Molecular basis of arsenite (As+3)-induced acute cytotoxicity in human cervical epithelial carcinoma cells

Background: Rapid industrialization is discharging toxic heavy metals into the environment, disturbing human health in many ways and causing various neurologic, cardiovascular, and dermatologic abnormalities and certain types of cancer. The presence of arsenic in drinking water from different urban and rural areas of the major cities of Pakistan, for example, Lahore, Faisalabad, and Kasur, was found to be beyond the permissible limit of 10 parts per billion set by the World Health Organization. Therefore the present study was initiated to examine the effects of arsenite (As+3) on DNA biosynthesis and cell death.Methods: After performing cytotoxic assays on a human epithelial carcinoma cell line, expression analysis was done by quantitative polymerase chain reaction, western blotting, and flow cytometry.Results: We show that As+3 ions have a dose- and time-dependent cytotoxic effect through the activation of the caspase-dependent apoptotic pathway. In contrast to previous research, the present study was designed to explore the early cytotoxic effects produced in human cells during exposure to heavy dosage of As+3 (7.5 μg/ml). Even treatment for 1 h significantly increased the mRNA levels of p21 and p27 and caspases 3, 7, and 9. It was interesting that there was no change in the expression levels of p53, which plays an important role in G2/M phase cell cycle arrest.Conclusion: Our results indicate that sudden exposure of cells to arsenite (As+3) resulted in cytotoxicity and mitochondrial-mediated apoptosis resulting from up-regulation of caspases.Keywords: apoptosis; epithelial carcinoma; cytotoxicity; arsenite; caspases; Pakistan Responsible Editor: Amin Bredan, VIB Inflammation Research Center & Ghent University, Belgium

Clinical characteristics and molecular genetic analysis of 22 patients with neonatal diabetes from the South-Eastern region of Turkey: predominance of non-KATP channel mutations.

PublishedJournal ArticleResearch Support, Non-U.S. Gov'tThis is an open access article available at http://www.eje-online.org/content/172/6/697.BACKGROUND: Neonatal diabetes mellitus (NDM) is a rare form of monogenic diabetes and usually presents in the first 6 months of life. We aimed to describe the clinical characteristics and molecular genetics of a large Turkish cohort of NDM patients from a single centre and estimate an annual incidence rate of NDM in South-Eastern Anatolian region of Turkey. DESIGN AND METHODS: NDM patients presenting to Diyarbakir Children State Hospital between 2010 and 2013, and patients under follow-up with presumed type 1 diabetes mellitus, with onset before 6 months of age were recruited. Molecular genetic analysis was performed. RESULTS: Twenty-two patients (59% males) were diagnosed with NDM (TNDM-5; PNDM-17). Molecular genetic analysis identified a mutation in 20 (95%) patients who had undergone a mutation analysis. In transient neonatal diabetes (TNDM) patients, the genetic cause included chromosome 6q24 abnormalities (n=3), ABCC8 (n=1) and homozygous INS (n=1). In permanent neonatal diabetes (PNDM) patients, homozygous GCK (n=6), EIF2AK3 (n=3), PTF1A (n=3), and INS (n=1) and heterozygous KCNJ11 (n=2) mutations were identified. Pancreatic exocrine dysfunction was observed in patients with mutations in the distal PTF1A enhancer. Both patients with a KCNJ11 mutation responded to oral sulphonylurea. A variable phenotype was associated with the homozygous c.-331C>A INS mutation, which was identified in both a PNDM and TNDM patient. The annual incidence of PNDM in South-East Anatolian region of Turkey was one in 48 000 live births. CONCLUSIONS: Homozygous mutations in GCK, EIF2AK3 and the distal enhancer region of PTF1A were the commonest causes of NDM in our cohort. The high rate of detection of a mutation likely reflects the contribution of new genetic techniques (targeted next-generation sequencing) and increased consanguinity within our cohort.The genetic testing was funded by the Wellcome Trust (Senior Investigator Award to Profs S Ellard and A T Hattersley), and by Diabetes UK (Project funding to Dr D J Mackay). H Demirbilek was funded by European Society for Paediatric Endocrinology (ESPE) and The Scientific and Technological Research Council of Turkey (TUBITAK) for his 1 year clinical fellowship at University College London (UCL), Institute of Child Health, Great Ormond Street Hospital for Children, NHS Trust, Department of Paediatric Endocrinology

