Exploring the diversity of CFTR gene mutations in cystic fibrosis individuals of South Asia.

OBJECTIVE: Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CFTR gene. This study aimed to identify the spectrum of CFTR variants reported in individuals with CF from South Asia (ISA). DATA SOURCES AND STUDY SELECTION: We conducted a PubMed search for CFTR variants reported in ISA. Full text of original articles and case reports was read to compile data on reported variants. To gather additional data, we independently cross-referenced each variant with the CFTR Mutation Database and ClinVar. RESULTS: Our investigation identified a total of 92 CFTR variants reported across 30 articles. The most frequently tested, and reported variant was ΔF508 with a global frequency of 69.74%. Notably, we found 14 pathogenic CFTR mutations shared among ISA, originating from more than one South Asian country: ΔF508, 1525-1 G > A, G542X, S549N, R117H, S549R, R709X, V456A, Y569D, L1077P, 1161delC, 1898 + 1 G > T, G551D, and 2184insA. CONCLUSION: In summary, the higher prevalence of consanguinity and the limited availability of CF diagnostic resources in South Asia considerably contribute to the prevalence of genetic disorders like CF. The spectrum of CFTR mutations exhibits noticeable variations within South Asian and other populations. The inclusion of current study-enlisted CFTR gene variants is highly recommended for CF disease genetic testing in South Asia which may aid in achieving a precise diagnosis, enhancing disease management, and discovering drugs for currently untreatable genetic variants. It is also imperative to conduct a comprehensive study in this region, especially in previously unexplored countries such as Nepal, Bhutan, Maldives, and Bangladesh.

Prevalence of GCKR rs1260326 Variant in Subjects with Obesity Associated NAFLD and T2DM: A Case-Control Study in South Punjab, Pakistan.

The glucokinase regulatory protein (GCKR) regulates glycogen metabolism and insulin secretion, and the GCKR rs1260326 is a putative single nucleotide polymorphism (SNP) associated with metabolic disorders including nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (T2DM). This study was conducted to investigate the genetic association of the GCKR rs1260326 in NAFLD and T2DM in our population. NAFLD (n = 103), T2DM (n = 100), and control (n = 100) samples were collected and genotyped for GCKR rs1260326 by tetra-arm PCR. The genetic variant GCKR rs1260326 was significantly linked with NAFLD and T2DM, while the GCKR rs1260326 was significantly associated with the progression of obesity only in NAFLD subjects. The frequency of the C allele (mutant) was higher in both NAFLD (f = 0.69) and T2DM (f = 0.66) subjects as compared to healthy controls of NAFLD (0.52) and T2DM (f = 0.32). The frequency of the C allele was also positively linked with the progression of obesity in both diseases. The frequency of the C allele was 0.66, 0.67, and 0.74 in NAFLD normal weight, overweight, and obese subjects, respectively, while the frequency of the C allele was 0.60, 0.60, and 0.74 in T2DM in normal weight, overweight, and obese subjects, respectively. Homozygous mutant (CC) was 53% in both NAFLD and T2DM subjects, while heterozygous mutant (CT) was 15.53% in NAFLD and 22% in T2DM subjects. Wild-type allele (TT) was 31.06% in NAFLD and 25% in T2DM subjects. In conclusion, the GCKR rs1260326 is a highly prevalent SNP in NAFLD and T2DM subjects, which possibly contributed to obesity, insulin resistance, and metabolic disorders in our population.

Potential of GJA8 gene variants in predicting age-related cataract: A comparison of supervised machine learning methods.

Cataracts are the problems associated with the crystallins proteins of the eye lens. Any perturbation in the conformity of these proteins results in a cataract. Age-related cataract is the most common type among all cataracts as it accounts for almost 80% of cases of senile blindness worldwide. This research study was performed to predict the role of single nucleotide polymorphisms (SNPs) of the GJA8 gene with age-related cataracts in 718 subjects (400 age-related cataract patients and 318 healthy individuals). A comparison of supervised machine learning classification algorithm including logistic regression (LR), random forest (RF) and Artificial Neural Network (ANN) were presented to predict the age-related cataracts. The results indicated that LR is the best for predicting age-related cataracts. This successfully developed model after accounting different genetic and demographic factors to predict cataracts will help in effective disease management and decision-making medical practitioner and experts.

Seasonality, epidemiology and phylogeny of Theileria ovis with a note on hematological and biochemical changes in asymptomatic infected goats from Pakistan.

