POLQ seals post-replicative ssDNA gaps to maintain genome stability in BRCA-deficient cancer cells.

POLQ is a key effector of DSB repair by microhomology-mediated end-joining (MMEJ) and is overexpressed in many cancers. POLQ inhibitors confer synthetic lethality in HR and Shieldin-deficient cancer cells, which has been proposed to reflect a critical dependence on the DSB repair pathway by MMEJ. Whether POLQ also operates independent of MMEJ remains unexplored. Here, we show that POLQ-deficient cells accumulate post-replicative ssDNA gaps upon BRCA1/2 loss or PARP inhibitor treatment. Biochemically, cooperation between POLQ helicase and polymerase activities promotes RPA displacement and ssDNA-gap fill-in, respectively. POLQ is also capable of microhomology-mediated gap skipping (MMGS), which generates deletions during gap repair that resemble the genomic scars prevalent in POLQ overexpressing cancers. Our findings implicate POLQ in mutagenic post-replicative gap sealing, which could drive genome evolution in cancer and whose loss places a critical dependency on HR for gap protection and repair and cellular viability.

Quantitative, titratable and high-throughput reporter assays to measure DNA double strand break repair activity in cells.

Repair of DNA damage is essential for the maintenance of genome stability and cell viability. DNA double strand breaks (DSBs) constitute a toxic class of DNA lesion and multiple cellular pathways exist to mediate their repair. Robust and titratable assays of cellular DSB repair (DSBR) are important to functionally interrogate the integrity and efficiency of these mechanisms in disease models as well as in response to genetic or pharmacological perturbations. Several variants of DSBR reporters are available, however these are often limited by throughput or restricted to specific cellular models. Here, we describe the generation and validation of a suite of extrachromosomal reporter assays that can efficiently measure the major DSBR pathways of homologous recombination (HR), classical nonhomologous end joining (cNHEJ), microhomology-mediated end joining (MMEJ) and single strand annealing (SSA). We demonstrate that these assays can be adapted to a high-throughput screening format and that they are sensitive to pharmacological modulation, thus providing mechanistic and quantitative insights into compound potency, selectivity, and on-target specificity. We propose that these reporter assays can serve as tools to dissect the interplay of DSBR pathway networks in cells and will have broad implications for studies of DSBR mechanisms in basic research and drug discovery.

Targeting ATR in Cancer Medicine.

As a key component of the DNA Damage Response, the Ataxia telangiectasia and Rad3-related (ATR) protein is a promising druggable target that is currently widely evaluated in phase I-II-III clinical trials as monotherapy and in combinations with other rational antitumor agents, including immunotherapy, DNA repair inhibitors, chemo- and radiotherapy. Ongoing clinical studies for this drug class must address the optimization of the therapeutic window to limit overlapping toxicities and refine the target population that will most likely benefit from ATR inhibition. With advances in the development of personalized treatment strategies for patients with advanced solid tumors, many ongoing ATR inhibitor trials have been recruiting patients based on their germline and somatic molecular alterations, rather than relying solely on specific tumor subtypes. Although a spectrum of molecular alterations have already been identified as potential predictive biomarkers of response that may sensitize to ATR inhibition, these biomarkers must be analytically validated and feasible to measure robustly to allow for successful integration into the clinic. While several ATR inhibitors in development are poised to address a clinically unmet need, no ATR inhibitor has yet received FDA-approval. This chapter details the underlying rationale for targeting ATR and summarizes the current preclinical and clinical landscape of ATR inhibitors currently in evaluation, as their regulatory approval potentially lies close in sight.

SOMANZ position paper on the management of nausea and vomiting in pregnancy and hyperemesis gravidarum.

This is a brief summary of the Society of Obstetric Medicine of Australia and New Zealand (SOMANZ) evidence-based guideline for the management of nausea and vomiting of pregnancy (NVP) and hyperemesis gravidarum (HG). The full guideline and executive summary including auditable outcomes are freely available on the SOMANZ website [https://www.somanz.org/guidelines.asp]. The guideline includes a proposed SOMANZ definition of NVP and HG and evidence-based practical advice regarding the investigation and management of NVP, HG and associated conditions including thyroid dysfunction. A practical algorithm for assessment and management as well as an individual patient management plan and self-assessment tools are included.

SOMANZ guidelines for the investigation and management sepsis in pregnancy.

