Molecular Characterization of Lineage-IV Peste Des Petits Ruminants Virus and the Development of In-House Indirect Enzyme-Linked Immunosorbent Assay (IELISA) for its Rapid Detection".

Peste des petits ruminants (PPRV), a highly contagious viral disease, causes significant economic losses concerning sheep and goats. Recently, PPR viruses (PPRVs), have adopted new hosts and lineage IV of PPRVs represents genetic diversity within the same lineage. 350 samples, including blood, swabs, and tissues from sheep/goats, were collected during the 2020-2021 disease outbreaks in Pakistan. These samples were analysed through RT-PCR and three isolates of PPRV with accession numbers, MW600920, MW600921, and MW600922, were submitted to GenBank, based on the partial N-gene sequencing. This analysis provides a better understanding of genetic characterizations and a targeted RT-PCR approach for rapid PPRV diagnosis. An IELISA test was developed using the semi-purified antigen MW600922 isolate grown in Vero cells. The PPRV isolates currently present high divergence with the Turkish strain; conversely, similarities equivalent to 99.73% were observed for isolates collected from Pakistan. The developed indirect ELISA (IELISA) test demonstrated antibody detection rates at dilutions of 1:200 for antibodies (serum) and 1:32 for antigens. In comparison to cELISA, high specificity (85.23%) and sensitivity (90.60%) rates were observed. In contrast to the virus neutralization test (VNT), IELISA was observed to be 100% specific and 82.14% sensitive in its results. Based on these results, serological surveys conducted for PPR antibodies using IELISA can be a more effective strategy on a larger scale. Furthermore, our results demonstrate a significant breakthrough in the research in terms of cost-effectiveness and storage efficiency, and the developed IELISA test is highly recommended for use in developing countries.

Peste des petits ruminants (PPRV) is a transboundary, highly contagious, and economically significant viral disease affecting small ruminants and wildlife. PPRV, a disease that only targets animals, is the focus of the Global Eradication Programme (PPRV GEP), which aims to eradicate the disease by 2030. Following the completion of the first phase of the GEP (2017­2021), Pakistan has initiated the second phase: PPRV presence and the implementation of a control strategy. Rapid and accurate laboratory diagnosis is vital to the disease's effective control and eradication. In the present study, we have improved diagnosis by reverse transcriptase polymerase chain reaction (RT-PCR), which not only can detect low viral concentrations but also contributes to the genetic analysis of lineage-IV viruses. However, the development of cost-effective indirect ELISA (iELISA) may allow for the analysis of serum samples obtained from larger populations of small ruminants.

Possible prognostic impact of PKCι genetic variants in prostate cancer.

BACKGROUND: Single nucleotide polymorphisms (SNPs) have been linked with prostate cancer (PCa) and have shown potential as prognostic markers for advanced stages. Loss of function mutations in PKCι have been linked with increased risk of malignancy by enhancing tumor cell motility and invasion. We have evaluated the impact of two coding region SNPs on the PKCι gene (PRKCI) and their prognostic potential. METHODS: Genotypic association of non-synonymous PKCι SNPs rs1197750201 and rs1199520604 with PCa was determined through tetra-ARMS PCR. PKCι was docked with interacting partner Par-6 to determine the effect of these variants on PKCι binding capabilities. Molecular dynamic simulations of PKCι docked with Par-6 were performed to determine variant effects on PKCι protein interactions. The possible impact of changes in PKCι protein interactions on epithelial cell polarity was hypothesized. RESULTS: PKCι rs1199520604 mutant genotype TT showed association with PCa (p = 0.0055), while rs1197750201 mutant genotype AA also showed significant association with PCa (P = 0.0006). The binding interaction of PKCι with Par-6 was altered for both variants, with changes in Van der Waals energy and electrostatic energy of docked structures. CONCLUSION: Genotypic analysis of two non-synonymous PKCι variants in association with PCa prognosis was performed. Both variants in the PB1 domain showed potential as a prognostic marker for PCa. In silico analysis of the effect of the variants on PKCι protein interactions indicated they may be involved in PCa progression through aberration of epithelial cell polarity pathways.

Body mass index and altered lipid profile as major risk markers for breast cancer progression: a cross-sectional study of postmenopausal women in Pakistan.