Caprine theileriosis, caused by Theileria ovis is a serious production issue, especially in the areas that depend on goats and sheep for milk, meat, and other economic benefits. Pakistan has a large goat population, but few reports have been documented from this country regarding PCR-based detection of T. ovis. The molecular prevalence of T. ovis, on a seasonal basis, in various goat breeds enrolled from Muzaffar Garh district of Punjab in Pakistan was determined from October 2018 to September 2019. In this study, 1084 goat blood samples were screened for the detection of T. ovis DNA through PCR-based amplification of 18S rRNA gene. Out of 1084 goats, 12 (1.11%) were infected with T. ovis. The parasite prevalence varied with the sampling seasons (Chi square test, P = 0.008), and the parasite prevalence was highest in goat blood samples collected in summer (2.39%) followed by winter (1.88%). DNA sequencing and BLAST analysis confirmed the presence of T. ovis, and the amplified isolates from the 18S rRNA gene of T. ovis were found to be highly conserved during phylogenetic analysis. Young goats (Fischer exact test, P = 0.022) were found more infected with T. ovis during the winter season. Infected goats had elevated white blood cell counts (Two-sample t-test, P = 0.04), blood urea nitrogen to Creatinine ratio (Two-sample t-test, P = 0.02) and decreased serum Creatinine (Two-sample t-test, P = 0.001) as compared to T. ovis negative goats. We report a relatively low molecular prevalence of T. ovis in goats from the Muzaffar Garh district. However, it is recommended that control measures to eradicate T. ovis infection in goats in this area should be taken.

Association of genotypes at rs438228855 in bovine SLC35A3 receptor gene of Pakistani cattle with the susceptibility to develop complex vertebral malformation.

The aim of this study was to report the genotype and allelic frequency at rs438228855 (G > T) in SLC35A3 receptor gene and its association with a complex vertebral malformation (CMV) in the enrolled Pakistani cattle. Our results indicated that allelic and genotype frequency at rs438228855 varied non-significantly (p > .05) among the three enrolled cattle breeds. GT (heterozygous) genotype was most abundant (0.54) followed by GG (wild type) genotype (0.45) while the mutant genotype (TT) was not observed among the enrolled cattle. It was observed that the Holstein Friesian breed had more GG (wild) than GT (heterozygous) genotypes while Sahiwal and cross cattle breed had more heterozygous (GT) combination at rs438228855 than the wild (GG) genotype. Significant variations in white blood cell count, % lymphocytes, red blood cell count, % monocytes, haemoglobin, mean corpuscular volume and mean corpuscular haemoglobin concentration were observed when compared between the enrolled cattle breeds. Most of the studied haematological parameters showed no association with the genotype at rs438228855. In conclusion, the heterozygosity at rs438228855 is not limited to the Holstein Friesian breed as local Sahiwal and crossbred cattle had also higher heterozygosity at rs438228855. We recommend that animals must be genotyped for rs438228855 before their selection as breeders to prevent economic losses.

Experimental Validation of MHC Class I and II Peptide-Based Potential Vaccine Candidates for Human Papilloma Virus Using Sprague-Dawly Models.

Human papilloma virus (HPV) causes cervical and many other cancers. Recent trend in vaccine design is shifted toward epitope-based developments that are more specific, safe, and easy to produce. In this study, we predicted eight immunogenic peptides of CD4+ and CD8+ T-lymphocytes (MHC class I and II as M1 and M2) including early proteins (E2 and E6), major (L1) and minor capsid protein (L2). Male and female Sprague Dawly rats in groups were immunized with each synthetic peptide. L1M1, L1M2, L2M1, and L2M2 induced significant immunogenic response compared to E2M1, E2M2, E6M1 and E6M2. We observed optimal titer of IgG antibodies (>1.25 g/L), interferon-γ (>64 ng/L), and granzyme-B (>40 pg/mL) compared to control at second booster dose (240 µg/500 µL). The induction of peptide-specific IgG antibodies in immunized rats indicates the T-cell dependent B-lymphocyte activation. A substantial CD4+ and CD8+ cell count was observed at 240 µg/500 µL. In male and female rats, CD8+ cell count for L1 and L2 peptide is 3000 and 3118, and CD4+ is 3369 and 3484 respectively compared to control. In conclusion, we demonstrated that L1M1, L1M2, L2M1, L2M2 are likely to contain potential epitopes for induction of immune responses supporting the feasibility of peptide-based vaccine development for HPV.

