Flagellar motility plays important role in Biofilm formation of Bacillus cereus and Yersinia enterocolitica.

Bacteria live either independently as planktonic cells or in organized surface associated colonies called as biofilms. Biofilms play an important role in increased pathogenesis of bacteria and it is assumed that motility is one of the contributing factors towards biofilm initiation. This study was planned to identify the role of flagella in biofilm formation by constructing flagellated (wild type) and physically disrupted variants (non-motile). Total 10 clinical bacterial strains were isolated and characterized. Morphological and biochemical study identified these strains as Enterobacter spp., Pseudomonas spp., Yersinia spp., Escherichia spp., Salmonella spp., Proteus spp., Staphylococcus spp., Streptococcus spp., Lactobacillus spp. and Bacillus spp. Among all strains, two strains including Yersinia spp and Bacillus spp. showed higher antibiotic resistance, hence studied at molecular and physiological level. Biofilm formation capacity of strains was analyzed using three methods including Congo red assay, Test tube assay and Liquid-interface coverslip assay. Afterwards, flagellar disintegration was induced by blending and centrifugation for 5, 10 and 15 minutes. 16S rRNA sequencing showed two strains as Bacillus cereus and Yersinia enterocolitica. Both strains produced significant biofilm by all three above mentioned methods. A motility test of these blended variants showed partial/diminished motility with increased blending time. The significant loss in biofilm formation after 15 minutes blending confirmed the important flagellar contribution to the initiation of biofilm formation. This biofilm defect observed in flagella paralysed/minus variants presumably may be due to defects in attachments to surface at early stages. This study indicated that flagellar motility is crucial initially for surface attachment and subsequently for biofilm formation.

Genetic association of lipid metabolism related SNPs with myocardial infarction in the Pakistani population.

Myocardial infarction (MI) is the major cardiovascular disease. This can be caused by mutual interaction of environmental and genetic factors. The current study was designed to investigate the role of lipid metabolism related genetic polymorphisms with the onset of MI in Punjabi population of Pakistan. A total of 384 subjects was studied from April 2011 to July 2012. To determine the genetic associations with MI, the single nucleotide polymorphisms (SNPs) were genotyped by sequencing, as well as one label extension method. Out of eight SNPs in four candidate genes, seven genetic variants were significantly (P < 0.05) associated with elevated risk of MI. In current study two SNPs rs662799 risk allele G (P = 0.03) and rs3135506 risk allele C (P = 0.05) of APOA5 were found to be associated with significant higher risk of triglyceride levels, irrespective of age, sex, obesity, diabetes, hypertension and smoking. Gene variants (rs1558861, rs662799 and rs10750097) in APOA5 showed almost complete linkage disequilibrium and their minor allele frequencies (0.34, 0.28, and 0.41 respectively) were more prevalent (P < 0.05) in cases than controls. We further revealed risk haplotypes (C-T-G-A, G-C-A-G; P = 0.001) and protective haplotypes (G-T-A-G, C-C-G-A; P = 0.005) between these four SNPs for the progression of MI. Current study confirms the correlation between lipid metabolism related SNPs with MI and supports the role of APOA5 in raising plasma triglyceride levels in Pakistanis. However further studies are needed for delineating the role of these SNPs.

Association Between Arsenic Toxicity, AS3MT Gene Polymorphism and Onset of Type 2 Diabetes.

Arsenic (As) exposure in drinking water has become a serious public health issue. AS3MT gene is involved in the metabolism of arsenic, so a single nucleotide polymorphism in this gene may lead to the development of type 2 diabetes in arsenic-exposed areas. This study aimed to evaluate the association of the AS3MT gene with the development of type 2 diabetes in highly arsenic-exposed areas of Punjab, Pakistan. Total 200 samples equal in number from high arsenic exposed-areas of Lahore (Nishtar) and Kasur (Mustafa Abad) were collected. rs11191439 was utilized as an influential variable to evaluate the association between arsenic metabolism and diabetes status to find a single nucleotide polymorphism in the AS3MT gene. We observed the arsenic level in drinking water of the arsenic-exposed selected areas 115.54 ± 1.23 µg/L and 96.88 ± 0.48 µg/L, respectively. The As level in the urine of diabetics (98.54 ± 2.63 µg/L and 56.38 ± 12.66 µg/L) was higher as compared to non-diabetics (77.58 ± 1.8 µg/L and 46.9 ± 8.95 µg/L) of both affected areas, respectively. Correspondingly, the As level in the blood of diabetics (6.48 ± 0.08 µg/L and 5.49 ± 1.43 µg/L) and non-diabetics (6.22 ± 0.12 µg/L and 5.26 ± 0.24 µg/L) in the affected areas. Genotyping showed significant differences in the frequencies of alleles among cases and controls. Nevertheless, notable disparities in genotype distribution were observed in SNPs rs11191439 (T/C) (P < 0.05) and when comparing T2D patients and non-diabetic control subjects. The AS3MT gene and clinical parameters show a significant association with the affected people with diabetes living in arsenic-exposed areas.

