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.

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.

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.

Liver fibrosis is initial stage of any chronic liver disease and its end stage is develops into cirrhosis. Chronic liver diseases are a crucial global health issue and the cause of approximately 2 million deaths per year worldwide. Cirrhosis is currently the 11^th most common cause of death globally. Mesenchymal stem cell (MSCs) treatment is the best way to treat acute and chronic liver disease. The aim of this study is to improve the therapeutic potential of MSCs combined with melatonin (MLT) to overcome CCl₄-induced liver fibrosis and also investigate the individual impact of melatonin and MSCs against CCl₄-induced liver impairment in animal model. Female BALB/c mice were used as CCL₄-induced liver fibrotic animal model. Five groups of animal model were made; negative control, Positive control, CCl₄+MSCs treated group, CCl₄+MLT treated group and CCl₄+MSCs+MLT treated group. Cultured MSCs from mice bone marrow were transplanted to CCl₄-induced liver injured mice model, individually as well as together with melatonin. Two weeks after MSCs and MLT administration, all groups of mice were sacrificed for examination. Morphological and Histopathological results showed that combined therapy of MSCs+MLT showed substantial beneficial impact on CCl₄-induced liver injured model, compared with MSCs and MLT individually. Biochemically, considerable reduction was observed in serum bilirubin and ALT levels of MLT+MSC treated mice, compared to other groups. PCR results shown down-regulation of Bax and up-regulation of Bcl-xl and Albumin, confirm a significant therapeutic effect of MSCs+MLT on CCI₄-induced liver fibrosis. From the results, it is concluded that combined therapy of MSCs and MLT show strong therapeutic effect on CCL₄-induced liver fibrosis, compared with MSCs and MLT individually.

Plasmodium vivax is the most common human malaria parasite in Asian countries including Pakistan. Present study was designed to explore the genetic diversity of plasmodium vivax genotypes based on Pvmsp-3α and Pvmsp-3βgenes using allelic specific nested PCR and RFLP assays markers from field isolates in district Mardan, Pakistan. Blood samples of 200 P. vivax malarial patients were collected after taking their written informed consent. Genetic diversity in nested PCR products was determined by Restriction Fragment Length Polymorphism (RFLP) utilizing Alu1 and PstI restriction enzymes for alpha and beta gene products digestion, respectively. For analysis the genetic diversity of the sub allelic variants of Pvmsp3α and Pvmsp3β genes, Chi-Square test was performed by utilizing Minitab programming software 18. The P value 0.05 was considered as statistically significant. For Pvmsp-3α genes after gel electrophoresis of digested products, four distinct genotypes were obtained from total of 50 samples; type A: 35 (70%) (1.5-2.0 kb), 12 of type B (24%) (1.5-1.7 kb), 2 of type C (4%) (0.5-1.5) and one for type D (2%) (0.5-0.65 kb) which could be characterized into 9 allelic pattern (A1-A4, B1-B3, C1, D), in which A3 remained the most predominant. For Pvmsp-3βgenes, three distinct genotypes were obtained from 50 samples; 40(80%) of type A (1.5-2.5 kb), 9 (18%) of type B (1.0-1.5kb) and 1(2%) of type C (0.65 kb) which could be characterized into 6 allelic patterns (A1-A3, B1-B2, and C1). Most dominant one in Type A was A1 alleles which were noted (46%), while in Type B, the most dominant were B1 (10%).This study is the first ever report of molecular epidemiology and genetic variation in Pvmsp-3α and Pvmsp-3β genes of P. vivax isolates by using PCR/RFLP from District Mardan and showed a remarkable level of genetic diversity in the studied genes of circulating parasites in the study area. The results of this study will contribute in future studies about the genetic structure of parasite and vaccine development against the malaria.