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Uncontrolled bleeding from traumatic injury remains the leading cause of preventable death with loss of balance between blood clotting (coagulation) and blood clot breakdown (fibrinolysis). A major limitation of existing hemostatic agents is that they require a functioning clotting system to control the bleeding and are largely based on gauze delivery scaffolds. Herein, a novel rapid wound sealant, composed of two recombinant snake venom proteins, the procoagulant ecarin, to rapidly initiate blood clotting and the antifibrinolytic textilinin, to prevent blood clot breakdown within a synthetic thermoresponsive hydrogel scaffold is developed. In vitro, it is demonstrated that clotting is rapidly initiated with only nanomolar concentrations of venom protein and clot breakdown is effectively inhibited by textilinin. A stable clot is formed within 60 s compared to normal clot formation in 8 min. In vivo studies reveal that the snake venom hydrogel rapidly controls warfarin-induced bleeding, reducing the bleed volume from 48% to 12% and has demonstrated immune compatibility. A new class of hemostatic agents that achieve formation of rapid and stable blood clots even in the presence of blood thinners is demonstrated here.

Silica dust particles are representative of air pollution and long-term inhalation of silicon-containing dust through the respiratory tract can cause pulmonary fibrosis. Epithelial-mesenchymal transformation (EMT) plays an important role in the development of fibrosis. This process can relax cell-cell adhesion complexes and enhance cell migration and invasion properties of these cells. Dysregulation of microRNA-34c (miR-34c) is highly correlated with organ fibrosis including pulmonary fibrosis. In this study, we found that miR-34c-5p could alleviate the occurrence and development of silica-mediated EMT. Fos-related antigen 1 was identified as a functional target of miR-34c-5p by bioinformatics analysis and the dual luciferase gene reporting assay. Importantly, chemically induced up-regulation of hsa-miR-34c-5p correlated inversely with the expression of Fra-1 and further exploration found that the miR-34c-5p/Fra-1 axis inhibits the activation of the phosphatase and tensin homolog deleted on chromosome 10/phosphatidylinositol-4,5-bisphosphate3-kinase/protein kinase B (PTEN/PI3K/AKT) signaling pathway. In addition, through interaction with PTEN/p53 it inhibits the proliferation and migration of human bronchial epithelial cells stimulated by silica, and promotes cell apoptosis, thereby preventing EMT. This finding provides a promising biomarker for the diagnosis and prognosis of pulmonary fibrosis. Furthermore, overexpression of miR-34c-5p represents a potential therapeutic approach.

Ataxia-telangiectasia (A-T) is caused by absence of the catalytic activity of ATM, a protein kinase that plays a central role in the DNA damage response, many branches of cellular metabolism, redox and mitochondrial homeostasis, and cell cycle regulation. A-T is a complex disorder characterized mainly by progressive cerebellar degeneration, immunodeficiency, radiation sensitivity, genome instability, and predisposition to cancer. It is increasingly recognized that the premature aging component of A-T is an important driver of this disease, and A-T is therefore an attractive model to study the aging process. This review outlines the current state of knowledge pertaining to the molecular and cellular signatures of aging in A-T and proposes how these new insights can guide novel therapeutic approaches for A-T.

Patients with ataxia-telangiectasia (A-T) lack a functional ATM kinase protein and exhibit defective repair of DNA double-stranded breaks and response to oxidative stress. We show that CRISPR/Cas9-assisted gene correction combined with piggyBac (PB) transposon-mediated excision of the selection cassette enables seamless restoration of functional ATM alleles in induced pluripotent stem cells from an A-T patient carrying compound heterozygous exonic missense/frameshift mutations, and from a patient with a homozygous splicing acceptor mutation of an internal coding exon. We show that the correction of one allele restores expression of ~ 50% of full-length ATM protein and ameliorates DNA damage-induced activation (auto-phosphorylation) of ATM and phosphorylation of its downstream targets, KAP-1 and H2AX. Restoration of ATM function also normalizes radiosensitivity, mitochondrial ROS production and oxidative-stress-induced apoptosis levels in A-T iPSC lines, demonstrating that restoration of a single ATM allele is sufficient to rescue key ATM functions. Our data further show that despite the absence of a functional ATM kinase, homology-directed repair and seamless correction of a pathogenic ATM mutation is possible. The isogenic pairs of A-T and gene-corrected iPSCs described here constitute valuable tools for elucidating the role of ATM in ageing and A-T pathogenesis.

The Mre11A/RAD50/NBN complex (MRN) is an essential regulator of the cellular damage response after DNA double-strand breaks (DSBs). More recent work has indicated that MRN may also impact on the duration of mitosis. We show here that RAD50-deficient fibroblasts exhibit a marked delay in mitotic progression that can be rescued by lentiviral transduction of RAD50. The delay was observed throughout all mitotic phases in live cell imaging using GFP-labeled H2B as a fluorescent marker. In complementation assays with RAD50 phosphorylation mutants, modifications at Ser635 had little effect on mitotic progression. By contrast with RAD50, fibroblast strains deficient in ATM or NBN did not show a significant slowing of mitotic progression. Ataxia-telangiectasia-like disorder (ATLD) fibroblasts with nuclease-deficient MRE11A (p.W210C) tended to show slower mitosis, though by far not as significant as RAD50-deficient cells. Inhibitor studies indicated that ATM kinase activity might not grossly impact on mitotic progression, while treatment with MRE11A inhibitor PFM39 modestly prolonged mitosis. Inhibition of ATR kinase significantly prolonged mitosis but this effect was mostly independent of RAD50 status. Taken together, our data unravel a mitotic role of RAD50 that can be separated from its known functions in DNA repair.

Toluene-diisocyanate (TDI) is mainly used in the manufacturing process of polyurethane foams, and is a potent inducer of occupational asthma characterized by airway inflammation and airway hyperreactivity. Thymic stromal lymphopoietin (TSLP) plays an important role in the development of asthma, and correlating with the differentiation of Th2 and Th17 cells. However, the role of TSLP in TDI-induced asthma remains unclear. In this study, 96 TDI-exposed workers as well as a mouse model of TDI-induced asthma were investigated. The air exposure assessment result of TDI in the workplace showed that workers were exposed to inhalation of a very high concentration of TDI, approximately 8 times the recommended level, leading to a decrease in pulmonary function and an increase in inflammatory cells, as well as TSLP and IgE levels in the supernatant of sputum obtained from exposed workers. In order to further investigate the role of TSLP in the pathogenesis of TDI-induced asthma, a mouse model of TDI-induced asthma was also employed. Histopathological analysis of mouse lung and bronchus showed an obvious infiltration of inflammatory cells around the bronchus. The levels of inflammatory cells, IFN-γ, IL-4 and IL-17 in bronchoalveolar lavage fluid (BALF), the expression levels of TSLP protein and ROR-γt and IL-17 mRNA in mouse lung tissues were also significantly increased. However, after treatment with TSLP neutralizing antibody (TSLP-Ab), the degree of pulmonary and bronchial inflammation in mice was significantly alleviated, and the levels of inflammatory cells, IFN-γ, IL-4 and IL-17 in BALF, and the expression levels of ROR-γt and IL-17 mRNA in lung tissue were significantly decreased. Our data shows that TSLP plays an important role in the pathogenesis of TDI-induced asthma, and that TSLP-Ab can effectively alleviate TDI-induced airway inflammation of asthma.