Ingraining Polio Vaccine Acceptance through Public Service Advertisements in the Digital Era: The Moderating Role of Misinformation, Disinformation, Fake News, and Religious Fatalism.

Recently, misinformation and disinformation, as well as fake news, have become global threats to public health owing to their role in spreading viral health hazard information. The growing explosive religious fatalistic views presented on social media and widespread misinformation, disinformation, and fake news can result in detrimental outcomes in adopting protective behavior. The moderating implications of misinformation and religious fatalism can be severe, leading to adverse effects on polio vaccine acceptance. Consequently, this research provides brief empirical evidence on the efficacy of risk communication strategies to address polio vaccine reluctance in a digital age landscape, an area that remains understudied. This research argues that the spread of misinformation, disinformation, fake news, and religious fatalism is not solely the bane of the polio vaccine, but rather represents the absence of risk communication strategies. The study opines that polio vaccine acceptance can be improved using risk communication strategies. Recognizing these risk factors and counter-risk communication strategies, this research tested a theoretical model using the cross-sectional survey design. Overall, data was collected from 2160 parents with children aged below five years. The results, based on structural equation modeling, revealed that public service advertisements are an effective tool to counter the inverse impacts of misinformation, disinformation, fake news, and religious fatalism. Furthermore, the inverse moderating role of misinformation, disinformation, fake news, and religious fatalism has been verified to potentially diminish polio vaccine acceptance. These results suggest that healthcare providers must identify and address all forms of digitally disseminated information that encumbers public health behaviors. Accordingly, this research recognized the utilization of evidence-based strategic communication campaigns to cultivate and encourage the literacy necessary to counter health hazard information, including misinformation. This study's findings will benefit health and other concerned authorities in utilizing strategic communication on different media platforms to reduce or eradicate the polio endemic.

First report on the probiotic potential of Mammaliicoccus sciuri isolated from raw goat milk.

Probiotics are considered effective microbial dietary supplements that provide beneficial effects to consumers, usually by restoring or improving gut microflora. Goat milk is one of the rich sources of probiotics as well as nutrients. Therefore, the primary aim of this research was to isolate and evaluate the potential of novel indigenous probiotic strains present in goat milk. Six different raw goat milk samples were collected from different areas of Multan, Pakistan. For bacterial characterization, samples were cultured and isolated on MRS agar plates for different morphological and biochemical tests. The probiotic potential of the six isolates, all of which were gram positive (G1, G2, G3, G4, G5, and G6) and five of which were catalase negative (all except G1), were assessed via a milk coagulation assay and antimicrobial activity, pH tolerance, phenol tolerance, and sodium chloride (NaCl) tolerance tests, which revealed that all the isolates coagulated in milk and showed protease and lipase activity, except G3. All six isolates showed tolerance against 0.2% phenol and 2-4% NaCl and were able to survive in both alkaline and acidic conditions. Only five isolates showed antimicrobial activity against indicator strain Aspergillus niger strain STA9, validating their probiotic nature. The most potent bile-tolerant and bacteriocin-producing isolate, G1, also showed γ-hemolytic activity and resistance to penicillin but showed susceptibility to other antibiotics. The lactic acid-producing (0.60% titratable acidity) G1 isolate was identified as a novel strain of Mammaliicoccus sciuri based on 16S rDNA sequencing. The above findings suggest that the potent M. sciuri GMN01 strain can serve as a potential probiotic strain. A potent probiotic strain isolated from raw goat milk could be utilized as a dietary supplement, and goat milk could become an alternative to other sources of milk, particularly cow milk. However, safety aspects of this strain require further investigation because the present safety tests are insufficient to conclude that the GMN01 isolate is safe.

Experimental Study of Potential CD8+ Trivalent Synthetic Peptides for Liver Cancer Vaccine Development Using Sprague Dawley Rat Models.

