Cell-free DNA as a potential diagnostic biomarker in academic stress: A case-control study in young adults.

Background: Stress is a pervasive issue in modern life, affecting both physical and mental health. Identifying biomarkers like cell-free DNA (cfDNA) could provide insights into stress response and help detect individuals at risk for stress-related disorders. Objective: The aim of this study is to investigate the potential use of cfDNA as a diagnostic biomarker in individuals experiencing stress. Methodology: A case-control analysis was conducted using convenient sampling on university participants (N = 285 cases, N = 500 controls) aged 18-24. The study assessed haematological and lipid profile parameters using the Sysmex XP-300TM automated analyzer and an automated biochemistry analyzer, and cfDNA was extracted using a standardized in house developed Phenol-Chloroform protocol and estimated using Agarose Gel Electrophoresis and Nanodrop. Statistical analysis was performed using SPSS ver. 21.0. Results: The results indicated a significant difference between stressed individuals and healthy controls in demographic, haematological and biochemical parameters. Specifically, stressed cases had significantly higher levels of cholesterol, LDL cholesterol, triglycerides, glucose, VLDL cholesterol, and lower levels of HDL compared to healthy controls. Stressed cases also showed significantly elevated levels of circulating cfDNA relative to healthy controls. Conclusion: These findings suggest that cfDNA may have potential as a diagnostic biomarker for stress.

In silico analysis of the Val66Met mutation in BDNF protein: implications for psychological stress.

The brain-derived neurotrophic factor (BDNF) involves stress regulation and psychiatric disorders. The Val66Met polymorphism in the BDNF gene has been linked to altered protein function and susceptibility to stress-related conditions. This in silico analysis aimed to predict and analyze the consequences of the Val66Met mutation in the BDNF gene of stressed individuals. Computational techniques, including ab initio, comparative, and I-TASSER modeling, were used to evaluate the functional and stability effects of the Val66Met mutation in BDNF. The accuracy and reliability of the models were validated. Sequence alignment and secondary structure analysis compared amino acid residues and structural components. The phylogenetic analysis assessed the conservation of the mutation site. Functional and stability prediction analyses provided mixed results, suggesting potential effects on protein function and stability. Structural models revealed the importance of BDNF in key biological processes. Sequence alignment analysis showed the conservation of amino acid residues across species. Secondary structure analysis indicated minor differences between the wild-type and mutant forms. Phylogenetic analysis supported the evolutionary conservation of the mutation site. This computational study suggests that the Val66Met mutation in BDNF may have implications for protein stability, structural conformation, and function. Further experimental validation is needed to confirm these findings and elucidate the precise effects of this mutation on stress-related disorders.

Natural compound targeting BDNF V66M variant: insights from in silico docking and molecular analysis.

Brain-Derived Neurotrophic Factor (BDNF) is a neurotrophin gene family gene that encodes proteins vital for the growth, maintenance, and survival of neurons in the nervous system. The study aimed to screen natural compounds against BDNF variant (V66M), which affects memory, cognition, and mood regulation. BDNF variant (V66M) as a target structure was selected, and Vitamin D, Curcumin, Vitamin C, and Quercetin as ligands structures were taken from PubChem database. Multiple tools like AUTODOCK VINA, BIOVIA discovery studio, PyMOL, CB-dock, IMOD server, Swiss ADEMT, and Swiss predict ligands target were used to analyze binding energy, interaction, stability, toxicity, and visualize BDNF-ligand complexes. Compounds Vitamin D3, Curcumin, Vitamin C, and Quercetin with binding energies values of - 5.5, - 6.1, - 4.5, and - 6.7 kj/mol, respectively, were selected. The ligands bind to the active sites of the BDNF variant (V66M) via hydrophobic bonds, hydrogen bonds, and electrostatic interactions. Furthermore, ADMET analysis of the ligands revealed they exhibited sound pharmacokinetic and toxicity profiles. In addition, an MD simulation study showed that the most active ligand bound favorably and dynamically to the target protein, and protein-ligand complex stability was determined. The finding of this research could provide an excellent platform for discovering and rationalizing novel drugs against stress related to BDNF (V66M). Docking, preclinical drug testing and MD simulation results suggest Quercetin as a more potent BDNF variant (V66M) inhibitor and forming a more structurally stable complex.