Introduction: Failure to obtain complete blood clotting in serum is a common laboratory problem. Our aim was to determine whether snake prothrombin activators are effective in clotting blood and producing quality serum for analyte measurement in anticoagulated patients. Materials and methods: Whole blood clotting was studied in a total of 64 blood samples (41 controls, 20 Warfarin patients, 3 anticoagulated patients using snake venom prothrombin activator (OsPA)) with plain tubes. Coagulation was analysed using a visual assay, Hyland-Clotek and thromboelastography. Healthy control blood was spiked with a range of anticoagulants to determine the effectiveness of OsPa-induced clotting. A paired analysis of a Dabigatran patient and a control investigated the effectiveness of the OsPA clotting tubes. Biochemical analytes (N = 31) were determined for 7 samples on chemistry and immunoassay analysers and compared with commercial tubes. Results: Snake venom prothrombin activators efficiently coagulated blood and plasma spiked with heparin and commonly used anticoagulants. Clotting was observed in the presence of anticoagulants whereas no clotting was observed in BDRST tubes containing 3 U/mL of heparin. Snake venom prothrombin activator enhanced heparinised blood clotting by shortening substantially the clotting time and improving significantly the strength of the clot. Comparison of 31 analytes from the blood of five healthy and two anticoagulated participants gave very good agreement between the analyte concentrations determined. Conclusions: Our results showed that the snake venom prothrombin activators OsPA and PtPA efficiently coagulated recalcified and fresh bloods with or without added anticoagulants. These procoagulants produced high quality serum for accurate analyte measurement.

Background ATM, the protein defective in the human genetic disorder, ataxia-telangiectasia (A-T) plays a central role in response to DNA double-strand breaks (DSBs) and in protecting the cell against oxidative stress. We showed that A-T cells are hypersensitive to metabolic stress which can be accounted for by a failure to exhibit efficient endoplasmic reticulum (ER)-mitochondrial signalling and Ca2+ transfer in response to nutrient deprivation resulting in mitochondrial dysfunction. The objective of the current study is to use an anaplerotic approach using the fatty acid, heptanoate (C7), a metabolic product of the triglyceride, triheptanoin to correct the defect in ER-mitochondrial signalling and enhance cell survival of A-T cells in response to metabolic stress. Methods We treated control cells and A-T cells with the anaplerotic agent, heptanoate to determine their sensitivity to metabolic stress induced by inhibition of glycolysis with 2- deoxyglucose (2DG) using live-cell imaging to monitor cell survival for 72 h using the Incucyte system. We examined ER-mitochondrial signalling in A-T cells exposed to metabolic stress using a suite of techniques including immunofluorescence staining of Grp75, ER-mitochondrial Ca2+ channel, the VAPB-PTPIP51 ER-mitochondrial tether complexes as well as proximity ligation assays between Grp75-IP3R1 and VAPB1-PTPIP51 to establish a functional interaction between ER and mitochondria. Finally, we also performed metabolomic analysis using LC-MS/MS assay to determine altered levels of TCA intermediates A-T cells compared to healthy control cells. Results We demonstrate that heptanoate corrects all aspects of the defective ER-mitochondrial signalling observed in A-T cells. Heptanoate enhances ER-mitochondrial contacts; increases the flow of calcium from the ER to the mitochondrion; restores normal mitochondrial function and mitophagy and increases the resistance of ATM-deficient cells and cells from A-T patients to metabolic stress-induced killing. The defect in mitochondrial function in ATM-deficient cells was accompanied by more reliance on aerobic glycolysis as shown by increased lactate dehydrogenase A (LDHA), accumulation of lactate, and reduced levels of both acetyl CoA and ATP which are all restored by heptanoate. Conclusions We conclude that heptanoate corrects metabolic stress in A-T cells by restoring ER-mitochondria signalling and mitochondrial function and suggest that the parent compound, triheptanoin, has immense potential as a novel therapeutic agent for patients with A-T., Highlights • The fatty acid heptanoate rescues cell killing after nutrient deprivation in A-T cells. • Heptanoate enhances endoplasmic reticulum (ER)-mitochondrial contacts. • Normal mitochondrial function and mitophagy are restored in A-T cells by heptanoate. • Heptanoate shifts A-T cells to more reliance on oxidative phosphorylation.

The mechanism of the sterile inflammatory response in the respiratory tract induced by exposure to sterile particles has not been fully elucidated. The aim of our study is to explore the earlier events in initiating inflammatory response at molecular and cellular level in primary cultured human airway epithelial cells (AEC) exposed to silica particles in order to provide information for earlier diagnosis and prevention of silica particle-induced toxicity as well as possible information on the genesis of silicosis. We isolated primary AEC from three healthy adults and treated them with silica particles at different concentrations for 48 h. We found evidence for silica-induced inflammasome activation by the co-localization of Caspase-1 and NLRP3, as well as increased levels of IL-1β and IL-18. Lactate dehydrogenase and NucGreen analysis proved the occurrence of pyroptosis. High throughput mRNA sequencing showed that the inflammatory response and NF-κB signaling pathways were significantly enriched in gene ontology and Kyoto encyclopedia of genes and genomes analysis, and pyroptosis-related genes were up-regulated. The miR-455-3p and five lncRNAs (LOC105375913, NEAT1, LOC105375181, LOC100506098, and LOC105369370) were verified as key factors related to the mechanism by ceRNA network analysis. LOC105375913 was first discovered to be associated with inflammation in AEC. These data suggest that microcrystalline silica can induce significant inflammation and pyroptosis in human primary AEC through NLRP3 inflammasome pathway and NF-κB signaling pathway at both the gene and protein levels, and the possible mechanism could be miR-455-3p mediated ceRNA hypothesis. Our data provide a method for the studies of the respiratory toxicity of fine particulate matter and the pathogenesis of early silicosis. The miR-455-3p and five lncRNAs related ceRNA network might be the toxicity mechanism of microcrystalline silica particles to AEC.

We describe here the purification and cloning of a codon-optimized form of the snake prothrombin activator ecarin from the saw scaled viper (Echis carinatus) expressed in mammalian cells. Expression of recombinant ecarin (rEcarin) was carried out in human embryonic kidney cells (HEK) cells under conditions for the development and performance of a novel and scalable recombinant snake ecarin to industry standards. Clotting performance of the rEcarin was established in recalcified citrated whole blood, plasma, and fresh whole blood and found to be comparable to native ecarin (N-Ecarin). Furthermore, hemolysis was observed with N-Ecarin at relatively high doses in both recalcified citrated and fresh whole blood, while clotting was not observed with rEcarin, providing an important advantage for the recombinant form. In addition, rEcarin effectively clotted both recalcified citrated whole blood and fresh whole blood containing different anticoagulants including heparin, warfarin, dabigatran, Fondaparinux, rivaroxaban and apixaban, forming firm clots in the blood collection tubes. These results demonstrate that rEcarin efficiently clots normal blood as well as blood spiked with high concentrations of anticoagulants and has great potential as an additive to blood collection tubes to produce high quality serum for analyte analysis in diagnostic medicine.

Ataxia Telangiectasia is a rare autosomal recessive disorder caused by a mutated ATM gene. The most debilitating symptom of Ataxia Telangiectasia is the progressive neurodegeneration of the cerebellum, though the molecular mechanisms driving this degeneration remains unclear. Here we describe the generation and validation of an induced pluripotent stem cell (iPSC) line from an olfactory biopsy from a patient with Ataxia Telangiectasia. Sequencing identified two previously unreported disease-causing mutations in the ATM gene. This line can be used to generate 2D and 3D patient-specific neuronal models enabling investigations into the mechanisms underlying neurodegeneration.

