DNA methylation analysis of Sox2 regulatory regions SRR1 and SRR2 in undifferentiated and differentiated mouse embryonic stem cells

One hallmark of embryonic stem cells (ES) is their ability to renew themselves indefinitely and still retain the potential to develop into any specialized cell type once triggered by specific exogenous signals. This very versatile nature has made them an attractive model to study developmental events occurring during embryogenesis and also to employ them for regenerative medicine. The idea to exploit their developmental potential for intended therapeutic applications requires a detailed knowledge of the molecular regulation of differentiation. Thus pluripotent embryonic stem cells can be employed to investigate the molecular framework regulating pluripotency. The major aim of this research endeavour is to explore the role of DNA methylation in regulation of endogenous Sox2 transcription factor in context of mouse ES cells following their transition from the pluripotent to a differentiated state. An insight into molecular regulatory mechanisms will shed light on developmental programming and also aid in refining of methodologies for differentiation and nuclear reprogramming increasing their chances of success and efficiency. Mouse embryonic stem cells were differentiated towards osteogenic and neural cell types through the formation of embryoid bodies (EBs) – cellular aggregates partially recapitulating the early embryonic development. These EBs were then disaggregated and single cells plated in medium containing supplements to promote osteogenic or neural differentiation while control cells were grown in medium lacking those factors. Cells were harvested undifferentiated and at different time points during differentiation. Molecular characterization was carried out by expression profiling of lineage specific genes and proteins using RT-PCR and immunofluorescence respectively. DNA methylation analysis of two regulatory regions of Sox2 i.e. SRR1 and SRR2 was carried out by MS-PCR and bisulphite sequencing. Embryonic stem cells were observed to be differentiating as evidenced by changes in cellular morphologies and lineage-specific markers expression. Two regulatory regions of Sox2, namely SRR1 and SRR2, were found to be methylated by methylation sensitive PCR at all time-points chosen for analysis in differentiating cells. Three individual CpGs in SRR2 region were then analysed further by bisulphite sequencing which appeared unmethylated in both undifferentiated and differentiated embryonic stem cells. This hints towards the possible role of DNA methylation in regulating the expression of Sox2 in differentiating embryonic stem cells and need further investigation

DNA methylation analysis of Sox2 regulatory regions SRR1 and SRR2 in undifferentiated and differentiated mouse embryonic stem cells

One hallmark of embryonic stem cells (ES) is their ability to renew themselves indefinitely and still retain the potential to develop into any specialized cell type once triggered by specific exogenous signals. This very versatile nature has made them an attractive model to study developmental events occurring during embryogenesis and also to employ them for regenerative medicine. The idea to exploit their developmental potential for intended therapeutic applications requires a detailed knowledge of the molecular regulation of differentiation. Thus pluripotent embryonic stem cells can be employed to investigate the molecular framework regulating pluripotency. The major aim of this research endeavour is to explore the role of DNA methylation in regulation of endogenous Sox2 transcription factor in context of mouse ES cells following their transition from the pluripotent to a differentiated state. An insight into molecular regulatory mechanisms will shed light on developmental programming and also aid in refining of methodologies for differentiation and nuclear reprogramming increasing their chances of success and efficiency. Mouse embryonic stem cells were differentiated towards osteogenic and neural cell types through the formation of embryoid bodies (EBs) – cellular aggregates partially recapitulating the early embryonic development. These EBs were then disaggregated and single cells plated in medium containing supplements to promote osteogenic or neural differentiation while control cells were grown in medium lacking those factors. Cells were harvested undifferentiated and at different time points during differentiation. Molecular characterization was carried out by expression profiling of lineage specific genes and proteins using RT-PCR and immunofluorescence respectively. DNA methylation analysis of two regulatory regions of Sox2 i.e. SRR1 and SRR2 was carried out by MS-PCR and bisulphite sequencing. Embryonic stem cells were observed to be differentiating as evidenced by changes in cellular morphologies and lineage-specific markers expression. Two regulatory regions of Sox2, namely SRR1 and SRR2, were found to be methylated by methylation sensitive PCR at all time-points chosen for analysis in differentiating cells. Three individual CpGs in SRR2 region were then analysed further by bisulphite sequencing which appeared unmethylated in both undifferentiated and differentiated embryonic stem cells. This hints towards the possible role of DNA methylation in regulating the expression of Sox2 in differentiating embryonic stem cells and need further investigation

Plasma PD-L1 as a biomarker in the clinical management of glioblastoma multiforme—a retrospective cohort study