Background: Liver cancer (LC) is the most devastating disease affecting a large set of populations in the world. The mortality due to LC is escalating, indicating the lack of effective therapeutic options. Immunotherapeutic agents may play an important role against cancer cells. As immune cells, especially T lymphocytes, which are part of cancer immunology, the design of vaccine candidates for cytotoxic T lymphocytes may be an effective strategy for curing liver cancer. Results: In our study, based on an immunoinformatics approach, we predicted potential T cell epitopes of MHC class I molecules using integrated steps of data retrieval, screening of antigenic proteins, functional analysis, peptide synthesis, and experimental in vivo investigations. We predicted the binding affinity of epitopes LLECADDRADLAKY, VSEHRIQDKDGLFY, and EYILSLEELVNGMY of LC membrane-bounded extracellular proteins including butyrophilin-like protein-2 (BTNL2), glypican-3 (GPC3), and serum albumin (ALB), respectively, with MHC class I molecules (allele: HLA-A∗01:01). These T cell epitopes rely on the level of their binding energy and antigenic properties. We designed and constructed a trivalent immunogenic model by conjugating these epitopes with linkers to activate cytotoxic T cells. For validation, the nonspecific hematological assays showed a significant rise in the count of white blood cells (5 × 109/l), lymphocytes (13 × 109/l), and granulocytes (5 × 109/l) compared to the control after administration of trivalent peptides. Specific immunoassays including granzyme B and IgG ELISA exhibited the significant concentration of these effector molecules in blood serum, indicating the activity of cytotoxic T cells. Granzyme concentration increased to 1050 pg/ml at the second booster dose compared to the control (95 pg/ml), while the concentration of IgG raised to 6 g/l compared to the control (2 g/l). Conclusion: We concluded that a potential therapeutic trivalent vaccine can activate and modulate the immune system to cure liver cancer on the basis of significant outcomes of specific and nonspecific assays.

Molecular detection and prevalence of Theileria ovis and Anaplasma marginale in sheep blood samples collected from Layyah district in Punjab, Pakistan.

Theileria ovis and Anaplasma marginale are intracellular pathogens affecting a wide range of animals, causing huge economic losses worldwide. The present study reports the molecular evidence of Theileria ovis and Anaplasma marginale in sheep blood samples (N = 218) collected from Layyah district in Punjab (Pakistan), where economy heavily relies on livestock. A 520 base pair fragment specific for 18S ribosomal RNA gene of Theileria ovis was PCR amplified in 23/218 (10.6%) sheep blood samples, while for Anaplasma marginale, a 265 base pair fragment specific for msp1b gene was generated in 15/218 (6.9%) sheep blood samples. Two blood samples were found co-infected (0.9%) with both parasites. Amplified PCR products of both parasites were confirmed by DNA sequencing and submitted to GenBank. Prevalence of both Theileria ovis (p = 0.3) and Anaplasma marginale (p = 0.4) varied non-significantly among the investigated sheep breeds. Tick burden on dogs present with sheep herds was found associated with Theileria ovis infection in sheep (p = 0.05). It was observed that lambs (p = 0.009), sheep in small herds (p = 0.04), and tick burden on dogs present with sheep herds (p = 0.01) were associated with Anaplasma marginale infection in sheep during the present study. In conclusion, we are reporting a higher prevalence of Theileria ovis than Anaplasma marginale in blood samples of sheep collected from Layyah district. Tick-infested dogs were found to be risk factors for the transmission of both pathogens in sheep, and tick control strategies should be extended to dogs associated with sheep herds in this area.

Single nucleotide polymorphism (rs7543472) in EPHA2 gene is associated with age-related cataract in subjects enrolled from Multan in southern Punjab: A case-control study.

OBJECTIVES: To examine whether Ephrin type A receptor 2 gene polymorphisms are associated with susceptibility to age-related cataract. METHODS: The case-control study was conducted from January to May 2014 in Multan, Pakistan, and comprised patients of age-related cataract enrolled from Nishtar Hospital, Multan, and age-matched healthy controls without any type of cataract from the local population. A questionnaire was used to gather clinical and epidemiological data. Deoxyribonucleic acid was extracted from blood samples, and analysis of rs11260867, rs3568293 and rs7543472 single nucleotide polymorphisms was performed by using tetra amplification-refractory mutation system polymerase chain reaction protocol. Data was analysed using SPSS 17. RESULTS: Of the 230 subjects, 129(%) were patients and 101(%) were controls. Among the three polymorphisms analysed, rs7543472 was associated with age-related cataract. Among the epidemiological and clinical factors, age, diabetes, blood pressure, smoking, radiation exposure, steroids usage and use of tranquilisers were associated with age-related cataract (p<0.05 each). CONCLUSIONS: Polymorphism rs7543472 was found to be associated with age-related cataract.