SOMANZ (Society of Obstetric Medicine Australia and New Zealand) has written a guideline to provide evidence-based guidance for the investigation and care of women with sepsis in pregnancy or the postpartum period. The guideline is evidence-based and incorporates recent changes in the definition of sepsis. The etiology, investigation and treatment of bacterial, viral and non-infective causes of sepsis are discussed. Obstetric considerations relevant to anaesthetic and intensive care treatment in sepsis are also addressed. A multi-disciplinary group of clinicians with experience in all aspects of the care of pregnant women have contributed to the development of the guidelines. This is an executive summary of the guidelines.

Fabrication and Application of Isotopically Labelled Gold Arrays for Multiplexed Peptide Analysis.

A new array-based technology for the simultaneous capture, chemical labelling and mass spectrometry analysis of peptides is presented. Isotopically labelled self-assembled monolayer (SAM) gold arrays are constructed and used simultaneously to capture and label a range of peptides. The array-immobilised, labelled peptides were released by MALDI ablation, analysed by MALDI mass spectrometry and readily identified as labelled peptides from their characteristic isotope pattern. This new solid-phase array platform has the advantage of minimal sample manipulation and is suitable for multiple analyses of single protein digests on a single MALDI target plate.

Understanding factors that influence protective glove use among automotive spray painters.

Dermal contact with isocyanate-based coatings may lead to systemic respiratory sensitization. The most common isocyanates found in sprayed automotive coatings are monomeric and oligomeric 1,6-hexamethylene diisocyanate (HDI) and isophorone diisocyanate (IPDI). Most spray painters use thin (4-5 mil) latex gloves that are not effective at preventing dermal exposures when spraying isocyanate paints. Personal interviews with collision repair industry personnel and focus groups with spray painters were held to characterize risk awareness, to examine perceptions and challenges concerning protective glove use and selection, and to generate ideas for protective glove use interventions. The most popular gloves among spray painters were thin (4-5 mil) and thick (14 mil) latex. We found that medium to thick (6-8 mil) nitrile were not always perceived as comfortable and were expected to be more expensive than thin (4-5 mil) latex gloves. Of concern is the user's difficulty in distinguishing between nitrile and latex gloves; latex gloves are now sold in different colors including blue, which has traditionally been associated with nitrile gloves. Even though spray painters were familiar with the health hazards related to working with isocyanate paints, most were not always aware that dermal exposure to isocyanates could contribute to the development of occupational asthma. There is a need for more research to identify dermal materials that are protective against sprayed automotive coatings. Automotive spray painters and their employers need to be educated in the selection and use of protective gloves, specifically on attributes such as glove material, color, and thickness.

Assessment of DNA Double Strand Break Repair Activity Using High-throughput and Quantitative Luminescence-based Reporter Assays.

The repair of DNA double strand breaks (DSBs) is crucial for the maintenance of genome stability and cell viability. DSB repair (DSBR) in cells is mediated through several mechanisms: homologous recombination (HR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and single strand annealing (SSA). Cellular assays are essential to measure the proficiency and modulation of these pathways in response to various stimuli. Here, we present a suite of extrachromosomal reporter assays that each measure the reconstitution of a nanoluciferase reporter gene by one of the four major DSBR pathways in cells. Upon transient transfection into cells of interest, repair of pathway-specific reporter substrates can be measured in under 24 h by the detection of Nanoluciferase (NanoLuc) luminescence. These robust assays are quantitative, sensitive, titratable, and amenable to a high-throughput screening format. These properties provide broad applications in DNA repair research and drug discovery, complementing the currently available toolkit of cellular DSBR assays.

Ataxia-Telangiectasia Mutated Loss-of-Function Displays Variant and Tissue-Specific Differences across Tumor Types.

PURPOSE: Mutations in the ATM gene are common in multiple cancers, but clinical studies of therapies targeting ATM-aberrant cancers have yielded mixed results. Refinement of ATM loss of function (LOF) as a predictive biomarker of response is urgently needed. EXPERIMENTAL DESIGN: We present the first disclosure and preclinical development of a novel, selective ATR inhibitor, ART0380, and test its antitumor activity in multiple preclinical cancer models. To refine ATM LOF as a predictive biomarker, we performed a comprehensive pan-cancer analysis of ATM variants in patient tumors and then assessed the ATM variant-to-protein relationship. Finally, we assessed a novel ATM LOF biomarker approach in retrospective clinical data sets of patients treated with platinum-based chemotherapy or ATR inhibition. RESULTS: ART0380 had potent, selective antitumor activity in a range of preclinical cancer models with differing degrees of ATM LOF. Pan-cancer analysis identified 10,609 ATM variants in 8,587 patient tumors. Cancer lineage-specific differences were seen in the prevalence of deleterious (Tier 1) versus unknown/benign (Tier 2) variants, selective pressure for loss of heterozygosity, and concordance between a deleterious variant and ATM loss of protein (LOP). A novel ATM LOF biomarker approach that accounts for variant classification, relationship to ATM LOP, and tissue-specific penetrance significantly enriched for patients who benefited from platinum-based chemotherapy or ATR inhibition. CONCLUSIONS: These data help to better define ATM LOF across tumor types in order to optimize patient selection and improve molecularly targeted therapeutic approaches for patients with ATM LOF cancers.

