Evaluation of the spatial distribution of γH2AX following ionizing radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as γH2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of γH2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of γH2AX as molecular marker of DSBs, a disparity in γ-irradiation-induced γH2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of γH2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between γH2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of γH2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3Dmodeling.

Quantitation of gammaH2AX foci in tissue samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming γH2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear γH2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of γH2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of γH2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, γH2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of γH2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of γH2AX foci in mouse tissues.

Circulating antibodies against Plasmodium falciparum histidine-rich proteins 2 interfere with antigen detection by rapid diagnostic tests

Background Rapid diagnostic tests (RDTs) for detection of Plasmodium falciparum infection that target P. falciparum histidine-rich protein 2 (PfHRP2), a protein that circulates in the blood of patients infected with this species of malaria, are widely used to guide case management. Understanding determinants of PfHRP2 availability in circulation is therefore essential to understanding the performance of PfHRP2-detecting RDTs. Methods The possibility that pre-formed host anti-PfHRP2 antibodies may block target antigen detection, thereby causing false negative test results was investigated in this study. Results Anti-PfHRP2 antibodies were detected in 19/75 (25%) of plasma samples collected from patients with acute malaria from Cambodia, Nigeria and the Philippines, as well as in 3/28 (10.7%) asymptomatic Solomon Islands residents. Pre-incubation of plasma samples from subjects with high-titre anti-PfHRP2 antibodies with soluble PfHRP2 blocked the detection of the target antigen on two of the three brands of RDTs tested, leading to false negative results. Pre-incubation of the plasma with intact parasitized erythrocytes resulted in a reduction of band intensity at the highest parasite density, and a reduction of lower detection threshold by ten-fold on all three brands of RDTs tested. Conclusions These observations indicate possible reduced sensitivity for diagnosis of P. falciparum malaria using PfHRP2-detecting RDTs among people with high levels of specific antibodies and low density infection, as well as possible interference with tests configured to detect soluble PfHRP2 in saliva or urine samples. Further investigations are required to assess the impact of pre-formed anti-PfHRP2 antibodies on RDT performance in different transmission settings.

Activation of membrane-bound proteins and receptor systems: A link between tissue kallikrein and the KLK-related peptidases

The 15 members of the kallikrein-related serine peptidase (KLK) family have diverse tissue-specific expression profiles and roles in a range of cellular processes, including proliferation, migration, invasion, differentiation, inflammation and angiogenesis that are required in both normal physiology as well as pathological conditions. These roles require cleavage of a range of substrates, including extracellular matrix proteins, growth factors, cytokines as well as other proteinases. In addition, it has been clear since the earliest days of KLK research that cleavage of cell surface substrates is also essential in a range of KLK-mediated cellular processes where these peptidases are essentially acting as agonists and antagonists. In this review we focus on these KLK-regulated cell surface receptor systems including bradykinin receptors, proteinase-activated receptors, as well as the plasminogen activator, ephrins and their receptors, and hepatocyte growth factor/Met receptor systems and other plasma membrane proteins. From this analysis it is clear that in many physiological and pathological settings KLKs have the potential to regulate multiple receptor systems simultaneously; an important issue when these peptidases and substrates are targeted in disease.

Analysing the similarity of proteins based on a new approach to empirical mode decomposition

The function of a protein can be partially determined by the information contained in its amino acid sequence. It can be assumed that proteins with similar amino acid sequences normally have closer functions. Hence analysing the similarity of proteins has become one of the most important areas of protein study. In this work, a layered comparison method is used to analyze the similarity of proteins. It is based on the empirical mode decomposition (EMD) method, and protein sequences are characterized by the intrinsic mode functions (IMFs). The similarity of proteins is studied with a new cross-correlation formula. It seems that the EMD method can be used to detect the functional relationship of two proteins. This kind of similarity method is a complement of traditional sequence similarity approaches which focus on the alignment of amino acids

Néstor-Guillermo Progeria Syndrome: a biochemical insight into Barrier-to-Autointegration Factor 1, alanine 12 threonine mutation