First Report Regarding the Simultaneous Molecular Detection of Anaplasma marginale and Theileria annulata in Equine Blood Samples Collected from Southern Punjab in Pakistan.

AIM: The present study was designed to check the molecular detection of Anaplasma marginale and Theileria annulata in blood samples of horses and donkeys collected from Dera Ghazi Khan District in Punjab and to document their phylogenetic origin and their association with studied epidemiological factors (sex and age) and complete blood count parameters, if any. METHODS AND RESULTS: A total of 195 blood samples were collected from apparently healthy horses (N = 141) and donkeys (N = 54). A. marginale DNA was detected by PCR in 4.9% (7/141) horse and in 9.2% (5/54) of donkey blood samples. Prevalence of T. annulata was 5.6% (8/141) and 11.1% (6/54) in horse and donkey samples, respectively. While 1.4% (N = 2) horses and 3.7% (N = 2) donkeys were found co-infected with both parasites. Representative amplicon for both parasites was confirmed by DNA sequenced and partial DNA sequence of the major surface protein-1b encoding gene of A. marginale and cytochrome b gene from T. annulata were submitted to the GenBank database under the accession number MK792344-MK792348. Epidemiological data analysis revealed that female horses were more prone to A. marginale (P = 0.02) while female donkeys were more susceptible to A. marginale (P < 0.001) and T. annulata (P < 0.001) infection. It was observed that horse and donkey infected either with Anaplasma marginale or Theileria annulata had significantly disturbed red and white blood cell counts and their associated parameters. CONCLUSION: This is a first ever study regarding molecular detection of A. marginale and T. annulata in equine blood samples from Pakistan. We recommend that this multiplex PCR protocol should be used for the detection of Anaplasma marginale and Theileria annulata in livestock for their proper diagnosis and treatment.

Association of single nucleotide polymorphism in CD28(C/T-I3 + 17) and CD40 (C/T-1) genes with the Graves' disease.

OBJECTIVE: To find out a correlation between the single nucleotide polymorphisms in cluster of differentiation 28 and cluster of differentiation 40 genes with Graves' disease, if any. METHODS: This case-control study was conducted at the Multan Institute of Nuclear Medicine and Radiotherapy, Multan, Pakistan, and comprised blood samples of Graves' disease patients and controls. Various risk factors were also correlated either with the genotype at each single-nucleotide polymorphism or with various combinations of genotypes studied during present investigation. RESULTS: Of the 160 samples, there were 80(50%) each from patients and controls. Risk factor analysis revealed that gender (p=0.008), marital status (p<0.001), education (p<0.001), smoking (p<0.001), tri-iodothyronine (P <0.001), thyroxin (p<0.001) and thyroid-stimulating hormone (p<0.000) levels in blood were associated with Graves' disease. CONCLUSIONS: Both single-nucleotide polymorphisms in both genes were not associated with Graves' disease, either individually or in any combined form.

Association of Single Nucleotide Polymorphisms in XRCC1 (194) and XPD (751) with Age-related cataract.

PURPOSE: Age-related cataract (ARC) is a multifactorial disease and different risk factors, including genetic and environmental, are responsible for the development of its various types. The aim of this study was to find out a correlation, if any, between ARC and the single nucleotide polymorphisms (SNPs) in DNA repair genes XRCC1 (X-ray repair cross-complementary-1) [Arg194Trp (rs1799782)] and XPD (xerodermapigmentosa complementation group D) [Lys751Gln (rs13,181]. METHOD: The genotype at these two SNPs was analyzed in 260 subjects (125 control and 135 patients) from Southern Punjab population (Pakistan) by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method. Genotype at both analyzed codons was correlated either individually or in various combinations with the studied epidemiological factors known to be associated with ARC. RESULTS: Our results indicated that both SNPs Arg194Trp in XRCC1 (P = 0.967) and Lys751Gln in XPD (P = 0.995) were not associated with ARC whether they were analyzed individually or in combined form (P > 0.05). Analysis of epidemiological factors revealed that age (P < 0.001), cast of subjects (P = 0.001), diabetes (P < 0.001), hypertension (P = 0.001), smoking habit (P = 0.01), drug abuse (P < 0.05), steroid use (P = 0.001) and body weight (P < 0.001) can influence the incidence of ARC in enrolled subjects. After applying Binary logistic regression it was found that the weight (P < 0.01), family history (P = 0.05), drug abuse (P = 0.05), smoking (P < 0.05) and steroid use (P < 0.05) has a significant association with the phenotype of the subjects. All epidemiological factors were also studied in association with various genotypic combinations of both SNPS, diabetes was the only factor that had a significant association (P < 0.001) association with ARC. Hypertension (P = 0.01), body weight (P < 0.05) and cast (P < 0.001) were found associated with ARC when epidemiological factors were individually correlated with ARC. Result of the two proportion test indicated that gender had no influence on the incidence of disease. CONCLUSION: It is concluded that studied SNPs in XRCC1 and XPD have no association with the incidence of age related cataract in the analyzed group of subjects.