BACKGROUND: In Pakistan, the death rate for post-menopausal women with breast cancer is significant due to late detection and delayed referral to proper facilities. There are a few reports on Pakistan's epidemiology and breast cancer risk factors. There are modifiable and non-modifiable risk factors associated with the development of breast carcinoma; of which body mass index (BMI), central obesity, and lipid profile are considered as major risk markers. METHODS: This was a cross-sectional analytical study. A total of 384 women constituted the present study sample. Purposive sampling was used to collect 192 confirmed new breast cancer cases throughout the study. By using basic random sampling, an equal number of controls were chosen. Studied parameters included age, fasting blood sugar, cholesterol, triglyceride, serum high-density lipoprotein, cholesterol, serum low-density lipoprotein cholesterol, weight, height, BMI, waist circumference, and waist-to-hip ratio. The inclusion criteria of this study were post-menopausal women (45-65 years) in Pakistan. The confirmation of breast carcinoma was done through histopathology. Breast cancer occurrence was taken as a dependent variable, whereas BMI, central obesity, and lipid profile were taken as independent variables. RESULTS: Studied risk factors (cholesterol, BMI, and central obesity) significantly correlated with breast cancer. Cholesterol has a significantly high positive correlation (0.646) with breast cancer. BMI has a positive significant correlation (0.491) with breast cancer, and central obesity has a low but positive significant correlation (0.266) with breast cancer. Moreover, the binary logistic regression model also showed a significant association between biochemical factors and breast cancer occurrence. Regression analysis depicted a linear relationship between a dependent variable (breast cancer occurrence) and independent variables (central obesity, cholesterol, BMI). CONCLUSION: Postmenopausal overweight (central obesity), increased BMI and high cholesterol levels are major risk factors for breast cancer. Moreover, high total cholesterol proved to be the most significant risk marker for the occurrence of breast cancer in post-menopausal women of Pakistan.

Evaluation of therapeutic efficacy of Emustil drops for ocular discomfort and tear film osmolarity using different treatment management modes under dry environmental conditions.

BACKGROUND: We aimed to check the efficacy of Emustil (oil in water emulsion) drops on tear film index and ocular surface dynamics in dry environments through protection and relief treatment modalities. METHODS: The subjects were exposed to a dry environment using a Controlled Environment Chamber (CEC) where the relative humidity (RH) was 5% and the temperature was 21 °C and screened for ocular symptoms, tear osmolarity, ocular surface temperature (OST) and tear production using ocular Surface Disease Index questionnaire (OSDI), OcuSense TearLab Osmometer, FLIR System ThermaCAM P620 and Schirmer strips/phenol red test respectively. Tear production was calculated by the Tear Function Index test (TFI). RESULTS: The mean tear film osmolarity decreased significantly from 296.8 mOsm/l at 40% RH to 291 mOsm/l at 5%. (p = 0.01). Instillation of Emustil resulted in a significant increase in tear osmolarity in the relief method compared with osmolarity seen at 5% RH when no drop was used. The mean PRT value decreased from 26 ± 9 in normal conditions (40% RH) to 22 ± 4 mm in dry conditions (5% RH). Emustil drops did not induce any significant change in tear production in the PRT test. No significant change was found in OST following exposure to 5% RH. OST did not show a statistically significant change with the emulsion when used for relief (p > 0.05). The mean score of ocular discomfort observed was 70 at 5% RH. Still, the instillation of the oil-in-water emulsion (Emustil) resulted in a noticeable decrease in visual discomfort to 37 (p = 0.00) in protection and 59 in relief (p = 0.05). Emustil drops substantially improved tear film parameters under a desiccating environment, however, tear film parameters respond differently to the management modalities. In the protection method, tear film osmolarity was protected against a dry environment, while in the relief mode, tear production was improved. CONCLUSION: CEC allows for a thorough evaluation of tear film parameters and dry eye treatment protocols in labs, providing greater confidence when applying them to patients. In addition, our study showed that Emustil not only provides protection and relief for dry eyes but also helps to maintain ocular homeostasis in desiccating environments. This indicates a promising potential for improving dry eye treatment protocols.

Determination of IL-6 Gene Promoter Polymorphism in Patients with Hepatitis C and Its Impact on RNA Secondary Structure.

Background and Objectives: A polymorphism in the promoter region of the IL-6 gene would influence the level of IL-6 expression in patients with HCV, resulting in a pro-inflammatory response. Few studies have shown the association between -174G>C (rs1800795) and -1363G>T (rs2069827) polymorphisms and HCV infection, and their results have been contradictory. There are no data published in our population to study such an IL-6 stimulus against HCV infection and its impact on RNA secondary structure. Therefore, we isolated human subjects from the province of Punjab, Pakistan. The objective was to screen for IL-6 gene promoter polymorphisms -174G/C and -1363G/T and those correlated with serum concentrations of IL-6 in patients with HCV and compared with a control. Materials and Methods: In conventional PCR, measurement of serum IL-6 by CLIA and statistical analysis were performed to observe the genotype association studies. By integrating bioinformatics and computational tools, our study aimed to provide a comprehensive understanding of how variations in the promoter region of IL-6 may have functional implications on gene expression. Results: The -174G>C and -1363G>T genotypes in the promoter region of patients with HCV were in strong allelic association (Δ = 0.97, p < 0.001). Interestingly, the bioinformatics analysis was well aligned with our experimental data. Conclusions: Based on the data, it can be inferred that IL-6 gene promoter polymorphisms are important in the dysregulation of IL-6 levels in patients with HCV.

Efficacy of hydroxypropyl-guar drops in improving tear film index and ocular surface dynamics using two treatment methods under a controlled desiccating environment.

