Stromal Microenvironment Shapes the Intratumoral Architecture of Pancreatic Cancer.

Single-cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single-cell phenotypes have not been well defined. Combining single-cell RNA and protein analytics in studying the role of stromal cancer-associated fibroblasts (CAFs) in modulating heterogeneity in pancreatic cancer (pancreatic ductal adenocarcinoma [PDAC]) model systems, we have identified significant single-cell population shifts toward invasive epithelial-to-mesenchymal transition (EMT) and proliferative (PRO) phenotypes linked with mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling. Using high-content digital imaging of RNA in situ hybridization in 195 PDAC tumors, we quantified these EMT and PRO subpopulations in 319,626 individual cancer cells that can be classified within the context of distinct tumor gland "units." Tumor gland typing provided an additional layer of intratumoral heterogeneity that was associated with differences in stromal abundance and clinical outcomes. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.

Consent document translation expense hinders inclusive clinical trial enrolment.

Patients from historically under-represented racial and ethnic groups are enrolled in cancer clinical trials at disproportionately low rates in the USA1-3. As these patients often have limited English proficiency4-7, we hypothesized that one barrier to their inclusion is the cost to investigators of translating consent documents. To test this hypothesis, we evaluated more than 12,000 consent events at a large cancer centre and assessed whether patients requiring translated consent documents would sign consent documents less frequently in studies lacking industry sponsorship (for which the principal investigator pays the translation costs) than for industry-sponsored studies (for which the translation costs are covered by the sponsor). Here we show that the proportion of consent events for patients with limited English proficiency in studies not sponsored by industry was approximately half of that seen in industry-sponsored studies. We also show that among those signing consent documents, the proportion of consent documents translated into the patient's primary language in studies without industry sponsorship was approximately half of that seen in industry-sponsored studies. The results suggest that the cost of consent document translation in trials not sponsored by industry could be a potentially modifiable barrier to the inclusion of patients with limited English proficiency.

Cell morphology best predicts tumorigenicity and metastasis in vivo across multiple TNBC cell lines of different metastatic potential.

BACKGROUND: Metastasis is the leading cause of death in breast cancer patients. For metastasis to occur, tumor cells must invade locally, intravasate, and colonize distant tissues and organs, all steps that require tumor cell migration. The majority of studies on invasion and metastasis rely on human breast cancer cell lines. While it is known that these cells have different properties and abilities for growth and metastasis, the in vitro morphological, proliferative, migratory, and invasive behavior of these cell lines and their correlation to in vivo behavior is poorly understood. Thus, we sought to classify each cell line as poorly or highly metastatic by characterizing tumor growth and metastasis in a murine model of six commonly used human triple-negative breast cancer xenografts, as well as determine which in vitro assays commonly used to study cell motility best predict in vivo metastasis. METHODS: We evaluated the liver and lung metastasis of human TNBC cell lines MDA-MB-231, MDA-MB-468, BT549, Hs578T, BT20, and SUM159 in immunocompromised mice. We characterized each cell line's cell morphology, proliferation, and motility in 2D and 3D to determine the variation in these parameters between cell lines. RESULTS: We identified MDA-MB-231, MDA-MB-468, and BT549 cells as highly tumorigenic and metastatic, Hs578T as poorly tumorigenic and metastatic, BT20 as intermediate tumorigenic with poor metastasis to the lungs but highly metastatic to the livers, and SUM159 as intermediate tumorigenic but poorly metastatic to the lungs and livers. We showed that metrics that characterize cell morphology are the most predictive of tumor growth and metastatic potential to the lungs and liver. Further, we found that no single in vitro motility assay in 2D or 3D significantly correlated with metastasis in vivo. CONCLUSIONS: Our results provide an important resource for the TNBC research community, identifying the metastatic potential of 6 commonly used cell lines. Our findings also support the use of cell morphological analysis to investigate the metastatic potential and emphasize the need for multiple in vitro motility metrics using multiple cell lines to represent the heterogeneity of metastasis in vivo.

Combining avidin with CD63 improves basophil activation test accuracy in classifying peanut allergy.

BACKGROUND: Conventional basophil activation tests (BATs) measure basophil activation by the increased expression of CD63. Previously, fluorophore-labeled avidin, a positively-charged molecule, was found to bind to activated basophils, which tend to expose negatively charged granule constituents during degranulation. This study further compares avidin versus CD63 as basophil activation biomarkers in classifying peanut allergy. METHODS: Seventy subjects with either a peanut allergy (N = 47), a food allergy other than peanut (N = 6), or no food allergy (N = 17) were evaluated. We conducted BATs in response to seven peanut extract (PE) concentrations (0.01-10,000 ng/mL) and four control conditions (no stimulant, anti-IgE, fMLP (N-formylmethionine-leucyl-phenylalanine), and anti-FcεRI). We measured avidin binding and CD63 expression on basophils with flow cytometry. We evaluated logistic regression and XGBoost models for peanut allergy classification and feature identification. RESULTS: Avidin binding was correlated with CD63 expression. Both markers discriminated between subjects with and without a peanut allergy. Although small by percentage, an avidin+ /CD63- cell subset was found in all allergic subjects tested, indicating that the combination of avidin and CD63 could allow a more comprehensive identification of activated basophils. Indeed, we obtained the best classification accuracy (97.8% sensitivity, 96.7% specificity) by combining avidin and CD63 across seven PE doses. Similar accuracy was obtained by combining PE dose of 10,000 ng/mL for avidin and PE doses of 10 and 100 ng/mL for CD63. CONCLUSIONS: Avidin and CD63 are reliable BAT activation markers associated with degranulation. Their combination enhances the identification of activated basophils and improves the classification accuracy of peanut allergy.

Clustering plasma concentration-time curves: applications of unsupervised learning in pharmacogenomics.

Pharmaceutical researchers are continually searching for techniques to improve both drug development processes and patient outcomes. An area of recent interest is the potential for machine learning (ML) applications within pharmacology. One such application not yet given close study is the unsupervised clustering of plasma concentration-time curves, hereafter, pharmacokinetic (PK) curves. In this paper, we present our findings on how to cluster PK curves by their similarity. Specifically, we find clustering to be effective at identifying similar-shaped PK curves and informative for understanding patterns within each cluster of PK curves. Because PK curves are time series data objects, our approach utilizes the extensive body of research related to the clustering of time series data as a starting point. As such, we examine many dissimilarity measures between time series data objects to find those most suitable for PK curves. We identify Euclidean distance as generally most appropriate for clustering PK curves, and we further show that dynamic time warping, Fréchet, and structure-based measures of dissimilarity like correlation may produce unexpected results. As an illustration, we apply these methods in a case study with 250 PK curves used in a previous pharmacogenomic study. Our case study finds that an unsupervised ML clustering with Euclidean distance, without any subject genetic information, is able to independently validate the same conclusions as the reference pharmacogenomic results. To our knowledge, this is the first such demonstration. Further, the case study demonstrates how the clustering of PK curves may generate insights that could be difficult to perceive solely with population level summary statistics of PK metrics.

The relationship between tumour dosimetry, response, and overall survival in patients with unresectable Neuroendocrine Neoplasms (NEN) treated with 177Lu DOTATATE (LuTate).

Peptide Receptor Radionuclide Therapy (PRRT) delivers targeted radiation to Somatostatin Receptor (SSR) expressing Neuroendocrine Neoplasms (NEN). We sought to assess the predictive and prognostic implications of tumour dosimetry with respect to response by 68 Ga DOTATATE (GaTate) PET/CT molecular imaging tumour volume of SSR (MITVSSR) change and RECIST 1.1, and overall survival (OS). METHODS: Patients with gastro-entero-pancreatic (GEP) NEN who received LuTate followed by quantitative SPECT/CT (Q-SPECT/CT) the next day (Jul 2010 to Jan 2019) were retrospectively reviewed. Single time-point (STP) lesional dosimetry was performed for each cycle using population-based pharmacokinetic modelling. MITVSSR and RECIST 1.1 were measured at 3-months post PRRT. RESULTS: Median of 4 PRRT cycles were administered to 90 patients (range 2-5 cycles; mean 27.4 GBq cumulative activity; mean 7.6 GBq per cycle). 68% received at least one cycle with radiosensitising chemotherapy (RSC). RECIST 1.1 partial response was 24%, with 70% stable and 7% progressive disease. Cycle 1 radiation dose in measurable lesions was associated with local response (odds ratio 1.5 per 50 Gy [95% CI: 1.1-2.0], p = 0.002) when adjusted by tumour grade and RSC. Median change in MITVSSR was -63% (interquartile range -84 to -29), with no correlation with radiation dose to the most avid lesion on univariable or multivariant analyses (5.6 per 10 Gy [95% CI: -1.6, 12.8], p = 0.133). OS at 5-years was 68% (95% CI: 56-78%). Neither baseline MITVSSR (hazard ratio 1.1 [95% CI: 1.0, 1.2], p = 0.128) nor change in baseline MITVSSR (hazard ratio 1.0 [95% CI: 1.0, 1.1], p = 0.223) were associated with OS when adjusted by tumour grade and RSC but RSC was (95% CI: 0.2, 0.8, p = 0.012). CONCLUSION: Radiation dose to tumour during PRRT was predictive of radiologic response but not survival. Survival outcomes may relate to other biological factors. There was no evidence that MITVSSR change was associated with OS, but a larger study is needed.

Trends in Involuntary Psychiatric Hospitalization in British Columbia: Descriptive Analysis of Population-Based Linked Administrative Data from 2008 to 2018.

INTRODUCTION: Involuntary psychiatric hospitalization occurs when someone with a serious mental disorder requires treatment without their consent. Trends vary globally, and currently, there is limited data on involuntary hospitalization in Canada. We examine involuntary hospitalization trends in British Columbia, Canada, and describe the social and clinical characteristics of people ages 15 and older who were involuntarily hospitalized between 2008/2009 and 2017/2018. METHOD: We used population-based linked administrative data to examine and compare trends in involuntary and voluntary hospitalizations for mental and substance use disorders. We described patient characteristics (sex/gender, age, health authority, income, urbanity/rurality, and primary diagnosis) and tracked the count of involuntarily hospitalized people over time by diagnosis. Finally, we examined population-based prevalence over time by age and sex/gender. RESULTS: Involuntary hospitalizations among British Columbians ages 15 and older rose from 14,195 to 23,531 (65.7%) between 2008/2009 and 2017/2018. Apprehensions involving police increased from 3,502 to 8,009 (128.7%). Meanwhile, voluntary admissions remained relatively stable, with a minimal increase from 17,651 in 2008/2009 to 17,751 in 2017/2018 (0.5%). The most common diagnosis for involuntary patients in 2017/2018 was mood disorders (25.1%), followed by schizophrenia (22.3%), and substance use disorders (18.8%). From 2008/2009 to 2017/2018, the greatest increase was observed for substance use disorders (139%). Over time, population-based prevalence increased most rapidly among women ages 15-24 (162%) and men ages 15-34 (81%) and 85 and older (106%). CONCLUSION: Findings highlight the need to strengthen the voluntary care system for mental health and substance use, especially for younger adults, and people who use substances. They also signal a need for closer examination of the use of involuntary treatment for substance use disorders, as well as further research exploring forces driving police involvement and its implications.

First-in-Human Clinical Trial to Assess the Safety, Tolerability and Pharmacokinetics of Single Doses of NTM-1633, a Novel Mixture of Monoclonal Antibodies against Botulinum Toxin E.