Molecular Detection and Prevalence of Hepatozoon canis in Dogs from Punjab (Pakistan) and Hematological Profile of Infected Dogs.

The intraleukocytic parasite, Hepatozoon canis, causes the sometimes fatal tick borne disease canine hepatozoonosis. In this study, dogs from Islamabad, Lahore, and Multan Districts of the Punjab region of Pakistan were surveyed to investigate the presence and prevalence of H. canis infection and to determine the effects of the parasite on hematological parameters. Blood samples were collected from 151 domestic dogs (149 pet, 2 stray) of both sexes and varying ages. Data on sex, age, tick infestation, and clinical factors (body temperature, mucous membrane status, and presence of hematuria and vomiting) were collected. Using PCR, 18 dogs (11.9%) were found positive for the presence of H. canis DNA. Partial sequences of the 18S rRNA gene shared 99-100% similarity with the corresponding H. canis isolates. This epidemiological survey revealed higher prevalence of H. canis in Islamabad (11/49, 22.4%) compared to Lahore (3/52, 5.8%) and Multan (4/50, 8%) in Pakistan. No investigated epidemiological or clinical factors were found to be associated with the presence of H. canis (p > 0.05) in dogs. H. canis positive dogs exhibited higher minimum inhibitory dilution (p = 0.04), mixed inclusion (p = 0.008) and relative distribution width of red blood cells (p = 0.02), and lower hematocrit (p = 0.03) and mean hemoglobin content (p = 0.03) than did dogs in which H. canis was not detected. We are recommending this PCR-based protocol to the veterinary practitioners for the detection and/or confirmation of H. canis in dogs suspected for hepatozoonosis to improve their health status.

Report - Antibacterial activity of sea buckthorn (Hippophae rhamnoides L.) against methicillin resistant Staphylococcus aureus (MRSA).

Objective of the present study was to investigate the antibacterial activity of Sea buckthorn (Hippophae rhamnoides L.) berries and leaves against methicillin resistant Staphylococcus aureus (MRSA) by using the standard disc diffusion method. Chloroform, n-hexane and aqueous extract of the plant parts were used. Doses of 2mg/ml, 4 mg/ml and 6mg/ml were tested against the microorganism, and the zone of inhibition was compared against the standard drug vancomycin. Results indicated that n-hexane and chloroform extracts of berries and n-hexane extract leaves showed significant (p<0.05) antibacterial activity comparable with vancomycin. It was concluded from the study that extracts berries and leaves of Hippophae rhamnoides have antibacterial activity against MRSA.

Foot-and-mouth disease: overview of motives of disease spread and efficacy of available vaccines.

Control and prevention of foot and mouth disease (FMD) by vaccination remains unsatisfactory in endemic countries. Indeed, consistent and new FMD epidemics in previously disease-free countries have precipitated the need for a worldwide control strategy. Outbreaks in vaccinated animals require that a new and safe vaccine be developed against foot and mouth virus (FMDV). FMDV can be eradicated worldwide based on previous scientific information about its spread using existing and modern control strategies.

Association of endothelial nitric oxide synthase (eNOS) gene polymorphism (Glu 298 Asp) with coronary artery disease in subjects from Multan, Pakistan.

