ≤ 10-6 and bit score ≥100. The PathDIP putatively identified that these proteins enriched in a total of 68 metabolic pathways by a significant P-value (P < 0.005). The PSORTb analysis predicted that 26 out of these proteins are cytosolic, 1 predicted to be periplasmic protein, and 1 predicted to be localized in the cytoplasmic membrane. IEDB data analysis predicted many S.typhi and human homologs epitopes as a good binder of human HLA, i.e. DRB1*01:01, DPA1*03:01/DPB1*04:02, and DQA1*01:02/DQB1*06:02 with IC50 < 50 nM. Finally, the docking data demonstrated that homolog lead epitopes promisingly interact with HLA and immune TLR4 receptors by exhibiting the best docking scores and molecular interactions. The analyses ultimately identified several potential candidate proteins and peptides that could cause S.typhi infection-mediated autoimmune diseases in humans.

Genomic miscellany and allelic frequencies of Plasmodium falciparum msp-1, msp-2 and glurp in parasite isolates.

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.

Dispersal into the Qinghai-Tibet plateau: evidence from the genetic structure and demography of the alpine plant Triosteum pinnatifidum.

Triosteum pinnatifidum Maxim., an alpine plant, is traditionally used for several medicinal purposes. Here, both chloroplast DNA sequences and nuclear low copy sequence markers were used to investigate the genetic diversity and population structure of T. pinnatifidum. Materials were collected from thirteen localities in the northeast Qinghai-Tibet Plateau (QTP) and adjacent highlands and advanced analytical toolkits were used to access their origin and range shifts. The results revealed a higher level of population differentiation based on chloroplast DNA (cpDNA) concatenated sequences compared with the nuclear DNA sequences (F ST = 0.654 for cpDNA, F ST = 0.398 for AT103), indicating that pollen flow was still extensive in T. pinnatifidum. A decline in haplotype variation was observed from the plateau edge and adjoining highlands toward the platform of the QTP. The hypothesis "dispersal into the QTP," proposing that T. pinnatifidum experienced migration from the plateau edge and adjacent highlands to the platform, was supported. These results were in line with the hypothesis that multiple refugia exist on the plateau edge and adjacent highlands rather than on the plateau platform. Our unimodal mismatch distribution, star-like network supported a recent expansion in T. pinnatifidum.

In Silico Drug Designing for ala438 Deleted Ribosomal Protein S1 (RpsA) on the Basis of the Active Compound Zrl15.

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., zrl15. 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.

Phyto-ecological studies and distribution pattern of plant species and communities of Dhirkot, Azad Jammu and Kashmir, Pakistan.

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.

Analysing the essential proteins set of Plasmodium falciparum PF3D7 for novel drug targets identification against malaria.

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.

Chlamydia trachomatis core genome data mining for promising novel drug targets and chimeric vaccine candidates identification.

Chlamydia trachomatis is involved in most sexually transmitted diseases. The species has emerged as a major public health threat due to its multidrug-resistant capabilities, and new therapeutic target inferences have become indispensable to combat its pathogenesis. However, no commercial vaccine is yet available to treat the C. trachomatis infection. In this study, we used the publicly available complete genome sequences of C. trachomatis and performed comparative proteomics and reverse vaccinology analyses to explore novel drug and vaccine targets against this devastating pathogen. We identified 713 core proteins from 71 C. trachomatis complete genome sequences and prioritized them based on their cellular essentiality, virulence, and available antibiotic resistance. The analyses led to the identification of 16 pathogen-specific proteins with no resolved 3D structures, though holding significant druggable potential. The sequences of the three shortlisted candidates' membrane proteins were used for designing vaccine constructs. The antigenicity, toxicity, and solubility profile-based lead epitopes were prioritized for multi-epitope-based vaccine constructs in combination with specific linkers, PADRE sequences, and molecular adjuvants for immunogenicity enhancement. The molecular-level interactions of the prioritized vaccine construct with human immune cells HLA and TLR4/MD were validated by molecular docking and molecular dynamic simulation analyses. Furthermore, the cloning and expression potential of the lead vaccine construct was predicted in the E. coli cloning vector system. Additional testing and experimental validation of these multi-epitope constructs appear promising against C. trachomatis-mediated infection.