Isolation and biochemical characterizations of the bacteria (Acidovorax avenae subsp. avenae) associated with red stripe disease of sugarcane

Studies on Acidovorax avenae subsp. avenae, associated with red stripe disease of sugarcane was conducted in the Department of Plant Pathology, Pir Mehr Ali Shah Arid Agriculture University Rawalpindi during 2009 to 2010, in collaboration with Shakarganj Sugar Research Institute (SSRI), Jhang, Pakistan. Red stripe of sugarcane were recently observed on promising clones of sugarcane planted in autumn 2009 at Ashaba Research Farm of SSRI. Bacteria were isolated from diseased plants. These isolates yielded off white convex colonies on potato dextrose agar (PDA) media at 29°C with 1.7 to 1.9 mm diameter and were yellow on yeast extract dextrose chalk agar (YDC) media at 27°C with 1.8 to 2.0 mm diameter. The bacteria were rod shape measuring 0.5 to 0.6 × 1.4 to 1.6 μm on PDA and 0.6 to 0.7 × 1.5 to 1.7 μm on YDC. Bacterial culture was stored at different temperature levels for 150 days. Reisolation of bacterial culture which was stored at 4°C showed best result on YDC at 27°C after 150 days, whereas it showed positive result after 120 days on PDA at 29°C. Bacteria were gram negative, citrate utilization was positive, oxidase was negative, catalase was positive and urease was negative. Morphological appearance and biochemical characterizations identified the bacteria as A. avenae subsp. Avenae. In vitro screening for the efficacy of various antibiotics to inhibit the growth of A. avenae subsp. avenae on YDC media showed that ampicillin and vancomycin were most effective. Artificial inoculation on sugarcane against red stripe disease was observed. Observations were made upto six weeks for disease development. Out of 27 varieties, 16 were found resistant, four moderately resistant, five moderately susceptible and two susceptible.Key words: Sugarcane, yeast extract dextrose chalk agar (YDC), potato dextrose agar (PDA), Acidovorax avenae subsp. avenae, biochemical characterization, antibiotics

Family History of Prostate Cancer and Survival Outcomes in the UK Genetic Prostate Cancer Study.

BACKGROUND: A family history (FH) of prostate cancer (PrCa) is associated with an increased likelihood of PrCa diagnosis. Conflicting evidence exists regarding familial PrCa and clinical outcomes among PrCa patients, including all-cause mortality/overall survival (OS), PrCa-specific survival (PCSS), aggressive histology, and stage at diagnosis. OBJECTIVE: To determine how the number, degree, and age of a PrCa patient's affected relatives are associated with OS and PCSS of those already diagnosed with PrCa. DESIGN, SETTING, AND PARTICIPANTS: The UK Genetic Prostate Cancer Study is a longitudinal, multi-institutional, observational study collecting baseline and follow-up clinical data since 1992. We examined OS and PCSS in 16340 men by degree and number of relatives with prostate and genetically related cancers (breast, ovarian, and colorectal). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary outcome was all-cause mortality among PrCa patients. The risk of death with respect to FH was assessed by calculating hazard ratios from Cox proportional hazard regression models, adjusting for relevant factors. RESULTS AND LIMITATIONS: A stronger FH was inversely associated with the risk of all-cause and PrCa-specific mortality. This association was greater in those with an increasing number (p-trend < 0.001) and increasing closeness (p-trend < 0.001) of the diagnosed relatives. Patients with at least one first-degree relative were at a lower risk of all-cause mortality than those with no FH (hazard ratio = 0.82 [95% confidence interval 0.75-0.89]). The population is largely of European ancestry, and this may cause an issue with representation and generalisation. Data are missing on epidemiological risk factors for death such as smoking and on comorbidities. Recall of family members' diagnoses may affect the classification of FH in unconfirmed cases. CONCLUSIONS: Based on the investigation of the type and timing of relatives' cancers, it is likely that reductions in mortality are due almost completely to a greater awareness of the disease. This study provides information for clinicians guiding patients and their relatives based on their familial risk. It shows the importance of screening and awareness programmes, which are likely to improve survival among men with an FH. PATIENT SUMMARY: We were interested in how a family history of prostate cancer affects survival in prostate cancer patients. We studied 16340 patients, categorised them according to the strength of their family history, and found that the stronger their family history, the better they did in terms of overall survival. We looked at the type and timing of patients' diagnoses compared with those of their relatives and found that this effect is likely to be explained by awareness, which indicates the importance of screening and awareness programmes

