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.

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.

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.

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.

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.

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.

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.

"Can I Live": Black American Adolescent Boys' Reports of Police Abuse and the Role of Religiosity on Mental Health.

State sanctioned violence aimed at Black individuals and communities is an issue that has pervaded American history and society since before the establishment of the United States. For Black males, anticipating and preparing for involuntary police contact, unfortunately, is an inevitable part of life. The purpose of this study is to examine the impact of reports of police abuse on mental health and perceived racial out-group perceptions and the protective role of religiosity among a nationally representative sample of Black American adolescent boys (Mage = 14.98). Linear multiple regression was used to determine the interactive effects of subjective religiosity and reported police abuse on Black American adolescent boys. Higher reports of subjective religiosity were associated with lower depressive symptomatology. Reports of police abuse were associated with lower public regard beliefs (belief that society views Black Americans less favorably). Results highlight the impact experiencing police abuse has on Black adolescent boys and we conclude with implications, areas for future research and intervention points.

Fluoride occurrence in the lower East African Rift System, Southern Malawi.

Countries located on the East African Rift System (EARS) are vulnerable to fluoride in their groundwater; a vulnerability for the developing country of Malawi at the southern rift periphery that is not well characterised. Groundwater fluoride occurrence in Malawi is documented here to better understand and manage fluoride risks posed. Available literature and Gov't of Malawi archive fluoride data spanning some fifty years have been collated and augmented by our own 2016-18 surveys of groundwater quality in Southern Malawi, targeting deep-sourced springs. In total, fluoride data for 1365 borehole, spring and hot spring samples were assembled. Statistically, 83% of samples were below the 1.5 mg/l WHO limit, concentrations in the 1.5-6 mg/l range between former (pre-1993) and current WHO guidelines at 14%, and those with fluoride above the current Malawi (former WHO) 6 mg/l guideline, at 3%. A lower occurrence than in other zones of the EARS, but indicative of a need for a Malawi Gov't management policy revision and associated management strategies endorsed by several documented incidences of dental fluorosis in proximity to high fluoride groundwater. Increased fluoride is related to increased groundwater temperatures signifying the importance of geothermal groundwater provenance. Temperature data may indeed be used as a proxy indicator of fluoride risk; samples with a temperature >32 °C, contained >6 mg/l fluoride. Structural geological controls appear to allow deep geothermal groundwaters to come to the near surface, as evidenced by increased fluoride in springs and boreholes close to faulted areas. Hydrochemical evaluation shows that fluoride concentrations are influenced by fluorite equilibration and sensitivity to calcium and pH. Recommendations are made to further document the occurrence of fluoride and enhance management of risks due to fluoride in drinking water in Malawi. With fluoride as a key indicator within Sustainable Development Goal number 6, the current Malawi standard and waters with concentration between 1.5 and 6 mg/l will come under increased scrutiny and pose a key challenge to assessment and management efforts.

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.

Ketonuria Is Associated with Changes to the Abundance of Roseburia in the Gut Microbiota of Overweight and Obese Women at 16 Weeks Gestation: A Cross-Sectional Observational Study.

The gut microbiome in pregnancy has been associated with various maternal metabolic and hormonal markers involved in glucose metabolism. Maternal ketones are of particular interest due to the rise in popularity of low-carbohydrate diets. We assessed for differences in the composition of the gut microbiota in pregnant women with and without ketonuria at 16 weeks gestation. Fecal samples were obtained from 11 women with fasting ketonuria and 11 matched controls. The samples were analyzed to assess for differences in gut microbiota composition by 16S rRNA sequencing. Supervised hierarchical clustering analysis showed significantly different beta-diversity between women with and without ketonuria, but no difference in the alpha-diversity. Group comparisons and network analysis showed that ketonuria was associated with an increased abundance of the butyrate-producing genus Roseburia. The bacteria that contributed the most to the differences in the composition of the gut microbiota included Roseburia, Methanobrevibacter, Uncl. RF39, and Dialister in women with ketonuria and Eggerthella, Phascolarctobacterium, Butyricimonas, and Uncl. Coriobacteriaceae in women without ketonuria. This study found that the genus Roseburia is more abundant in the gut microbiota of pregnant women with ketonuria. Roseburia is a butyrate producing bacterium and may increase serum ketone levels.

A Xenopus oocyte model system to study action potentials.

Action potentials (APs) are the functional units of fast electrical signaling in excitable cells. The upstroke and downstroke of an AP is generated by the competing and asynchronous action of Na+- and K+-selective voltage-gated conductances. Although a mixture of voltage-gated channels has been long recognized to contribute to the generation and temporal characteristics of the AP, understanding how each of these proteins function and are regulated during electrical signaling remains the subject of intense research. AP properties vary among different cellular types because of the expression diversity, subcellular location, and modulation of ion channels. These complexities, in addition to the functional coupling of these proteins by membrane potential, make it challenging to understand the roles of different channels in initiating and "temporally shaping" the AP. Here, to address this problem, we focus our efforts on finding conditions that allow reliable AP recordings from Xenopus laevis oocytes coexpressing Na+ and K+ channels. As a proof of principle, we show how the expression of a variety of K+ channel subtypes can modulate excitability in this minimal model system. This approach raises the prospect of studies on the modulation of APs by pharmacological or biological means with a controlled background of Na+ and K+ channel expression.

Prevalence of maternal urinary ketones in pregnancy in overweight and obese women.

BACKGROUND: Ketonuria may be associated with adverse fetal outcomes. This study aimed to determine the prevalence of ketonuria at three time points in pregnancy and to assess whether ketonuria correlates with a clinical indication for performing a urine test. METHODS: Women had fasting urinary ketone levels measured at 16 and 28 weeks gestation and random ketone levels measured close to 36 weeks gestation. All ketone levels in the third trimester were recorded along with the clinical indication for the test. RESULTS: One hundred and eighty-seven women were included in the study. Twenty-two per cent of women had ketonuria at either 16 or 28 weeks gestation and 8% at 36 weeks gestation. Ketonuria was significantly more likely if a test was performed for a clinical indication (p = 0.0002). CONCLUSION: Ketonuria in pregnancy is common affecting at least one in five women. Ketonuria is more common in women who have a clinical indication for performing a urine test.

Increasing the involvement of diverse populations in genomics-based health care-lessons from haemoglobinopathies.

Integrating genomic medicine into health care delivery poses significant challenges to health professionals. To draw clinical benefit from genomic information, there is a need to build an evidence-based relationship between genotype and the physical expression of that genomic information. The work presented here uses preliminary work in the field of haemoglobinopathies to address two important challenges: to ensure that health care professionals in low- and middle-income countries are actively involved in the processes that will support genomic medicine, and that equity and diversity concerns are met so that clinical services can have relevance across all population and sub-population groups. Haemoglobinopathies provide an opportunity for gaining a better understanding of how long-standing genetic knowledge can be leveraged to determine if genomic-based services can be beneficial in low-resource settings. The Global Globin 2020 Challenge (GG2020) is an international initiative that uses haemoglobinopathies as an entry point to achieving growth in the quality and quantity of curated inputs into internationally recognised databases, harmonising the sharing of variant information within and between countries for better health care delivery and ensuring that storing, curation and sharing of variant information become an integral part of health care. Early findings from GG2020 indicate that paying attention to population diversity is an integral part of prevention and control of haemoglobinopathies.

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.