Investigation and application of γH2AX as a potential biomarker of DNA double strand breaks in insect and human cells

Double strand breaks (DSBs) are one of the most biologically significant DNA damage lesions. Replication stress, endogenous reactive oxygen species, exogenous sources of DNA damage such as ionizing radiation (IR), and genotoxic compounds are key causes of DNA breaks in living systems. An early known response to DNA DSBs in the cell is the phosphorylation of the C-terminal of the core histone protein H2AX (termed γH2AX when phosphorylated). It is accepted that with the passage of time, the level of γH2AX declines as repair of DSBs is completed; however, DSBs can remain unrepaired and may result in persistent γH2AX signals and the knowledge of persistent γH2AX signals remain relatively unexplored. DNA damage has been associated with some agerelated diseases, including the neurodegenerative disorder, Alzheimer’s disease (AD). The aim of this PhD thesis was to (i) investigate IR-induced persistent γH2AX responses in Queensland fruit fly (Q-fly) (Bactrocera tryoni), and human buccal cell as a model system (ii) quantify endogenous γH2AX levels in buccal cells and lymphocytes of individuals with mild cognitive impairment (MCI) and AD relative to healthy controls in the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing (AIBL) and the South Australian Alzheimer’s Nutrition and DNA Damage (SAND) studies. Persistent and dose-dependent γH2AB (a homologue of γH2AX) signals were detected and quantified either by Western blot or laser scanning cytometry (LSC) for up to 17 days post-IR exposure in adult Q-flies (when irradiated as pupae). In human buccal cells irradiated (up to 4 Gy), LSC and visual scoring demonstrated a significant increase in γH2AX (n=6 individuals). Twenty-four hours after IR exposure the γH2AX levels remained significantly higher than baseline. The frequency of visually scored γH2AX in human buccal cell nuclei showed a strong positive correlation (up to r=0.999) with automated LSC scored γH2AX signals. In the SAND study, the endogenous γH2AX levels were significantly higher in lymphocytes from AD (n=20) compared to MCI (n=18) and controls (n=40). Plasma homocysteine, creatinine, and chitinase-3-like protein 1 (CHI3L1) were positively correlated with lymphocyte γH2AX signals, whilst glomerular filtration rate (GFR) was negatively correlated. In buccal cells, the endogenous γH2AX levels were significantly elevated in AD (n=16), compared to MCI (n=18) and controls (n=17), from the AIBL study. Nuclear circularity (irregular nuclear shapes) was significantly higher in buccal cell nuclei from AD compared to MCI and controls and there was a positive correlation between nuclear circularity and γH2AX signals. The elevated γH2AX levels in lymphocytes and buccal cells of AD patients may indicate defects in the efficiency of repairing the chronic endogenous DNA DSBs contributing to the accumulation of unrepaired or persistent DSBs. The measurement of persistent and endogenous γH2AX may have application in radiation biodosimetry as well as a potential biomarker in AD.Thesis (Ph.D.)--University of Adelaide, School of Agriculture, Food and Wine, 201

Evaluation of gammah2ax in buccal cells as a molecular biomarker of DNA damage in Alzheimer’s disease in the AIBL study of ageing

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. In response to double-stranded breaks (DSBs) in chromosomal DNA, H2AX (a member of histone H2A family) becomes phosphorylated to form γH2AX. Although increased levels of γH2AX have been reported in the neuronal nuclei of Alzheimer’s disease (AD) patients, the understanding of γH2AX responses in buccal nuclei of individuals with mild cognitive impairment (MCI) and AD remain unexplored. In the current study, endogenous γH2AX was measured in buccal cell nuclei from MCI (n = 18) or AD (n = 16) patients and in healthy controls (n = 17) using laser scanning cytometry (LSC). The γH2AX level was significantly elevated in nuclei of the AD group compared to the MCI and control group, and there was a concomitant increase in P-trend for γH2AX from the control group through MCI to the AD group. Receiver-operating characteristic curves were carried out for different γH2AX parameters; γH2AX in nuclei resulted in the greatest area under the curve value of 0.7794 (p = 0.0062) with 75% sensitivity and 70% specificity for the identification of AD patients from control. In addition, nuclear circularity (a measure of irregular nuclear shape) was significantly higher in the buccal cell nuclei from the AD group compared with the MCI and control groups. Additionally, there was a positive correlation between the nuclear circularity and γH2AX signals. The results indicated that increased DNA damage is associated with AD

Diarrhea in young children from low-income countries leads to large-scale alterations in intestinal microbiota composition

Acknowledgments This work was funded in part by the William and Melinda Gates Foundation, award 42917 to JPN and OCS; US National Institutes of Health grants 5R01HG005220 to HCB, 5R01HG004885 to MP; US National Science Foundation Graduate Research Fellowship award DGE0750616 to JNP; AWW and JP are funded by The Wellcome Trust (Grant No. WT098051).Peer reviewedPublisher PD

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

γH2AX is a biomarker of modulated cytostatic drug resistance

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Persistent γH2AX : a promising molecular marker of DNA damage and aging

One of the earliest cellular responses to DNA double strand breaks (DSBs) is the phosphorylation of the core histone protein H2AX (termed γH2AX). Persistent γH2AX is the level of γH2AX above baseline, measured at a given time-point beyond which DNA DSBs are normally expected to be repaired (usually persist for days to months). This review summarizes the concept of persistent γH2AX in the context of exogenous source induced DNA DSBs (e.g. ionizing radiation (IR), chemotherapeutic drugs, genotoxic agents), and endogenous γH2AX levels in normal aging and accelerated aging disorders. Summary of the current literature demonstrates the following (i) γH2AX persistence is a common phenomenon that occurs in humans and animals; (ii) nuclei retain persistent γH2AX foci for up to several months after IR exposure, allowing for retrospective biodosimetry; (iii) the combination of various radiosensitizing drugs with ionizing radiation exposure leads to persistent γH2AX response, thus enabling the potential for monitoring cancer patients' response to chemotherapy and radiotherapy as well as tailoring cancer treatments; (iv) persistent γH2AX accumulates in telomeric DNA and in cells undergoing cellular senescence; and (v) increased endogenous γH2AX levels may be associated with diseases of accelerated aging. In summary, measurement of persistent γH2AX could potentially be used as a marker of radiation biodosimetry, evaluating sensitivity to therapeutic genotoxins and radiotherapy, and exploring the association of unrepaired DNA DSBs on telomeres with diseases of accelerated aging.19 page(s

Survey of culture, GoldenGate assay, Universal Biosensor assay, and 16S rRNA gene sequencing as alternative methods of bacterial pathogen detection

Cultivation-based assays combined with PCR or enzyme-linked immunosorbent assay (ELISA)-based methods for finding virulence factors are standard methods for detecting bacterial pathogens in stools; however, with emerging molecular technologies, new methods have become available. The aim of this study was to compare four distinct detection technologies for the identification of pathogens in stools from children under 5 years of age in The Gambia, Mali, Kenya, and Bangladesh. The children were identified, using currently accepted clinical protocols, as either controls or cases with moderate to severe diarrhea. A total of 3,610 stool samples were tested by established clinical culture techniques: 3,179 DNA samples by the Universal Biosensor assay (Ibis Biosciences, Inc.), 1,466 DNA samples by the GoldenGate assay (Illumina), and 1,006 DNA samples by sequencing of 16S rRNA genes. Each method detected different proportions of samples testing positive for each of seven enteric pathogens, enteroaggregative Escherichia coli (EAEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shigella spp., Campylobacter jejuni, Salmonella enterica, and Aeromonas spp. The comparisons among detection methods included the frequency of positive stool samples and kappa values for making pairwise comparisons. Overall, the standard culture methods detected Shigella spp., EPEC, ETEC, and EAEC in smaller proportions of the samples than either of the methods based on detection of the virulence genes from DNA in whole stools. The GoldenGate method revealed the greatest agreement with the other methods. The agreement among methods was higher in cases than in controls. The new molecular technologies have a high potential for highly sensitive identification of bacterial diarrheal pathogens.7 page(s