An octameric PqiC toroid stabilises the outer-membrane interaction of the PqiABC transport system.

The E. coli Paraquat Inducible (Pqi) Pathway is a putative Gram-negative phospholipid transport system. The pathway comprises three components: an integral inner membrane protein (PqiA), a periplasmic spanning MCE family protein (PqiB) and an outer membrane lipoprotein (PqiC). Interactions between all complex components, including stoichiometry, remain uncharacterised; nevertheless, once assembled into their quaternary complex, the trio of Pqi proteins are anticipated to provide a continuous channel between the inner and outer membranes of diderms. Here, we present X-ray structures of both the native and a truncated, soluble construct of the PqiC lipoprotein, providing insight into its biological assembly, and utilise neutron reflectometry to characterise the nature of the PqiB-PqiC-membrane interaction. Finally, we employ phenotypic complementation assays to probe specific PqiC residues, which imply the interaction between PqiB and PqiC is less intimate than previously anticipated.

A Bayesian multivariate factor analysis model for causal inference using time-series observational data on mixed outcomes.

Assessing the impact of an intervention by using time-series observational data on multiple units and outcomes is a frequent problem in many fields of scientific research. Here, we propose a novel Bayesian multivariate factor analysis model for estimating intervention effects in such settings and develop an efficient Markov chain Monte Carlo algorithm to sample from the high-dimensional and nontractable posterior of interest. The proposed method is one of the few that can simultaneously deal with outcomes of mixed type (continuous, binomial, count), increase efficiency in the estimates of the causal effects by jointly modeling multiple outcomes affected by the intervention, and easily provide uncertainty quantification for all causal estimands of interest. Using the proposed approach, we evaluate the impact that Local Tracing Partnerships had on the effectiveness of England's Test and Trace programme for COVID-19.

Comparative transmission of SARS-CoV-2 Omicron (B.1.1.529) and Delta (B.1.617.2) variants and the impact of vaccination: national cohort study, England.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant (B.1.1.529) rapidly replaced Delta (B.1.617.2) to become dominant in England. Our study assessed differences in transmission between Omicron and Delta using two independent data sources and methods. Omicron and Delta cases were identified through genomic sequencing, genotyping and S-gene target failure in England from 5-11 December 2021. Secondary attack rates for named contacts were calculated in household and non-household settings using contact tracing data, while household clustering was identified using national surveillance data. Logistic regression models were applied to control for factors associated with transmission for both methods. For contact tracing data, higher secondary attack rates for Omicron vs. Delta were identified in households (15.0% vs. 10.8%) and non-households (8.2% vs. 3.7%). For both variants, in household settings, onward transmission was reduced from cases and named contacts who had three doses of vaccine compared to two, but this effect was less pronounced for Omicron (adjusted risk ratio, aRR 0.78 and 0.88) than Delta (aRR 0.62 and 0.68). In non-household settings, a similar reduction was observed only in contacts who had three doses vs. two doses for both Delta (aRR 0.51) and Omicron (aRR 0.76). For national surveillance data, the risk of household clustering, was increased 3.5-fold for Omicron compared to Delta (aRR 3.54 (3.29-3.81)). Our study identified increased risk of onward transmission of Omicron, consistent with its successful global displacement of Delta. We identified a reduced effectiveness of vaccination in lowering risk of transmission, a likely contributor for the rapid propagation of Omicron.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infectivity by Viral Load, S Gene Variants and Demographic Factors, and the Utility of Lateral Flow Devices to Prevent Transmission.

BACKGROUND: How severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectivity varies with viral load is incompletely understood. Whether rapid point-of-care antigen lateral flow devices (LFDs) detect most potential transmission sources despite imperfect clinical sensitivity is unknown. METHODS: We combined SARS-CoV-2 testing and contact tracing data from England between 1 September 2020 and 28 February 2021. We used multivariable logistic regression to investigate relationships between polymerase chain reaction (PCR)-confirmed infection in contacts of community-diagnosed cases and index case viral load, S gene target failure (proxy for B.1.1.7 infection), demographics, SARS-CoV-2 incidence, social deprivation, and contact event type. We used LFD performance to simulate the proportion of cases with a PCR-positive contact expected to be detected using 1 of 4 LFDs. RESULTS: In total, 231 498/2 474 066 (9%) contacts of 1 064 004 index cases tested PCR-positive. PCR-positive results in contacts independently increased with higher case viral loads (lower cycle threshold [Ct] values), for example, 11.7% (95% confidence interval [CI] 11.5-12.0%) at Ct = 15 and 4.5% (95% CI 4.4-4.6%) at Ct = 30. B.1.1.7 infection increased PCR-positive results by ~50%, (eg, 1.55-fold, 95% CI 1.49-1.61, at Ct = 20). PCR-positive results were most common in household contacts (at Ct = 20.1, 8.7% [95% CI 8.6-8.9%]), followed by household visitors (7.1% [95% CI 6.8-7.3%]), contacts at events/activities (5.2% [95% CI 4.9-5.4%]), work/education (4.6% [95% CI 4.4-4.8%]), and least common after outdoor contact (2.9% [95% CI 2.3-3.8%]). Contacts of children were the least likely to test positive, particularly following contact outdoors or at work/education. The most and least sensitive LFDs would detect 89.5% (95% CI 89.4-89.6%) and 83.0% (95% CI 82.8-83.1%) of cases with PCR-positive contacts, respectively. CONCLUSIONS: SARS-CoV-2 infectivity varies by case viral load, contact event type, and age. Those with high viral loads are the most infectious. B.1.1.7 increased transmission by ~50%. The best performing LFDs detect most infectious cases.

Overall Survival with Ribociclib plus Endocrine Therapy in Breast Cancer.

BACKGROUND: An earlier analysis of this phase 3 trial showed that the addition of a cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor to endocrine therapy provided a greater benefit with regard to progression-free survival than endocrine therapy alone in premenopausal or perimenopausal patients with advanced hormone-receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancer. Here we report the results of a protocol-specified interim analysis of the key secondary end point of overall survival. METHODS: We randomly assigned patients to receive either ribociclib or placebo in addition to endocrine therapy (goserelin and either a nonsteroidal aromatase inhibitor or tamoxifen). Overall survival was evaluated with the use of a stratified log-rank test and summarized with the use of Kaplan-Meier methods. RESULTS: A total of 672 patients were included in the intention-to-treat population. There were 83 deaths among 335 patients (24.8%) in the ribociclib group and 109 deaths among 337 patients (32.3%) in the placebo group. The addition of ribociclib to endocrine therapy resulted in significantly longer overall survival than endocrine therapy alone. The estimated overall survival at 42 months was 70.2% (95% confidence interval [CI], 63.5 to 76.0) in the ribociclib group and 46.0% (95% CI, 32.0 to 58.9) in the placebo group (hazard ratio for death, 0.71; 95% CI, 0.54 to 0.95; P = 0.00973 by log-rank test). The survival benefit seen in the subgroup of 495 patients who received an aromatase inhibitor was consistent with that in the overall intention-to-treat population (hazard ratio for death, 0.70; 95% CI, 0.50 to 0.98). The percentage of patients who received subsequent antineoplastic therapy was balanced between the groups (68.9% in the ribociclib group and 73.2% in the placebo group). The time from randomization to disease progression during receipt of second-line therapy or to death was also longer in the ribociclib group than in the placebo group (hazard ratio for disease progression or death, 0.69; 95% CI, 0.55 to 0.87). CONCLUSIONS: This trial showed significantly longer overall survival with a CDK4/6 inhibitor plus endocrine therapy than with endocrine therapy alone among patients with advanced hormone-receptor-positive, HER2-negative breast cancer. No new concerns regarding toxic effects emerged with longer follow-up. (Funded by Novartis; MONALEESA-7 ClinicalTrials.gov number, NCT02278120.).

ATM kinase inhibitor AZD0156 in combination with irinotecan and 5-fluorouracil in preclinical models of colorectal cancer.

BACKGROUND: AZD0156 is an oral inhibitor of ATM, a serine threonine kinase that plays a key role in DNA damage response (DDR) associated with double-strand breaks. Topoisomerase-I inhibitor irinotecan is used clinically to treat colorectal cancer (CRC), often in combination with 5-fluorouracil (5FU). AZD0156 in combination with irinotecan and 5FU was evaluated in preclinical models of CRC to determine whether low doses of AZD0156 enhance the cytotoxicity of irinotecan in chemotherapy regimens used in the clinic. METHODS: Anti-proliferative effects of single-agent AZD0156, the active metabolite of irinotecan (SN38), and combination therapy were evaluated in 12 CRC cell lines. Additional assessment with clonogenic assay, cell cycle analysis, and immunoblotting were performed in 4 selected cell lines. Four colorectal cancer patient derived xenograft (PDX) models were treated with AZD0156, irinotecan, or 5FU alone and in combination for assessment of tumor growth inhibition (TGI). Immunofluorescence was performed on tumor tissues. The DDR mutation profile was compared across in vitro and in vivo models. RESULTS: Enhanced effects on cellular proliferation and regrowth were observed with the combination of AZD0156 and SN38 in select models. In cell cycle analysis of these models, increased G2/M arrest was observed with combination treatment over either single agent. Immunoblotting results suggest an increase in DDR associated with irinotecan therapy, with a reduced effect noted when combined with AZD0156, which is more pronounced in some models. Increased TGI was observed with the combination of AZD0156 and irinotecan as compared to single-agent therapy in some PDX models. The DDR mutation profile was variable across models. CONCLUSIONS: AZD0156 and irinotecan provide a rational and active combination in preclinical colorectal cancer models. Variability across in vivo and in vitro results may be related to the variable DDR mutation profiles of the models evaluated. Further understanding of the implications of individual DDR mutation profiles may help better identify patients more likely to benefit from treatment with the combination of AZD0156 and irinotecan in the clinical setting.

Comparative symptomatology of infection with SARS-CoV-2 variants Omicron (B.1.1.529) and Delta (B.1.617.2) from routine contact tracing data in England.

Symptoms are currently used as testing indicators for SARS-CoV-2 in England. In this study, we analysed national contact tracing data for England (NHS Test and Trace) for the period 1 December to 28 December 2021 to explore symptom differences between the variants, Delta and Omicron. We found that at least one of the symptoms currently used as indicators (fever, cough and loss of smell and taste) were reported in 61.5% of Omicron cases and 72.2% in Delta cases, suggesting that these symptoms are less predictive of Omicron infections. Nearly 40% of Omicron infections did not report any of the three key indicative symptoms, reinforcing the importance of the entire spectrum of symptoms for targeted testing. After adjusting for potential confounding factors, fever and cough were more commonly associated with Omicron infections compared to Delta, showing the importance of considering age and vaccination status when assessing symptom profiles. Sore throat was also more commonly reported in Omicron infections, and loss of smell and taste more commonly reported in Delta infections. Our study shows the value of continued monitoring of symptoms associated with SARS-CoV-2, as changes may influence the effectiveness of testing policy and case ascertainment approaches.

Workplace exposures associated with COVID-19: evidence from a case-control study with multiple sampling periods in England, August-October 2020.

We investigated risk factors associated with COVID-19 by conducting a retrospective, frequency-matched case-control study, with three sampling periods (August-October 2020). We compared cases completing routine contact tracing to asymptomatic population controls. Multivariable analyses estimated adjusted odds ratios (aORs) for non-household community settings. Meta-analyses using random effects provided pooled odds ratios (pORs). Working in healthcare (pOR 2.87; aORs 2.72, 2.81, 3.08, for study periods 1-3 respectively), social care (pOR 4.15; aORs 2.46, 5.06, 5.41, for study periods 1-3 respectively) or hospitality (pOR 2.36; aORs 2.01, 2.54, 2.63, for study periods 1-3 respectively) were associated with increased odds of being a COVID-19 case. Additionally, working in bars, pubs and restaurants, warehouse settings, construction, educational settings were significantly associated. While definitively determining where transmission occurs is impossible, we provide evidence that in certain sectors, the impact of mitigation measures may only be partial and reinforcement of measures should be considered in these settings.