Refinement of the critical genomic region for congenital hyperinsulinism in the Chromosome 9p deletion syndrome

Version 2; peer review: 3 approved. Available from F1000 Research via the DOI in this recordBackground: Large contiguous gene deletions at the distal end of the short arm of chromosome 9 result in the complex multi-organ condition chromosome 9p deletion syndrome. A range of clinical features can result from these deletions with the most common being facial dysmorphisms and neurological impairment. Congenital hyperinsulinism is a rarely reported feature of the syndrome with the genetic mechanism for the dysregulated insulin secretion being unknown. Methods: We studied the clinical and genetic characteristics of 12 individuals with chromosome 9p deletions who had a history of neonatal hypoglycaemia. Using off-target reads generated from targeted next-generation sequencing of the genes known to cause hyperinsulinaemic hypoglycaemia (n=9), or microarray analysis (n=3), we mapped the minimal shared deleted region on chromosome 9 in this cohort. Targeted sequencing was performed in three patients to search for a recessive mutation unmasked by the deletion. Results: In 10/12 patients with hypoglycaemia, hyperinsulinism was confirmed biochemically. A range of extra-pancreatic features were also reported in these patients consistent with the diagnosis of the Chromosome 9p deletion syndrome. The minimal deleted region was mapped to 7.2 Mb, encompassing 38 protein-coding genes. In silico analysis of these genes highlighted SMARCA2 and RFX3 as potential candidates for the hypoglycaemia. Targeted sequencing performed on three of the patients did not identify a second disease-causing variant within the minimal deleted region. Conclusions: This study identifies 9p deletions as an important cause of hyperinsulinaemic hypoglycaemia and increases the number of cases reported with 9p deletions and hypoglycaemia to 15 making this a more common feature of the syndrome than previously appreciated. Whilst the precise genetic mechanism of the dysregulated insulin secretion could not be determined in these patients, mapping the deletion breakpoints highlighted potential candidate genes for hypoglycaemia within the deleted region.Wellcome TrustRoyal Societ

Retromer and Its Role in Regulating Signaling at Endosomes.

The retromer complex is a key element of the endosomal protein sorting machinery being involved in trafficking of proteins from endosomes to the Golgi and also endosomes to the cell surface. There is now accumulating evidence that retromer also has a prominent role in regulating the activity of many diverse signaling proteins that traffic through endosomes and this activity has profound implications for the functioning of many different cell and tissue types from neuronal cells to cells of the immune system to specialized polarized epithelial cells of the retina. In this review, the protein composition of the retromer complex will be described along with many of the accessory factors that facilitate retromer-mediated endosomal protein sorting to detail how retromer activity contributes to the regulation of several distinct signaling pathways

Identification of Genes with Rare Loss of Function Variants Associated with Aggressive Prostate Cancer and Survival.

BACKGROUND: Prostate cancer (PrCa) is a substantial cause of mortality among men globally. Rare germline mutations in BRCA2 have been validated robustly as increasing risk of aggressive forms with a poorer prognosis; however, evidence remains less definitive for other genes. OBJECTIVE: To detect genes associated with PrCa aggressiveness, through a pooled analysis of rare variant sequencing data from six previously reported studies in the UK Genetic Prostate Cancer Study (UKGPCS). DESIGN, SETTING, AND PARTICIPANTS: We accumulated a cohort of 6805 PrCa cases, in which a set of ten candidate genes had been sequenced in all samples. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We examined the association between rare putative loss of function (pLOF) variants in each gene and aggressive classification (defined as any of death from PrCa, metastatic disease, stage T4, or both stage T3 and Gleason score ≥8). Secondary analyses examined staging phenotypes individually. Cox proportional hazards modelling and Kaplan-Meier survival analyses were used to further examine the relationship between mutation status and survival. RESULTS AND LIMITATIONS: We observed associations between PrCa aggressiveness and pLOF mutations in ATM, BRCA2, MSH2, and NBN (odds ratio = 2.67-18.9). These four genes and MLH1 were additionally associated with one or more secondary analysis phenotype. Carriers of germline mutations in these genes experienced shorter PrCa-specific survival (hazard ratio = 2.15, 95% confidence interval 1.79-2.59, p = 4 × 10-16) than noncarriers. CONCLUSIONS: This study provides further support that rare pLOF variants in specific genes are likely to increase aggressive PrCa risk and may help define the panel of informative genes for screening and treatment considerations. PATIENT SUMMARY: By combining data from several previous studies, we have been able to enhance knowledge regarding genes in which inherited mutations would be expected to increase the risk of more aggressive PrCa. This may, in the future, aid in the identification of men at an elevated risk of dying from PrCa

