Addition of pomegranate juice (Punica granatum) in tris‐based extender improves post‐thaw quality,motion dynamics and in vivo fertility of Nili Ravi buffalo (Bubalus bubalis) bull spermatozoa

The aim of the present study was to determine the protective effects of pomegranate juice in tris‐based extender on semen parameters, computer‐assisted sperm analysis (CASA) motion characteristics and field fertility of post‐thawed Nili Ravi buffalo (Bubalus bubalis) bull spermatozoa. Two consecutive ejaculates/collection from each of the five adult Nili Ravi buffalo bulls were collected with artificial vagina at 42°C for a period of 7 weeks, diluted in extender containing different concentrations of pomegranate juice (0.0%, 2.5%, 5%, 7.5% and 10%). Diluted samples were packed and frozen in 0.54 ml French straws. The addition of 10% pomegranate juice in extender significantly improved post‐thaw sperm morphology (%), motilities (CASA total motility, progressive motility (%) as well as VAP, VSL, VCL, STR, DAP, DSL) compared to the control group (p < 0.05). Plasma membrane, acrosome membrane and DNA integrity were significantly higher in extender with 10% pomegranate juice than the control group (p < 0.05). Field fertility rate (60.39% vs. 46.53%) was higher (p < 0.05) in extender with 10% pomegranate juice as compared to the control. It is therefore concluded that the addition of 10% pomegranate juice in tris‐based extender improves post‐thaw semen parameters, CASA motion dynamics and field fertility in Nili Ravi buffaloes.     

From Molecular Pathology of COVID-19 to Nigella Sativum as a Treatment Option:Scientific Based Evidence of Its Myth or Reality

COVID-19 virus is a causative agent of viral pandemic in human beings which specifically targets respiratory system of humans and causes viral pneumonia.This un...     

Antisperm contraceptive vaccines: where we are and where we are going?

This is a review of current status and future perspectives on the development of antisperm contraceptive vaccines (CV) and immunocontraceptives. The development of antisperm CV is an exciting proposition. There is a strong rationale and recent data indicating that this proposition can translate into reality. The search for novel sperm-specific antigens/genes, that can be used for CV, continues using various recent developing technologies. Various approaches of proteomics, genomics, reproductive biology, mucosal immunity and vaccinology and several novel technologies such as gene knockout technology, phage display technology, antibody engineering, differential display technique, subtractive hybridization, and hybridoma technology are being used to delineate sperm-specific antigens and construct CV. Various sperm antigens/genes have been delineated, cloned, and sequenced from various laboratories. Vaccination with these sperm antigens (recombinant/synthetic peptide/DNA) causes a reversible contraceptive effect in females and males of various animal species, by inducing a systemic and local antisperm antibody response. The efficacy is enhanced by combination vaccination, including peptides based on various sperm antigens. Several human novel scFv antibodies with unique complementarity-determining regions (CDRs), that react with specific well-defined fertility-related sperm antigens, have been synthesized. These human infertility-related antibodies may find application in the development of novel immunocontraceptives. Besides finding the novel sperm antigens, the present and future focus is on enhancing the immunogenicity, bioefficacy, and on obliterating the inter-individual variability of the immune response, and proceeding for primate and human clinical trials. Multi-epitope vaccines combining sperm proteins involved in various steps of fertilization cascade have been found to enhance the immunogenicity and bioefficacy of the contraceptive effect. The in vitro synthesis of infertility-related human scFv antibodies may provide unique once-a-month immunocontraceptives, the first of its kind, for human use. The multi-epitope CV and preformed engineered human antibodies of defined specificity may obliterate the concern related to inter-individual variability of the immune response.     

The changing epidemiology pattern and frequency distribution of hepatitis C virus in Pakistan

Information regarding the changing pattern in hepatitis C virus (HCV) genotypes/subtypes and resulting disease outcome is not well known. The specific objective of this study was to find out the frequency distribution of HCV genotypes and changing pattern of various HCV genotypes overtime in well-characterized Pakistani HCV isolates. The genotype distribution of HCV from all the four provinces of Pakistan was tracked for a period of 10 years (2000–2009) on total 20,552 consecutive anti-HCV and HCV RNA positive patients sample using type-specific genotyping assay. Of these, 16,891 (82.2%) samples were successfully genotyped. Of these 11,189 (54.4%) were males and 9363 (45.55%) were females. Of the successfully genotyped samples, 12,537 (74.2%) were with 3a, 1834 (10.9%) with 3b, 50 (0.24%) with 3c, 678 (3.3%) with 1a, 170 (0.83%) with 1b, 49 (0.24%) with 1c, 431 (2.1%) with 2a, 48 (0.23%) with 2b, 3 (0.01%) with 2c, 13 (0.06%) with 5a, 12 (0.06%) with 6a, 101 (0.49%) with 4, and 965 (4.7%) were with mixed-genotype infection. A changing pattern of HCV genotypes prevalence was observed in Pakistan overtime, with an increase in the relative proportion of genotype 3a and mixed genotypes and a decrease of genotypes 3b, 2b, 4, 5a and 2a. This changed HCV genotype pattern might have direct impact on HCV disease outcome and new therapeutic strategies may be needed.     