BACKGROUND Ataxia-telangiectasia (A-T) is a rare genetic disorder characterized by a distinct range of clinical manifestations, including progressive ataxia, immunodeficiency, and radiosensitivity. METHODS Clinical data, laboratory results, and genetic data were collected from forty-three A-T patients. Whole-exome sequencing and Sanger sequencing were done for the patients clinically diagnosed as suffering from A-T. Based on the phenotype severity of the disease, patients were divided into severe and mild subgroups. RESULTS The median (IQR) age of diagnosis in this cohort was 5 (3-7) years, and various types of clinical manifestations, including fever (P =.005), lower respiratory tract infection (P = .033), diarrhea (P = .014), and hepatosplenomegaly (P = .032), were significantly higher among patients diagnosed with the severe phenotype. Our results showed a correlation between phenotype severity and mutation type. The chance of having severe phenotype in patients who have severe mutations, including frameshift and nonsense, was 7.3 times higher than in patients who were categorized in the mild genotype group (odds ratio = 7.3, P = .006). Thirty-four types of mutations including 9 novel mutations were observed in our study. CONCLUSION Molecular analysis provides the opportunity for accurate diagnosis and timely management in A-T patients with chronic progressive disease, especially infections and the risk of malignancies. This study characterizes for the first time the broad spectrum of mutations and phenotypes in Iranian A-T patients, which is required for carrier detection and reducing the burden of disease in the future using the patients' families and for the public healthcare system.

Venoms act with remarkable specificity upon a broad diversity of physiological targets. Venoms are composed of proteins, peptides, and small molecules, providing the foundation for the development of novel therapeutics. This study assessed the effect of venom from the red-bellied black snake (Pseudechis porphyriacus) on human primary leukocytes using bead-based flow cytometry, mixed lymphocyte reaction, and cell viability assays. We show that venom treatment had a significant immunosuppressive effect, inhibiting the secretion of interleukin (IL)-2 and tumor necrosis factor (TNF) from purified human T cells by 90% or greater following stimulation with mitogen (phorbol 12-myristate 13-acetate and ionomycin) or via cluster of differentiation (CD) receptors, CD3/CD28. In contrast, venom treatment did not inhibit TNF or IL-6 release from antigen-presenting cells stimulated with lipopolysaccharide. The reduced cytokine release from T cells was not associated with inhibition of T cell proliferation or reduction of cell viability, consistent with an anti-inflammatory mechanism unrelated to the cell cycle. Deconvolution of the venom using reverse-phase HPLC identified four fractions responsible for the observed immunosuppressive activity. These data suggest that compounds from P. porphyriacus venom may be potential drug leads for T cell-associated conditions such as graft versus host disease, rheumatoid arthritis, and inflammatory bowel disease.

Respiratory disease is a major cause of morbidity and mortality in patients with ataxia-telangiectasia (A-T) who are prone to recurrent sinopulmonary infections, bronchiectasis, pulmonary fibrosis, and pulmonary failure. Upper airway infections are common in patients and S. pneumoniae is associated with these infections. We demonstrate here that the upper airway microbiome in patients with A-T is different from that to healthy controls, with S. pneumoniae detected largely in patients only. Patient-specific airway epithelial cells and differentiated air-liquid interface cultures derived from these were hypersensitive to infection which was at least in part due to oxidative damage since it was partially reversed by catalase. We also observed increased levels of the pro-inflammatory cytokines IL-8 and TNF-α (inflammasome-independent) and a decreased level of the inflammasome-dependent cytokine IL-β in patient cells. Further investigation revealed that the ASC-Caspase 1 signalling pathway was defective in A-T airway epithelial cells. These data suggest that the heightened susceptibility of these cells to S. pneumoniae infection is due to both increased oxidative damage and a defect in inflammasome activation, and has implications for lung disease in these patients.

The protein defective in the human genetic disorder ataxia-telangiectasia, ATM, plays a central role in responding to DNA double strand breaks and other lesions to protect the genome against DNA damage and in this way minimize the risk of mutations that can lead to abnormal cellular behaviour. Its function in normal cells is to protect the cell against genotoxic stress but inadvertently it can assist cancer cells by providing resistance against chemotherapeutic agents and thus favouring tumour growth and survival. However, it is now evident that ATM also functions in a DNA damage-independent fashion to protect the cell against other forms of stress such as oxidative and nutrient stress and this non-canonical mechanism may also be relevant to cancer susceptibility in individuals who lack a functional ATM gene. Thus the use of ATM inhibitors to combat resistance in tumours may extend beyond a role for this protein in the DNA damage response. Here, we provide some background on ATM and its activation and investigate the efficacy of ATM inhibitors in treating cancer.

Background Incomplete blood clotting or latent clotting in serum is a common laboratory problem, especially for patients on anticoagulant therapy or when serum tubes are centrifuged before clotting is completed. We describe a novel approach to producing high-quality serum using snake venom prothrombin activator complex (OsPA) as an additive in blood collection tubes for non-anticoagulated (normal) individuals. Methods Plasma clotting assays were performed using a Hyland-Clotek instrument. Blood clotting was visually observed, and thromboelastography was also performed to determine the important parameters of coagulation. Thrombin generation was assayed using the chromogenic substrate S-2238, and biochemical analytes in the serum were determined on chemistry and immunoassay analysers. Fibrinogen was determined by either ELISA or Clauss fibrinogen assay. Results We initially showed that OsPA had strong coagulation activity in clotting not only recalcified citrated plasma and recalcified citrated whole blood, but also fresh whole blood in a clinical setting. The use of TEG clearly showed improved speed of clotting and generation of a firmer clot. We also showed that the use of OsPA to produce serum did not interfere with the determination of commonly measured biochemical analytes. The underlying clotting mechanism involves a burst of thrombin production at the initial stages of the clotting process upon contact with prothrombin in blood. Conclusions These results demonstrate rapid generation of high-quality serum, contributing to faster turnaround times with standardised quality samples, for accurate analyte determinations in normal individuals.

Ataxia-telangiectasia (A-T) is a rare autosomal recessive syndrome characterized by progressive cerebellar ataxia, oculocutaneous telangiectasia, immunodeficiency and cancer predisposition, caused by mutations in the ataxia telangiectasia mutated (ATM) gene. The clinical and immunological manifestations of A-T are very heterogeneous, especially at an early age, leading to frequent misdiagnosis. Cutaneous granulomas with unknown pathogenesis occur uncommonly in a minority of A-T patients. We herein report an unusual case of a 13-year-old girl with A-T who presented severe clinical manifestations, including multiple granulomatous lesions of the skin and a class switch defect phenotype. This patient is the first Iranian A-T case with cutaneous granulomatosis and immunodeficiency. In addition, the literature on skin granulomas in all previously reported A-T patients is reviewed indicating an increased frequency of elevated IgM level and female dominancy in this selected group of patients.

The use of emerging nanotechnologies, such as plasmonic nanoparticles in diagnostic applications, potentially offers opportunities to revolutionize disease management and patient healthcare. Despite worldwide research efforts in this area, there is still a dearth of nanodiagnostics which have been successfully translated for real-world patient usage due to the predominant sole focus on assay analytical performance and lack of detailed investigations into clinical performance in human samples. In a bid to address this pressing need, we herein describe a comprehensive clinical verification of a prospective label-free surface-enhanced Raman scattering (SERS) nanodiagnostic assay for prostate cancer (PCa) risk stratification. This contribution depicts a roadmap of (1) designing a SERS assay for robust and accurate detection of clinically validated PCa RNA targets; (2) employing a relevant and proven PCa clinical biomarker model to test our nanodiagnostic assay; and (3) investigating the clinical performance on independent training ( n = 80) and validation ( n = 40) cohorts of PCa human patient samples. By relating the detection outcomes to gold-standard patient biopsy findings, we established a PCa risk scoring system which exhibited a clinical sensitivity and specificity of 0.87 and 0.90, respectively [area-under-curve of 0.84 (95% confidence interval: 0.81-0.87) for differentiating high- and low-risk PCa] in the validation cohort. We envision that our SERS nanodiagnostic design and clinical verification approach may aid in the individualized prediction of PCa presence and risk stratification and may overall serve as an archetypical strategy to encourage comprehensive clinical evaluation of nanodiagnostic innovations.

