A CAR enhancer increases the activity and persistence of CAR T cells.

Although chimeric antigen receptor (CAR) T cell therapies have demonstrated promising clinical outcomes, durable remissions remain limited. To extend the efficacy of CAR T cells, we develop a CAR enhancer (CAR-E), comprising a CAR T cell antigen fused to an immunomodulatory molecule. Here we demonstrate this strategy using B cell maturation antigen (BCMA) CAR T cells for the treatment of multiple myeloma, with a CAR-E consisting of the BCMA fused to a low-affinity interleukin 2 (IL-2). This selectively induces IL-2 signaling in CAR T cells upon antigen-CAR binding, enhancing T cell activation and antitumor activity while reducing IL-2-associated toxicities. We show that the BCMA CAR-E selectively binds CAR T cells and increases CAR T cell proliferation, clearance of tumor cells and development of memory CAR T cells. The memory cells retain the ability to re-expand upon restimulation, effectively controlling tumor growth upon rechallenge. Mechanistic studies reveal the involvement of both CAR and IL-2 receptor endodomains in the CAR-E mechanism of action. The CAR-E approach avoids the need for specific engineering and enables CAR T cell therapy with lower cell doses.

Microbiota metabolized Bile Acids accelerate Gastroesophageal Adenocarcinoma via FXR inhibition.

Background: The incidence of Barrett esophagus (BE) and Gastroesophageal Adenocarcinoma (GEAC) correlates with obesity and a diet rich in fat. Bile acids (BA) support fat digestion and undergo microbial metabolization in the gut. The farnesoid X receptor (FXR) is an important modulator of the BA homeostasis. The capacity of inhibiting cancer-related processes when activated, make FXR an appealing therapeutic target. In this work, we assess the role of diet on the microbiota-BA axis and evaluate the role of FXR in disease progression. Results: Here we show that high fat diet (HFD) accelerated tumorigenesis in L2-IL1B mice (BE- and GEAC- mouse model) while increasing BA levels and enriching gut microbiota that convert primary to secondary BA. While upregulated in BE, expression of FXR was downregulated in GEAC in mice and humans. In L2-IL1B mice, FXR knockout enhanced the dysplastic phenotype and increased Lgr5 progenitor cell numbers. Treatment of murine organoids and L2-IL1B mice with the FXR agonist obeticholic acid (OCA) deacelerated GEAC progression. Conclusion: We provide a novel concept of GEAC carcinogenesis being accelerated via the diet-microbiome-metabolome axis and FXR inhibition on progenitor cells. Further, FXR activation protected with OCA ameliorated the phenotype in vitro and in vivo, suggesting that FXR agonists have potential as differentiation therapy in GEAC prevention.

Stool Protein Mass Spectrometry Identifies Biomarkers for Early Detection of Diffuse-type Gastric Cancer.

There is a high unmet need for early detection approaches for diffuse gastric cancer (DGC). We examined whether the stool proteome of mouse models of gastric cancer (GC) and individuals with hereditary diffuse gastric cancer (HDGC) have utility as biomarkers for early detection. Proteomic mass spectrometry of the stool of a genetically engineered mouse model driven by oncogenic KrasG12D and loss of p53 and Cdh1 in gastric parietal cells [known as Triple Conditional (TCON) mice] identified differentially abundant proteins compared with littermate controls. Immunoblot assays validated a panel of proteins, including actinin alpha 4 (ACTN4), N-acylsphingosine amidohydrolase 2 (ASAH2), dipeptidyl peptidase 4 (DPP4), and valosin-containing protein (VCP), as enriched in TCON stool compared with littermate control stool. Immunofluorescence analysis of these proteins in TCON stomach sections revealed increased protein expression compared with littermate controls. Proteomic mass spectrometry of stool obtained from patients with HDGC with CDH1 mutations identified increased expression of ASAH2, DPP4, VCP, lactotransferrin (LTF), and tropomyosin-2 relative to stool from healthy sex- and age-matched donors. Chemical inhibition of ASAH2 using C6 urea ceramide was toxic to GC cell lines and GC patient-derived organoids. This toxicity was reversed by adding downstream products of the S1P synthesis pathway, which suggested a dependency on ASAH2 activity in GC. An exploratory analysis of the HDGC stool microbiome identified features that correlated with patient tumors. Herein, we provide evidence supporting the potential of analyzing stool biomarkers for the early detection of DGC. Prevention Relevance: This study highlights a novel panel of stool protein biomarkers that correlate with the presence of DGC and has potential use as early detection to improve clinical outcomes.