Outbreak of STEC O157:H7 linked to a milk pasteurisation failure at a dairy farm in England, 2019.

In November 2019, an outbreak of Shiga toxin-producing Escherichia coli O157:H7 was detected in South Yorkshire, England. Initial investigations established consumption of milk from a local dairy as a common exposure. A sample of pasteurised milk tested the next day failed the phosphatase test, indicating contamination of the pasteurised milk by unpasteurised (raw) milk. The dairy owner agreed to immediately cease production and initiate a recall. Inspection of the pasteuriser revealed a damaged seal on the flow divert valve. Ultimately, there were 21 confirmed cases linked to the outbreak, of which 11 (52%) were female, and 12/21 (57%) were either <15 or >65 years of age. Twelve (57%) patients were treated in hospital, and three cases developed haemolytic uraemic syndrome. Although the outbreak strain was not detected in the milk samples, it was detected in faecal samples from the cattle on the farm. Outbreaks of gastrointestinal disease caused by milk pasteurisation failures are rare in the UK. However, such outbreaks are a major public health concern as, unlike unpasteurised milk, pasteurised milk is marketed as 'safe to drink' and sold to a larger, and more dispersed, population. The rapid, co-ordinated multi-agency investigation initiated in response to this outbreak undoubtedly prevented further cases.

Predicting stress and mental wellbeing among doctoral researchers.

BACKGROUND: Although mental health in higher education is increasingly recognised as a public health issue, postgraduate research students are often overlooked. Recent studies indicate a high prevalence of mental distress in this population. AIMS: This study assesses the experience of doctoral researchers and identifies factors influencing mental wellbeing and perceived stress. METHODS: A cross-sectional study examined how key demographic, individual and contextual factors related to stress and mental wellbeing in a sample of 431 doctoral researchers in the United Kingdom. RESULTS: Respondents gave positive reports about their supervisory relationship and identified feeling confidently prepared for their work. Family support, good general health, sleep and low levels of self-depreciation predicted stronger mental wellbeing and lower levels of stress. Students who were confident about their future career and felt well prepared for their studies were less stressed and those who were achievement orientated had better mental wellbeing. CONCLUSIONS: Focused attention on exploring career options and building confidence may help reduce stress among doctoral researchers. Taking steps to tackle the imposter phenomenon may help further. These could include addressing fear of failure, improving confidence in research ability and clarifying the role of doctoral researchers within the wider academic community.

Understanding how the university curriculum impacts student wellbeing: a qualitative study.

There is increasing pressure within universities to address student mental health. From a whole university or settings-based perspective, this could include curriculum-embedded approaches. There is little research about how this should work or what approaches might be most effective. Semi -structured interviews were conducted with fifty-seven undergraduate students from five disciplines (Psychology, English studies, Nursing, International Politics, and War Studies) to understand students' perspectives. Students reflected on wellbeing module content and, more broadly, on curriculum processes (teaching, pedagogy, assessment) within their degree. Reflexive thematic analysis was applied to transcripts, generating three themes: embedding wellbeing in the curriculum; assessment, challenge, and academic support; and social connection and interaction. The findings provide evidence for teaching, pedagogy, and assessment practices supporting higher education student wellbeing. These align with recommended good teaching practices, such as considering appropriate assessment methods followed by effective feedback. Students saw the benefits of being academically challenged if scaffolded appropriately. Strong peer connection, teacher-student interaction, and communication were crucial to learning and wellbeing. These findings provide implications for future curriculum design that can support learning and wellbeing.

The impact of clinical simulation on the development of advanced practice skills.