Glioblastoma (GBM) is a highly fatal disease with a 5 year survival rate of less than 22%. One of the most effective treatment regimens to date is the use of radiotherapy which induces lethal DNA double-strand breaks to prevent tumour growth. However, recurrence occurs in the majority of patients and is in-part a result of robust radioresistance mechanisms. In this study, we demonstrate that the multifunctional cytokine, interleukin-6 (IL-6), confers a growth advantage in GBM cells but does not have the same effect on normal neural progenitor cells. Further analysis showed IL-6 can promote radioresistance in GBM cells when exposed to ionising radiation. Ablation of the Ataxia-telangiectasia mutated serine/threonine kinase that is recruited and activated by DNA double-strand breaks reverses the effect of radioresistance and re-sensitised GBM to DNA damage thus leading to increase cell death. Our finding suggests targeting the signaling cascade of DNA damage response is a potential therapeutic approach to circumvent IL-6 from promoting radioresistance in GBM.

The phosphoinositide-3-kinase like kinases are a family of very large protein kinases. These PI3-kinase like kinase (PIKK) proteins have well-established roles in detection and repair of damage to the genome, regulation of the transcriptome and cellular metabolism. Recently there has emerged, evidence for links between these proteins and inflammation. While some of these links come from an increased understanding of the impacts of damage to the cell on inflammatory responses, others suggest that PIKK proteins also have direct roles in regulation of immune responses. Particularly evident is the link between DNA damage and innate immune response pathways. Here, we review recent findings on the PIKK family of proteins and how they impact on inflammation, particularly activation of the innate immune system.

Background Despite significant endeavor having been applied to identify effective therapies to treat glioblastoma (GBM), survival outcomes remain intractable. The greatest nonsurgical benefit arises from radiotherapy, though tumors typically recur due to robust DNA repair. Patients could therefore benefit from therapies with the potential to prevent DNA repair and synergize with radiotherapy. In this work, we investigated the potential of salinomycin to enhance radiotherapy and further uncover novel dual functions of this ionophore to induce DNA damage and prevent repair. Methods In vitro primary GBM models and ex vivo GBM patient explants were used to determine the mechanism of action of salinomycin by immunoblot, flow cytometry, immunofluorescence, immunohistochemistry, and mass spectrometry. In vivo efficacy studies were performed using orthotopic GBM animal xenograft models. Salinomycin derivatives were synthesized to increase drug efficacy and explore structure-activity relationships. Results Here we report novel dual functions of salinomycin. Salinomycin induces toxic DNA lesions and prevents subsequent recovery by targeting homologous recombination (HR) repair. Salinomycin appears to target the more radioresistant GBM stem cell–like population and synergizes with radiotherapy to significantly delay tumor formation in vivo. We further developed salinomycin derivatives which display greater efficacy in vivo while retaining the same beneficial mechanisms of action. Conclusion Our findings highlight the potential of salinomycin to induce DNA lesions and inhibit HR to greatly enhance the effect of radiotherapy. Importantly, first-generation salinomycin derivatives display greater efficacy and may pave the way for clinical testing of these agents.

Ataxia with oculomotor apraxia type 2 (AOA2) is a rare autosomal recessive cerebellar ataxia characterized by onset between 10 and 20 years of age and a range of neurological features that include progressive cerebellar atrophy, axonal sensorimotor neuropathy, oculomotor apraxia in a majority of patients, and elevated serum alpha-fetoprotein (AFP). AOA2 is caused by mutation of the SETX gene which encodes senataxin, a DNA/RNA helicase involved in transcription regulation, RNA processing, and DNA maintenance. Disruption of senataxin in rodents led to defective spermatogenesis and sterility in males uncovering a key role for senataxin in male germ cell survival. Here, we report the first clinical and cellular evidence of impaired spermatogenesis in AOA2 patients. We assessed sperm production in three AOA2 patients and testicular pathology in one patient and compared the findings to those of Setx-knockout mice. Sperm production was impaired in all patients assessed (3/3, 100%). Analyses of testicular biopsies from an AOA2 patient recapitulate features of the histology seen in Setx-knockout mice, strongly suggesting an underlying mechanism centering on DNA-damage-mediated germ cell apoptosis. These findings support a role for senataxin in human reproductive function and highlight a novel clinical feature of AOA2 that extends the extra-neurological roles of senataxin. This raises an important reproductive counseling issue for clinicians, and fertility specialists should be aware of SETX mutations as a possible diagnosis in young male patients presenting with oligospermia or azoospermia since infertility may presage the later onset of neurological manifestations in some individuals.

Snake venom prothrombin activators such as Ecarin are readily assayed by continuous spectrophotometric monitoring of p-nitroaniline production in a one step assay containing prothrombin and a p-nitroanilide peptide substrate for thrombin. The coupled reactions result in accelerating p-nitroaniline (pNA) production over the course of the assay giving non-linear progress curves, from which initial velocities are not readily obtained. Most studies therefore resort to approximate estimates of activity, based on the absorbance reached at an arbitrary time. A simple kinetic analysis of the coupled reactions shows that the early points of such curves should be fitted by second order polynomials, representing the accelerating reaction rate in μmol pNA/min/min. The first derivative of the polynomial then gives the increasing velocity of pNA production in μmol pNA/min over the time course of the assay. We demonstrate here that, with the substrate S2238, these rates can be converted to absolute thrombin concentrations using the Michaelis-Menten equation, substituted with values for kcat and Km. These thrombin concentrations increase linearly over the time course of the assay allowing the activity to be expressed in units, defined as μmol product/min, most commonly used to report enzyme activity.

Background Atorvastatin and metformin are known energy restricting mimetic agents that act synergistically to produce molecular and metabolic changes in advanced prostate cancer (PCa). This trial seeks to determine whether these drugs favourably alter selected parameters in men with clinically-localized, aggressive PCa. Methods/design This prospective phase II randomized, controlled window trial is recruiting men with clinically significant PCa, confirmed by biopsy following multiparametric MRI and intending to undergo radical prostatectomy. Ethical approval was granted by the Royal Brisbane and Women's Hospital Human and The University of Queensland Medical Research Ethics Committees. Participants are being randomized into four groups: metformin with placebo; atorvastatin with placebo; metformin with atorvastatin; or placebo alone. Capsules are consumed for 8 weeks, a duration selected as the most appropriate period in which histological and biochemical changes may be observed while allowing prompt treatment with curative intent of clinically significant PCa. At recruitment and prior to RP, participants provide blood, urine and seminal fluid. A subset of participants will undergo 7Tesla magnetic resonance spectroscopy to compare metabolites in-vivo with those in seminal fluid and biopsied tissue. The primary end point is biochemical evolution, defined using biomarkers (serum prostate specific antigen; PCA3 and citrate in seminal fluid and prostatic tissue). Standard pathological assessment will be undertaken. Discussion This study is designed to assess the potential synergistic action of metformin and atorvastatin on PCa tumour biology. The results may determine simple methods of tumour modulation to reduce disease progression.