Association of APO B gene polymorphisms with the development of myocardial infarction in Pakistani population.

Myocardial infarction (MI) is when a blood clot in the coronary artery obstructs blood flow to a specific part of the heart, leading to the death of myocardium in that area. The development of MI is influenced by various environmental factors, genetic components, and their interactions, even though the exact cause has not been fully established. This is the first case-control study examining the possible association between the human Apo B gene and MI in the Punjab region of Pakistan. The study included 100 patients and 50 healthy individuals. Genomic DNA was isolated from blood samples using manual extraction methods. Subsequently, primers were optimized, and genotyping was performed using PCR, followed by DNA sequencing and RFLP analysis. The research focused on two specific APO B gene SNPs, codon 4154 G/A (rs1801701) and codon 2488 G/A (rs1042031). Both SNPs involved the substitution of guanine with adenine. It was found that individuals carrying the minor allele A of SNP rs1801701 (p < 0.001) and the minor allele A of rs1042031 (p < 0.001) had a significantly higher risk of developing MI. Additionally, haplotype analysis revealed that the AA haplotype (comprising both rs1801701 and rs1042031 SNPs) was associated with a substantially increased risk of MI (OR = 3.845). In conclusion, the study provides evidence supporting the association between specific mutations in the APOB gene and the risk of myocardial infarction in the Pakistani population.

Therapeutic Potential of Green Synthesized Silver Nanoparticles for Promoting Wound-Healing Process in Diabetic Mice.

Diabetes is a serious metabolic disorder characterized by abnormal glucose levels in the body. Delayed wound healing is a severe diabetes complication. Nanotechnology represents the latest advancement in treating diabetic wounds through nanoparticles (NPs). In this study, silver nanoparticles (AgNPs) were synthesized using a green method involving cucumber pulp extract. The synthesis was confirmed using techniques including ultraviolet-visible spectrophotometry (UV-Vis), Fourier-transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive X-ray (EDX). To evaluate wound-healing properties, mouse models were utilized with wounds induced by excision on the dorsal surface. An ointment containing silver nanoparticles was applied to assess its healing potential. Additionally, antibacterial and antioxidant activities were examined using agar well diffusion and DPPH scavenging methods, respectively. The results demonstrated that the ointment prepared with green synthesized AgNPs effectively healed the wounds within 15 days, while also exhibiting antibacterial and antioxidant properties. Therefore, it can be concluded that due to its efficacy in biological activities, silver nanoparticles can be employed in the treatment of diabetic wounds.

Vaccine Adjuvants: Selection Criteria, Mechanism of Action Associated with Immune Responses and Future Directions.

The most effective method to minimize the prevalence of infectious diseases is vaccination. Vaccines enhance immunity and provide protection against different kinds of infections. Subunit vaccines are safe and less toxic, but due to their lower immunogenicity, they need adjuvants to boost the immune system. Adjuvants are small particles/molecules integrated into a vaccine to enhance the immunogenic feedback of antigens. They play a significant role to enhance the potency and efficiency of vaccines. There are several types of adjuvants with different mechanisms of action; therefore, improved knowledge of their immunogenicity will help develop a new generation of adjuvants. Many trials have been designed using different kinds of vaccine adjuvants to examine their safety and efficacy, but in practice, only a few have entered in animal and human clinical trials. However, for the development of safe and effective vaccines, it is important to have adequate knowledge of the side effects and toxicity of different adjuvants. The current review discussed the adjuvants which are available for producing modern vaccines as well as some new classes of adjuvants in clinical trials.

Molecular evolution, virology and spatial distribution of HCV genotypes in Pakistan: A meta-analysis.