Differential gene expression of chemokines in KRAS and BRAF mutated colorectal cell lines:Role of cytokines

AIM:To study KRAS/BRAF mutations in colorectalcancer(CRC)that influences the efficacy of treatment.To develop strategies for overcoming combination of treatment.METHODS:Five colonic cell-lines were investigated:DLD-1 with KRAS(G13D)mutation,HT 29 and Colo205 with BRAF(V600E)mutation as well as the wild type(Wt)cell-lines Caco2 and Colo-320.DLD-1(KRAS),HT-29(BRAF)and Caco2(Wt)cell lines were treated with cytokines(TNFα50 ng,IL-1β1 ng and IFNγ50ng)and harvested at different time points(1-24 h).KRAS inhibition was performed by the siRNA-approach.Two colorectal cancer cells DLD-1 and Caco2 were used for KRAS inhibition.About 70%confluency were confirmed before transfection with small interferring RNA(siRNA)oligonucleotides.All the synthetic siRNA sequences were designed in our laboratory.Total RNA and protein was isolated from the cells for RT-PCR and Western blotting.Densitometry of the Western blotting was analyzed with the Image J software(NIH).Results are shown as mean±SD.RESULTS:RT-PCR analysis in non-stimulated cells showed a low basal expression of TNFαand IL-1βin the DLD-1 KRAS-mutated cell-line,compared to Caco2wild type.No detection was found for IL-6 and IFNγin any of the studied cell lines.In contrast,pro-angiogenic chemokines(CXCL1,CXCL8)showed a high constitutive expression in the mutated cell-lines DLD-1(KRAS),HT-29 and Colo205(BRAF),compared to wild type(Caco2).The anti-angiogenic chemokine(CXCL10)showed a high basal expression in wild-type,compared to mutated cell-lines.KRAS down-regulation by siRNA showed a significant decrease in CXCL1 and CXCL10gene expression in the DLD-1(KRAS)cell-line in comparison to wild type(Caco2)at 72 h after KRAS silencing.In contrast,the specific KRAS inhibition resulted in an up-regulation of CXCL1 and CXCL10.The results of our study show a higher expression of pro-angiogenic chemokines at basal level in mutated cell-lines,which was further increased by cytokine treatment.CONCLUSION:To summarize,basal chemokine gene expression for pro-angiogenic chemokines was high in mutated as compared to wild type cell-lines.This reflects the likely existence of a different microenvironment in tumours consistent of wild type or mutated cells.This may help to rationalize the choice of molecular targets for suitable therapeutic investigation in clinical studies.     

ERBB4 exonic deletions on chromosome 2q34 in patients with intellectual disability or epilepsy

ERBB4 encodes the tyrosine kinase receptor HER4, a critical regulator of normal cell function and neurodevelopmental processes in the brain. One of the key ligands of HER4 is neureglin-1 (NRG1), and the HER4-NRG1 signalling pathway is essential in neural crest cell migration, and neuronal differentiation. Pharmacological inactivation of HER4 has been shown to hasten the progression of epileptogenesis in rodent models, and heterozygous ERBB4 null mice are shown to have cognitive deficits and delayed motor development. Thus far there is only a single case report in the literature of a heterozygous ERBB4 deletion in a patient with intellectual disability (ID). We identified nine subjects from five unrelated families with chromosome 2q34 deletions, resulting in heterozygous intragenic loss of multiple exons of ERBB4, associated with either non-syndromic ID or generalised epilepsy. In one family, the deletion segregated with ID in five affected relatives. Overall, this case series further supports that haploinsufficiency of ERBB4 leads to non-syndromic intellectual disability or epilepsy.Subject terms: Chromosome abnormality, Genetic testing, Neurodevelopmental disorders     

Clinicopathologic evaluation of subgroups of diffuse large B cell lymphoma by immunohistochemistry

Diffuse large B cell lymphoma (DLBCL) has become an emerging epidemic in recent years. Striking heterogeneity in its clinical, biological and treatment responses prompted us to identify variation in our study group. The aim was to classify the DLBCL into prognosis-based subgroups according to the WHO classification and to evaluate their relation to clinical parameters (age, gender, anatomic location and B symptoms), as well as bcl 2 and Ki 67 status. Patients and Methods: A cross sectional study was carried out on 42 DLBCL patients, classified histologically and immunophenotypically into germinal center B cell like (GCB) or non-GCB type. Immunohistochemistry (IHC) was performed using antibodies against CD 10, MUM-1 and bcl 6; additionally anti-apoptotic protein bcl 2 and proliferative marker Ki 67 (using cutoff value of 70%) were also assayed by IHC. Results: Of the total 27/42 (64%) were males and 15/42 (36%) females, with a mean age of 44.1±15 years. 15/42 (36%) cases were of GCB type as compared to 27/42 (64%) of non GCB type. Extranodal involvement and B symptoms were seen in 18/27 (66.6%) and 20/27(74%) of the non GCB type, whereas bcl 2 protein expression and Ki 67 proliferative index (PI) <70% were each noted in 22/27 (81.4%). Conclusion: We document an astonishingly high number of non-GCB type DLBCL in our population. It is alarming to see such an aggressive tumor proliferating in our region. Significant association of non-GCB type with extranodal origin, B symptoms and low Ki 67 PI (<70%) is another concern.