Pseudechis australis is one of the most venomous and lethal snakes in Australia. Numerous phospholipase A2 (PLA2) isoforms constitute a major portion of its venom, some of which have previously been shown to exhibit not only enzymatic, but also haemolytic, neurotoxic and anticoagulant activities. Here, we have purified a potent anticoagulant PLA2 (identified as PA11) from P. australis venom to investigate its phospholipase, anticoagulant, haemolytic and cytotoxic activities and shown that addition of 11 nM PA11 resulted in a doubling of the clotting time of recalcified whole blood. We have also demonstrated that PA11 has high PLA2 enzymatic activity (10.9 × 10(4) Units/mg), but low haemolytic activity (0.6% of red blood cells hydrolysed in the presence of 1 nM PA11). PA11 at a concentration lower than 600 nM is not cytotoxic towards human cultured cells. Chemical modification experiments using p-bromophenacyl bromide have provided evidence that the catalytic histidine of PA11 is critical for the anticoagulant activity of this PLA2. PA11 that was subjected to trypsin digestion without previous reduction and alkylation of the disulfide bonds maintained enzymatic and anticoagulant activity, suggesting that proteolysis alone cannot abolish these properties. Consistent with these results, administration of PA11 by gavage in a rabbit stasis thrombosis model increased the clotting time of recalcified citrated whole blood by a factor of four. These data suggest that PA11 has potential to be developed as an anticoagulant in a clinical setting.

The recognition, signalling and repair of DNA double strand breaks (DSB) involves the participation of a multitude of proteins and post-translational events that ensure maintenance of genome integrity. Amongst the proteins involved are several which when mutated give rise to genetic disorders characterised by chromosomal abnormalities, cancer predisposition, neurodegeneration and other pathologies. ATM (mutated in ataxia-telangiectasia (A-T) and members of the Mre11/Rad50/Nbs1 (MRN complex) play key roles in this process. The MRN complex rapidly recognises and locates to DNA DSB where it acts to recruit and assist in ATM activation. ATM, in the company of several other DNA damage response proteins, in turn phosphorylates all three members of the MRN complex to initiate downstream signalling. While ATM has hundreds of substrates, members of the MRN complex play a pivotal role in mediating the downstream signalling events that give rise to cell cycle control, DNA repair and ultimately cell survival or apoptosis. Here we focus on the interplay between ATM and the MRN complex in initiating signaling of breaks and more specifically on the adaptor role of the MRN complex in mediating ATM signalling to downstream substrates to control different cellular processes.

Purpose: Roberts syndrome (RBS) is a rare, recessively transmitted developmental disorder characterized by growth retardation, craniofacial abnormalities, and truncation of limbs. All affected individuals to date have mutations in the ESCO2 (establishment of cohesion 2) gene, a key regulator of the cohesin complex, which is involved in sister chromatid cohesion and DNA double-strand break (DSB) repair. Here we characterize DNA damage responses (DDRs) for the first time in an RBS-affected family.Methods and Materials: Lymphoblastoid cell lines were established from an RBS family, including the proband and parents carrying ESCO2 mutations. Various DDR assays were performed on these cells, including cell survival, chromosome break, and apoptosis assays; checkpoint activation indicators; and measures of DNA breakage and repair.Results: Cells derived from the RBS-affected individual showed sensitivity to ionizing radiation (IR) and mitomycin Ceinduced DNA damage. In this ESCO2 compound heterozygote, other DDRs were also defective, including enhanced IR-induced clastogenicity and apoptosis; increased DNA DSB induction; and a reduced capacity for repairing IR-induced DNA DSBs, as measured by gamma-H2AX foci and the comet assay.Conclusions: In addition to its developmental features, RBS can be, like ataxia telangiectasia, considered a DDR-defective syndrome, which contributes to its cellular, molecular, and clinical phenotype. (C) 2018 Elsevier Inc. All rights reserved.

© 2018 Elsevier Ltd Background: Previous trials have found similar early outcomes after robot-assisted laparoscopic prostatectomy and open radical retropubic prostatectomy. We report functional and oncological postoperative outcomes up to 24 months after surgery for these two surgical techniques. Methods: In this randomised controlled phase 3 study, men who had newly diagnosed clinically localised prostate cancer and who had chosen surgery as their treatment approach, and were aged between 35 years and 70 years were eligible and recruited from the Royal Brisbane and Women's Hospital (Brisbane, QLD, Australia). Participants were randomly assigned (1:1) to have either robot-assisted laparoscopic prostatectomy or open radical retropubic prostatectomy. Randomisation was computer generated and occurred in blocks of ten. This was an open trial; however, study investigators involved in data analysis were masked to each patient's surgical treatment. Primary outcomes were urinary function (urinary domain of Expanded Prostate Cancer Index Composite [EPIC]) and sexual function (sexual domain of EPIC and International Index of Erectile Function Questionnaire [IIEF]) at 6 months, 12 months, and 24 months and oncological outcome (biochemical recurrence and imaging evidence of progression). The trial was powered to assess health-related and domain-specific quality-of-life outcomes over 24 months. All analyses were done on a per-protocol basis. The trial was registered with the Australian New Zealand Clinical Trials Registry, number ACTRN12611000661976. Findings: Between Aug 23, 2010, and Nov 25, 2014, 326 men were enrolled, of whom 163 were randomly assigned to robot-assisted laparoscopic prostatectomy and 163 to open radical retropubic prostatectomy. 18 withdrew (12 assigned to radical retropubic prostatectomy and six assigned to robot-assisted laparoscopic prostatectomy); thus, 151 in the radical retropubic prostatectomy group and 157 in the robot-assisted laparoscopic prostatectomy group proceeded to surgery. At the 24-month follow-up time point, 150 men remained in the robot-assisted laparoscopic prostatectomy group and 146 remained in the open radical retropubic prostatectomy group. Urinary function scores did not differ significantly between robot-assisted laparoscopic prostatectomy and open radical retropubic prostatectomy at 6 months post-surgery (88·68 [95% CI 86·79–90·58] vs 88·45 [86·54–90·36]; p1

The MRE11/RAD50/NBS1 (MRN) complex plays a central role as a sensor of DNA double strand breaks (DSB) and is responsible for the efficient activation of ataxia-telangiectasia mutated (ATM) kinase. Once activated ATM in turn phosphorylates RAD50 and NBS1, important for cell cycle control, DNA repair and cell survival. We report here that MRE11 is also phosphorylated by ATM at S676 and S678 in response to agents that induce DNA DSB, is dependent on the presence of NBS1, and does not affect the association of members of the complex or ATM activation. A phosphosite mutant (MRE11S676AS678A) cell line showed decreased cell survival and increased chromosomal aberrations after radiation exposure indicating a defect in DNA repair. Use of GFP-based DNA repair reporter substrates in MRE11S676AS678A cells revealed a defect in homology directed repair (HDR) but single strand annealing was not affected. More detailed investigation revealed that MRE11S676AS678A cells resected DNA ends to a greater extent at sites undergoing HDR. Furthermore, while ATM-dependent phosphorylation of Kap1 and SMC1 was normal in MRE11S676AS678A cells, there was no phosphorylation of Exonuclease 1 consistent with the defect in HDR. These results describe a novel role for ATM-dependent phosphorylation of MRE11 in limiting the extent of resection mediated through Exonuclease 1.