Beyond the genomics blueprint: the 4th Human Variome Project Meeting, UNESCO, Paris, 2012.

The 4th Biennial Meeting of the Human Variome Project Consortium was held at the headquarters of the United Nations Educational, Scientific and Cultural Organization (UNESCO) in Paris, 11-15 June 2012. The Human Variome Project, a nongovernmental organization and an official partner of UNESCO, enables the routine collection, curation, interpretation, and sharing of information on all human genetic variation. This meeting was attended by more than 180 delegates from 39 countries and continued the theme of addressing issues of implementation in this unique project. The meeting was structured around the four main themes of the Human Variome Project strategic plan, "Project Roadmap 2012-2016": setting normative function, behaving ethically, sharing knowledge, and building capacity. During the meeting, the members held extensive discussions to formulate an action plan in the key areas of the Human Variome Project. The actions agreed on were promulgated at the Project's two Advisory Council and Scientific Advisory Committee postconference meetings.

The Human Variome Project Beijing meeting.

The Human Variome Project Beijing Meeting, a joint meeting of the Human Variome Project Consortium and the Human Variome Project Chinese Node, was held in Beijing, 8th-12th of December, 2011. The aim of the Human Variome Project is to ensure that all information on genetic variation can be collected, curated, interpreted and shared freely and openly. The meeting officially welcomed the Human Variome Project Chinese Node as a partner of the Human Variome Project and focused on those areas where collaborations between China and the global Human Variome Project Consortium are required to develop and extend the coverage of international gene/disease specific databases.

Identification of barriers to the prevention and treatment of heat-related illness in Latino farmworkers using activity-oriented, participatory rural appraisal focus group methods.

BACKGROUND: Heat-related illness (HRI) is an important cause of non-fatal illness and death in farmworkers. We sought to identify potential barriers to HRI prevention and treatment in Latino farmworkers. METHODS: We conducted three semi-structured focus group discussions with 35 Latino farmworkers in the Central Washington, USA area using participatory rural appraisal techniques. Interviews were audio taped and transcribed in Spanish. Three researchers reviewed and coded transcripts and field notes, and investigator triangulation was used to identify relevant themes and quotes. RESULTS: Although the majority of participants in our study reported never receiving formal HRI training, most participants were aware that extreme heat can cause illness and were able to accurately describe HRI symptoms, risk factors, and certain prevention strategies. Four main observations regarding farmworkers' HRI-relevant beliefs and attitudes were identified: 1) farmworkers subscribe to varying degrees to the belief that cooling treatments should be avoided after heat exposure, with some believing that such treatments should be avoided after heat exposure, and others encouraging the use of such treatments; 2) the desire to lose weight may be reflected in behaviors that promote increased sweating; 3) highly caffeinated energy drinks are preferred to increase work efficiency and maintain alertness; and 4) the location of drinking water at work (e.g. next to restrooms) and whether water is clean, but not necessarily chemically-treated, are important considerations in deciding whether to drink the water provided at worksites. CONCLUSIONS: We identified potential barriers to HRI prevention and treatment related to hydration, certain HRI treatments, clothing use, and the desire to lose weight among Latino farmworkers. Strategies to address potential barriers to HRI prevention and treatment in this population may include engineering, administrative, and health education and health promotion strategies at individual, workplace, community, and societal levels. Although farmworkers in our study were able to describe HRI risk factors, reported practices were not necessarily consistent with reported knowledge. Further study of potential knowledge-behavior gaps may uncover opportunities for additional HRI prevention strategies. Farmworkers and employers should be included in the development and evaluation of interventions to prevent HRI.

Small-Molecule Polθ Inhibitors Provide Safe and Effective Tumor Radiosensitization in Preclinical Models.