AIM: This study aimed to assess the efficacy of hp-guar eye drops on tear film index and ocular surface dynamics under desiccating conditions using protection and relief treatment modalities. METHODOLOGY: The 12 normal, non-dry eye participants were subjected to adverse environmental conditions using a Controlled Environment Chamber (CEC) where the relative humidity (RH) was 5% and the ambient temperature was 21 °C. The participants were screened for ocular symptoms, tear osmolarity, ocular surface temperature (OST), tear production using the Ocular Surface Disease Index questionnaire (OSDI), OcuSense TearLab Osmometer, FLIR System ThermaCAM P620, and Schirmer strips. Tear production was calculated by the Tear Function Index test (TFI). RESULTS: The mean tear film osmolarity decreased significantly from 296 mOsm/L at 40% RH to 285 mOsm/L at 5% RH (p = 0.01). Conflicting responses were seen for osmolarity in protection and relief. Mean tear osmolarity was significantly higher in the protection method in comparison to the relief method (p = 0.005). The mean TFI increased from 557 at 40% to 854 at 5% (p = 0.02). A significant increase in TFI was observed in the relief method in comparison with both 40% (p = 0.001) and 5% (p = 0.04). In the relief method, the mean TFI score went up to 1139 when hp-guar was installed. A significant improvement in ocular comfort was experienced in both the protection (p = 0.041) and relief (p = 0.010) methods at 5% RH. The instillation of hp-guar drops in the relief method resulted in a significant reduction in OST. The mean OST dropped to 33.01 ºC, significantly lower than the recorded OST for both normal (p = 0.040) and dry (p = 0.014) environmental conditions. CONCLUSION: Hp-guar drops significantly improve tear film parameters under a desiccating environment, however, tear film parameters respond differently to the management modalities. In the protection method, tear film osmolarity was protected against a dry environment, while in the relief mode, an improvement in tear production and a decrease in ocular surface temperature were seen. Hp-guar performance could be maximized for the management of exposure to adverse environments by using a treatment protocol that targets the most affected parameters in each group of patients. Using CEC has the potential to provide researchers with a readily available method to evaluate the efficiency of tear supplementation.

Mutation pattern of cancer suppressor p53 gene in factory workers exposed to polypropylene and impact on their reproductive hormones.

BACKGROUND: Polypropylene is a thermoplastic polymer playing the role of an endocrine disruptor that interferes with the union, emission, transport or elimination of normal hormones. Epidemiological information indicated the relation of endocrine-disturbing chemicals with prostate cancer, testis tumor and diminished fertility. p53 is a key tumor silencer gene. The present study aimed to evaluate luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels and the risk of p53 mutations as a result of exposure to polypropylene in non-tumorous adult male factory workers. METHODS: In total, 150 (controls = 35, workers = 115) subjects were recruited. Groups were maintained according to the tenure of exposure G1 (1-5 years), G2 (6-10 years), G3 (11-15 years) and G4 (16-20 years). Concentrations of LH and FSH were determined through an enzyme-linked immunosorbent assay. Genotyping analysis was performed by polymerase chain reaction based gel electrophoresis followed by DNA sequencing. The structural and functional impact of the mutation on the p53 structure was evaluated using 50-ns molecular dynamics (MD) simulations and protein-DNA docking. RESULTS: Mean plasma LH levels were significantly decreased in G1 (p > 0.05) as well as the G2, G3 and G4 (p > 0.001) groups. Similarly, FSH levels were significant decrease in G1 (p > 0.05), G2 (p > 0.01), G3 (p > 0.001) and G4 (p > 0.001) compared to the control group. Sequencing results found three variants i.e. g.13450 T>G, g.13430C>T and g.13737G>A. One of them was predicted to be disease-causing others are polymorphisms. MD simulation of missense mutation R273H showed no structural impact on the protein structure in MD simulation, but it resulted in weaker binding of p53 with the DNA that might lower the gene expression of cell cycle regulatory proteins. CONCLUSIONS: These findings predict decreased fertility and risk of malignancies in the future. The spectrum of p53 mutations as a result of polypropylene exposure in the Pakistani population has not been investigated before. Further studies and meta-analyses are required to elucidate the role of different plasticizers in reproduction and cancer-causing risk factors in a larger population.

Non-synonymous SNPs variants of PRKCG and its association with oncogenes predispose to hepatocellular carcinoma.

BACKGROUND: PRKCG encodes PKC γ, which is categorized under the classical protein kinase C family. No studies have specifically established the relationship between PRKCG nsSNPs with structural and functional variations in PKC γ in the context of hepatocellular carcinoma (HCC). The present study aims to uncover this link through in-silico and experimental studies. METHODS: The 3D structure of PKC γ was predicted. Molecular Dynamic (MD) Simulations were run and estimates were made for interactions, stability, conservation and post-translational alterations between wild and mutant structures. The association of PRKCG levels with HCC survival rate was determined. Genotyping analyses were conducted to investigate the deleterious PRKCG nsSNP association with HCC. mRNA expression of PKC γ, HIF-1 alpha, AKT, SOCS3 and VEGF in the blood of controls and HCC patients was analyzed and a genetic cascade was constructed depicting these interactions. RESULTS: The expression level of studied oncogenes was compared to tumour suppressor genes. Through Alphafold, the 3D structure of PKC γ was explored. Fifteen SNPs were narrowed down for in-silico analyses that were identified in exons 5, 10 and 18 and the regulatory and kinase domain of PKC γ. Root mean square deviation and fluctuation along with the radius of gyration unveiled potential changes between the wild and mutated variant structures. Mutant genotype AA (homozygous) corresponding to nsSNP, rs386134171 had more frequency in patients with OR (2.446), RR (1.564) and P-values (< 0.0029) that highlights its significant association with HCC compared to controls in which the wild genotype GG was found more prevalent. CONCLUSION: nsSNP rs386134171 can be a genetic marker for HCC diagnosis and therapeutic studies. This study has laid down a road map for future studies to be conducted on HCC.