COVID-19 vaccines and adverse events of special interest: A multinational Global Vaccine Data Network (GVDN) cohort study of 99 million vaccinated individuals.

BACKGROUND: The Global COVID Vaccine Safety (GCoVS) Project, established in 2021 under the multinational Global Vaccine Data Network™ (GVDN®), facilitates comprehensive assessment of vaccine safety. This study aimed to evaluate the risk of adverse events of special interest (AESI) following COVID-19 vaccination from 10 sites across eight countries. METHODS: Using a common protocol, this observational cohort study compared observed with expected rates of 13 selected AESI across neurological, haematological, and cardiac outcomes. Expected rates were obtained by participating sites using pre-COVID-19 vaccination healthcare data stratified by age and sex. Observed rates were reported from the same healthcare datasets since COVID-19 vaccination program rollout. AESI occurring up to 42 days following vaccination with mRNA (BNT162b2 and mRNA-1273) and adenovirus-vector (ChAdOx1) vaccines were included in the primary analysis. Risks were assessed using observed versus expected (OE) ratios with 95 % confidence intervals. Prioritised potential safety signals were those with lower bound of the 95 % confidence interval (LBCI) greater than 1.5. RESULTS: Participants included 99,068,901 vaccinated individuals. In total, 183,559,462 doses of BNT162b2, 36,178,442 doses of mRNA-1273, and 23,093,399 doses of ChAdOx1 were administered across participating sites in the study period. Risk periods following homologous vaccination schedules contributed 23,168,335 person-years of follow-up. OE ratios with LBCI > 1.5 were observed for Guillain-Barré syndrome (2.49, 95 % CI: 2.15, 2.87) and cerebral venous sinus thrombosis (3.23, 95 % CI: 2.51, 4.09) following the first dose of ChAdOx1 vaccine. Acute disseminated encephalomyelitis showed an OE ratio of 3.78 (95 % CI: 1.52, 7.78) following the first dose of mRNA-1273 vaccine. The OE ratios for myocarditis and pericarditis following BNT162b2, mRNA-1273, and ChAdOx1 were significantly increased with LBCIs > 1.5. CONCLUSION: This multi-country analysis confirmed pre-established safety signals for myocarditis, pericarditis, Guillain-Barré syndrome, and cerebral venous sinus thrombosis. Other potential safety signals that require further investigation were identified.

Fast and efficient template-mediated synthesis of genetic variants.

Efficient methods for the generation of specific mutations enable the study of functional variations in natural populations and lead to advances in genetic engineering applications. Here, we present a new approach, mutagenesis by template-guided amplicon assembly (MEGAA), for the rapid construction of kilobase-sized DNA variants. With this method, many mutations can be generated at a time to a DNA template at more than 90% efficiency per target in a predictable manner. We devised a robust and iterative protocol for an open-source laboratory automation robot that enables desktop production and long-read sequencing validation of variants. Using this system, we demonstrated the construction of 31 natural SARS-CoV2 spike gene variants and 10 recoded Escherichia coli genome fragments, with each 4 kb region containing up to 150 mutations. Furthermore, 125 defined combinatorial adeno-associated virus-2 cap gene variants were easily built using the system, which exhibited viral packaging enhancements of up to 10-fold compared with wild type. Thus, the MEGAA platform enables generation of multi-site sequence variants quickly, cheaply, and in a scalable manner for diverse applications in biotechnology.

Resurrecting essential amino acid biosynthesis in mammalian cells.

Major genomic deletions in independent eukaryotic lineages have led to repeated ancestral loss of biosynthesis pathways for nine of the twenty canonical amino acids. While the evolutionary forces driving these polyphyletic deletion events are not well understood, the consequence is that extant metazoans are unable to produce nine essential amino acids (EAAs). Previous studies have highlighted that EAA biosynthesis tends to be more energetically costly, raising the possibility that these pathways were lost from organisms with access to abundant EAAs. It is unclear whether present-day metazoans can reaccept these pathways to resurrect biosynthetic capabilities that were lost long ago or whether evolution has rendered EAA pathways incompatible with metazoan metabolism. Here, we report progress on a large-scale synthetic genomics effort to reestablish EAA biosynthetic functionality in mammalian cells. We designed codon-optimized biosynthesis pathways based on genes mined from Escherichia coli. These pathways were de novo synthesized in 3 kilobase chunks, assembled in yeasto and genomically integrated into a Chinese hamster ovary (CHO) cell line. One synthetic pathway produced valine at a sufficient level for cell viability and proliferation. 13C-tracing verified de novo biosynthesis of valine and further revealed build-up of pathway intermediate 2,3-dihydroxy-3-isovalerate. Increasing the dosage of downstream ilvD boosted pathway performance and allowed for long-term propagation of second-generation cells in valine-free medium at 3.2 days per doubling. This work demonstrates that mammalian metabolism is amenable to restoration of ancient core pathways, paving a path for genome-scale efforts to synthetically restore metabolic functions to the metazoan lineage.