Botulism is a rare, life-threatening paralytic disease caused by botulinum neurotoxin (BoNT). Available treatments including an equine antitoxin and human immune globulin are given postexposure and challenging to produce and administer. NTM-1633 is an equimolar mixture of 3 human IgG monoclonal antibodies, E1, E2, and E3, targeting BoNT serotype E (BoNT/E). This first-in-human study assessed the safety, tolerability, pharmacokinetics (PK), and immunogenicity of NTM-1633. This double-blind, single-center, placebo-controlled dose escalation study randomized 3 cohorts of healthy volunteers to receive a single intravenous dose of NTM-1633 (0.033, 0.165, or 0.330 mg/kg) or saline placebo. Safety monitoring included physical examinations, clinical laboratory studies, and vital signs. Blood sampling was performed at prespecified time points for PK and immunogenicity analyses. Twenty-four subjects received study product (18 NTM-1633; 6 placebo), and no deaths were reported. An unrelated serious adverse event was reported in a placebo subject. Adverse events in the NTM-1633 groups were generally mild and similar in frequency and severity to the placebo group, and no safety signal was identified. NTM-1633 has a favorable PK profile with a half-life >10 days for the 0.330 mg/kg dose and an approximately linear relationship with respect to maximum concentration and area under the concentration-time curve (AUC0→t). NTM-1633 also demonstrated low immunogenicity. NTM-1633 is well tolerated at the administered doses. The favorable safety, PK, and immunogenicity profile supports further development as a treatment for BoNT/E intoxication and postexposure prophylaxis.

The relationship between outpatient service use and emergency department visits among people treated for mental and substance use disorders: analysis of population-based administrative data in British Columbia, Canada.

BACKGROUND: Research findings on the association between outpatient service use and emergency department (ED) visits for mental and substance use disorders (MSUDs) are mixed and may differ by disorder type. METHODS: We used population-based linked administrative data in British Columbia, Canada to examine associations between outpatient primary care and psychiatry service use and ED visits among people ages 15 and older, comparing across people treated for three disorder categories: common mental disorders (MDs) (depressive, anxiety, and/or post-traumatic stress disorders), serious MDs (schizophrenia spectrum and/or bipolar disorders), and substance use disorders (SUDs) in 2016/7. We used hurdle models to examine the association between outpatient service use and odds of any ED visit for MSUDs as well count of ED visits for MSUDs, stratified by cohort in 2017/8. RESULTS: Having had one or more MSUD-related primary care visit was associated with lower odds of any ED visit among people treated for common MDs and SUDs but not people treated for serious MDs. Continuity of primary care was associated with slightly lower ED use in all cohorts. One or more outpatient psychiatrist visits was associated with lower odds of ED visits among people treated for serious MDs and SUDs, but not among people with common MDs. CONCLUSION: Findings highlight the importance of expanded access to outpatient specialist mental health services, particularly for people with serious MDs and SUDs, and collaborative models that can support primary care providers treating people with MSUDs.

Cerebrospinal Fluid Pterins, Pterin-Dependent Neurotransmitters, and Mortality in Pediatric Cerebral Malaria.

BACKGROUND: Cerebral malaria (CM) pathogenesis remains incompletely understood. Having shown low systemic levels of tetrahydrobiopterin (BH4), an enzymatic cofactor for neurotransmitter synthesis, we hypothesized that BH4 and BH4-dependent neurotransmitters would likewise be low in cerebrospinal fluid (CSF) in CM. METHODS: We prospectively enrolled Tanzanian children with CM and children with nonmalaria central nervous system conditions (NMCs). We measured CSF levels of BH4, neopterin, and BH4-dependent neurotransmitter metabolites, 3-O-methyldopa, homovanillic acid, and 5-hydroxyindoleacetate, and we derived age-adjusted z-scores using published reference ranges. RESULTS: Cerebrospinal fluid BH4 was elevated in CM (n = 49) compared with NMC (n = 51) (z-score 0.75 vs -0.08; P < .001). Neopterin was increased in CM (z-score 4.05 vs 0.09; P < .001), and a cutoff at the upper limit of normal (60 nmol/L) was 100% sensitive for CM. Neurotransmitter metabolite levels were overall preserved. A higher CSF BH4/BH2 ratio was associated with increased odds of survival (odds ratio, 2.94; 95% confidence interval, 1.03-8.33; P = .043). CONCLUSION: Despite low systemic BH4, CSF BH4 was elevated and associated with increased odds of survival in CM. Coma in malaria is not explained by deficiency of BH4-dependent neurotransmitters. Elevated CSF neopterin was 100% sensitive for CM diagnosis and warrants further assessment of its clinical utility for ruling out CM in malaria-endemic areas.