This article considers the findings of a qualitative research study into the impact of simulation on the development of advanced clinical practitioners' skills and knowledge. STUDY AIM: To explore simulated learning through the eyes of trainee and trained advanced clinical practitioners (ACPs) and consider its potential in supporting their development. METHOD: This qualitative research study explored the experiences of trained and trainee ACP volunteers undertaking a structured simulated event provided by a local acute hospital trust simulation team. A questionnaire (n=10) and a focus group (n=4) acted as the data gathering tools. RESULTS: Although simulation can be daunting for the participants, the overwhelming outcome was positive. Participants stated that they gained confidence and suggested that simulation offered a safe place to practise the challenging scenarios that occur in the clinical environment. Additionally, they emphasised that simulation provided a place to network and receive constructive feedback that was non-judgemental, and which helped them to develop clinical knowledge and appreciate their limitations. CONCLUSION: Simulation is a valuable addition to the education and development of ACPs. It should be considered for inclusion within the educational curriculum as a supplement to theoretical knowledge and to the structured clinical supervision provided within the clinical environment.

Surface-tethered planar membranes containing the ß-barrel assembly machinery: a platform for investigating bacterial outer membrane protein folding.

The outer membrane of Gram-negative bacteria presents a robust physicochemical barrier protecting the cell from both the natural environment and acting as the first line of defense against antimicrobial materials. The proteins situated within the outer membrane are responsible for a range of biological functions including controlling influx and efflux. These outer membrane proteins (OMPs) are ultimately inserted and folded within the membrane by the ß-barrel assembly machine (Bam) complex. The precise mechanism by which the Bam complex folds and inserts OMPs remains unclear. Here, we have developed a platform for investigating Bam-mediated OMP insertion. By derivatizing a gold surface with a copper-chelating self-assembled monolayer, we were able to assemble a planar system containing the complete Bam complex reconstituted within a phospholipid bilayer. Structural characterization of this interfacial protein-tethered bilayer by polarized neutron reflectometry revealed distinct regions consistent with known high-resolution models of the Bam complex. Additionally, by monitoring changes of mass associated with OMP insertion by quartz crystal microbalance with dissipation monitoring, we were able to demonstrate the functionality of this system by inserting two diverse OMPs within the membrane, pertactin, and OmpT. This platform has promising application in investigating the mechanism of Bam-mediated OMP insertion, in addition to OMP function and activity within a phospholipid bilayer environment.

A Meta-Analytical Assessment of the Aggregation Parameter of the Binary Power Law for Characterizing Spatial Heterogeneity of Plant Disease Incidence.

The binary power law (BPL) is often used to characterize spatial heterogeneity of disease incidence. A hierarchical mixed model, coupled with multiple imputation to randomly generate any missing standard errors, was used to conduct a meta-analysis of >200 published values of the estimated aggregation (b) parameter of the BPL. Approximately 50% of estimated b values ranged from 1.1 to 1.3. Moderator variable analysis showed that the number of individuals per sampling unit (n) had a strong positive effect on b, with a linear relation between estimated b and ln(n). Estimated expected value of b for the population of published regressions at a reference n of 15 was 1.22. The increase in the variance due to the imputations was only 0.03, and the efficiency exceeded 0.98. Results were confirmed with an alternative mixed model that considered a range of possible within-trial correlations of the estimated b values and with a random-coefficient mixed model fitted to the subset of the data. Cropping system, dispersal mode, and pathogen type all had significant effects on b, with annuals having larger expected value than woody perennials, soilborne and rain-splashed dispersed pathogens having the largest expected values for dispersal mode, and bacteria and oomycetes having the largest expected values for pathogen type. However, there was considerable variation within each of the levels of the moderators, and the differences of expected values from smallest to largest were small, ≤0.16. Results are discussed in relation to previously published findings from stochastic simulations.

Functional characterization of the mucus barrier on the Xenopus tropicalis skin surface.