Background and objectivesGlioblastoma multiforme (GBM) is the most aggressive, malignant, and therapy-resistant tumor of the brain. Blockade therapy targeting the programmed cell death protein 1 (PD-1)/programmed death ligand (PD-L1) axis is currently under investigation for the clinical management of the GBM. This study has quantified the plasma levels of PD-L1 as a biomarker for the clinical management of GBM.MethodsA cohort (n = 128) of Pakistani adult glioblastoma patients together with age- and sex-matched healthy controls was used for quantification of pre-surgery levels of plasma PD-L1. PD-L1 protein and mRNA were measured by PD-L1 platinum enzyme-linked immunosorbent assay and quantitative real-time PCR, respectively. Receiver operating characteristic (ROC) curve analysis was used to compute area under the curve (AUC) for specificity and sensitivity analyses. The Kaplan–Meier survival analysis was employed to compute overall survival.ResultsPD-L1 protein and mRNA were significantly higher in GBM compared to the healthy controls (p < 0.0001). Mean PD-L1 concentration for the GBM was found to be 48.98 ± 2.290 pg/ml compared to 27.63 ± 1.281 pg/ml for controls. Gene expression analysis showed statistically significant upregulation (p < 0.0001) of PD-L1 in blood of GBM compared to healthy controls. Plasma PD-L1 showed an AUC of 0.840 (p < 0.0001; 95% CI = 0.7716 to 0.9090) where a cutoff value higher than 46 pg/ml demonstrated 100% specificity and 57.81% sensitivity. Higher pre-surgery levels of PD-L1 were found to be associated with overall poor survival [p < 0.0001; HR (log-rank) = 0.08; 95% CI = 0.04 to 0.15]. Age, gender, and ethnic background were not found to be associated with plasma PD-L1 levels.ConclusionThe study concludes that blood-based measurements of PD-L1 in GBM can be a promising prognostic marker and therapeutic target besides a rapid and relatively non-invasive screening tool for routine clinical management. Future work extending the analysis to larger cohorts through multi-center collaborations involving pre-treatment and post-treatment groups is required to fully explore the usefulness of circulating PD-L1 for effective clinical applications

Utility of mitochondrial COI gene for identification of wild ungulate species of conservational importance from Pakistan

Most of the ungulates of Pakistan are either threatened or endangered species and their solitary and inaccessible life style makes them difficult to study. Therefore, estimating biodiversity, monitoring illegal trades and detecting commercial food frauds involving these species is a challenge for zoologists and conservation biologists. Here, we have attempted to exploit the discriminating power of mitochondrial COI gene to identify and to generate barcodes of the wild ungulate species of conservational importance found in Pakistan. 86 specimens of 19 wild ungulate species found in Pakistan were analyzed for their COI sequences. This is the first generated molecular data for many of these endemic and nearly endemic species. Intra and interspecific distances revealed distinct barcode gap for each species and a Neighborhood-joining tree able to discriminate all species into their respective clades. In conclusion, mtCOI is a powerful discriminatory tool for the taxonomic classification of ungulates especially for species that are inaccessible and require noninvasive sampling

Epithelioid inflammatory myofibroblastic sarcoma: the youngest case reported

Epithelioid inflammatory myofibroblastic sarcoma (EIMS) is a rare variant of the inflammatory myofibroblastic tumor. It has an aggressive clinical course and a high rate of recurrence. EIMS primarily affects children and young adults. Hereby, we report this entity in a 4-month-old infant who presented with an abdominal mass. Imaging studies revealed a large hypodense mesentery-based lesion involving the right half and mid-region of the abdomen. The mass with an attached segment of the small bowel was excised in toto. Grossly, a large encapsulated tumor was identified arising from the mesentery of the small bowel. The histological examination showed a tumor consisting of epithelioid to spindle cells loosely arranged in a myxoid background with numerous blood vessels and lymphoplasmacytic inflammatory infiltrate. On immunohistochemistry, the tumor cells showed positivity for ALK1 (nuclear), desmin, SMA, CD68, and focal positivity for CD30. A final diagnosis of EIMS of the small intestine was rendered. To the best of our knowledge, this case is the youngest reported case in literature

In vitro and in silico study for plant growth promotion potential of indigenous Ochrobactrum ciceri and Bacillus australimaris

Insights into plant and bacterial associations, along with their genome mining, have paved the way for the improvement of the agriculture sector. Initially, 69 morphologically different bacterial strains were isolated from 6 different extreme environment samples. For in vitro screening of plant growth-promoting bacteria, auxin, hydrogen cyanide, and antibiotic production along with phosphate solubilization potential was estimated. Only 15 strains exhibited noteworthy production of plant growth-promoting compounds. Ochrobactrum ciceri CS-10 fostered Triticum aestivum and Zea mays seedling root growth remarkably (104.12 and 159%, respectively), while Bacillus australimaris TP-10 significantly increased the number of leaves in T. aestivum (166.66%) and Z. mays (133.33%) seedlings. These strains also boosted seedling biochemical traits, like indole acetic acid, peroxidase, and soluble protein content. Particularly, O. ciceri elevated peroxidase content greatly in T. aestivum (249.7%) and Z. mays (260.5%). Scanning electron micrographs of inoculated roots revealed the aggregation of cells at the roots of Z. mays, whereas single cells/micro-colonies were observed on T. aestivum roots. For in silico analysis, AntiSMASH was used for genome mining of the environmental Ochrobactrum sp. and B. australimaris reference genome. This genome mining unveiled diverse gene clusters encoding; terpenes, beta-lactones, acyl-amino-acids, aryl polyenes, lanthipeptide, and siderophores, etc. Two common biosynthetic gene clusters (terpenes and beta-lactones) were identified in these strains, which can act as plant growth promoters. This symbiotic plant–bacteria relationship has promising ecological and economic implications, offering avenues for beneficial applications