Safety, Tolerability, and Pharmacokinetics of NTM-1632, a Novel Mixture of Three Monoclonal Antibodies against Botulinum Toxin B.

Botulism is a rare, life-threatening paralytic disease caused by Clostridium botulinum neurotoxin (BoNT). Available treatments, including an equine antitoxin and human immune globulin, are given postexposure and challenging to produce and administer. NTM-1632 is an equimolar mixture of 3 human IgG monoclonal antibodies, B1, B2, and B3, targeting BoNT serotype B (BoNT/B). This first-in-human study assessed the safety, tolerability, pharmacokinetics (PK), and immunogenicity of NTM-1632. This double-blind, single-center, placebo-controlled dose escalation study randomized 3 cohorts of healthy volunteers to receive a single intravenous dose of NTM-1632 (0.033, 0.165, or 0.330 mg/kg) or saline placebo. Safety monitoring included physical examinations, clinical laboratory studies, and vital signs. Blood sampling was performed at prespecified time points for PK and immunogenicity analyses. Twenty-four subjects received study product (18 NTM-1632; 6 placebo), and no deaths or serious adverse events were reported. Adverse events in the NTM-1632 groups were generally mild and similar in frequency and severity to the placebo group, and no safety signal was identified. NTM-1632 has a favorable PK profile with a half-life of >20 days for the 0.330-mg/kg dose and an approximately linear relationship with respect to maximum concentration and area under the concentration-time curve (AUC0→t). NTM-1632 demonstrated low immunogenicity with only a few treatment-emergent antidrug antibody responses in the low and middle dosing groups and none at the highest dose. NTM-1632 is well tolerated at the administered doses. The favorable safety, PK, and immunogenicity profile of NTM-1632 supports further clinical development as a treatment for BoNT/B intoxication and postexposure prophylaxis. (This study has been registered at ClinicalTrials.gov under identifier NCT02779140.).

Trap Assays of the Walnut Twig Beetle, Pityophthorus juglandis Blackman (Coleoptera: Curculionidae: Scolytinae), Reveal an Effective Semiochemical Repellent Combination.

Thousand cankers disease (TCD), is an invasive insect-disease complex caused by the walnut twig beetle, Pityophthorus juglandis, and fungal pathogen, Geosmithia morbida. Semiochemical interruption is a viable option for protecting walnut trees from P. juglandis attack. The goal of this study was to test beetle responses to potential repellent compounds. The results of five, flight-intercept assays are reported. Assays 1-3 tested four compounds at variable release rates: (S)-(-)-verbenone, (R)-(+)-verbenone, racemic chalcogran, and racemic trans-conophthorin. Trapping results indicated that the highest release rate tested for each compound was the most effective in reducing the number of beetles caught. (S)-(-)-Verbenone was the least effective, reducing P. juglandis trap catches by 66%. (R)-(+)-Verbenone reduced the number of P. juglandis by 84%. Neither enantiomer of verbenone performed as well as chalcogran or trans-conophthorin, which both reduced the number of beetles caught by ca. 98%. Following individual assays, the most effective compounds were tested in subtractive-combination assays. Combinations of high release rates for (R)-(+)-verbenone, trans-conophthorin, and two stereoisomers of limonene (tested in a previous study) were tested in two assays. The subtractive-combination assays were inconclusive in that trap catches were similar across all treatments. All combination treatments were highly effective, achieving approximately 99% reduction in the number of beetles caught. Based on the trapping results, commercial availability, and cost of the semiochemicals tested, we conclude that a combination of (R)-(+)-limonene, trans-conophthorin, and (R)-(+)-verbenone constitutes an effective tool for reducing P. juglandis trap catches.