Mucosal surfaces represent critical routes for entry and exit of pathogens. As such, animals have evolved strategies to combat infection at these sites, in particular the production of mucus to prevent attachment and to promote subsequent movement of the mucus/microbe away from the underlying epithelial surface. Using biochemical, biophysical, and infection studies, we have investigated the host protective properties of the skin mucus barrier of the Xenopus tropicalis tadpole. Specifically, we have characterized the major structural component of the barrier and shown that it is a mucin glycoprotein (Otogelin-like or Otogl) with similar sequence, domain organization, and structural properties to human gel-forming mucins. This mucin forms the structural basis of a surface barrier (∼6 µm thick), which is depleted through knockdown of Otogl. Crucially, Otogl knockdown leads to susceptibility to infection by the opportunistic pathogen Aeromonas hydrophila To more accurately reflect its structure, tissue localization, and function, we have renamed Otogl as Xenopus Skin Mucin, or MucXS. Our findings characterize an accessible and tractable model system to define mucus barrier function and host-microbe interactions.

The epidemiology and management of clusters of invasive meningococcal disease in England, 2010-15.

BACKGROUND: Guidance for public health management of invasive meningococcal disease (IMD) in in England recommends the use of antibiotic chemoprophylaxis and vaccination. We summarized clinical and epidemiological data collected during routine management of IMD clusters in England. METHODS: Data on epidemiology and operational decisions for public health management were reviewed for clusters between April 2010 and December 2015. RESULTS: Clusters were generally 2-3 cases (53/58; 91%) within a single age band <18-years. Nurseries (n = 20, 34%), households/social networks (n = 14, 24%) and schools (n = 10, 17%) were the commonest settings. Chemoprophylaxis alone was used in 36 (58%) clusters, including most serogroup B clusters (31/41; 76%). Chemoprophylaxis and vaccination was used in a further 20 (32%) clusters. Vaccine was delivered promptly (<7 days). Four clusters had cases with onset post-chemoprophylaxis; no clusters recorded cases with onset post-vaccination. No pattern was observed between interventions and setting/population at risk, and interventions were consistent with national guidance. Challenges to management included logistical issues related to intervention delivery. CONCLUSIONS: Public health management of IMD clusters presents challenges in decision-making and implementation of interventions. Nonetheless, few cases were observed following intervention. Responses were consistent with national guidance. A systematic data collection tool should be developed to support future evaluation.

On the Binormal Predictive Receiver Operating Characteristic Curve for the Joint Assessment of Positive and Negative Predictive Values.

The predictive receiver operating characteristic (PROC) curve is a diagrammatic format with application in the statistical evaluation of probabilistic disease forecasts. The PROC curve differs from the more well-known receiver operating characteristic (ROC) curve in that it provides a basis for evaluation using metrics defined conditionally on the outcome of the forecast rather than metrics defined conditionally on the actual disease status. Starting from the binormal ROC curve formulation, an overview of some previously published binormal PROC curves is presented in order to place the PROC curve in the context of other methods used in statistical evaluation of probabilistic disease forecasts based on the analysis of predictive values; in particular, the index of separation (PSEP) and the leaf plot. An information theoretic perspective on evaluation is also outlined. Five straightforward recommendations are made with a view to aiding understanding and interpretation of the sometimes-complex patterns generated by PROC curve analysis. The PROC curve and related analyses augment the perspective provided by traditional ROC curve analysis. Here, the binormal ROC model provides the exemplar for investigation of the PROC curve, but potential application extends to analysis based on other distributional models as well as to empirical analysis.

Information Graphs Incorporating Predictive Values of Disease Forecasts.

Diagrammatic formats are useful for summarizing the processes of evaluation and comparison of forecasts in plant pathology and other disciplines where decisions about interventions for the purpose of disease management are often based on a proxy risk variable. We describe a new diagrammatic format for disease forecasts with two categories of actual status and two categories of forecast. The format displays relative entropies, functions of the predictive values that characterize expected information provided by disease forecasts. The new format arises from a consideration of earlier formats with underlying information properties that were previously unexploited. The new diagrammatic format requires no additional data for calculation beyond those used for the calculation of a receiver operating characteristic (ROC) curve. While an ROC curve characterizes a forecast in terms of sensitivity and specificity, the new format described here characterizes a forecast in terms of relative entropies based on predictive values. Thus it is complementary to ROC methodology in its application to the evaluation and comparison of forecasts.