Elucidating the role of missense SNP of protein kinase C epsilon in HCV-induced hepatocellular carcinoma.

BACKGROUND: The protein kinase C (PKC) family of serine/threonine kinases contains more than ten isozymes that are involved in multiple signaling pathways, including cell cycle regulation and carcinogenesis. The PKCε isozyme is an oncogene known to be upregulated in various signaling pathways involved in hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC). However, there is no known association of missense SNPs in PKCε with this disease, which can be a potential biomarker for early diagnosis and treatment. This research reveals a novel missense SNP in PKCε that is associated with HCV-induced HCC in the Pakistani population. METHODS: The PKCε SNP with amino acid substitution of E14K was chosen for wet lab analysis. Tetra ARMS-PCR was employed for the identification of high-risk SNP in PKCε of HCV-induced HCC patients. Liver function testing was also performed for comparison between the liver condition of the HCC patient and control group, and the viral load of HCC patient samples was evaluated to determine any alteration in the viral infectivity between different genotypes of the selected high-risk PKCε variant SNP. RESULTS: Frequency distribution of the homozygous GG genotype was found to be highest among HCV-induced HCC patients and was also found to be significantly associated with disease development and progression. The p values of comparative data obtained for the other two genotypes, heterozygous AG and homozygous AA, of the SNP also showed the significance of the data for these alleles. Still, their odds ratio and relative risk analysis did not indicate their association with HCV-induced HCC. CONCLUSION: The distribution of a genotype GG of PKCε has been found in HCV- induced HCC patients. Therefore, these PKCε SNP have the potential to be biomarkers for HCV-induced HCC. Further investigation using a larger sample size would provide additional insight into these initial data and open a new avenue for a better prognosis of this disease.

Exploring the prognostic significance of PKCε variants in cervical cancer.

BACKGROUND: Protein Kinase C-epsilon (PKCε) is a member of the novel subfamily of PKCs (nPKCs) that plays a role in cancer development. Studies have revealed that its elevated expression levels are associated with cervical cancer. Previously, we identified pathogenic variations in its different domains through various bioinformatics tools and molecular dynamic simulation. In the present study, the aim was to find the association of its variants rs1553369874 and rs1345511001 with cervical cancer and to determine the influence of these variants on the protein-protein interactions of PKCε, which can lead towards cancer development and poor survival rates. METHODS: The association of the variants with cervical cancer and its clinicopathological features was determined through genotyping analysis. Odds ratio and relative risk along with Fisher exact test were calculated to evaluate variants significance and disease risk. Protein-protein docking was performed and docked complexes were subjected to molecular dynamics simulation to gauge the variants impact on PKCε's molecular interactions. RESULTS: This study revealed that genetic variants rs1553369874 and rs1345511001 were associated with cervical cancer. Smad3 interacts with PKCε and this interaction promotes cervical cancer angiogenesis; therefore, Smad3 was selected for protein-protein docking. The analysis revealed PKCε variants promoted aberrant interactions with Smad3 that might lead to the activation of oncogenic pathways. The data obtained from this study suggested the prognostic significance of PRKCE gene variants rs1553369874 and rs1345511001. CONCLUSION: Through further in vitro and in vivo validation, these variants can be used at the clinical level as novel prognostic markers and therapeutic targets against cervical cancer.

Evaluation of different treatment modalities on the efficacy of hydroxypropyl Guar (HP-Guar) formulation on tear film stability (TFS) in subjects exposed to adverse environmental conditions.

The study aimed to assess the efficacy of hydroxypropyl guar (HP) formulation (Systane) to protect tear film parameters under desiccating environment using protection and relief treatment modalities. The subjects were exposed to adverse environmental conditions using a Controlled Environment Chamber (CEC) where the relative humidity (RH) was 5% and the ambient temperature was 21 °C and screened for Tear break-up time (TBUT), Tear film evaporation rate (TFER) and lipid layer thickness (LLT) using the HIRCAL grid, Servomed EP3 Evaporimeter and Keeler's TearScope-Plus respectively. Significant improvement in LLT was noticed in the protection modality. The mean tear film evaporation rate doubled after exposure to the humidity of 5% to a value of 105.37 g/m2/h (0.29 µl/min). All subjects displayed a significant reduction in non-invasive tear break-up time (NITBUT) with a mean NITBUT of 7.7 s after exposure to a desiccating environment for 15 min. A significant increase in NITBUT after the instillation of the drops was recorded in both methods. The results obtained from this study showed that a solution containing HP-Guar significantly improves tear film parameters under a desiccating environment. Apart from the tear evaporation rate, all tear parameters showed improvement after the use of HP-Guar eye drops. It is evident that tear film parameters respond differently to the management modalities and using CEC has the potential to provide researchers with a readily available method to evaluate the efficiency of tear supplementation.

