Imran Riaz Malik

Genetic polymorphisms of apolipoprotein B gene (APOB) may result into serum proteomic perturbance in Coronary Artery Disease (CAD). The current case–control cohort of Pakistani subjects was designed to analyze the genetic influence of APOB rs1042031, (G/T) genotype on serum proteome. Subjects were categorized into two groups: CAD patients (n = 480) and healthy individuals (n = 220). For genotyping, tetra ARMS-PCR was carried out and validated through sequencing, whereas LC/MS-based proteomic analysis of serum samples was performed through label-free quantification. In initial step of genotyping, the frequencies of each genotype GG, GT, and TT were 70%, 27%, and 30% in CAD patients, while in control group, the subjects were 52%, 43%, and 5%, respectively, in CAD patients. The genotypic frequencies in patients vs. control groups found significantly different (p = 0.004), and a strong association of dominant alleles GG with the CAD was observed in both dominant (OR: 2.4 (1.71–3.34), p = 0.001) and allelic genetic models (OR: 2.0 (1.45–2.86), p = 0.001). In second step of label-free quantitation, a total of 40 significant proteins were found with altered expression in CAD patients. The enriched Gene Ontology (GO) terms of molecular functions and pathways of these protein showed upregulated pathways as follows: chylomicron remodeling and assembly, complement cascade activation, plasma lipoprotein assembly, apolipoprotein-A receptor binding, and metabolism of fat-soluble vitamins in G allele carrier of rs1042031 (G > T) vs. mutant T-allele carriers. This study provides better understanding of CAD pathobiology by proteogenomics of APOB. It evidences the influence of APOB rs1042031-dominant (GG) genotype with CAD patients.

The intake of various types and amounts of dietary fats influences metabolic and cardiovascular health. Hence, this study evaluated the impact of routinely consumed Pakistani dietary fats on their cardiometabolic impact. For this, we made four groups of mice, each comprising 5 animals: (1) C-ND: Control mice on a normal diet, (2) HFD-DG: High-fat diet mice on a normal diet plus 10% (w/w) desi ghee, (3) HFD-O: Mice on normal diet plus 10% (w/w) plant oil (4) HFD-BG: Mice on normal diet plus 10% (w/w) banaspati ghee. Mice were fed for 16 weeks, and blood, liver, and heart samples were collected for biochemical, histological, and electron microscopic analysis. The physical factors indicated that mice fed on HFD gained more body weight than the C-ND group. Blood parameters do not show significant differences, but overall, the glucose and cholesterol concentrations were raised in the mice fed with a fat-rich diet, with the highest concentrations in the HFD-BG group. The mice fed with HFD-BG and HFD-O had more lipid droplets in the liver, compared to HFD-DG and C-ND.

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.

To guard consumers from meat adulteration, there is a dire obligation to encompass a sensitive, simple, and precise way for the identification of meat animal species. Molecular techniques have hoisted the expectations of developing authentic and reliable methods for testing the origin of meat species. This study explains the utilization of Polymerase Chain Reaction and Restriction Fragment Length Polymorphism along with mitochondrial cytochrome c oxidase subunit I gene to identify six meat species i.e., buffalo (Bubalus bubalis), cow (Bos indicus), cat (Felis catus), goat (Capra hircus), donkey (Equus asinus) and sheep (Ovis aries). Universal primers were utilized to amplify a specific region (approx. 710 bp) encoding the mitochondrial COI gene in each species and after insilico analysis two restriction enzymes (TasI and TruI) were selected to digest all desired amplicons. Restriction analysis on 3% agarose gel uncovered unique species-specific restriction profiles. The level of COI variation by utilizing TasI and TruI was found to be adequate to produce effectively analyzable species-specific profiles that could recognize all species unambiguously. This study suggested that PCR-RFLP is a swift and reliable scheme in favor of recognition and discrimination of analyzed meat species by mitochondrial COI gene and can be employed in food control laboratories. Keywords: PCR-RFLP; Mitochondrial COI gene; Molecular markers.