Mutual Information as a Performance Measure for Binary Predictors Characterized by Both ROC Curve and PROC Curve Analysis.

The predictive receiver operating characteristic (PROC) curve differs from the more well-known receiver operating characteristic (ROC) curve in that it provides a basis for the evaluation of binary diagnostic tests using metrics defined conditionally on the outcome of the test rather than metrics defined conditionally on the actual disease status. Application of PROC curve analysis may be hindered by the complex graphical patterns that are sometimes generated. Here we present an information theoretic analysis that allows concurrent evaluation of PROC curves and ROC curves together in a simple graphical format. The analysis is based on the observation that mutual information may be viewed both as a function of ROC curve summary statistics (sensitivity and specificity) and prevalence, and as a function of predictive values and prevalence. Mutual information calculated from a 2 × 2 prediction-realization table for a specified risk score threshold on an ROC curve is the same as the mutual information calculated at the same risk score threshold on a corresponding PROC curve. Thus, for a given value of prevalence, the risk score threshold that maximizes mutual information is the same on both the ROC curve and the corresponding PROC curve. Phytopathologists and clinicians who have previously relied solely on ROC curve summary statistics when formulating risk thresholds for application in practical agricultural or clinical decision-making contexts are thus presented with a methodology that brings predictive values within the scope of that formulation.

Preclinical Evaluation of AZ12601011 and AZ12799734, Inhibitors of Transforming Growth Factor ß Superfamily Type 1 Receptors.

The transforming growth factor ß (TGFß) superfamily includes TGFß, activins, inhibins, and bone morphogenetic proteins (BMPs). These extracellular ligands have essential roles in normal tissue homeostasis by coordinately regulating cell proliferation, differentiation, and migration. Aberrant signaling of superfamily members, however, is associated with fibrosis as well as tumorigenesis, cancer progression, metastasis, and drug-resistance mechanisms in a variety of cancer subtypes. Given their involvement in human disease, the identification of novel selective inhibitors of TGFß superfamily receptors is an attractive therapeutic approach. Seven mammalian type 1 receptors have been identified that have context-specific roles depending on the ligand and the complex formation with the type 2 receptor. Here, we characterize the biologic effects of two transforming growth factor ß receptor 1 (TGFBR1) kinase inhibitors designed to target TGFß signaling. AZ12601011 [2-(2-pyridinyl)-4-(1H-pyrrolo[3,2-c]pyridin-1-yl)-6,7-dihydro-5H-cyclopenta[d]pyrimidine]; structure previously undisclosed] and AZ12799734 [4-({4-[(2,6-dimethyl-3-pyridinyl)oxy]-2-pyridinyl}amino)benzenesulfonamide] (IC50 = 18 and 47 nM, respectively) were more effective inhibitors of TGFß-induced reporter activity than SB-431542 [4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide] (IC50 = 84 nM) and LY2157299 [4-[2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]quinoline-6-carboxamide monohydrate]] (galunisertib) (IC50 = 380 nM). AZ12601011 inhibited phosphorylation of SMAD2 via the type 1 receptors activin A receptor type 1B (ALK4), TGFBR1, and activin A receptor type 1C (ALK7). AZ12799734, however, is a pan TGF/BMP inhibitor, inhibiting receptor-mediated phosphorylation of SMAD1 by activin A receptor type 1L, bone morphogenetic protein receptor type 1A, and bone morphogenetic protein receptor type 1B and phosphorylation of SMAD2 by ALK4, TGFBR1, and ALK7. AZ12601011 was highly effective at inhibiting basal and TGFß-induced migration of HaCaT keratinocytes and, furthermore, inhibited tumor growth and metastasis to the lungs in a 4T1 syngeneic orthotopic mammary tumor model. These inhibitors provide new reagents for investigating in vitro and in vivo pathogenic processes and the contribution of TGFß- and BMP-regulated signaling pathways to disease states.