Evaluation of Therapeutic Capability of Emustil Drops against Tear Film Complications under Dry Environmental Conditions in Healthy Individuals.

Background and Objectives: Dry eye disease (DED) is a multifactorial ailment of the tears and ocular surface. The purpose of this study was to assess the tear film physiology under controlled dry environmental conditions and compare the efficacy of oil-in-water emulsion drops on tear film parameters in protection and relief treatment modalities under low-humidity conditions. Emustil eye drops were used after exposure to a low-humidity environment in the relief method, whereas, in the protection method, the drops were applied before exposure to low humidity. Materials and Methods: 12 normal male subjects (mean age 34.0 ± 7.0 years) were exposed to ultra-dry environmental conditions. A number of tear film measurements were carried out under desiccating environmental conditions in a controlled environment chamber (CEC), where the chamber temperature sat at 21 °C with a relative humidity (RH) of 5%. Keeler's TearScope Plus and an HIRCAL grid were used to assess the tear break-up time and lipid layer thickness (LLT), and the evaporation rate was evaluated using a Servomed EP3 Evaporimeter. Results: LLT measurements showed that the dry environment affected LLT significantly (p = 0.031). The median grade of LLT dropped from grade 3 (50-70 nm) at 40% RH to grade 2 (13-50 nm) at 5% RH. A significant increase in LLT was seen after both modes of treatment, with a median LLT grade of 3 when the Emustil was used for both protection (p = 0.004) and relief (p = 0.016). The mean tear evaporation rate in normal environmental conditions (40%) was 40.46 ± 11.80 g/m2/h (0.11 µL/min) and increased sharply to 83.77 ± 20.37 g/m2/h (0.25 µL/min) after exposure to the dry environment. A minimal decrease in tear film evaporation rate was seen in relief; however, statistical tests showed that the decrease in tear film evaporation rate was not significant. Mean NITBUT dropped from 13.6 s at 40% RH to 6.6 s at 5% RH (p = 0.002). All NITBUT measurements at 5% RH (with or without the instillation of Emustil) were significantly lower than those at 40%. The instillation of Emustil at 5% RH resulted in a significant improvement in NITBUT for protection (p = 0.016) but this was not the case for relief (p = 0.0.56). Conclusions: A control environmental chamber (CEC) enables the analysis of tear film parameters comparable to those found in dry eye patients. This enables us to examine the capability of oil in emulsion drops to manage tear film disruption in healthy individuals. This study suggests that using Emustil oil-in-water emulsion before exposure to a dry environment should be advocated for people who work in dry environments.

Protecting Tear-Film Stability under Adverse Environmental Conditions Using a Mucomimetic with a Non-Newtonian Viscosity Agent.

Background and Objectives: Tamarind-seed polysaccharide (TSP) and hyaluronic acid (HA) have mucoadhesive properties that improve drug absorption and delay in drug elimination from the ocular surface. We aimed to evaluate TSP/HA-containing formulation for its efficiency in dry-eye symptoms induced by adverse environments and the interaction between mucomimic polymer and tear-film parameters. Materials and Methods: The participants were exposed to 5% relative humidity (RH) in a Controlled Environment Chamber (CEC) under constant room temperature (21 °C). Tear-film parameters were assessed at 40% RH and 5% RH. Rohto Dry Eye Relief drops were used in the two treatment modalities, protection (drops instilled before exposure to the dry environment) and relief (drops instilled after exposure to the dry environment). The HIRCAL grid, Servomed EP3 Evaporimeter, and Keeler's TearScope-Plus were used to screen for non-invasive tear break-up time (NITBUT), tear evaporation rate, and lipid-layer thickness (LLT) using protection and relief treatment methodology. Results: LLT was found to be significantly thinner at 5% RH compared with at 40% RH (p = 0.007). The median LLT dropped from 50-70 nm (grade 3) at 40% RH to 10-50 nm (grade 2) at 5% RH. TSP/HA eye drops significantly augment LLT in both treatment modalities, protection (p = 0.01) and relief (p = 0.004) at 5% RH. The mean evaporation rate doubled from 40.93 at 40% RH to 82.42 g/m2/h after exposure to 5% RH. In protection mode, the TSP/HA allowed the average evaporation rate to be much lower than when no TSP/HA was used at 5% RH (p < 0.008). No alteration in evaporation rate was recorded when the TSP/HA drop was used after exposure (relief). The mean NITBUT was reduced from 13 s in normal conditions to 6 s in the dry environment. Instillation of TSP/HA eye drops resulted in significant improvement (p = 0.006) in tear stability, where the NITBUT increased to 8 s in both protection (before exposure) and relief (after exposure) (p = 0.001). Although improved, these values were still significantly lower than NITBUT observed at 40% RH. Conclusions: Significant protection of tear-film parameters was recorded post instillation of TSP/HA eye drop under a desiccating environment. Both treatment methods (protection and relief) were shown to be effective. The presence of TSP/HA enhances the effectiveness of teardrops in protecting the tear-film parameters when exposed to adverse environments.