Anthropological analyses of 30 insertion/deletion autosomal markers in five major ethnic groups of Pakistan.

We investigated the forensic efficacy of the 30 insertion/deletion (Indel) markers included in the Qiagen Investigator® DIPplex kit in 529 Pakistani individuals from five major subpopulations in Pakistan (Punjabi, Pashtun, Sindhi, Saraiki, and Baloch). In the Sindhi population, the distribution of HLD81 and HLD97 alleles deviated from Hardy-Weinberg equilibrium after Bonferroni correction. The combined match probability ranged from 2.0E-12 (Pashtun and Baloch) to 1.0E-12 (Sindhi), and the mean paternity exclusion power varied from 0.995 (Punjabi, Sindhi, and Saraiki) to 0.996 (Pashtun and Baloch). The high combined power of discrimination (0.999 999 999 999 97) and low combined match probability (1.7E-12) for all subpopulations studied support the utility of the 30 Indel markers for forensic identification in the studied subpopulations. The allele frequencies of the Indel markers in the Pakistani subpopulations were compared with those from 18 other populations. The results show that the populations clustered according to geography. The subpopulations investigated in this work showed a close genetic relationship with others from Pakistan, as well as with South Central Asian and Middle Eastern populations. The results suggest that the Investigator® DIPplex kit can be useful as a supplementary tool for human identification in the five Pakistani subpopulations investigated in this study. Supplemental data for this article is available online at https://doi.org/10.1080/20961790.2021.1933366 .

Analysis of Skin Pigmentation and Genetic Ancestry in Three Subpopulations from Pakistan: Punjabi, Pashtun, and Baloch.

Skin pigmentation is one of the most prominent and variable phenotypes in humans. We compared the alleles of 163 SNPs and indels from the Human Pigmentation (HuPi) AmpliSeq™ Custom panel, and biogeographic ancestry with the quantitative skin pigmentation levels on the upper arm, lower arm, and forehead of 299 Pakistani individuals from three subpopulations: Baloch, Pashtun, and Punjabi. The biogeographic ancestry of each individual was estimated using the Precision ID Ancestry Panel. All individuals were mainly of mixed South-Central Asian and European ancestry. However, the Baloch individuals also had an average proportion of Sub-Saharan African ancestry of approximately 10%, whereas it was <1% in the Punjabi and Pashtun individuals. The pairwise genetic distances between the Pashtun, Punjabi, and Baloch subpopulations based on the ancestry markers were statistically significantly different. Individuals from the Pashtun subpopulation had statistically significantly lower skin pigmentation than individuals from the Punjabi and Baloch subpopulations (p < 0.05). The proportions of European and Sub-Saharan African ancestry and five SNPs (rs1042602, rs10831496, rs1426654, rs16891982, and rs12913832) were statistically significantly associated with skin pigmentation at either the upper arm, lower arm or forehead in the Pakistani population after correction for multiple testing (p < 10-3). A model based on four of these SNPs (rs1426654, rs1042602, rs16891982, and rs12913832) explained 33% of the upper arm skin pigmentation. The four SNPs and the proportions of European and Sub-Saharan African ancestry explained 37% of the upper arm skin pigmentation. Our results indicate that the four likely causative SNPs, rs1426654, rs1042602, rs16891982, and rs12913832 located in SLC24A5, TYR, SLC45A2, and HERC2, respectively, are essential for skin color variation in the admixed Pakistani subpopulations.

Forensic application and genetic diversity of 21 autosomal STR loci in five major population groups of Pakistan.