In vitro differentiation effect of CCL4-induced liver injured mice serum on bone marrow-derived mesenchymal stem cells toward hepatocytes like cells.

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 (CCl4) induced liver injured mice serum on MSCs differentiation toward hepatocytes in vitro. Male Balb/c mice were treated for liver injury with CCl4 as determined through biochemical tests spectrophotometrically and different growth factors (EGF, HGF) quantification through Sandwich ELISA in both normal and CCl4-induced liver injured mice serum. Mice bone marrow derived-MSCs at second passage were treated with normal and CCl4-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 CCl4 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 CCl4-induced liver injured mice serum. RT-PCR results of CCl4-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 CCl4-induced liver injured serum treatment can differentiate MSCs into hepatocyte-like cells in vitro.

Frequency of Hyperemesis Gravidarum and associated risk factors among pregnant women.

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.

Potential druggable proteins and chimeric vaccine construct prioritization against Brucella melitensis from species core genome data.

The Brucella melitensis chronic infection and drug resistance emerged as a severe health problem in humans and domestic cattle. The pathogens fast genome sequences availability fetched the possibility to address novel therapeutics targets in a rationale way. We acquired the core genes set from 56 B. melitensis publically available complete genome sequences. A stringent bioinformatics layout of comparative genomics and reverse vaccinology was followed to identify potential druggable proteins and multi-epitope vaccine constructs from core genes. The 23 proteins were shortlisted as novel druggable targets based on their role in pathogen-specific metabolic pathways, non-homologous to human and human gut microbiome proteins and their druggability potential. Furthermore, potential chimeric vaccine constructs were generated from lead T and B-cell overlapped epitopes in combination with immune enhancer adjuvants and linkers sequences. The molecular docking and MD simulation analyses ensured stable molecular interaction of a finally prioritized vaccine construct with human immune cells receptors.

Antioxidant Potential, Phytochemicals Composition, and Metal Contents of Datura alba.

This study investigated the phytochemical characteristics and antioxidant activity in leaves, roots, stem, flower, and seed parts of Datura alba (D. alba). The study also assessed the heavy metal (Cr, Mn, Zn, and Cu) accumulation in each part of the plant. Among the phytochemicals, alkaloids were found only in leaves while tannins, flavonoids, and phenols were present in all parts of the plant. For antioxidant activity, free radical scavenging assay for 2,2-diphenyl-1-picrylhydrazyl (DPPH) was performed using ascorbic acid as the standard. Higher activity was shown by stem extract in methanol and leaf extract in n-hexane, ethyl acetate, and chloroform. Furthermore, all the target heavy metals were detected in all plant sections with the highest concentration of Zn in leaves and Cu in stem, root, flower, and seed. Due to stronger antioxidant potential and phytochemical composition, D. alba could prove as valuable prospect in pharmaceutical formulations by taking part in the antioxidant defense system against generation of free radicals.

Immunoinformatics and structural vaccinology driven prediction of multi-epitope vaccine against Mayaro virus and validation through in-silico expression.

The Mayaro virus (MAYV) belongs to genus "Alphavirus" and family "Togaviridae". MAYV has distribution in the Amazonia, Central and Northeastern regions of Brazil. The abundance of mosquito vector Haemagogus janthinomys has major role in the outbreaks of arthralgia disease in Brazil. Vaccination or immunization is an alternative approach for the protection against this disease. To search the effective candidate for vaccine against Mayaro virus, various immunoinformatics tools were used to predict both the B and T cell epitopes from five structural polyproteins (capsid, E2, 6K, E3and E1). A multi subunit vaccine was designed and the final sequence was modeled for docking with TLR-3. Human b defensin based on previous studies was used as linker. The docked complexes of vaccine-TLR-3 were then subjected to dynamics stability and RMSD and RMSF results suggested that the complexes are stable. Further, to validate our final vaccine construct, in silico cloning was carried out using E. coli as host. The CAI value of 0.96 suggests that the vaccine construct properly expresses in the host. The current findings will be useful for the future experimental validations to ratify the immunogenicity and safety of the supposed structure of vaccine, and ultimately to treat the Mayaro virus, associated infections.