The alarmins high mobility group box protein 1 and S100A8/A9 display different inflammatory profiles after acute knee injury.

OBJECTIVE: To compare the concentrations of high mobility group box 1 protein (HMGB1) and S100A8/A9 in synovial fluid between patients with knee injuries and osteoarthritis (OA), and knee healthy subjects. To investigate associations of alarmin levels with different joint injuries and with biomarkers of inflammation, Wnt signaling, complement system, bone and cartilage degradation. METHODS: HMGB1 and S100A8/A9 were measured in synovial fluid by immunoassays in patients with knee injuries, with OA and from knee healthy subjects, and were related to time from injury and with biomarkers obtained from previous studies. Hierarchical cluster and enrichment analyses of biomarkers associated to HMGB1 and S100A8/A9 were performed. RESULTS: The synovial fluid HMGB1 and S100A8/A9 concentrations were increased early after knee injury; S100A8/A9 levels were negatively associated to time after injury and was lower in the old compared to recent injury group, while HMGB1 was not associated to time after injury. The S100A8/A9 levels were also increased in OA. The initial inflammatory response was similar between the alarmins, and HMGB1 and S100A8/A9 shared 9 out of 20 enriched pathways. The alarmins displayed distinct response profiles, HMGB1 being associated to cartilage biomarkers while S100A8/A9 was associated to proinflammatory cytokines. CONCLUSIONS: HMGB1 and S100A8/A9 are increased as an immediate response to knee trauma. While they share many features in inflammatory and immunoregulatory mechanisms, S100A8/A9 and HMGB1 are associated to different downstream responses, which may have impact on the OA progression after acute knee injuries.

Probing immune infiltration dynamics in cancer by in vivo imaging.

Cancer immunotherapies typically aim to stimulate the accumulation and activity of cytotoxic T-cells or pro-inflammatory antigen-presenting cells, reduce immunosuppressive myeloid cells or regulatory T-cells, or elicit some combination of effects thereof. Notwithstanding the encouraging results, immunotherapies such as PD-1/PD-L1-targeted immune checkpoint blockade act heterogeneously across individual patients. It remains challenging to predict and monitor individual responses, especially across multiple sites of metastasis or sites of potential toxicity. To address this need, in vivo imaging of both adaptive and innate immune cell populations has emerged as a tool to quantify spatial leukocyte accumulation in tumors non-invasively. Here we review recent progress in the translational development of probes for in vivo leukocyte imaging, focusing on complementary perspectives provided by imaging of T-cells, phagocytic macrophages, and their responses to therapy.

A survey of UK and Irish surgeons' attitudes, behaviours and barriers to change for environmental sustainability.

INTRODUCTION: Surgery is a major contributor to the large environmental impact of healthcare, demanding urgent attention. To date there are no data on the attitudes and behaviours of surgeons towards climate change, or perceived barriers towards sustainable practice. METHODS: We invited surgeons and surgical trainees in the UK and Ireland to participate in an online survey (developed in accordance with the CHERRIES checklist) conducted between June and November 2020 and disseminated via the Royal College of Surgeons of England, Edinburgh and Ireland, the Association of Surgeons in Training and through local communication. RESULTS: We received 130 responses, across 14 surgical specialties. The majority of respondents (122/130; 94%) were concerned about the threat of climate change. Most respondents had instigated more sustainable practices in their personal lives (113/130; 87%) and, to a lesser extent, at work (73/130; 56%). Surgeons were willing to make changes to their clinical practice (107/130; 82%), but the main perceived barrier to improving sustainability was a lack of leadership (92/130; 70%). Surgeons welcomed greater leadership and guidance from national bodies (118/130; 91%) and more monitoring and regulation (113/130; 87%). CONCLUSIONS: The surgeons who responded to our survey are concerned about climate change and willing to engage in efforts to transition to more sustainable practice, but would welcome greater support, guidance and leadership.

Prognostic gene expression signature for high-grade serous ovarian cancer.

BACKGROUND: Median overall survival (OS) for women with high-grade serous ovarian cancer (HGSOC) is ∼4 years, yet survival varies widely between patients. There are no well-established, gene expression signatures associated with prognosis. The aim of this study was to develop a robust prognostic signature for OS in patients with HGSOC. PATIENTS AND METHODS: Expression of 513 genes, selected from a meta-analysis of 1455 tumours and other candidates, was measured using NanoString technology from formalin-fixed paraffin-embedded tumour tissue collected from 3769 women with HGSOC from multiple studies. Elastic net regularization for survival analysis was applied to develop a prognostic model for 5-year OS, trained on 2702 tumours from 15 studies and evaluated on an independent set of 1067 tumours from six studies. RESULTS: Expression levels of 276 genes were associated with OS (false discovery rate < 0.05) in covariate-adjusted single-gene analyses. The top five genes were TAP1, ZFHX4, CXCL9, FBN1 and PTGER3 (P < 0.001). The best performing prognostic signature included 101 genes enriched in pathways with treatment implications. Each gain of one standard deviation in the gene expression score conferred a greater than twofold increase in risk of death [hazard ratio (HR) 2.35, 95% confidence interval (CI) 2.02-2.71; P < 0.001]. Median survival [HR (95% CI)] by gene expression score quintile was 9.5 (8.3 to -), 5.4 (4.6-7.0), 3.8 (3.3-4.6), 3.2 (2.9-3.7) and 2.3 (2.1-2.6) years. CONCLUSION: The OTTA-SPOT (Ovarian Tumor Tissue Analysis consortium - Stratified Prognosis of Ovarian Tumours) gene expression signature may improve risk stratification in clinical trials by identifying patients who are least likely to achieve 5-year survival. The identified novel genes associated with the outcome may also yield opportunities for the development of targeted therapeutic approaches.

Dairy and related nutrient intake and risk of uterine leiomyoma: a prospective cohort study.

STUDY QUESTION: Is there an association between consumption of dairy foods and related nutrients and risk of uterine leiomyoma? SUMMARY ANSWER: While dairy consumption was not consistently associated with uterine leiomyoma risk, intake of yogurt and calcium from foods may reduce risk of uterine leiomyoma. WHAT IS KNOWN ALREADY: Two studies have examined the association between dairy intake and uterine leiomyoma risk with inconsistent results. Dairy foods have been inversely associated with inflammation and tumorigenesis, suggesting that vitamins and minerals concentrated in these dietary sources may influence uterine leiomyoma risk. STUDY DESIGN, SIZE, DURATION: A prospective cohort study was carried out using data collected from 81 590 premenopausal women from 1991 to 2009 as part of the Nurses' Health Study II cohort. PARTICIPANTS/MATERIALS, SETTING, METHODS: Diet was assessed with a validated food frequency questionnaire every 4 years. Cases were restricted to self-reported ultrasound or hysterectomy-confirmation uterine leiomyoma. Cox proportional hazards models were used to calculate hazard ratios (HRs) and 95% CI. MAIN RESULTS AND THE ROLE OF CHANCE: Eight thousand one hundred and forty-two cases of ultrasound or hysterectomy-confirmed uterine leiomyoma were diagnosed over an 18-year period. When compared to participants who consumed two servings a week of total dairy foods, participants who consumed four or more servings had a borderline significant 8% reduced risk of uterine leiomyoma (HR = 0.92, 95% CI = 0.85, 1.00; ptrend = 0.19). When the association between specific dairy foods and uterine leiomyoma was examined, the relation between dairy-food intake and uterine leiomyoma appeared to be driven primarily by yogurt consumption (HR for 2+ servings/day = 0.76; 95% CI = 0.55, 1.04 compared to <=4 servings/week; ptrend = 0.03); however, there was a small number of cases in the 2+ servings/day group (n = 39). Of the nutrients examined, the association was strongest for calcium from foods (HR fifth quintile = 0.92, 95% CI = 0.86, 0.99; ptrend = 0.04). LIMITATIONS, REASONS FOR CAUTION: Some cases of uterine leiomyoma were likely misclassified, particularly those that were asymptomatic. It is possible that dairy product constituents reduce uterine leiomyoma symptomology rather than development, giving the appearance of a protective effect on leiomyoma development: no data on uterine leiomyoma symptomology were available. We did not have vitamin and mineral concentrations from actual blood levels. Similarly, there is the potential for misclassification of participants based on predicted 25(OH)D, and changes in vitamin D supplementation over time may have impacted prediction models for 25(OH)D. Further, some error in the self-reporting of dietary intake is expected. Given our prospective design, it is likely that these misclassifications were non-differential with respect to the outcome, likely biasing estimates toward the null. WIDER IMPLICATIONS OF THE FINDINGS: While no clear association between overall dairy consumption and uterine leiomyoma risk was observed, our findings suggest that intake of yogurt and calcium from foods may reduce risk of uterine leiomyoma. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by research grant HD081064 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development. The Nurses' Health Study II is supported by the Public Health Service grant UM1 CA176726 from the National Cancer Institute, NIH, U.S. Department of Health and Human Services. H.R.H. is supported by the National Cancer Institute, National Institutes of Health (K22 CA193860). There are no conflicts of interest to declare.

Early stage blockade of the alarmin HMGB1 reduces cartilage destruction in experimental OA.

OBJECTIVE: The alarmin HMGB1 is an endogenous molecule that is released into the extracellular space upon trauma or cell activation. Extracellular HMGB1 initiates innate immune responses and besides mediating inflammation, has osteoclast-activating features and mediates pain, all important features in OA. The aim of this study was to examine the involvement of HMGB1 in experimental OA and to explore the effect of local anti-HMGB1-therapy on disease progression. METHOD: OA was induced in mice by surgical destabilization of knee joints and HMGB1 expression and localization was assessed by immunohistochemistry. For therapy evaluation, HMGB1-neutralizing antibodies were injected intraarticularly, alone or encapsulated in an injectable hyaluronan-based delivery vehicle. Human primary chondrocytes were stimulated with rHMGB1 and analyzed by qPCR and cytometric bead-array. RESULTS: HMGB1 immunostaining of mouse OA joints demonstrated intra- and pericellular expression in chondrocytes, overlapping with proteoglycan depleted areas. Intra-articular injection of anti-HMGB1 antibodies had cartilage-protective effects, comparable to treatment with a TNF inhibitor. Direct and vehicle-based delivery had similar ameliorating effects and the effect of a single, early injection could not be enhanced by repeated injections. In vitro stimulation of chondrocytes with rHMGB1 affected chondrocyte function by inducing protein expression of IL6 and IL8 and downregulating mRNA of COL2A1. CONCLUSIONS: Our results suggest that the alarmin HMGB1 might be a new target for OA therapy development as we could observe an aberrant HMGB1 expression in mouse OA joints, stimulation of chondrocytes with rHMGB1 induced cytokine production and decreased matrix production and finally that HMGB1 blockade suppressed disease progression.

Genome-wide association and epidemiological analyses reveal common genetic origins between uterine leiomyomata and endometriosis.

Uterine leiomyomata (UL) are the most common neoplasms of the female reproductive tract and primary cause for hysterectomy, leading to considerable morbidity and high economic burden. Here we conduct a GWAS meta-analysis in 35,474 cases and 267,505 female controls of European ancestry, identifying eight novel genome-wide significant (P < 5 × 10-8) loci, in addition to confirming 21 previously reported loci, including multiple independent signals at 10 loci. Phenotypic stratification of UL by heavy menstrual bleeding in 3409 cases and 199,171 female controls reveals genome-wide significant associations at three of the 29 UL loci: 5p15.33 (TERT), 5q35.2 (FGFR4) and 11q22.3 (ATM). Four loci identified in the meta-analysis are also associated with endometriosis risk; an epidemiological meta-analysis across 402,868 women suggests at least a doubling of risk for UL diagnosis among those with a history of endometriosis. These findings increase our understanding of genetic contribution and biology underlying UL development, and suggest overlapping genetic origins with endometriosis.

Hernia prevention: practice patterns and surgeons' attitudes about abdominal wall closure and the use of prophylactic mesh.