PURPOSE: DNA polymerase theta (Polθ, encoded by the POLQ gene) is a DNA repair enzyme critical for microhomology mediated end joining (MMEJ). Polθ has limited expression in normal tissues but is frequently overexpressed in cancer cells and, therefore, represents an ideal target for tumor-specific radiosensitization. In this study we evaluate whether targeting Polθ with novel small-molecule inhibitors is a feasible strategy to improve the efficacy of radiotherapy. EXPERIMENTAL DESIGN: We characterized the response to Polθ inhibition in combination with ionizing radiation in different cancer cell models in vitro and in vivo. RESULTS: Here, we show that ART558 and ART899, two novel and specific allosteric inhibitors of the Polθ DNA polymerase domain, potently radiosensitize tumor cells, particularly when combined with fractionated radiation. Importantly, noncancerous cells were not radiosensitized by Polθ inhibition. Mechanistically, we show that the radiosensitization caused by Polθ inhibition is most effective in replicating cells and is due to impaired DNA damage repair. We also show that radiosensitization is still effective under hypoxia, suggesting that these inhibitors may help overcome hypoxia-induced radioresistance. In addition, we describe for the first time ART899 and characterize it as a potent and specific Polθ inhibitor with improved metabolic stability. In vivo, the combination of Polθ inhibition using ART899 with fractionated radiation is well tolerated and results in a significant reduction in tumor growth compared with radiation alone. CONCLUSIONS: These results pave the way for future clinical trials of Polθ inhibitors in combination with radiotherapy.

Human Variome Project country nodes: documenting genetic information within a country.

The Human Variome Project (http://www.humanvariomeproject.org) is an international effort aiming to systematically collect and share information on all human genetic variation. The two main pillars of this effort are gene/disease-specific databases and a network of Human Variome Project Country Nodes. The latter are nationwide efforts to document the genomic variation reported within a specific population. The development and successful operation of the Human Variome Project Country Nodes are of utmost importance to the success of Human Variome Project's aims and goals because they not only allow the genetic burden of disease to be quantified in different countries, but also provide diagnosticians and researchers access to an up-to-date resource that will assist them in their daily clinical practice and biomedical research, respectively. Here, we report the discussions and recommendations that resulted from the inaugural meeting of the International Confederation of Countries Advisory Council, held on 12th December 2011, during the 2011 Human Variome Project Beijing Meeting. We discuss the steps necessary to maximize the impact of the Country Node effort for developing regional and country-specific clinical genetics resources and summarize a few well-coordinated genetic data collection initiatives that would serve as paradigms for similar projects.

The Real-Life Outcome of VACOped Boot in the Management of Diabetic Foot Ulcers.

The total contact cast (TCC) is considered the gold standard treatment to off-load diabetic foot ulcers (DFUs); however, the use of TCC can be limited due to various reasons such as underlying infections, ischemia, and patient's reluctance. Removable cast walkers are used in such cases, and the VACOped boot is one such device. The aim of this study was to analyze the results of the VACOped boot in the treatment of DFUs in real life. Case records of all patients with DFUs treated with a VACOped from 2011 to 2017 were reviewed retrospectively. Eighty-three episodes of ulcerations in 42 subjects were identified, of which 48 (57.8%) healed in a median duration of 17.5 (95% confidence interval = 15-33) weeks with the use of the VACOped and 35 (42.2%) discontinued its use. The median duration of healing with the VACOped of 17.5 weeks appears to be longer, but this cohort included patients with underlying infection and ischemia, which are often excluded in the clinical trials of off-loading. Our data show that the VACOped application is preferred by many patients and seems to be equally effective to other removable cast walkers.

Discovery, Characterization, and Structure-Based Optimization of Small-Molecule In Vitro and In Vivo Probes for Human DNA Polymerase Theta.

Human DNA polymerase theta (Polθ), which is essential for microhomology-mediated DNA double strand break repair, has been proposed as an attractive target for the treatment of BRCA deficient and other DNA repair pathway defective cancers. As previously reported, we recently identified the first selective small molecule Polθ in vitro probe, 22 (ART558), which recapitulates the phenotype of Polθ loss, and in vivo probe, 43 (ART812), which is efficacious in a model of PARP inhibitor resistant TNBC in vivo. Here we describe the discovery, biochemical and biophysical characterization of these probes including small molecule ligand co-crystal structures with Polθ. The crystallographic data provides a basis for understanding the unique mechanism of inhibition of these compounds which is dependent on stabilization of a "closed" enzyme conformation. Additionally, the structural biology platform provided a basis for rational optimization based primarily on reduced ligand conformational flexibility.

Global Globin Network Consensus Paper: Classification and Stratified Roadmaps for Improved Thalassaemia Care and Prevention in 32 Countries.