Association of CTLA-4 and IL-4 polymorphisms in viral induced liver cancer.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most prevalent types of cancer and is responsible for close to one million annual deaths globally. In Pakistan, HCC accounts for 10.7% of cancer incidence. Prior studies indicated an association between interleukin 4 (IL-4) and cytotoxic T lymphocyte protein 4 (CTLA-4) gene polymorphisms in many types of cancers, including HCC that are either hepatitis B virus (HBV)- or hepatitis C Virus (HCV)-induced. The association of IL-4 and CTLA-4 genetic polymorphisms with HCV-induced HCC is not yet determined in the Pakistani population. Therefore, this research is designed to investigate the implication of IL-4 and CTLA-4 gene polymorphisms by determining the association of IL-4 -590 C/T (rs2243250) and CTLA-4 + 49 A/G (rs231775) with HCC in Pakistan. METHODS: Different bioinformatics tools were employed to determine the pathogenicity of these polymorphisms. Samples were collected from HCV-induced HCC patients, followed by DNA extraction and ARMS-PCR analysis. RESULTS: The SNP analysis results indicated a positive association of IL-4 -590C/T and CTLA-4 + 49A/G gene polymorphisms with HCV-induced HCC in Pakistan. The CTLA-4 polymorphism might enhance therapeutic efficiency of HCC chemotherapy medicines. The IL-4 polymorphism might introduce new transcription factor binding site in IL-4 promoter region. CONCLUSION: This study delineated risk factor alleles in CTLA-4 and IL-4 genes associated with HCV-mediated HCC among Pakistani patients that may have application to serve as genetic markers for pre- and early diagnosis and prognosis of HCC in HCV patients.

The influence of paternal overweight on sperm chromatin integrity, fertilization rate and pregnancy outcome among males attending fertility clinic for IVF/ICSI treatment.

BACKGROUND: Low and middle-income countries are facing a rapid increase in obesity and overweight burden, particularly in urban settings. Being overweight in men is associated with infertility and a higher risk to have a low sperm count or no sperm in their ejaculate. Despite potential limitations, this is one of few studies conducted to determine the potential risk of paternal overweight on sperm standard parameters, sperm chromatin integrity and assisted conception outcome including fertilization, embryo quality, cleavage rate, reduce blastocyst development, implantation, and cumulative live birth rate (CLBR). METHODS: A cross-sectional study of 750 infertile couples undergoing assisted reproduction technique at a single reproductive medicine center of Salma Kafeel Medical Centre Islamabad. Sperm from men undergoing ART were analyzed for chromatin integrity using sperm chromatin dispersion assay (SCD), Chromomycin A3 staining (CMA3), and toluidine blue (TB) staining, while other semen parameters were assessed on same day includes; standard semen parameters, reactive oxygen species (ROS), sperm deformity index (SDI), teratozoospermic index (TZI), and hypo-osmatic swelling test (HOST). Paternal body mass index (BMI) < 24.5-20 kg/m2 served as the reference group, while the male patients with BMI > 24.5-30 kg/m2 were considered to be overweight. RESULTS: In the analysis of the percentage of spermatozoa with chromatin maturity (CMA3) and chromatin integrity (TB) was reduced significantly in overweight men (p < 0.01) compared with a reference group. Increase in paternal BMI correlate with the increase in sperm chromatin damage (SCD r = 0.282, TB r = 0.144, p < 0.05), immaturity (CMA3, r = 0.79, p < 0.05) and oxidative stress (ROS) (r = 0.282, p < 0.001). Peri-fertilization effects were increased in oocytes fertilization in couples with overweight men (FR = 67%) compared with normal-weight men (FR = 74.8%), similarly, after univariant regression paternal weight remain predictor of sperm chromatin maturity, successful fertilization and CLBR. In the embryo, developmental stage number of the embryo in cleavage was higher in normal weight men, while day 3 (D3) embryos, percent good quality embryo D3, and blastocyst formation rate were compared able between the groups. The paternal overweight group had significant (p < 0.001) increased neonatal birth weight (2952.14 ± 53.64gm; within normal range) when compared with the reference group (2577.24 ± 30.94gm) following assisted reproductive technology (ART). CLBR was higher (p < 0.05) in normal weight men compared to couples with overweight male partners. CLBR per embryo transfer and per 2PN was a statistically significant (p < 0.05) difference between the two groups. An inverse association was observed in the linear regression model between paternal BMI with fertilization rate and CLBR. CONCLUSION: The present study demonstrated the impact of paternal overweight on male reproductive health, as these patients had a higher percentage of immature sperm (CMA3) with impaired chromatin integrity (SCD, TB) in their semen and had decreased fertilization rate, CLBR following assisted reproductive treatments. The present study supports that paternal overweight should be regarded as one of the predictors for fertilization, CLBR and useful for counseling, to consider body mass index not only in women but also for men, in those couples opting for ART treatment, and warrant a poor reproductive outcome in overweight men.

Vitamin D status and its correlation to depression.

BACKGROUND: Vitamin D can influence more than 200 genes in various tissues showing its credibility among the fat-soluble vitamins. Vitamin D deficiency is directly proportional to major clinical conditions such as cardiovascular diseases, diabetes, malignancy, and multiple sclerosis. This study was conducted to determine the vitamin D level of individuals and its association with depression. METHODS: Vitamin D levels of 100 healthy and 100 depressed subjects were determined. The isolated subjects were screened on the Beck Depression Inventory (BDI) scale and divided into three groups according to their age. Group-I comprised subjects of age 20 years and below, Group-II included subjects of age 21 to 60, and Group-III comprised subjects of ≥ 61 years of age. A sufficient level of vitamin D in normal subjects was noted, while mild deficiency of vitamin D status was observed in depressed subjects. RESULTS: Our study has reported a higher percentage of vitamin D deficiency in the Peshawar region. The results of our study indicated that depression was common in individuals having vitamin D deficiency. CONCLUSIONS: The study showed a very high frequency of vitamin D deficiency in subjects with depression in Peshawar, Pakistan. The deficiency of vitamin D was observed more in females as compared to males. Further studies should explicate whether the highly widespread vitamin D deficiency could be cost-effectively treated as part of preventive or treatment interventions for depression.

Association of endothelial nitric oxide synthase gene variants with preeclampsia.

BACKGROUND: Preeclampsia (PE) is a complex pregnancy hypertensive disorder with multifaceted etiology. The endothelial nitric oxide synthase (eNOS) gene and nitric oxide (NO) levels has been reported to be associated with PE predisposition in various populations. Therefore, present study was designed to investigate the role of NO levels and eNOS gene variants in preeclamptic women in Pakistan. METHODS: A total of 600 women were evaluated, 188 of PE with mild features, 112 of PE with severe features and 300 normotensive pregnant women. NO levels were detected by Greiss reaction method and genotyping following sequencing was conducted for eNOS gene variants. Further insilico studies were performed to get insights into the structural and functional impact of identifies mutation on eNOS protein as well as on protein regulation. RESULTS: Reduced concentrations of NO were reported in all PE groups (p < 0.05) as compared to controls. The frequency of c.894 T (p.298Asp) and g.-786C alleles were significantly associated with PE. In addition, novel homozygous variant g.2051G > A was also significantly associated with PE when compared to normotensive women. Dynamic simulation studies revealed that Glu298Asp mutation destabilize the protein molecule and decrease the overall stability of eNOS protein. Molecular docking analysis of mutant promoter with transcription factors STAT3 and STAT6 proposed changes in protein regulation upon these reported mutations in upstream region of the gene. CONCLUSION: Considering the results of current study, the functional alterations induced by these variants may influence the bioavailability of NO and represents a genetic risk factor for increased susceptibility to PE. However, large studies or meta-analysis are necessary to validate these findings.

Preeclampsia (PE) is a complex pregnancy hypertensive disorder with multifaceted etiology characterized by increased hypertension and proteinuria after 20 weeks of gestation. The present study was directed to determine the role of eNOS in susceptibility to PE and the association of c.894G > T (p.(Glu298Asp), intron 4b/4a, g.-786 T > C and other possible variants of eNOS gene with preeclampsia in Pakistani population. Computational analysis of identified variants in the coding and non-coding region of the eNOS gene was also conducted to determine the change in gene regulation and further protein stability. A total of 600 women were evaluated, 188 with mild and 112 with PE with severe features PE with 300 normotensive pregnant women. NO levels and genotyping following sequencing was conducted for eNOS gene variants. Further insilico studies were performed to get insights into the structural and functional impact of identifies mutation on eNOS protein as well as on protein regulation. Data from the current study suggest that there might be other risk variants of the eNOS gene (g.2051G > A and g.1861G > A) and lower levels of serum NO that confers in an increased risk of PE. The detailed computational investigation further confirmed the deformities and changes in protein flexibility upon Glu298Asp. These structural alterations might be associated with preeclampsia. Variants in the promoter region of the eNOS gene further validate the change in gene regulation for the onset of disease. Identification of key structural and functional features in eNOS protein and gene regulatory region might be used for designing specific drugs for therapeutic purpose.

Chronic exposure of bisphenol S (BPS) affect hypothalamic-pituitary-testicular activities in adult male rats: possible in estrogenic mode of action.

BACKGROUND: The industrial revolution has resulted in increased synthesis and the introduction of a variety of compounds into the environment and their potentially hazardous effects have been observed in the biota. The present study was aimed to evaluate the potential endocrine-disrupting effects of chronic exposure to the low concentrations of bisphenol S (BPS) in male rats. METHODS: Weaning male Sprague-Dawley rats (22 days old) were either exposed to water containing 0.1% ethanol for control or different concentrations of BPS (0.5, 5, and 50 µg/L) in drinking water for 48 weeks in the chronic exposure study. After completion of the experimental period, animals were dissected and different parameters (hormone concentrations, histology of testis and epididymis, oxidative stress and level of antioxidant enzymes in the testis, daily sperm production (DSP), and sperm parameters) were determined. RESULTS: Results of the present study showed a significant alteration in the gonadosomatic index (GSI) and relative reproductive organ weights. Oxidative stress in the testis was significantly elevated while sperm motility, daily sperm production, and the number of sperm in epididymis were reduced. Plasma testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) concentrations were reduced and estradiol levels were high in the 50 µg/L-exposed group. Histological observations involved a significant reduction in the epithelial height of the testis along with disrupted spermatogenesis, an empty lumen of the seminiferous tubules, and the caput region of the epididymis. CONCLUSION: These results suggest that exposure to 5 and 50 µg/L of BPS for the chronic duration started from an early age can induce structural changes in testicular tissue architecture and endocrine alterations in the male reproductive system which may lead to infertility in males.