Background: Hepatitis C, caused by the Hepatitis C Virus (HCV), is the second most common form of viral hepatitis. The geographical distribution of HCV genotypes can be quite complex, making it challenging to ascertain the most prevalent genotype in a specific area. Methods: To address this, a review was conducted to determine the prevalence of HCV genotypes across various provinces and as a whole in Pakistan. The scientific literature regarding the prevalence, distribution, genotyping, and epidemiology of HCV was gathered from published articles spanning the years 1996-2020. Results: Genotype 1 accounted for 5.1% of the patients, with its predominant subtype being 1a at 4.38%. The frequencies of its other subtypes, 1b and 1c, were observed to be 1.0% and 0.31% respectively. Genotype 2 had a frequency of 2.66%, with the most widely distributed subtype being 2a at 2.11% of the patients. Its other subtypes, 2b and 2c, had frequencies of 0.17% and 0.36% respectively. The most prevalent genotype among all isolates was 3 (65.35%), with the most frequent subtype being 3a (55.15%), followed by 3b (7.18%). The prevalence of genotypes 4, 5, and 6 were scarce in Pakistan, with frequencies of 0.97%, 0.08%, and 0.32% respectively. The prevalence of untypeable and mixed genotypes was 21.34% and 3.53% respectively. Estimating genotypes proves to be a productive method in assisting with the duration and selection of antiviral treatment. Different HCV genotypes can exhibit variations in their response to specific antiviral treatments. Different genotypes may have distinct natural histories, including variations in disease progression and severity. Some genotypes may lead to more rapid liver damage, while others progress more slowly. Conclusions: This information can guide screening and testing strategies, helping to identify individuals at higher risk of developing severe complications. Studying the distribution of HCV genotypes in a population can provide valuable insights into the transmission dynamics of the virus.

Bacillus licheniformis (MN900686) Mediated Synthesis, Characterization and Antimicrobial Potential of Silver Nanoparticles.

The use of bacteria in the synthesis of silver nanoparticles (AgNPs) emerges as an ecofriendly and exciting approach. In the present study, we reported the biosynthesis of AgNPs by using culture supernatant of the bacteria Bacillus licheniformis (MN900686). The biogenically synthesized AgNPs were confirmed by the change in the color of the culture filtrate from yellow to brown after the addition of AgNO3. Further characterization performed by means of UV vis-spectroscopy showed absorption peak at 414 nm which confirmed the formation of AgNPs. Fourier Transfer infrared (FTIR) confirmed the involvement of biological molecules in the formation of nanoparticles (NPs). The SEM revealed that the NPs have approximately 38 nm size. The agar well diffusion assay was used to determine antibacterial activity while tube dilution method was used to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The human pathogenic bacterial strains i.e., P. aeruginosa (MN900691) and B. subtilis (MN900684), were used as test strains. The anti-bacterial assay against test strains revealed that these NPs showed concentration dependent increased zone of inhibition (ZOI). The maximum ZOI at 25 µL of AgNPs was 20 mm against B. subtilis after 24 hours of incubation. One-way ANOVA test showed significant ZOI (p ≤ 0.05) against B. subtilis. The MIC was ranged from 4.3-6.6 µg/mL while MBC ranged from 8.3 to 6.6 µg/mL. Overall, this study suggested that the biogenically synthesized NPs are an effective alternative source of antimicrobials against pathogenic bacteria.

Pathophysiology and antioxidant status of patients with fibromyalgia.

Fibromyalgia syndrome (FMS) is characterized by systemic pain of unknown etiology and is often accompanied by various psychological symptoms. Research on different parameters in fibromyalgia (FM) indicates that multifactors are involved in its pathophysiology; such as genetic factors, substance P, serotonin, hypothalamic pituitary adrenal axis (HPA), muscles metabolic dysfunction, reactive oxygen species (ROS) and reactive nitrogen species (RNS). Oxidative stress has also been implicated in the pathophysiology of FM; therefore, supplementation with antioxidants may be important in modulation of the effects of ROS in patients with FM.

Impact of Interplay between Obese Gut Microbiota and Diet in Developing Obesity in Synthetic Community Mice.

This study aims at investigating the effects of cultured gut microbiota (GM) of obese human coupled high fat diet (HFD) or chow diet (CD) in development of obesity in mice. 20 stool samples were collected from obese patients and isolated bacteria were identified morphologically and biochemically. Identified isolates were mixed in equal proportions to synthesize obese GM. In vivo study was performed using obese GM combined with HFD/CD using mouse model for three months. Albino mice were treated with ampicillin from one week prior to birth until weaning of the pups at seven weeks of age and then inoculated with obese GM. Sixteen mice were divided into four groups: i.e. group 1 (G1) mice fed with CD, group 2 (G2) mice with HFD, group 3 (G3) mice with GM + HFD and group 4 (G4) mice with GM + CD. Mice from groups 3-4 were considered synthetic community (SC) mice due to transfer of synthesize human GM. 16S rRNA sequencing identified five abundant bacteria as Pseudomonas aeruginosa, Staphylococcus sp., Escherichia coli, Morganella morganii, and Klebsiella oxytoca (accession numbers: MZ150742-MZ150746). In vivo study indicated that GM combination with either HFD/CD caused significantly increased body weight in SC mice (BMI; Kg/m2) compared to HFD or CD fed mice groups. One way ANOVA revealed highly significant increase (p ≤ 0.001) in levels of total cholesterol (TC), triglycerides and low density lipoprotein (LDL) in GM coupled diet groups (G3-G4; SC mice) compared to significant increase in HFD group (G2) versus CD group (G1). Our study is first of its kind to report significant effects of using purified strains as obese GM plus diet (HFD/CD) in inducing obesity in SC mice and elevated serum liver parameters as metabolic indicators, hence providing strong evidence about significance of modified GM combination with HFD in developing obesity in SC mice.

Efficacy of a Novel Food Supplement (Ferfer®) Containing Microencapsulated Iron in Liposomal Form in Female Iron Deficiency Anemia.

Introduction Iron deficiency anemia (IDA) is a highly afflicting condition which affects young children of growing age and reproductive age women in countries of lower economies. Conventional oral iron salts have poor absorption and gastrointestinal side effects. Microencapsulated liposomal iron pyrophosphate is a novel compound with enhanced palatability, higher bioavailability, and consequently increased adherence among people with IDA. This study aims to assess the efficacy of microencapsulated iron pyrophosphate sachets in non-pregnant women with IDA. Methods It was a 12-week long, open label clinical trial conducted with 558 IDA women. Participants were advised one sachet of microencapsulated liposomal iron pyrophosphate (Ferfer®) twice daily. At baseline, and every four-week interval, serum hemoglobin levels and taste tolerability were assessed. Data was entered and analyzed using SPSS v. 24 (IBM Corp, Armonk, NY, USA). Results Four hundred and thirty-seven women completed the trial. The mean serum Hb level at baseline was 8.71 ± 2.24 which increased to 10.47 ± 1.69 by the end of 12 weeks (p < 0.001). Conclusion Treatment of IDA with microencapsulated liposomal iron pyrophosphate sachets significantly increases serum hemoglobin levels in non-pregnant women of reproductive age.

Complications of Diabetes: An Insight into Genetic Polymorphism and Role of Insulin.

BACKGROUND: Diabetes Mellitus (DM) is an advanced and chronic endocrine disorder characterized by an insufficiency of insulin secretion from pancreatic ß-cells and liver, adipose tissues, and skeletal muscles. OBJECTIVE: The main objective of this study is to understand the mechanism and genes which are responsible for the prevalence of diabetes. The study also covers various types of diabetic complications with special reference to insulin role and defects. METHODS: The scientific literature and patents were reviewed and analyzed based on their suitability and relevance to the theme of the study. The scientific literature was covered from the authentic databases such as Elsevier, Springer, and Bentham Science. The patents were reviewed from http://www.freepatentsonline.com. RESULTS: Glucokinase (ATP: D-glucose-6-phosphotransferase; GCK), initiates glycolysis and acts as a glucose sensor and metabolic signal producer in liver and pancreas. PCR-sequencing showed qualitative differences in diabetic patients in comparison to healthy subjects. Glucokinase is the most important component in glucose detection of pancreatic islet beta cells in diabetes because glucokinase mutations can be one of the most common single gene disorders described. It is known that a genetic variation of a human glucokinase gene, including a point mutation, causes MODY, the concentration of plasma glucose increased and it is supposed to be the cause of diabetes of the present study subjects. Owing to hyperglycemia and individual components of the insulin resistance (metabolic) syndrome, people with Type II DM are prone to the high threat for microvascular complications (including nephropathy, retinopathy, and neuropathy) and macrovascular complications (such as Ischemic Heart Disease). There were also significant differences (P < 0.0001) in glycation levels (0.90, 0.4838mole/mole), random blood sugar (348.8, 105.8mg/dL), cholesterol levels (235.3, 161.8mg/dL), low density lipoprotein in diabetic subjects (155.3, 28.46mg/dL) and in healthy donors. GCK gene mutations were found in 70% of the patients while 30% are non-mutated. CONCLUSION: In conclusion, lipids, glucose, and protein play an essential role in the initiation of AGE's or diabetic complications (Micro and Macrovascular Complications). The importance of the clinical results should also be recognized in the genetic analysis of heterogeneous disorders as NIDDM/ Type II DM.