2-D Electronic Spectroscopy on the light-dependent enzyme protochlorophyllide oxidoreductase

In photosynthesis, quantum coherence in light harvesting complexes could form the basis for the highly efficient propagation of excited states within the photosynthetic membranes of bacteria and plants. Recent studies on Photosystem II reaction centres extends the possible relevance of electronic coherence to the photochemical steps of photosynthesis; it was shown that coherent states are sufficiently long-lived, at room temperature, to persist during the initial steps of electron transfer. This extension of quantum coherence, from the energy transfer to the energy trapping steps of photosynthesis, made it timely to investigate if quantum coherence also plays a role in the formation of product states in enzyme catalysis. Given the short timescales involved it was necessary to study an enzyme where the catalytic cycle can be triggered by femtosecond light pulses, so protochlorophyllide oxidoreductase (POR) was selected as an ideal model system. 2-dimensional electronic spectroscopy (2DES) was used to investigate the presence of quantum coherence in the intermediates of the light-dependent reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) catalysed by POR

Chromosome 20p11.2 deletions cause congenital hyperinsulinism via the loss of FOXA2 or its regulatory elements

This is the final version. Available on open access from Springer Nature via the DOI in this recordData availability: All non-clinical data analyzed during this study are included in this article (and its Supplementary Information). The 20p11.2 variants reported in this study were uploaded to ClinVar (SUB14235415). Clinical and genotype data can be used to identify individuals and are therefore available only through collaboration to experienced teams working on approved studies examining the mechanisms, cause, diagnosis and treatment of diabetes and other beta cell disorders. Requests for collaboration will be considered by a steering committee following an application to the Genetic Beta Cell Research Bank (https://www.diabetesgenes.org/current-research/genetic-beta-cell-research-bank/). Contact by email should be directed to S. Flanagan ([email protected]). All requests for access to data will be responded to within 14 d. Accession codes and DOI numbers for all ChIP-seq, ATAC-seq, RNA-seq and scRNA-seq datasets are provided in Supplementary Table 2. We used the Genome Reference Consortium Human Build 37 (GRCh37) to annotate genetic data (accession number GCF_000001405.13). Details of this assembly are provided at https://www.ncbi.nlm.nih.gov/assembly/GCF_000001405.13/.Persistent congenital hyperinsulinism (HI) is a rare genetically heterogeneous condition characterised by dysregulated insulin secretion leading to life-threatening hypoglycaemia. For up to 50% of affected individuals screening of the known HI genes does not identify a disease-causing variant. Large deletions have previously been used to identify novel regulatory regions causing HI. Here, we used genome sequencing to search for novel large (>1 Mb) deletions in 180 probands with HI of unknown cause and replicated our findings in a large cohort of 883 genetically unsolved individuals with HI using off-target copy number variant calling from targeted gene panels. We identified overlapping heterozygous deletions in five individuals (range 3-8 Mb) spanning chromosome 20p11.2. The pancreatic beta-cell transcription factor gene, FOXA2, a known cause of HI was deleted in two of the five individuals. In the remaining three, we found a minimal deleted region of 2.4 Mb adjacent to FOXA2 that encompasses multiple non-coding regulatory elements that are in conformational contact with FOXA2. Our data suggests that the deletions in these three children may cause disease through the dysregulation of FOXA2 expression. These findings provide new insights into the regulation of FOXA2 in the beta-cell and confirm an aetiological role for chromosome 20p11.2 deletions in syndromic HI.Wellcome Trus