Phytochemical analysis and Evaluation of hepatoprotective effect of Maytenus royleanus leaves extract against anti-tuberculosis drug induced liver injury in mice.

BACKGROUND: Myrin®-p Forte is an anti-tuberclosis agent that can cause hepatic injuries in clinical settings. Maytenus royleanus (Celastraceae) is a medicinal plant, possesses antioxidant and anticancer activities. The hepatoprotective effect of the methanol extract of Maytenus royleanus leaves (MEM) against Myrin®-p Forte induced hepatotoxicity in mice was investigated. METHODS: Mice were randomly parted into six groups (n = 6). Fixed-dose combination of Myrin®-p Forte (13.5 mg/kg Rifampicin, 6.75 mg/kg Isoniazid, 36.0 mg/kg Pyrazinamide and 24.8 mg/kg Ethambutol; RIPE] was administered for 15 days to induce liver injury. In treatment groups MEM (200 mg/kg and 400 mg/kg doses) and Vitamin B6 (180mg/kg) were administered prior to RIPE. Control group received 2% DMSO. Serum liver function tests, DNA damage, tissue antioxidant enzymes and histopathological alterations were studied. HPLC analysis was performed to determine the chemical composition using standard compounds. RESULTS: The quercitin, gallic acid, luteolin, viteixin, apigenin, kaempherol, hyperoside and myricetin contents of all samples were determined by reverse-phase HPLC. Quercetin (0.217 mg/g dry weight) and luteolin (0.141 mg/g dry weight) were the major flavonoids identified in MEM. Myrin®-p Forte markedly (p < 0.05) deteriorated lipid profile and upregulated the concentration of LDH, AST, ALP, ALT and γ-GT in serum along with DNA fragmentation (37.13 ± 0.47%) and histopathological injuries in hepatic tissues of mice compared with the control group. Myrin®-p Forte increased (p < 0.001) lipid peroxidation and H2O2 while decreased (p < 0.001) the activity level of CAT, SOD, POD, GPx, GST, GSR, γ-GT and GSH. Co-administration of MEM (200 mg/kg; 400 mg/kg) or the vitamin B6 (180 mg/kg) to Myrin®-p Forte administered mice significantly ameliorated LDL, cholesterol, HDL and triglyceride content. Furthermore, MEM dose dependently corrected serum liver function tests, decrease % DNA fragmentation (17.82 ± 0.35 and 7.21 ± 0.32 respectively), DNA damage. MEM treated protect RIPE induced oxidative damage by enhancing antioxidants to oxidants balance. Histological examination comprehends biochemical findings. CONCLUSION: The antioxidant effects of MEM exerted the hepatoprotective potential against the Myrin®-p Forte induced hepatotoxicity in mice.

Screening and computational analysis of colorectal associated non-synonymous polymorphism in CTNNB1 gene in Pakistani population.

BACKGROUND: Colorectal cancer (CRC) is categorized by alteration of vital pathways such as ß-catenin (CTNNB1) mutations, WNT signaling activation, tumor protein 53 (TP53) inactivation, BRAF, Adenomatous polyposis coli (APC) inactivation, KRAS, dysregulation of epithelial to mesenchymal transition (EMT) genes, MYC amplification, etc. In the present study an attempt was made to screen CTNNB1 gene in colorectal cancer samples from Pakistani population and investigated the association of CTNNB1 gene mutations in the development of colorectal cancer. METHODS: 200 colorectal tumors approximately of male and female patients with sporadic or familial colorectal tumors and normal tissues were included. DNA was extracted and amplified through polymerase chain reaction (PCR) and subjected to exome sequence analysis. Immunohistochemistry was done to study protein expression. Molecular dynamic (MD) simulations of CTNNB1WT and mutant S33F and T41A were performed to evaluate the stability, folding, conformational changes and dynamic behaviors of CTNNB1 protein. RESULTS: Sequence analysis revealed two activating mutations (S33F and T41A) in exon 3 of CTNNB1 gene involving the transition of C.T and A.G at amino acid position 33 and 41 respectively (p.C33T and p.A41G). Immuno-histochemical staining showed the accumulation of ß-catenin protein both in cytoplasm as well as in the nuclei of cancer cells when compared with normal tissue. Further molecular modeling, docking and simulation approaches revealed significant conformational changes in the N-terminus region of normal to mutant CTNNB1 gene critical for binding with Glycogen synthase kinase 3-B (GSK3) and transducin containing protein1 (TrCp1). CONCLUSION: Present study on Pakistani population revealed an association of two non-synonymous polymorphisms in the CTNNB1 gene with colorectal cancer. These genetic variants led to the accumulation of the CTNNB1, a hallmark of tumor development. Also, analysis of structure to function alterations in CTNNB1 gene is crucial in understanding downstream biological events.