Background Premature aging syndromes recapitulate many aspects of natural aging and provide an insight into this phenomenon at a molecular and cellular level. The progeria syndromes appear to cause rapid aging through disruption of normal nuclear structure. Recently, a coding mutation (c.34G > A [p.A12T]) in the Barrier to Autointegration Factor 1 (BANF1) gene was identified as the genetic basis of Néstor-Guillermo Progeria syndrome (NGPS). This mutation was described to cause instability in the BANF1 protein, causing a disruption of the nuclear envelope structure. Results Here we demonstrate that the BANF1 A12T protein is indeed correctly folded, stable and that the observed phenotype, is likely due to the disruption of the DNA binding surface of the A12T mutant. We demonstrate, using biochemical assays, that the BANF1 A12T protein is impaired in its ability to bind DNA while its interaction with nuclear envelope proteins is unperturbed. Consistent with this, we demonstrate that ectopic expression of the mutant protein induces the NGPS cellular phenotype, while the protein localizes normally to the nuclear envelope. Conclusions Our study clarifies the role of the A12T mutation in NGPS patients, which will be of importance for understanding the development of the disease.

Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis

Systems-level identification and analysis of cellular circuits in the brain will require the development of whole-brain imaging with single-cell resolution. To this end, we performed comprehensive chemical screening to develop a whole-brain clearing and imaging method, termed CUBIC (clear, unobstructed brain imaging cocktails and computational analysis). CUBIC is a simple and efficient method involving the immersion of brain samples in chemical mixtures containing aminoalcohols, which enables rapid whole-brain imaging with single-photon excitation microscopy. CUBIC is applicable to multicolor imaging of fluorescent proteins or immunostained samples in adult brains and is scalable from a primate brain to subcellular structures. We also developed a whole-brain cell-nuclear counterstaining protocol and a computational image analysis pipeline that, together with CUBIC reagents, enable the visualization and quantification of neural activities induced by environmental stimulation. CUBIC enables time-course expression profiling of whole adult brains with single-cell resolution.

Basic personal values underlie and give coherence to political values: A cross national study in 15 countries

Do the political values of the general public form a coherent system? What might be the source of coherence? We view political values as expressions, in the political domain, of more basic personal values. Basic personal values (e.g., security, achievement, benevolence, hedonism) are organized on a circular continuum that reflects their conflicting and compatible motivations. We theorize that this circular motivational structure also gives coherence to political values. We assess this theorizing with data from 15 countries, using eight core political values (e.g., free enterprise, law and order) and ten basic personal values. We specify the underlying basic values expected to promote or oppose each political value. We offer different hypotheses for the 12 non-communist and three post-communist countries studied, where the political context suggests different meanings of a basic or political value. Correlation and regression analyses support almost all hypotheses. Moreover, basic values account for substantially more variance in political values than age, gender, education, and income. Multidimensional scaling analyses demonstrate graphically how the circular motivational continuum of basic personal values structures relations among core political values. This study strengthens the assumption that individual differences in basic personal values play a critical role in political thought.

Large-scale interlaboratory study to develop, analytically validate and apply highly multiplexed, quantitative peptide assays to measure cancer-relevant proteins in plasma

There is an increasing need in biology and clinical medicine to robustly and reliably measure tens-to-hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma, and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and 7 control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to sub-nanogram/mL sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and inter-laboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy isotope labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an inter-laboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality c`ontrol measures, enables sensitive, specific, reproducible and quantitative measurements of proteins and peptides in complex biological matrices such as plasma.

Different correlation of Sphingosine-1-Phosphate receptor 1 with receptor activator of nuclear factor kappa B ligand and regulatory T cells in rat periapical lesions

Introduction Sphingosine-1-phosphate receptor 1 (S1P1) is crucial for regulation of immunity and bone metabolism. This study aimed to investigate the expression of S1P1 in rat periapical lesions and its relationship with receptor activator of nuclear factor kappa B ligand (RANKL) and regulatory T (Treg) cells. Methods Periapical lesions were induced by pulp exposure in the first lower molars of 55 Wistar rats. Thirty rats were killed on days 0, 7, 14, 21, 28, and 35, and their mandibles were harvested for x-ray imaging, micro–computed tomography scanning, histologic observation, immunohistochemistry, enzyme histochemistry, and double immunofluorescence analysis. The remaining 25 rats were killed on days 0, 14, 21, 28, and 35, and mandibles were harvested for flow cytometry. Results The volume and area of the periapical lesions increased from day 0 to day 21 and then remained comparably stable after day 28. S1P1-positive cells were observed in the inflammatory periapical regions; the number of S1P1-positive cells peaked at day 14 and then decreased from day 21 to day 35. The distribution of S1P1-positive cells was positively correlated with the dynamics of RANKL-positive cells but was negatively correlated with that of Treg cells. Conclusions S1P1 expression was differentially correlated with RANKL and Treg cell infiltration in the periapical lesions and is therefore a contributing factor to the pathogenesis of such lesions.