Acute Coronary Syndrome (ACS) is the most common disease and cause of mortality in both genders across the world and certain risk factors i.e. age, gender, smoking, diabetes, hypertension, drugs usage, weight etc are known to be associated with the disease. The aim of this study was to find if there is any correlation exists between ACS and hereditary genetic defect in endothelial nitric oxide synthase (ecNOS) gene as eNOS generates Nitric oxide in blood vessels and regulates the vascular tone hence directly affecting the cardiovascular function. Single nucleotide polymorphism (SNP) (Glu 298 Asp) in ecNOS was determined in 280 subjects, from Southern Punjab (in Pakistan) population, including (160 ACS patients and 120 healthy controls) by PCR-RFLP method and genotype was correlated with various risk factors as well as with serum cholesterol and triglyceride levels. Our results indicated that the genotype Glu 298 Asp was not associated with ACS but when various studied parameters were compared among patients suffering from various forms of ACS and their healthy controls, it was observed that age (45-55 years) (P = 0.05), gender (male) (P < 0.001), education (P<0.001), family history (P=0.03), hypertension (P<0.001), diabetes (P<0.01) and smoking habit (P = 0.03) were the significantly different parameters among them and may be associated with the incidence of cardiovascular disease. Cholesterol (161.5±79 mg/dL) level was found to be higher in patients (P = 0.04) than controls while triglyceride remained unaffected (P = 0.87) in both groups.

A report on the high prevalence of Anaplasma sp. in buffaloes from two provinces in Pakistan.

The present study was designed to use a PCR-RFLP protocol for the molecular detection of Anaplasma sp. and to compare its prevalence in blood samples from buffaloes (Bubalus bubalis) from 2 provinces of Pakistan and to determine the risk factors associated with the spread of Anaplasma infection. A total of 281 blood samples were collected from adults and calves of buffaloes from 4 sampling sites in Southern Punjab (Bahawalnagar, Burewala, Layyah, and Multan districts) and 2 in Khyber Pukhtoon Khwa (Peshawer and Kohat districts) from randomly selected herds. Data on the characteristics of the animals (gender, age, tick presence or absence, prior treatment for Anaplasma infection) and the herd (location, size, dogs associated with the herds, tick burden of dogs associated with the herds) were collected through questionnaires. One hundred and sixteen blood samples (41% of total) produced the 577-base pairs DNA fragment specific for the 16S rRNA gene of Anaplasma sp. by PCR amplification. Twenty of the 116 Anaplasma sp.-positive PCR products were confirmed to be Anaplasma marginale upon restriction with BssNa1, specific to cut A. marginale sequences. ANOVA results revealed a highly significant association between sampling sites and prevalence of Anaplasma sp. (P<0.001) indicating that Anaplasma sp. prevalence was variable among all 6 sampling sites. Risk factor analysis indicated that the association of dogs with the herd was the only significant (P=0.029) risk factor associated with the spread of Anaplasma sp. in buffaloes while sex, age, presence of ticks on animals or herd size showed no association with Anaplasma infection.

Effects of glutathione-S-transferase polymorphisms on the risk of breast cancer: a population-based case-control study in Pakistan.

Cancer is widely accepted as one of the major health issues. Diet composition and exposure to environmental genotoxic and carcinogenic agents such as polycyclic aromatic hydrocarbons (PAHs) are among the causative factors for various types of cancers, including breast cancer. Low penetrance genes including glutathione S transferases (GST) in association with environmental factors can contribute greatly in the development of breast cancer. We were interested to investigate the association of the polymorphisms of GSTM1, GSTT1, GSTP1 and GSTO2 with the risk of breast cancer in the Pakistani population. One hundred women visiting the Department of Radiology and Oncology, Nishter Hospital, Multan with pathologically confirmed breast cancer, and 100 healthy volunteers from central Pakistan were enrolled in the present study. The strength of the association of various factors with breast cancer was measured by calculating odd ratios (ORs) which were determined by logistic regression. All P values cited are two-sided; differences resulting in a P value of less or equal to 0.05 were declared statistically significant. The Hardy Weinberg equilibrium was tested for the genotype proportions in the control group, as a measure of quality control. Those aged 36-45, in menopause or with a history of cancer in the family had a significantly higher prevalence of breast cancer compared with controls. The frequency of GSTM1 and GSTT1 was similar in both control and patients suggesting no association with the risk of cancer development, however GSTM1 and GSTT1 were significantly linked with the risk of breast cancer in smokers and in women with a history of breast cancer in the family respectively. Similarly women homozygous for GSTP1 or GSTO2 and with a history of breast cancer, or in menopause, were at greater risk of breast cancer than wild type or heterozygotes. Our data suggest that genetic differences in some GST genes may be linked with an increased susceptibility to breast cancer. Furthermore it also gives an insight into the interaction between the GST polymorphisms and pre-menopausal diagnosis of breast cancer.