Ataxia telangiectasia mutated (ATM) mediates DNA damage response by controling irradiation-induced foci formation, cell cycle checkpoint, and apoptosis. However, how upstream signaling regulates ATM is not completely understood. Here, we show that upon irradiation stimulation, ATM associates with and is phosphorylated by epidermal growth factor receptor (EGFR) at Tyr370 (Y370) at the site of DNA double-strand breaks. Depletion of endogenous EGFR impairs ATM-mediated foci formation, homologous recombination, and DNA repair. Moreover, pretreatment with an EGFR kinase inhibitor, gefitinib, blocks EGFR and ATM association, hinders CHK2 activation and subsequent foci formation, and increases radiosensitivity. Thus, we reveal a critical mechanism by which EGFR directly regulates ATM activation in DNA damage response, and our results suggest that the status of ATM Y370 phosphorylation has the potential to serve as a biomarker to stratify patients for either radiotherapy alone or in combination with EGFR inhibition.

Background: PCA3 is a long noncoding RNA (lncRNA) with unknown function, upregulated in prostate cancer. LncRNAs may be processed into smaller active species. We hypothesized this for PCA3. Methods: We computed feasible RNA hairpins within the BMCC1 gene (encompassing PCA3) and searched a prostate transcriptome for these. We measured expression using qRT-PCR in three cohorts of prostate cancer tissues (n = 60), exfoliated urinary cells (n = 484 with cancer and n = 166 controls), and in cell lines (n = 22). We used in silico predictions and RNA knockup to identify potential mRNA targets of short transcribed RNAs. Results: We predicted 13 hairpins, of which PCA3-shRNA2 was most abundant within the prostate transcriptome. PCA3-shRNA2 is located within intron 1 of PCA3 and appears regulated by androgens. Expression of PCA3-shRNA2 was upregulated in malignant prostatic tissues, exfoliated urinary cells from men with prostate cancer (13–273 fold change; t test P < 0.003), and closely correlated to PCA3 expression (r = 0.84–0.93; P < 0.001). Urinary PCA3-shRNA2 (C-index, 0.75–0.81) and PCA3 (C-index, 0.78) could predict the presence of cancer in most men. PCA3-shRNA2 knockup altered the expression of predicted target mRNAs, including COPS2, SOX11, WDR48, TEAD1, and Noggin. PCA3-shRNA2 expression was negatively correlated with COPS2 in patient samples (r = −0.32; P < 0.001). Conclusion: We identified a short RNA within PCA3, whose expression is correlated to PCA3, which may target mRNAs implicated in prostate biology. Impact: This short RNA is stable ex vivo, suggesting a role as a robust biomarker. We identify cytoplasmic enrichment of this RNA and potential targeting of mRNAs implicated in prostate carcinogenesis. Cancer Epidemiol Biomarkers Prev; 24(1); 268–75. ©2014 AACR.

ATM (ataxia-telangiectasia mutated) protein kinase is a key regulator of cellular responses to DNA damage and oxidative stress. DNA damage triggers complex cascade of signaling events leading to numerous posttranslational modification on multitude of proteins. Understanding the regulation of ATM kinase is therefore critical not only for understanding the human genetic disorder ataxia-telangiectasia and potential treatment strategies, but essential for deciphering physiological responses of cells to stress. These responses play an important role in carcinogenesis, neurodegeneration, and aging. We focus here on the identification of DNA damage inducible ATM phosphorylation sites to understand the importance of autophosphorylation in the mechanism of ATM kinase activation. We demonstrate the utility of using immunoprecipitated ATM in quantitative LC-MS/MS workflow with stable isotope dimethyl labeling of ATM peptides for identification of phosphorylation sites.

Reprogramming of cells enables generation of pluripotent stem cells and resulting progeny through directed differentiation, making this technology an invaluable tool for the study of human development and disease. Reprogramming occurs with a wide range of efficiency, a culmination of intrinsic and extrinsic factors including the tissue of origin, the passage number and culture history of the target cells. Another major factor affecting reprogramming is the methodology used and the quality of the reprogramming process itself, including for conventional viral-based approaches viral titer and subsequent viral transduction efficiency, including downstream transgene insertion and stoichiometry. Genetic background is an important parameter affecting the efficiency of the reprogramming process with reports that cells from individuals harboring specific mutations are more difficult to reprogram than control counterparts.Ataxia-Telangiectasia (A-T) fibroblasts underwent reprogramming at reduced efficiency in contrast to their controls. To optimize reprogramming of fibroblasts from patients with A-T, we examined the response of A-T cells to various cell culture conditions after lentiviral transduction with reprogramming factors Oc4/Sox2 (pSIN4-EF2-O2S) and Klf4/c-Myc (pSIN4-CMV-K2M). Parameters included media type (KSR or serum-containing DMEM), treatment with a p53 inhibitor (small-molecule cyclic pifithrin-α), and either a low or high concentration of bFGF. Post-transduction, equivalent numbers of cells from heterozygote and homozygote patients were plated and assessed at regular intervals for survival and proliferation. Our findings indicate that A-T cells responded favorably to the addition of FCS and gradual weaning away from their native media into KSR-containing stem cell media that produced suitable conditions for their reprogramming. We examined a range of properties to identify and isolate good quality iPSCs including the expression status of important stem cell transcription factors/surface proteins, methylation levels at stem cell associated regulatory loci, persistence of transgenes, karyotype status, and teratoma-forming ability.

During S-phase the cell replicates its DNA which is critical to maintaining the integrity of the genome and cell survival amidst damaging events. The cell is equipped with a series of checkpoints to slow progress throughout the cycle and facilitate DNA repair. Ataxia telangiectasia mutated (ATM), defective in the human genetic disorder ataxia-telangiectasia (A-T), is the key to initiating a signaling cascade activating the intra-S-phase checkpoint. This was first identified in A-T cells as radioresistant DNA synthesis using

The molecular pathogenesis of ataxia-telangiectasia (A-T) is not yet fully understood, and a versatile cellular model is required for in vitro studies. The occurrence of continuous neurogenesis and easy access make the multipotent adult stem cells from the olfactory mucosa within the nasal cavity a potential cellular model. We describe an efficient method to establish neuron-like cells from olfactory mucosa biopsies derived from A-T patients for the purpose of studying the cellular and molecular aspects of this debilitating disease.

6 1 Dame Roma Mitchell Cancer Research Laboratories and Adelaide Prostate Cancer Research 7 Centre, The University of Adelaide, South Australia, Australia; 2 Freemasons Foundation 8 Centre for Men’s Health, The University of Adelaide, South Australia, Australia; 3 University 9 of Queensland Centre for Clinical Research, Herston, Brisbane, Queensland, Australia; 10 4 Department of Urology, Royal Brisbane and Women’s Hospital, Brisbane, Queensland, 11 Australia; 5 Queensland Institute of Medical Research, Herston, Brisbane, Queensland, 12 Australia, 6 School of Population Health, The University of Queensland, Brisbane, 13 Queensland, Australia, 7 Department of Endocrinology, Princess Alexandra Hospital, 14 Brisbane, Queensland, Australia 15

The MRE11/RAD50/NBN (MRN) complex plays a key role in detecting DNA double-strand breaks, recruiting and activating ataxia-telangiectasia mutated and in processing the breaks. Members of this complex also act as adaptor molecules for downstream signaling to the cell cycle and other cellular processes. Somewhat more controversial are the results to support a role for MRN in the ataxia-telangiectasia and Rad3-related (ATR) activation and signaling. We provide evidence that RAD50 is required for ATR activation in mammalian cells in response to DNA replication stress. It is in turn phosphorylated at a specific site (S635) by ATR, which is required for ATR signaling through Chk1 and other downstream substrates. We find that RAD50 phosphorylation is essential for DNA replication restart by promoting loading of cohesin at these sites. We also demonstrate that replication stress-induced RAD50 phosphorylation is functionally significant for cell survival and cell cycle checkpoint activation. These results highlight the importance of the adaptor role for a member of the MRN complex in all aspects of the response to DNA replication stress.