OBJECTIVES: Investigation of genetic diversity of the 21 autosomal STR loci included in the GlobalFilerTM PCR Amplification Kit in 529 Pakistani individuals belonging to the Punjabi, Pashtun, Sindhi, Saraiki, and Baloch ethnic groups. Population genetic parameters and forensic informative metrics for each group were evaluated. RESULTS: SE33 showed the greatest power of discrimination in all populations studied. The combined match probability ranged from 8.06E-27 (Saraiki) to 1.05E-26 (Baloch), and the combined power of exclusion ranged from 0.99999999902 (Punjabi) to 0.99999999964 (Pashtun). D12S391 in the Baloch population and D2S441 in the Saraiki population showed deviation from Hardy-Weinberg equilibrium. CONCLUSION: Significant genetic distances were observed between the Punjabi, Pashtun, and Baloch populations. This study supports the utilization of the GlobalFilerTM STR kit for forensic applications in Pakistan.

Development of new PCR multiplex system by the simultaneous detection of 10 miniSTRs, SE33, Penta E, Penta D, and four Y-STRs.

The 18 loci multiplex system has been instigated for co-amplification and fluorescent detection of Amelogenin and 17 STRs, including 10 MiniSTRs (CSF1PO, D18S51, D7S820, D2S1338, TPOX, D13S317, FGA, D5S818, D21S11, D16S539), SE33, Penta E, Penta D, and four Y-STRs (DYS385a/b, DYS438, DYS392). This multiplex system was developed for the simultaneous analysis of compromised DNA samples, Y-amelogenin marker mutation, motherless paternity issues where single allele sharing occurs at autosomal STRs in unrelated individuals, and other complex forensic cases. Selection of loci, primers, and allelic ladders were designed and created in-house with a design strategy to work in this multiplex. The multiplex system was evaluated by sensitivity, specificity, stability, precision and accuracy, case-type samples, mixture studies, PCR-based and population distribution studies to establish the robustness and reliability of the system as the current requirements of the forensic case work. Among all the markers evaluated for this study, 209 alleles including 44 variants were observed with combined power of discrimination, combined power of exclusion, and the combined probability of matching calculated as 0.999999999999999999893916339344, 0.999993816173890, and 5.90019 × 10-19, respectively. Due to highly polymorphic characteristics of these loci particularly SE33 and Penta E which are most discriminatory (PD = 0.991 and 0.983, respectively) in the Pakistani population, this multiplex would be highly valuable for individual identification in complex forensic cases and paternity issues as well as population database.

Genetic distribution of 15 autosomal STR markers in the Punjabi population of Pakistan.

Genetic diversity of 15 autosomal short tandem repeat (STR) loci was evaluated in 713 unrelated individual samples of a Punjabi population of Pakistan. These loci were scrutinized to establish allelic frequencies and statistical parameters of forensic and paternity interests. A total of 165 alleles were observed with the corresponding allele frequencies ranging from 0.001 to 0.446. D2S1338 was found as the most informative locus while TPOX (0.611) was the least discriminating locus. The combined power of discrimination (CPD), the combined probability of exclusion (CPE), and cumulative probability of matching (CPM) were found equaled to 0.999999999999999998606227424808, 0.999995777557989, and 1.37543 × 10-18, respectively. All the loci followed the Hardy-Weinberg equilibrium after the Bonferroni correction (p < 0.0033) except one locus D3S1358. The study revealed that these STR loci are highly polymorphic, suitable for forensic and parentage analyses. In comparison to different populations (Asians and non-Asians), significant differences were recorded for these loci.

Genetic polymorphism of ten MiniSTR loci in the population of Punjab Pakistan for forensic application.

Ten MiniSTR loci were analyzed on 250 unrelated individuals of Punjabi population from Punjab province of Pakistan. The product amplification range is from 65 to 280 bp. Allele frequency for a total of 98 observed alleles is from 0.002 to 0.41. Forensic and paternity statistical parameters were investigated including combined power of discrimination (PD), combined power of exclusion (PE), and cumulative probability of matching (PM) equaled to 0.99999999999824, 0.999824, and 1.75931 × 10(-12), respectively. These MiniSTRs on the basis of high degree polymorphism and fragment size reduction will be highly informative in most of the forensic cases where DNA of the samples is degraded, mass disasters, and dead body identification. Significant differences were observed in all loci when comparing with same STR loci in previously published different world populations.