Kinetic and Cross-Sectional Studies on the Genesis of Hypoargininemia in Severe Pediatric Plasmodium falciparum Malaria.

The low bioavailability of nitric oxide (NO) and its precursor, arginine, contributes to the microvascular pathophysiology of severe falciparum malaria. To better characterize the mechanisms underlying hypoargininemia in severe malaria, we measured the plasma concentrations of amino acids involved in de novo arginine synthesis in children with uncomplicated falciparum malaria (UM; n = 61), children with cerebral falciparum malaria (CM; n = 45), and healthy children (HC; n = 109). We also administered primed infusions of l-arginine uniformly labeled with 13C6 and 15N4 to 8 children with severe falciparum malaria (SM; age range, 4 to 9 years) and 7 healthy children (HC; age range, 4 to 8 years) to measure the metabolic flux of arginine, hypothesizing that arginine flux is increased in SM. Using two different tandem mass spectrometric methods, we measured the isotopic enrichment of arginine in plasma obtained at 0, 60, 90, 120, 150, and 180 min during the infusion. The plasma concentrations of glutamine, glutamate, proline, ornithine, citrulline, and arginine were significantly lower in UM and CM than in HC (P ≤ 0.04 for all pairwise comparisons). Of these, glutamine concentrations were the most markedly decreased: median, 457 µM (interquartile range [IQR], 400 to 508 µM) in HC, 300 µM (IQR, 256 to 365 µM) in UM, and 257 µM (IQR, 195 to 320 µM) in CM. Arginine flux during steady state was not significantly different in SM than in HC by the respective mass spectrometric methods: 93.2 µmol/h/kg of body weight (IQR, 84.4 to 129.3 µmol/h/kg) versus 88.0 µmol/h/kg (IQR, 73.0 to 102.2 µmol/h/kg) (P = 0.247) by the two mass spectrometric methods in SM and 93.7 µmol/h/kg (IQR, 79.1 to 117.8 µmol/h/kg) versus 81.0 µmol/h/kg (IQR, 75.9 to 88.6 µmol/h/kg) (P = 0.165) by the two mass spectrometric methods in HC. A limited supply of amino acid precursors for arginine synthesis likely contributes to the hypoargininemia and NO insufficiency in falciparum malaria in children.

A HEART Pathway pitfall in an admitted patient.

This paper discusses a possible weakness of the HEART Pathway specific to patients identified as high risk, requiring admission for inpatient risk stratification. Emergency Department (ED) crowding is at an all-time high and the possibility that many of these patients will board in the ED for a period of time before they are transported to an inpatient ward is becoming more likely. Given troponins peak at 6 h after the initial cardiac injury, it is plausible an initial troponin could still remain negative upon arrival. Extending the HEART Pathway to include a 3-hour delta troponin for admitted patients boarded in the emergency department may help alert the patient's inpatient team of those requiring more aggressive evaluations or more timely interventions. The case discussed herein highlights the course of a patient who was admitted to a medicine floor for chest pain along the HEART Pathway. After remaining in the ED for 3 h following admission a second troponin was drawn that resulted in the diagnosis of a non-ST segment myocardial infarction. The patient then received further management in the ED and a change in admission to the Cardiac Care Unit instead of the medicine floor. The patient ultimately received a Coronary Artery Bypass Graft during admission. If the patient had not had the second troponin while in the ED this care would have been delayed.