Basic Religious Charities

Just over 44,000 registered charities filed their first Annual Information Statement (AIS) return with the Australian Charities and Not-for-profits Commission (ACNC) before the end of October 2014. Of these, 10,918 charities self-identified as Basic Religious Charities (BRCs). These are usually, but not always, unincorporated religious congregations which receive no or little government funding. Having a central agency for reporting, in the form of the ACNC, having access to information supplied in the AIS by registered organisations has allowed access to new measures of charities and their activities. In September 2014 the ACNC, in conjunction with Curtin University Not-for-profit Initiative, released a high-level report on the first AIS, and the data were also made available digitally through the Australian Government Data Repository. This factsheet builds on that report by focusing on BRCs.

Antioxidants in Athlete's Basic Nutrition: Considerations towards a Guideline for the Intake of Vitamin C and Vitamin E

Antioxidants in acute physical exercise and exercise training remain a hot topic in sport nutrition, exercise physiology and biology, in general (Jackson, 2008; Margaritis and Rousseau, 2008; Gomez-Cabrera et al., 2012; Nikolaidis et al., 2012). During the past few decades, antioxidants have received attention predominantly as a nutritional strategy for preventing or minimising detrimental effects of reactive oxygen and nitrogen species (RONS), which are generated during and after strenuous exercise (Jackson, 2008, 2009; Powers and Jackson, 2008). Antioxidant supplementation has become a common practice among athletes as a means to (theoretically) reduce oxidative stress, promote recovery and enhance performance (Peternelj and Coombes, 2011). However, until now, requirements of antioxidant micronutrients and antioxidant compounds for athletes training for and competing in different sport events, including marathon running, triathlon races or team sport events involving repeated sprinting, have not been determined sufficiently (Williams et al., 2006; Margaritis and Rousseau, 2008). Crucially, evidence has been emerging that higher dosages of antioxidants may not necessarily be beneficial in this context, but can also elicit detrimental effects by interfering with performance-enhancing (Gomez-Cabrera et al., 2008) and health-promoting training adaptations (Ristow et al., 2009). As originally postulated in a pioneering study on exercise-induced production of RONS by Davies et al. (1982) in the early 1980s, evidence has been increasing in recent years that RONS are not only damaging agents, but also act as signalling molecules for regulating muscle function (Reid, 2001; Jackson, 2008) and for initiating adaptive responses to exercise (Jackson, 2009; Powers et al., 2010). The recognition that antioxidants could, vice versa, interact with the signalling pathways underlying the responses to acute (and repeated) bouts of exercise has contributed important novel aspects to the continued discussion on antioxidant requirements for athletes. In view of the recent advances in this field, it is the aim of this report to examine the current knowledge of antioxidants, in particular of vitamins C and E, in the basic nutrition of athletes. While overviews on related topics including basic mechanisms of exercise-induced oxidative stress, redox biology, antioxidant defence systems and a summary of studies on antioxidant supplementation during exercise training are provided, this does not mean that this report is comprehensive. Several issues of the expanding and multidisciplinary field of antioxidants and exercise are covered elsewhere in this book and/or in the literature. Exemplarily, the reader is referred to reviews on oxidative stress (Konig et al., 2001; Vollaard et al., 2005; Knez et al., 2006; Powers and Jackson, 2008; Nikolaidis et al., 2012), redox-sensitive signalling and muscle function (Reid, 2001; Vollaard et al., 2005; Jackson, 2008; Ji, 2008; Powers and Jackson, 2008; Powers et al., 2010; Radak et al., 2013) and antioxidant supplementation (Williams et al., 2006; Peake et al., 2007; Peternelj and Coombes, 2011) in the context with exercise. Within the scope of the report, we rather aim to address the question regarding requirements of antioxidants, specifically vitamins C and E, during exercise training, draw conclusions and provide practical implications from the recent research.