The Global Globin Network (GGN) is a project-wide initiative of the Human Variome/Global Variome Project (HVP) focusing on haemoglobinopathies to build the capacity for genomic diagnosis, clinical services, and research in low- and middle-income countries. At present, there is no framework to evaluate the improvement of care, treatment, and prevention of thalassaemia and other haemoglobinopathies globally, despite thalassaemia being one of the most common monogenic diseases worldwide. Here, we propose a universally applicable system for evaluating and grouping countries based on qualitative indicators according to the quality of care, treatment, and prevention of haemoglobinopathies. We also apply this system to GGN countries as proof of principle. To this end, qualitative indicators were extracted from the IthaMaps database of the ITHANET portal, which allowed four groups of countries (A, B, C, and D) to be defined based on major qualitative indicators, supported by minor qualitative indicators for countries with limited resource settings and by the overall haemoglobinopathy carrier frequency for the target countries of immigration. The proposed rubrics and accumulative scores will help analyse the performance and improvement of care, treatment, and prevention of haemoglobinopathies in the GGN and beyond. Our proposed criteria complement future data collection from GGN countries to help monitor the quality of services for haemoglobinopathies, provide ongoing estimates for services and epidemiology in GGN countries, and note the contribution of the GGN to a local and global reduction of disease burden.

Predictive validity of a qualitative and quantitative Prechtl's General Movements Assessment at term age: Comparison between preterm infants and term infants with HIE.

AIM: To determine (1) if the General Movement Optimality Score (GMOS) at term age enhances prediction of motor impairment at 12 and 24 months of age in high-risk infants, when compared to a global General Movement Assessment (GMA), and (2) compare predictive validity for two high-risk populations: infants born preterm and infants born at term with hypoxic ischaemic encephalopathy who have received therapeutic hypothermia. METHODS: Fifty-nine extremely preterm or term age infants with hypoxic ischaemic encephalopathy underwent term age GMA. A GMA score of normal or abnormal, and a comparative numerical General Movement Optimality Score (GMOS, total values 5-42) were assigned. Neurology and motor assessment were carried out at age 12 and 24 months using standardised assessments; Alberta Infant Motor Scale, Bayley Scales of Infant and Toddler Development or Ages and Stages Questionnaire. Outcomes were recorded as normal, motor delayed or cerebral palsy. Motor outcome prediction at 12 and 24 months of age was calculated using the GMA and, using ROC analysis, GMOS cut-off scores were determined. RESULTS: At 12 and 24 months global GMA sensitivity for preterms was 80% and 100%, and for Term HIE was 100% at both ages. Specificity values for preterm infants at 12 and 24 months were 68.8% and 60% versus 28.8% and 21.4% for term HIE. Median GMOS scores were lower in the term HIE group than the preterm group in the normal and poor repertoire categories. Optimality cut off scores enhanced specificity, but values remained low. INTERPRETATION: At term age, specificity for identification of infants with later normal motor outcome is low. The GMOS may assist identification of infants with the highest probability of motor impairment, enabling targeted intervention during critical periods for neuroplasticity.

The effect of cannabis policies on treatment outcomes for cannabis use among U.S. adults.

BACKGROUND: Research has explored the impact of various medical cannabis policies on substance use treatment admission in recent years, but we know little about factors related to participants' treatment engagement and outcome. To fill this gap in the existing literature, this study used national data to examine the influence of cannabis policies (decriminalized, medical, and recreational) and referral sources (criminal justice vs. voluntary) on treatment completion and length of stay. METHODS: Data came from the Treatment Episode Data Set-Discharge (2006-2017) on adults 18+ whose primary drug at treatment admission was cannabis. Difference-in-difference analyses using logistic regression examined the effect of cannabis policies on outpatient treatment completion (yes/no; n = 2,192,807) and length of stay (more/fewer than 90 days; n = 1,863,585) in those with a criminal justice or voluntary referral source. RESULTS: Cannabis policy was not associated with treatment completion in either those with a criminal justice or voluntary referral source. Compared to individuals in states where cannabis use was strictly illegal, those in states with a decriminalization policy were less likely to stay in treatment for 91+ days regardless of the referral source. CONCLUSIONS: Cannabis policy appears to have a differential effect on treatment completion versus length of stay, with policy having no impact on successful treatment completion. Specifically, we found that decriminalization policies hinder treatment engagement past 90 days. In this sense, length of stay may be a more useful measure of treatment outcome for research than treatment completion moving forward. Furthermore, our study found that neither medical nor recreational policies affected length of stay or treatment completion, regardless of referral source.

Polθ inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance.

To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance.