Senataxin suppresses the antiviral transcriptional response and controls viral biogenesis.

The human helicase senataxin (SETX) has been linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS4) and ataxia with oculomotor apraxia (AOA2). Here we identified a role for SETX in controlling the antiviral response. Cells that had undergone depletion of SETX and SETX-deficient cells derived from patients with AOA2 had higher expression of antiviral mediators in response to infection than did wild-type cells. Mechanistically, we propose a model whereby SETX attenuates the activity of RNA polymerase II (RNAPII) at genes stimulated after a virus is sensed and thus controls the magnitude of the host response to pathogens and the biogenesis of various RNA viruses (e.g., influenza A virus and West Nile virus). Our data indicate a potentially causal link among inborn errors in SETX, susceptibility to infection and the development of neurologic disorders.

Integrated genome and transcriptome analyses reveal the mechanism of genome instability in ataxia with oculomotor apraxia 2.

Mutations in the SETX gene, which encodes Senataxin, are associated with the progressive neurodegenerative diseases ataxia with oculomotor apraxia 2 (AOA2) and amyotrophic lateral sclerosis 4 (ALS4). To identify the causal defect in AOA2, patient-derived cells and SETX knockouts (human and mouse) were analyzed using integrated genomic and transcriptomic approaches. A genome-wide increase in chromosome instability (gains and losses) within genes and at chromosome fragile sites was observed, resulting in changes to gene-expression profiles. Transcription stress near promoters correlated with high GCskew and the accumulation of R-loops at promoter-proximal regions, which localized with chromosomal regions where gains and losses were observed. In the absence of Senataxin, the Cockayne syndrome protein CSB was required for the recruitment of the transcription-coupled repair endonucleases (XPG and XPF) and RAD52 recombination protein to target and resolve transcription bubbles containing R-loops, leading to genomic instability. These results show that transcription stress is an important contributor to SETX mutation-associated chromosome fragility and AOA2.

Ataxia Telangiectasia patient-derived neuronal and brain organoid models reveal mitochondrial dysfunction and oxidative stress.

Ataxia Telangiectasia (AT) is a rare disorder caused by mutations in the ATM gene and results in progressive neurodegeneration for reasons that remain poorly understood. In addition to its central role in nuclear DNA repair, ATM operates outside the nucleus to regulate metabolism, redox homeostasis and mitochondrial function. However, a systematic investigation into how and when loss of ATM affects these parameters in relevant human neuronal models of AT was lacking. We therefore used cortical neurons and brain organoids from AT-patient iPSC and gene corrected isogenic controls to reveal levels of mitochondrial dysfunction, oxidative stress, and senescence that vary with developmental maturity. Transcriptome analyses identified disruptions in regulatory networks related to mitochondrial function and maintenance, including alterations in the PARP/SIRT signalling axis and dysregulation of key mitophagy and mitochondrial fission-fusion processes. We further show that antioxidants reduce ROS and restore neurite branching in AT neuronal cultures, and ameliorate impaired neuronal activity in AT brain organoids. We conclude that progressive mitochondrial dysfunction and aberrant ROS production are important contributors to neurodegeneration in AT and are strongly linked to ATM's role in mitochondrial homeostasis regulation.

Correction of ATM mutations in iPS cells from two ataxia-telangiectasia patients restores DNA damage and oxidative stress responses.

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.

miRNA-34c-5p targets Fra-1 to inhibit pulmonary fibrosis induced by silica through p53 and PTEN/PI3K/Akt signaling pathway.

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.

Microcrystalline silica particles induce inflammatory response via pyroptosis in primary human respiratory epithelial cells.

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-1ß 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.

Progress Curve Analysis of the one stage chromogenic assay for ecarin.

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.

Ataxia-telangiectasia: from a rare disorder to a paradigm for cell signalling and cancer.

First described over 80 years ago, ataxia-telangiectasia (A-T) was defined as a clinical entity 50 years ago. Although not encountered by most clinicians, it is a paradigm for cancer predisposition and neurodegenerative disorders and has a central role in our understanding of the DNA-damage response, signal transduction and cell-cycle control. The discovery of the protein A-T mutated (ATM) that is deficient in A-T paved the way for rapid progress on understanding how ATM functions with a host of other proteins to protect against genome instability and reduce the risk of cancer and other pathologies.

SMG1 heterozygosity exacerbates haematopoietic cancer development in Atm null mice by increasing persistent DNA damage and oxidative stress.

Suppressor of morphogenesis in genitalia 1 (SMG1) and ataxia telangiectasia mutated (ATM) are members of the PI3-kinase like-kinase (PIKK) family of proteins. ATM is a well-established tumour suppressor. Loss of one or both alleles of ATM results in an increased risk of cancer development, particularly haematopoietic cancer and breast cancer in both humans and mouse models. In mice, total loss of SMG1 is embryonic lethal and loss of a single allele results in an increased rate of cancer development, particularly haematopoietic cancers and lung cancer. In this study, we generated mice deficient in Atm and lacking one allele of Smg1, Atm-/- Smg1gt/+ mice. These mice developed cancers more rapidly than either of the parental genotypes, and all cancers were haematopoietic in origin. The combined loss of Smg1 and Atm resulted in a higher level of basal DNA damage and oxidative stress in tissues than loss of either gene alone. Furthermore, Atm-/- Smg1gt/+ mice displayed increased cytokine levels in haematopoietic tissues compared with wild-type animals indicating the development of low-level inflammation and a pro-tumour microenvironment. Overall, our data demonstrated that combined loss of Atm expression and decreased Smg1 expression increases haematopoietic cancer development.

A rat model of ataxia-telangiectasia: evidence for a neurodegenerative phenotype.

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.

Disruption of Spermatogenesis and Infertility in Ataxia with Oculomotor Apraxia Type 2 (AOA2).

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.

Next-generation rapid serum tube technology using prothrombin activator coagulant: fast, high-quality serum from normal samples.

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.

Robot-assisted laparoscopic prostatectomy versus open radical retropubic prostatectomy: 24-month outcomes from a randomised controlled study.

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<0·0001, p2<0·0001), 12 months post-surgery (90·76 [88·89-92·62] vs 91·53 [90·07-92·98]; p1<0·0001, p2<0·0001), or 24 months post-surgery (91·33 [89·64-93·03] vs 90·86 [89·01-92·70]; p1<0·0001, p2<0·0001). Sexual function scores were not significantly different between robot-assisted laparoscopic prostatectomy and open radical retropubic prostatectomy at 6 months post-surgery (EPIC: 37·40 [33·60-41·19] vs 38·63 [34·76-42·49], p1=0·0001, p2<0·0001; IIEF: 29·75 [26·66-32·84] vs 29·78 [26·41-33·16], p1<0·0001, p2<0·0001), 12 months post-surgery (EPIC: 42·28 [38·05-46·51] vs 42·51 [38·29-46·72], p1<0·0001, p2<0·0001; IIEF: 33·10 [29·59-36·61] vs 33·50 [29·87-37·13], p1=0·0002, p2<0·0001), or 24 months post-surgery (EPIC: 45·70 [41·17-50·23] vs 46·90 [42·20-51·60], p1=0·0003, p2<0·0001; IIEF: 33·95 [30·11-37·78] vs 33·89 [29·82-37·96], p1=0·0003, p2=0·0004). Equivalence testing on the difference between the proportion of biochemical recurrences between the two groups (13 [9%] in the open radical retropubic prostatectomy group vs four [3%] 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 prespecified margin of 10%. However, a superiority test showed that the two proportions were significantly different (p=0·0199). Equivalence testing on the proportion of patients who had imaging evidence of progression revealed that the two groups were not significantly different (p=0·2956). INTERPRETATION: Robot-assisted laparoscopic prostatectomy and open radical retropubic prostatectomy yielded similar functional outcomes at 24 months. We advise caution in interpreting the oncological outcomes of our study because of the absence of standardisation in postoperative management between the two trial groups and the use of additional cancer treatments. Clinicians and patients should view the benefits of a robotic approach as being largely related to its minimally invasive nature. FUNDING: Cancer Council Queensland.

PIKKing a way to regulate inflammation.

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.

A new model to study neurodegeneration in ataxia oculomotor apraxia type 2.

Ataxia oculomotor apraxia type 2 (AOA2) is a rare autosomal recessive cerebellar ataxia. Recent evidence suggests that the protein defective in this syndrome, senataxin (SETX), functions in RNA processing to protect the integrity of the genome. To date, only patient-derived lymphoblastoid cells, fibroblasts and SETX knockdown cells were available to investigate AOA2. Recent disruption of the Setx gene in mice did not lead to neurobehavioral defects or neurodegeneration, making it difficult to study the etiology of AOA2. To develop a more relevant neuronal model to study neurodegeneration in AOA2, we derived neural progenitors from a patient with AOA2 and a control by induced pluripotent stem cell (iPSC) reprogramming of fibroblasts. AOA2 iPSC and neural progenitors exhibit increased levels of oxidative damage, DNA double-strand breaks, increased DNA damage-induced cell death and R-loop accumulation. Genome-wide expression and weighted gene co-expression network analysis in these neural progenitors identified both previously reported and novel affected genes and cellular pathways associated with senataxin dysfunction and the pathophysiology of AOA2, providing further insight into the role of senataxin in regulating gene expression on a genome-wide scale. These data show that iPSCs can be generated from patients with the autosomal recessive ataxia, AOA2, differentiated into neurons, and that both cell types recapitulate the AOA2 cellular phenotype. This represents a novel and appropriate model system to investigate neurodegeneration in this syndrome.

Robot-assisted laparoscopic prostatectomy versus open radical retropubic prostatectomy: early outcomes from a randomised controlled phase 3 study.

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.

ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1.

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.

RAD50 phosphorylation promotes ATR downstream signaling and DNA restart following replication stress.

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.

Mutation of senataxin alters disease-specific transcriptional networks in patients with ataxia with oculomotor apraxia type 2.

Senataxin, encoded by the SETX gene, contributes to multiple aspects of gene expression, including transcription and RNA processing. Mutations in SETX cause the recessive disorder ataxia with oculomotor apraxia type 2 (AOA2) and a dominant juvenile form of amyotrophic lateral sclerosis (ALS4). To assess the functional role of senataxin in disease, we examined differential gene expression in AOA2 patient fibroblasts, identifying a core set of genes showing altered expression by microarray and RNA-sequencing. To determine whether AOA2 and ALS4 mutations differentially affect gene expression, we overexpressed disease-specific SETX mutations in senataxin-haploinsufficient fibroblasts and observed changes in distinct sets of genes. This implicates mutation-specific alterations of senataxin function in disease pathogenesis and provides a novel example of allelic neurogenetic disorders with differing gene expression profiles. Weighted gene co-expression network analysis (WGCNA) demonstrated these senataxin-associated genes to be involved in both mutation-specific and shared functional gene networks. To assess this in vivo, we performed gene expression analysis on peripheral blood from members of 12 different AOA2 families and identified an AOA2-specific transcriptional signature. WGCNA identified two gene modules highly enriched for this transcriptional signature in the peripheral blood of all AOA2 patients studied. These modules were disease-specific and preserved in patient fibroblasts and in the cerebellum of Setx knockout mice demonstrating conservation across species and cell types, including neurons. These results identify novel genes and cellular pathways related to senataxin function in normal and disease states, and implicate alterations in gene expression as underlying the phenotypic differences between AOA2 and ALS4.

Loss of caspase-2 augments lymphomagenesis and enhances genomic instability in Atm-deficient mice.

Caspase-2, the most evolutionarily conserved member of the caspase family, has been shown to be involved in apoptosis induced by various stimuli. Our recent work indicates that caspase-2 has putative functions in tumor suppression and protection against cellular stress. As such, the loss of caspase-2 enhances lymphomagenesis in Eµ-Myc transgenic mice, and caspase-2 KO (Casp2(-/-)) mice show characteristics of premature aging. However, the extent and specificity of caspase-2 function in tumor suppression is currently unclear. To further investigate this, ataxia telangiectasia mutated KO (Atm(-/-)) mice, which develop spontaneous thymic lymphomas, were used to generate Atm(-/-)Casp2(-/-) mice. Initial characterization revealed that caspase-2 deficiency enhanced growth retardation and caused synthetic perinatal lethality in Atm(-/-) mice. A comparison of tumor susceptibility demonstrated that Atm(-/-)Casp2(-/-) mice developed tumors with a dramatically increased incidence compared with Atm(-/-) mice. Atm(-/-)Casp2(-/-) tumor cells displayed an increased proliferative capacity and extensive aneuploidy that coincided with elevated oxidative damage. Furthermore, splenic and thymic T cells derived from premalignant Atm(-/-)Casp2(-/-) mice also showed increased levels of aneuploidy. These observations suggest that the tumor suppressor activity of caspase-2 is linked to its function in the maintenance of genomic stability and suppression of oxidative damage. Given that ATM and caspase-2 are important components of the DNA damage and antioxidant defense systems, which are essential for the maintenance of genomic stability, these proteins may synergistically function in tumor suppression by regulating these processes.