Haematological alterations under the anti-helminthic application in Pavo cristatus

ABSTRACTAnti-helminthic drugs (Albendazole and Levamisole) trials were conducted on Indian Peafowls (n = 20) kept at Jallo Wildlife Park, Lahore, Pakistan for 15. Sampling was conducted on days 7 and 15 of treatments. The results showed that the WBC count was significantly (P < 0.05) higher in both samples in response to Levamisole treatment. Haemoglobin (HGB) and Mean Corpuscular Haemoglobin Concentration (MCHC) concentration increased significantly (P < 0.05) in response to both treatments; however, Mean Corpuscular Haemoglobin (MCH) decreased significantly (P < 0.05) in Albendazole-treated birds on both the sampling days compared to the control. Red Blood Cell Distribution Width-Standard Deviation (RDW-SD) decreased significantly (P < 0.05) in all treatment groups. On the other hand, the prevalence of eggs per gram (EPG) was reduced by 20% after the application of Levamisole on days 7 and 15. It was concluded that both drugs had significant effects on WBCs, HGB, MCH, MCHC and RDW-SD

Purification and biochemical characterisation of acid phosphatase-I from seeds of <i>Nelumbo nucifera</i>

<div><p>Acid phosphatase-I (Apase-I) from seeds of <i>Nelumbo nucifera</i> was purified to electrophoretic homogeneity by combination of ammonium sulfate precipitation, size-exclusion and ion exchange chromatography. SDS-PAGE of purified Apase-I gave a single band with molecular mass of 80 kDa under reducing and non-reducing conditions, indicating that the enzyme was a monomer. The purified enzyme showed maximum activity at 50°C and at pH 5. The Km, Vmax and Kcat for <i>p</i>-nitrophenyl phosphate were 132 μM, 10 μmol/min/mg and 6.7/sec respectively. Apase-I activity was strongly inhibited by Zn²⁺, W²⁺; weakly inhibited by Cu²⁺, Mo²⁺ and Cr⁶⁺ and moderately activated by Mg²⁺. The enzyme was shown to be thermolabile as it lost 50% of its activity at 50°C after incubation for 1 hour. The amino acid analysis of enzyme revealed high proportion of acidic amino acids, which is very similar to that of tomato Apase-I and lower than potato Apase.</p></div

Oxidative Stress and Analysis of Selected SNPs of ACHE (rs 2571598), BCHE (rs 3495), CAT (rs 7943316), SIRT1 (rs 10823108), GSTP1 (rs 1695), and Gene GSTM1, GSTT1 in Chronic Organophosphates Exposed Groups from Cameroon and Pakistan

The detrimental effects of organophosphates (OPs) on human health are thought to be of systemic, i.e., irreversible inhibition of acetylcholinesterase (AChE) at nerve synapses. However, several studies have shown that AChE inhibition alone cannot explain all the toxicological manifestations in prolonged exposure to OPs. The present study aimed to assess the status of antioxidants malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH) (reduced), catalase, and ferric reducing antioxidant power (FRAP) in chronic OP-exposed groups from Cameroon and Pakistan. Molecular analysis of genetic polymorphisms (SNPs) of glutathione transferases (GSTM1, GSTP1, GSTT1), catalase gene (CAT, rs7943316), sirtuin 1 gene (SIRT1, rs10823108), acetylcholinesterase gene (ACHE, rs2571598), and butyrylcholinesterase gene (BCHE, rs3495) were screened in the OP-exposed individuals to find the possible causative association with oxidative stress and toxicity. Cholinesterase and antioxidant activities were measured by colorimetric methods using a spectrophotometer. Salting-out method was employed for DNA extraction from blood followed by restriction fragment length polymorphism (RFLP) for molecular analysis. Cholinergic enzymes were significantly decreased in OP-exposed groups. Catalase and SOD were decreased and MDA and FRAP were increased in OP-exposed groups compared to unexposed groups in both groups. GSH was decreased only in Pakistani OPs-exposed group. Molecular analysis of ACHE, BCHE, Catalase, GSTP1, and GSTM1 SNPs revealed a tentative association with their phenotypic expression that is level of antioxidant and cholinergic enzymes. The study concludes that chronic OPs exposure induces oxidative stress which is associated with the related SNP polymorphism. The toxicogenetics of understudied SNPs were examined for the first time to our understanding. The findings may lead to a newer area of investigation on OPs induced health issues and toxicogenetics