The contribution of social cognition in predicting social participation following moderate and severe TBI in youth.

Youth with moderate or severe traumatic brain injury (TBI) are at risk for reduced social participation after the injury, and the contribution of social cognition to these changes in functioning has been little studied. This study aimed to examine social participation and to measure the contribution of social and non-social cognitive functions to social participation impairment in youth (ages 12-21) who sustained moderate or severe TBI. Youth with TBI (n = 23) were compared to typically developing (TD) controls on self- and parent-rated social participation questionnaires. Direct testing of social cognition (mentalising, social knowledge, emotion recognition) and higher order cognitive abilities (intellectual abilities, attention and executive functions) was also conducted. Significant differences were found between the TBI participants and TD controls on social participation measures. Mentalising and problem-solving abilities revealed to be significant correlates of social participation as reported by youth with brain-injury and their parents. Overall, these results corroborate previous findings by showing that social participation is significantly reduced after TBI, and further shows that mentalising, which is not always considered during rehabilitation, is an important contributing factor. In addition to executive function measures, social cognition should therefore be systematically included in assessment following youth TBI for intervention and prevention purposes.

Methylene-linked bis-phenylbenzimidazoles - a new scaffold to target telomeric DNA/RNA hybrid duplex.

We report a series of novel methylene-linked bis-phenylbenzimidazoles intercalators that stabilize telomeric DNA/RNA hybrid (tDRH) structures by up to 7.2 °C at a 1 µM ligand concentration while having negligible affinity for DNA/DNA duplexes, although with a low affinity for quadruplex DNA. We have used molecular modelling studies to rationalize this selectivity, concluding that the methylene spacer between the terminal benzimidazole and phenylene moieties plays a key role in facilitating the bis-intercalating process. This scaffold may be used to develop chemical tools or new therapeutics to selectively target the telomeric DNA/RNA duplex without affecting normal genomic DNA.

Contact-dependent growth inhibition toxins exploit multiple independent cell-entry pathways.

Contact-dependent growth inhibition (CDI) systems function to deliver toxins into neighboring bacterial cells. CDI+ bacteria export filamentous CdiA effector proteins, which extend from the inhibitor-cell surface to interact with receptors on neighboring target bacteria. Upon binding its receptor, CdiA delivers a toxin derived from its C-terminal region. CdiA C-terminal (CdiA-CT) sequences are highly variable between bacteria, reflecting the multitude of CDI toxin activities. Here, we show that several CdiA-CT regions are composed of two domains, each with a distinct function during CDI. The C-terminal domain typically possesses toxic nuclease activity, whereas the N-terminal domain appears to control toxin transport into target bacteria. Using genetic approaches, we identified ptsG, metI, rbsC, gltK/gltJ, yciB, and ftsH mutations that confer resistance to specific CdiA-CTs. The resistance mutations all disrupt expression of inner-membrane proteins, suggesting that these proteins are exploited for toxin entry into target cells. Moreover, each mutation only protects against inhibition by a subset of CdiA-CTs that share similar N-terminal domains. We propose that, following delivery of CdiA-CTs into the periplasm, the N-terminal domains bind specific inner-membrane receptors for subsequent translocation into the cytoplasm. In accord with this model, we find that CDI nuclease domains are modular payloads that can be redirected through different import pathways when fused to heterologous N-terminal "translocation domains." These results highlight the plasticity of CDI toxin delivery and suggest that the underlying translocation mechanisms could be harnessed to deliver other antimicrobial agents into Gram-negative bacteria.

Identifying and Eliminating Sources of Recreational Water Quality Degradation along an Urban Coast.