PURPOSE: The penetration of hernia prevention techniques into surgical practice remains unknown. METHODS: A survey about knowledge/attitudes on hernia prevention was sent to the members of hernia societies. RESULTS: The 497 respondents were mostly from the US (47%) or Europe (40%). Most reported practicing, but not measuring their suture-to-wound length closure of > 4:1 (63%) and practicing but not measuring the number of stitches (58%). Reasons for not using short stitch closure were: does not apply to patient population (19%), not familiar enough with methods to correctly execute (25%), takes too long (13%), not reimbursed (4%), concerned about closure-related complications (27%), and other (22%). Regarding prophylactic mesh, respondents stated they were not familiar with literature (11%), familiar with literature but would not use (24%), familiar with literature and interested in use (45%), familiar with literature and using (15%), and other (5%). CONCLUSIONS: There appears to be some application of hernia prevention principles related to fascial closure; however, the use of prophylactic mesh still appears to be controversial.

Influence of anti-inflammatory diet and smoking on mortality and survival in men and women: two prospective cohort studies.

BACKGROUND: The associations between an anti-inflammatory diet and both all-cause and cause-specific mortality have been studied previously; however, the influence of an anti-inflammatory diet on survival time has not been investigated. Moreover, the potential modification of these associations by smoking status remains unclear. OBJECTIVE: The aims of this study were to examine the associations between an anti-inflammatory diet index (AIDI) and all-cause and cause-specific mortality, to determine the association between the AIDI and differences in survival time and to assess effect modification by smoking status. METHODS: The study population included 68 273 Swedish men and women (aged 45-83 years) at baseline. The anti-inflammatory potential of the diet was estimated using the validated AIDI, which includes 11 potential anti-inflammatory and five potential pro-inflammatory foods. Cox proportional hazards and Laplace regression were used to estimate hazard ratios and differences in survival time. RESULTS: During 16 years of follow-up (1 057 959 person-years), 16 088 deaths [5980 due to cardiovascular disease (CVD) and 5252 due to cancer] were recorded. Participants in the highest versus lowest quartile of the AIDI had lower risks of all-cause (18% reduction, 95% CI: 14-22%), CVD (20%, 95% CI: 14-26%) and cancer (13%, 95% CI: 5-20%) mortality. The strongest inverse associations between the highest and lowest quartiles of AIDI and risk of mortality were observed in current smokers: 31%, 36% and 22% lower risks of all-cause, CVD and cancer mortality, respectively. The difference in survival time between current smokers in the lowest AIDI quartile and never smokers in the highest quartile was 4.6 years. CONCLUSION: Adherence to a diet with high anti-inflammatory potential may reduce all-cause, CVD and cancer mortality and prolong survival time especially amongst smokers.

Analyzing Multilevel Factors Underlying Adolescent Smoking Behaviors: The Roles of Friendship Network, Family Relations, and School Environment.

BACKGROUND: This study investigates the extent to which friendship network, family relations, and school context are related to adolescent cigarette smoking. Friendship network is measured in terms of delinquent peers; family relations in terms of parental supervision; and school environment in terms of objective (eg, antismoking policy) and subjective (eg, school attachment) characteristics. METHODS: Findings are based on the secondary analysis of the health behavior in school-aged children, 2009-2010. Two-level hierarchical generalized linear models are estimated using hierarchical linear modeling 7. RESULTS: At the student level, ties to delinquent friends is significantly related to higher odds of smoking, while greater parental supervision is associated with lower odds. At the school level, antismoking policy and curriculum independently lower smoking behavior. Better within-class peer relations, greater school attachment, and higher academic performance are also negatively related to smoking. Last, the positive association between delinquent friends and smoking is weaker in schools with a formally enacted antismoking policy. However, this association is stronger in schools with better peer relations. CONCLUSIONS: Adolescent smoking behavior is embedded in a broader ecological setting. This research reveals that a proper understanding of it requires comprehensive analysis that incorporates factors measured at individual (student) and contextual (school) levels.

Fruit and vegetable consumption and risk of endometriosis.