The human genetic disorder ataxia telangiectasia (A-T) is characterised by neurodegeneration, immunodeficiency, radiosensitivity, cell cycle checkpoint defects, genomic instability and cancer predisposition. Progressive cerebellar ataxia represents the most debilitating aspect of this disorder. At present, there is no therapy available to cure or prevent the progressive symptoms of A-T. While it is possible to alleviate some of the symptoms associated with immunodeficiency and deficient lung function, neither the predisposition to cancer nor the progressive neurodegeneration can be prevented. Significant effort has focused on improving our understanding of various clinical, genetic and immunological aspects of A-T; however, little attention has been directed towards identifying altered brain structure and function using MRI. To date, most imaging studies have reported radiological anomalies in A-T. This review outlines the clinical and biological features of A-T along with known radiological imaging anomalies. In addition, we briefly discuss the advent of high-resolution MRI in conjunction with diffusion-weighted imaging, which enables improved investigation of the microstructural tissue environment, giving insight into the loss in integrity of motor networks due to abnormal neurodevelopmental or progressive neurodegenerative processes. Such imaging approaches have yet to be applied in the study of A-T and could provide important new information regarding the relationship between mutation of the ataxia telangiectasia mutated (ATM) gene and the integrity of motor circuitry.

A randomised trial of robotic and open prostatectomy commenced in October 2010 and is progressing well. Clinical and quality of life outcomes together with economic costs to individuals and the health service are being examined critically to compare outcomes.

Defects in the recognition and/or repair of damage to DNA are responsible for a sub-group of autosomal recessive ataxias. Included in this group is a novel form of ataxia with oculomotor apraxia characterised by sensitivity to DNA damaging agents, a defect in p53 stabilisation, oxidative stress and resistance to apoptosis. We provide evidence here that the defect in this patient's cells is at the level of the mitochondrion. Mitochondrial membrane potential was markedly reduced in cells from the patient and ROS levels were elevated. This was accompanied by lipid peroxidation of mitochondrial proteins involved in electron transport and RNA synthesis. However, no gross changes or alteration in composition or activity of mitochondrial electron transport complexes was evident. Sequencing of mitochondrial DNA revealed a mutation, I349T, in the mitochondrial cytochrome b gene. These results describe a patient with an apparently novel form of AOA characterised by a defect at the level of the mitochondrion.

Ataxia-telangiectasia (A-T), an autosomal recessive disease caused by mutations in the ATM gene is characterised by cerebellar atrophy and progressive neurodegeneration which has been poorly recapitulated in Atm mutant mice. Consequently, pathways leading to neurodegeneration in A-T are poorly understood. We describe here the generation of an Atm knockout rat model that does not display cerebellar atrophy but instead paralysis and spinal cord atrophy, reminiscent of that seen in older patients and milder forms of the disorder. Loss of Atm in neurons and glia leads to accumulation of cytosolic DNA, increased cytokine production and constitutive activation of microglia consistent with a neuroinflammatory phenotype. Rats lacking ATM had significant loss of motor neurons and microgliosis in the spinal cord, consistent with onset of paralysis. Since short term treatment with steroids has been shown to improve the neurological signs in A-T patients we determined if that was also the case for Atm-deficient rats. Betamethasone treatment extended the lifespan of Atm knockout rats, prevented microglial activation and significantly decreased neuroinflammatory changes and motor neuron loss. These results point to unrepaired damage to DNA leading to significant levels of cytosolic DNA in Atm-deficient neurons and microglia and as a consequence activation of the cGAS-STING pathway and cytokine production. This in turn would increase the inflammatory microenvironment leading to dysfunction and death of neurons. Thus the rat model represents a suitable one for studying neurodegeneration in A-T and adds support for the use of anti-inflammatory drugs for the treatment of neurodegeneration in A-T patients.

Summary Background The absence of trial data comparing robot-assisted laparoscopic prostatectomy and open radical retropubic prostatectomy is a crucial knowledge gap in uro-oncology. We aimed to compare these two approaches in terms of functional and oncological outcomes and report the early postoperative outcomes at 12 weeks. Method In this randomised controlled phase 3 study, men who had newly diagnosed clinically localised prostate cancer and who had chosen surgery as their treatment approach, were able to read and speak English, had no previous history of head injury, dementia, or psychiatric illness or no other concurrent cancer, had an estimated life expectancy of 10 years or more, and were aged between 35 years and 70 years were eligible and recruited from the Royal Brisbane and Women's Hospital (Brisbane, QLD). Participants were randomly assigned (1:1) to receive either robot-assisted laparoscopic prostatectomy or radical retropubic prostatectomy. Randomisation was computer generated and occurred in blocks of ten. This was an open trial; however, study investigators involved in data analysis were masked to each patient's condition. Further, a masked central pathologist reviewed the biopsy and radical prostatectomy specimens. Primary outcomes were urinary function (urinary domain of EPIC) and sexual function (sexual domain of EPIC and IIEF) at 6 weeks, 12 weeks, and 24 months and oncological outcome (positive surgical margin status and biochemical and imaging evidence of progression at 24 months). The trial was powered to assess health-related and domain-specific quality of life outcomes over 24 months. We report here the early outcomes at 6 weeks and 12 weeks. The per-protocol populations were included in the primary and safety analyses. This trial was registered with the Australian New Zealand Clinical Trials Registry (ANZCTR), number ACTRN12611000661976. Findings Between Aug 23, 2010, and Nov 25, 2014, 326 men were enrolled, of whom 163 were randomly assigned to radical retropubic prostatectomy and 163 to robot-assisted laparoscopic prostatectomy. 18 withdrew (12 assigned to radical retropubic prostatectomy and six assigned to robot-assisted laparoscopic prostatectomy); thus, 151 in the radical retropubic prostatectomy group proceeded to surgery and 157 in the robot-assisted laparoscopic prostatectomy group. 121 assigned to radical retropubic prostatectomy completed the 12 week questionnaire versus 131 assigned to robot-assisted laparoscopic prostatectomy. Urinary function scores did not differ significantly between the radical retropubic prostatectomy group and robot-assisted laparoscopic prostatectomy group at 6 weeks post-surgery (74·50 vs 71·10; p=0·09) or 12 weeks post-surgery (83·80 vs 82·50; p=0·48). Sexual function scores did not differ significantly between the radical retropubic prostatectomy group and robot-assisted laparoscopic prostatectomy group at 6 weeks post-surgery (30·70 vs 32·70; p=0·45) or 12 weeks post-surgery (35·00 vs 38·90; p=0·18). Equivalence testing on the difference between the proportion of positive surgical margins between the two groups (15 [10%] in the radical retropubic prostatectomy group vs 23 [15%] in the robot-assisted laparoscopic prostatectomy group) showed that equality between the two techniques could not be established based on a 90% CI with a Δ of 10%. However, a superiority test showed that the two proportions were not significantly different (p=0·21). 14 patients (9%) in the radical retropubic prostatectomy group versus six (4%) in the robot-assisted laparoscopic prostatectomy group had postoperative complications (p=0·052). 12 (8%) men receiving radical retropubic prostatectomy and three (2%) men receiving robot-assisted laparoscopic prostatectomy experienced intraoperative adverse events. Interpretation These two techniques yield similar functional outcomes at 12 weeks. Longer term follow-up is needed. In the interim, we encourage patients to choose an experienced surgeon they trust and with whom they have rapport, rather than a specific surgical approach. Funding Cancer Council Queensland.