Restoration of highly degraded urban coastal waters often requires large-scale, complex projects, but in the interim, smaller-scale efforts can provide immediate improvements to water quality conditions for visitor use. We examined short-term efforts to improve recreational water quality near the Grand Calumet River (GC) in the Laurentian Great Lakes. Identified as an Area of Concern (AOC) by the International Joint Commission, the GC has experienced years of industrial and municipal waste discharges, and as a result, coastal beaches have some of the highest rates of beach closings (>70%) in the United States. Project objectives were to identify sources of microbial contamination and to evaluate a short-term management solution to decrease beach closings: during 2015 (partial) and 2016 (season-long), canines were used to deter gull presence. Water samples were analyzed for in 2015 and 2016, and fecal sources were evaluated using microbial source tracking markers (2015): human ( HF183, ), gull (Gull2), and dog (DogBact). Hydrometeorological conditions were simultaneously measured. Results indicated that human, gull, and canine fecal sources were present, with gulls being the dominant source. densities were highly correlated with number of gulls present, Gull2 marker, and turbidity. Gull deterrence decreased and Gull2 marker detection during 2015, but numbers rebounded after program completion. The full-season program in 2016 resulted in lower densities and fewer beach closings. Large-scale restoration efforts are underway at this location, but short-term, small-scale projects can be useful for reducing beach closings and restoring ecosystem services.

Trends in Breastfeeding Among Infants Enrolled in the Special Supplemental Nutrition Program for Women, Infants and Children - New York, 2002-2015.

Breastfeeding is widely accepted as the optimal method of infant feeding (1,2). New York Special Supplemental Nutrition Program for Women, Infants and Children (WIC) has prioritized the promotion of breastfeeding. To assess breastfeeding trends among New York WIC infants, indicators for measuring breastfeeding practices reported by the New York Pediatric Nutrition Surveillance System (PedNSS) during 2002-2015 were examined. The prevalence of breastfeeding initiation increased from 62.0% (2002) to 83.4% (2015), exceeding the Healthy People 2020 (HP2020)* objective of 81.9% in 2014, with improvements among all racial/ethnic groups. The percentage of New York WIC infants who breastfed for ≥6 and ≥12 months increased from 30.2% and 15.0% (2002) to 39.5% and 22.8% (2015), respectively. The prevalence of exclusive breastfeeding for ≥3 and ≥6 months increased from 8.9% and 2.9% (2006) to 14.3% and 8.0% (2015), respectively. Despite improvements in breastfeeding initiation, increasing the duration of breastfeeding and of exclusive breastfeeding among infants enrolled in the New York WIC program remains challenging. Identifying targeted strategies to support continued and exclusive breastfeeding should remain priorities for the New York WIC program.

Airway exposure to multi-walled carbon nanotubes disrupts the female reproductive cycle without affecting pregnancy outcomes in mice.

BACKGROUND: The use of multiwalled carbon nanotubes (MWCNT) is increasing due to a growing use in a variety of products across several industries. Thus, occupational exposure is also of increasing concern, particularly since airway exposure to MWCNTs can induce sustained pulmonary acute phase response and inflammation in experimental animals, which may affect female reproduction. This proof-of-principle study therefore aimed to investigate if lung exposure by intratracheal instillation of the MWCNT NM-400 would affect the estrous cycle and reproductive function in female mice. RESULTS: Estrous cycle regularity was investigated by comparing vaginal smears before and after exposure to 67 µg of NM-400, whereas reproductive function was analyzed by measuring time to delivery of litters after instillation of 2, 18 or 67 µg of NM-400. Compared to normal estrous cycling determined prior to exposure, exposure to MWCNT significantly prolonged the estrous cycle during which exposure took place, but significantly shortened the estrous cycle immediately after the exposed cycle. No consistent effects were seen on time to delivery of litter or other gestational or litter parameters, such as litter size, sex ratio, implantations and implantation loss. CONCLUSION: Lung exposure to MWCNT interfered with estrous cycling. Effects caused by MWCNTs depended on the time of exposure: the estrous stage was particularly sensitive to exposure, as animals exposed during this stage showed a higher incidence of irregular cycling after exposure. Our data indicates that MWCNT exposure may interfere with events leading to ovulation.