In the current study, APOB (rs1052031) genotype-guided proteomic analysis was performed in a cohort of Pakistani population. A total of 700 study subjects, including Coronary Artery Disease (CAD) patients (n = 480) and healthy individuals (n = 220) as a control group were included in the study. Genotyping was carried out by using tetra primer-amplification refractory mutation system-based polymerase chain reaction (T-ARMS-PCR) whereas mass spectrometry (Orbitrap MS) was used for label free quantification of serum samples. Genotypic frequency of GG genotype was found to be 90.1%, while 6.4% was for GA genotype and 3.5% was for AA genotypes in CAD patients. In the control group, 87.2% healthy subjects were found to have GG genotype, 11.8% had GA genotype, and 0.9% were with AA genotypes. Significant (p = 0.007) difference was observed between genotypic frequencies in the patients and the control group. The rare allele AA was found to be strongly associated with the CAD [OR: 4 (1.9–16.7)], as compared to the control group in recessive genetic model (p = 0.04). Using label free proteomics, altered expression of 60 significant proteins was observed. Enrichment analysis of these protein showed higher number of up-regulated pathways, including phosphatidylcholine-sterol O-acyltransferase activator activity, cholesterol transfer activity, and sterol transfer activity in AA genotype of rs562338 (G>A) as compared to the wild type GG genotype. This study provides a deeper insight into CAD pathobiology with reference to proteogenomics, and proving this approach as a good platform for identifying the novel proteins and signaling pathways in relation to cardiovascular diseases.

Background: Hepatitis B virus (HBV) is among the smallest DNA viruses resulting in ~800,000 deaths each year. Pakistan is considered a country affected by HBV. In Pakistan, the most dominant genotype is D. HBV is an enveloped virus of 3.2 kb. The study's goal was to express hepatitis B surface antigen in a bacterial host to produce a recombinant protein.Method: Blood samples were collected in EDTA coated vacutainer from patients after their consent. DNA was extracted from serum through the phenol-chloroform method; Hepatitis B surface gene was cloned in TA cloning vector, subclone in pET 28a expression vector. An expression vector containing the Surface gene was then transformed into a competent bacterial host BL21 and inducted with IPTG at 0.1-0.2mM concentration for expression. The expressed proteins (soluble and pellet form) were analyzed on SDS PAGE.Results: Hepatitis B Surface gene of 681bp after PCR were detected under UV light then successfully cloned and subcloned in pET 28 expression vector. The restricted fragment indicating the gene of interest was 681bp when analyzed on 1.2% Agarose gel under UV light. The required protein of 25kDa was obtained in soluble form when detected on 12% SDS PAGE after staining with Coomassie Blue dye.Conclusion: Hepatitis B surface gene was successfully expressed in both insoluble and pellet forms using E.coli. The expression of surface protein needs to maximize through optimizing conditions to be used as potent candidate for vaccine production to prevent hepatitis B infection.Keywords: Hepatitis B virus; Surface gene; Cloning vector; pET expression vector

Plant growth enhancement using plant beneficial bacteria has been viewed in the sustainable agriculture as an alternative to chemical fertilizers. Actinobacteria, among the group of important plant-associated bacteria, have been widely studied for its plant growth promotion activities. Actinobacteria are considered as a limelight among agriculturists for their beneficial aspects toward plants. They are naturally occurring spore-forming bacteria inhabiting the soil and known for their plant growth-promoting and biocontrol properties. The mechanisms behind these activities include nitrogen fixation, phosphate solubilization, siderophore production, and other attributes such as antifungal production of metabolites, phytohormones, and volatile organic compound. All these activities not only enhance the plant growth but also provide resistance in plants to withstand unfavorable conditions of the environment. Hence, this chapter emphasizes on the plant growth traits of actinobacteria and how far it was studied for enhanced growth and bio-fortification.