Glioblastoma multiforme (GBM) is the most common form of brain tumor with a poor prognosis and resistance to radiotherapy. Recent evidence suggests that glioma-initiating cells play a central role in radioresistance through DNA damage checkpoint activation and enhanced DNA repair. To investigate this in more detail, we compared the DNA damage response in nontumor forming neural progenitor cells (NPC) and glioma-initiating cells isolated from GBM patient specimens. As observed for GBM tumors, initial characterization showed that glioma-initiating cells have long-term self-renewal capacity. They express markers identical to NPCs and have the ability to form tumors in an animal model. In addition, these cells are radioresistant to varying degrees, which could not be explained by enhanced nonhomologous end joining (NHEJ). Indeed, NHEJ in glioma-initiating cells was equivalent, or in some cases reduced, as compared with NPCs. However, there was evidence for more efficient homologous recombination repair in glioma-initiating cells. We did not observe a prolonged cell cycle nor enhanced basal activation of checkpoint proteins as reported previously. Rather, cell-cycle defects in the G1–S and S-phase checkpoints were observed by determining entry into S-phase and radioresistant DNA synthesis following irradiation. These data suggest that homologous recombination and cell-cycle checkpoint abnormalities may contribute to the radioresistance of glioma-initiating cells and that both processes may be suitable targets for therapy. Mol Cancer Ther; 11(9); 1863–72. ©2012 AACR.

Pluripotent stem cells can differentiate into every cell type of the human body. Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) therefore provides an opportunity to gain insight into the molecular and cellular basis of disease. Because the cellular DNA damage response poses a barrier to reprogramming, generation of iPSCs from patients with chromosomal instability syndromes has thus far proven to be difficult. Here we demonstrate that fibroblasts from patients with ataxia-telangiectasia (A-T), a disorder characterized by chromosomal instability, progressive neurodegeneration, high risk of cancer, and immunodeficiency, can be reprogrammed to bona fide iPSCs, albeit at a reduced efficiency. A-T iPSCs display defective radiation-induced signaling, radiosensitivity, and cell cycle checkpoint defects. Bioinformatic analysis of gene expression in the A-T iPSCs identifies abnormalities in DNA damage signaling pathways, as well as changes in mitochondrial and pentose phosphate pathways. A-T iPSCs can be differentiated into functional neurons and thus represent a suitable model system to investigate A-T-associated neurodegeneration. Collectively, our data show that iPSCs can be generated from a chromosomal instability syndrome and that these cells can be used to discover early developmental consequences of ATM deficiency, such as altered mitochondrial function, that may be relevant to A-T pathogenesis and amenable to therapeutic intervention.

Snake venoms are attractive for drug discovery and development, with a number of therapeutics derived from snake venom either in clinical use or in development. Recognising this opportunity, Australian biopharmaceutical company QRxPharma Ltd and its subsidiary Venomics Pty Ltd (VPL) has partnered with the University of Queensland (UQ) to screen and develop drug candidates from Australian elapid snake venoms. VPL has three haemostasis candidates in early preclinical development. Textilinin-1 (Q8008) is a 7 kDa potent and selective plasmin inhibitor that has application as an anti-fibrinolytic agent to reduce blood loss associated with complex surgeries. Haempatch™ (Q8009) is a Factor Xa-like protein that displays potent procoagulant effects and is being developed as a topical haemostatic agent to reduce blood loss resulting from surgery or trauma. CoVase™ (V0801) is a procoagulant cofactor that may have application as a systemic anti-bleeding agent in the treatment of internal bleeding and non-compressible haemorrhage. This review focuses on drug discovery from Australian elapid snake venoms, with emphasis on the QRxPharma/VPL drug discovery project undertaken in collaboration with UQ and candidates at further stages of development.

Ataxia-telangiectasia, mutated (ATM) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signaling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoints, initiating DNA repair, and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here, we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach to identify cytoplasmic proteins altered in their phosphorylation state in control and ataxia-telangiectasia (A-T) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites, including 6,686 high-confidence sites mapping to 2,536 unique proteins. A total of 62 differentially phosphorylated peptides were identified; of these, 43 were phosphorylated in control but not in A-T cells, and 19 varied in their level of phosphorylation. Motif enrichment analysis of phosphopeptides revealed that consensus ATM serine glutamine sites were overrepresented. When considering phosphorylation events, only observed in control cells (not observed in A-T cells), with predicted ATM sites phosphoSerine/phosphoThreonine glutamine, we narrowed this list to 11 candidate ATM-dependent cytoplasmic proteins. Two of these 11 were previously described as ATM substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens, and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (oxidative stress responsive 1(OSR1), HDGF, and ccdc82) as ATM dependent after H 2O 2 exposure, and another protein (S100A11) demonstrated ATM-dependence for translocation from the cytoplasm to the nucleus. These data provide new insights into the activation of ATM by oxidative stress through identification of novel substrates for ATM in the cytoplasm.

Cancer incidence in schizophrenia is not increased commensurate with higher rates of risk exposures. Here we report an investigation of the DNA damage response, an anti-tumorigenic defence, in immortalised lymphoblasts from patients with schizophrenia.Unirradiated and irradiated (5Gy) lymphoblasts from schizophrenia patients (n = 28) and healthy controls (n = 28) were immunostained for the phosphorylated histone variant H2AX (γH2AX), an index of DNA double-strand breaks. Flow cytometry was used to assess cell cycle distribution and γH2AX immunofluorescence. Rate of DNA repair was quantified by determining the temporal change in γH2AX values following irradiation.In unirradiated lymphoblasts, γH2AX levels were significantly increased in the schizophrenia group compared with controls (effect size = 0.86). This increase was most evident in patients with cognitive deficits. In irradiated lymphoblasts, peak radiation-induced γH2AX levels were significantly reduced in patients. No differences between patients and controls were found in the rate of DNA repair or in cell cycle distribution.The significant differences in DNA damage response signalling observed involve modification of histone variant H2AX and thereby implicate regulatory processes determining chromatin structure in dividing lymphoblasts from patients with schizophrenia. The role that aberrant DNA damage response signalling plays in protecting patients from cancer is unclear.

ATM is the most significant molecule involved in monitoring the genomic integrity of the cell. Any damage done to DNA relentlessly challenges the cellular machinery involved in recognition, processing and repair of these insults. ATM kinase is activated early to detect and signal lesions in DNA, arrest the cell cycle, establish DNA repair signaling and faithfully restore the damaged chromatin. ATM activation plays an important role as a barrier to tumorigenesis, metabolic syndrome and neurodegeneration. Therefore, studies of ATM-dependent DNA damage signaling pathways hold promise for treatment of a variety of debilitating diseases through the development of new therapeutics capable of modulating cellular responses to stress. In this review, we have tried to untangle the complex web of ATM signaling pathways with the purpose of pinpointing multiple roles of ATM underlying the complex phenotypes observed in AT patients.

Malignant myoepithelioma of the breast (MMB) is a rare and often aggressive disease with poor prognosis. Little is known regarding its optimal treatment and progression. We describe the clinical history of a woman following excision of a benign adenomyoepithelioma which recurred years later as a radioresistant malignant myoepithelioma with high levels of ataxia telangiectasia mutated protein and mutant p53 (Cys135Phe). MMB requires close follow-up and aggressive treatment. If adjuvant radiotherapy is adopted to improve local control, minimal postoperative delay and higher doses than for standard post-mastectomy radiation are recommended.