Association between Genetic Polymorphism and Risk of von Willebrand Disease in Pakistan.

von Willebrand disease (VWD) is an inherited, genetically and clinically heterogeneous hemorrhagic disorder. The most common cause of this disease is mutation in the gene that encodes protein von Willebrand factor (VWF) which is responsible for blood clotting. The current study was designed to investigate the role of genetic polymorphisms with the onset of VWD in population of Pakistan. Three exonic variants (c.3445T>C; c.4975C>T; c.7603C>T) from VWF gene were used for the genotyping purpose. The current study employed a case-control association design involving 43 VWD patients and 100 healthy controls from Pakistani population. The genetic reason of VWD was investigated using the allele specific PCR. The significant (P < 0.05) allelic association was found between all three exonic variants and VWD. The CT genotype of these variants was noticed to be associated with significantly higher risk of VWD [odds ratio (95% CI): 14.7 (4.546-47.98), 26.71 (7.281-97.98), and 21.5 (5.806-80.01) for c.3445T>C, c.4975C>T, and c.7603C>T, resp.] while genotypes CC (c.4975C>T) and TT (c.3445T>C and c.7603C>T) were having protective effect against the disease. However, replicated studies are needed for elaborating the role of these SNPs.

Frequency of Group B Streptococci in Pregnant Women in a Tertiary Care Hospital.

OBJECTIVE: To determine the frequency and risk factors of Group B Streptococci (GBS) in pregnant patients in third trimester in a tertiary care hospital in Lahore. STUDY DESIGN: Cross-sectional, prospective study. PLACE AND DURATION OF STUDY: Lady Willingdon Hospital, Lahore, from October 2014 to March 2015. METHODOLOGY: Sterile lower vaginal swabs were taken from 200 women aged 20 years and over, in third trimester, with no history of vaginal bleeding, ruptured membrane, recent intake of antibiotics or chronic illness. These swabs were cultured for detection of GBS. The risk factors of GBS and its frequency were noted in the pregnant population. Quantitative and qualitative data was analyzed by SPSS version 20. Chi-square test was applied to see association between diagnosis of GBS and other categorical variables. P-value ≤0.05 was considered as statistically significant. RESULTS: In this study, the mean age of all the females was 26.36 ± 4.32 years and mean duration of pregnancy was 35.54 ± 2.65 weeks. Frequency of GBS in pregnant women was found as 14%. We observed significant association of GBS with parity and previous history of miscarriage (p-value = 0.033 and 0.010 respectively). Moreover, significant association between vaginal discharge and GBS was also found (p = 0.027). CONCLUSION: GBS is present in a small but significant number of pregnant women in our setting and it has association with multiparity, vaginal discharge during pregnancy, and previous history of miscarriage.

Epidemiology and Management Cost of Myocardial Infarction in North Punjab, Pakistan.

BACKGROUND: Coronary heart disease (CHD) is an important cause of morbidity and mortality in Pakistan. The temporal trends in the risk factors for myocardial infarction (MI) and the impact of socioeconomic status on these risk factors remain ambiguous. OBJECTIVES: The objectives of the present analysis were to investigate the potential association between various risk factors and MI in North Punjab, Pakistan, and to assess the status of the control of the risk factors associated with MI in this population. PATIENTS AND METHODS: The present study included 515 patients admitted to the coronary care units or equivalent cardiology wards of the participating hospitals between 2011 and 2012 in North Punjab, Pakistan. The analysis was focused on identifying the socioeconomic status, lifestyle, family history of MI, and risk factors (i.e. hypertension, diabetes, smoking, and hyperlipidemia). A structured questionnaire was designed to collect data. The lipid profile was recorded from the investigation chart of every patient. For statistical analysis, the Kruskal Wallis, Mann-Whitney U, Wilcoxon, and chi-square tests were used. RESULTS: MI was common in the males at the age of 41 - 60 years as compared to the females (P = 0.015). Patients with a positive parental history of CHD experienced MI at a younger age (P = 0.0001) at a body mass index (BMI) ≤ 25 kg/m(2). Sedentary lifestyle (70%) and smoking (60%) had a male predominance. Hypertension accounted for nearly 37%, hyperlipidemia 26%, and diabetes 19.4% of the rural and urban subjects (P < 0.01). High-density lipoprotein cholesterol decreased (up to 34 mg/dl), while low-density lipoprotein cholesterol and hypertension increased with age. The mean monthly cost of medicines and physicians' fees per patient was 2381.132 Pakistani Rupees (24.24 USD). CONCLUSIONS: Higher BMI, positive family history, smoking, hypertension, hyperlipidemia, and diabetes were the strong predictors of MI in North Punjab, Pakistan. Preventive efforts are needed to start early in life and continue throughout the life course.