STUDY QUESTION: Is there an association between intake of fruits and vegetables and risk of laparoscopically confirmed endometriosis? SUMMARY ANSWER: Higher intake of fruits, particularly citrus fruits, is associated with a lower risk of endometriosis. WHAT IS KNOWN ALREADY: Two case-control studies have examined the associations between fruit and vegetable intake and endometriosis risk with contrasting results. Diets rich in fruits and vegetables include higher levels of pro-vitamin A nutrients (alpha-carotene, beta-carotene, beta-cryptoxanthin) and women with endometriosis have been reported to have lower intake of vitamin A than women without endometriosis. STUDY DESIGN SIZE, DURATION: A prospective cohort study using data collected from 70 835 premenopausal women from 1991 to 2013 as part of the Nurses' Health Study II cohort. PARTICIPANTS/MATERIALS, SETTING, METHODS: Diet was assessed with a validated food frequency questionnaire (FFQ) every 4 years. Cases were restricted to laparoscopically confirmed endometriosis. Cox proportional hazards models were used to calculate rate ratios (RR) and 95% CI. MAIN RESULTS AND THE ROLE OF CHANCE: During 840 012 person-years of follow-up, 2609 incident cases of laparoscopically confirmed endometriosis were reported (incidence rate = 311 per 100 000 person-years). We observed a non-linear inverse association between higher fruit consumption and risk of laparoscopically confirmed endometriosis (Psignificance of the curve = 0.005). This inverse association was particularly evident for citrus fruits. Women consuming ≥1 servings of citrus fruits/day had a 22% lower endometriosis risk (95% CI = 0.69-0.89; Ptrend = 0.004) compared to those consuming <1 serving/week. No association was observed between total vegetable intake and endometriosis risk. However, women consuming ≥1 servings/day cruciferous vegetables had a 13% higher risk of endometriosis (95% CI = 0.95-1.34; Ptrend = 0.03) compared to those consuming <1 serving/week. Of the nutrients examined, only beta-cryptoxanthin intake was significantly associated with lower endometriosis risk (RR fifth quintile = 0.88; 95% CI = 0.78-1.00; Ptrend = 0.02). LIMITATIONS REASONS FOR CAUTION: Some error in the self-reporting of dietary intake is expected, however, use of a validated FFQ and examining diet prospectively across multiple time points, make it unlikely that this non-differential misclassification strongly influenced the results. WIDER IMPLICATIONS OF THE FINDINGS: Our findings suggest that a higher intake of fruits, particularly citrus fruits, is associated with a lower risk of endometriosis, and beta-cryptoxanthin in these foods may partially explain this association. In contrast to what we hypothesized, consumption of some vegetables increased endometriosis risk which may indicate a role of gastrointestinal symptoms in both the presentation and exacerbation of endometriosis-related pain; however, it is not clear what components of these foods might underlie the observed associations. Future studies examining dietary patterns that consider different combinations of food intake may help clarify these associations. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by research grants HD4854, HD52473 and HD57210 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development, and grant P30 DK046200 from the National Institute of Diabetes and Digestive and Kidney Diseases. The Nurses' Health Study II is supported by the Public Health Service grant UM1 CA176726 from the National Cancer Institute, National Institutes of Health. HRH is supported by the National Cancer Institute, National Institutes of Health (K22 CA193860). No competing interests. TRIAL REGISTRATION NUMBER: n/a.

Long and irregular menstrual cycles, polycystic ovary syndrome, and ovarian cancer risk in a population-based case-control study.

Long and irregular menstrual cycles, a hallmark of polycystic ovary syndrome (PCOS), have been associated with higher androgen and lower sex hormone binding globulin levels and this altered hormonal environment may increase the risk of specific histologic subtypes of ovarian cancer. We investigated whether menstrual cycle characteristics and self-reported PCOS were associated with ovarian cancer risk among 2,041 women with epithelial ovarian cancer and 2,100 controls in the New England Case-Control Study (1992-2008). Menstrual cycle irregularity, menstrual cycle length, and PCOS were collected through in-person interview. Unconditional logistic regression models were used to calculate odds ratios (OR) and 95% confidence intervals (95% CIs) for ovarian cancer risk overall, and polytomous logistic regression to evaluate whether risk differed between histologic subtypes. Overall, we observed no elevation in ovarian cancer risk for women who reported periods that were never regular or for those reporting a menstrual cycle length of >35 days with ORs of 0.87 (95% CI = 0.69-1.10) and 0.83 (95% CI = 0.44-1.54), respectively. We observed no overall association between self-reported PCOS and ovarian cancer (OR = 0.97; 95% CI = 0.61-1.56). However, we observed significant differences in the association with menstrual cycle irregularity and risk of ovarian cancer subtypes (pheterogeneity = 0.03) as well as by BMI and OC use (pinteraction < 0.01). Most notable, menstrual cycle irregularity was associated with a decreased risk of high grade serous tumors but an increased risk of serous borderline tumors among women who had never used OCs and those who were overweight. Future research in a large collaborative consortium may help clarify these associations.

Intracellular distribution and stability of a luminescent rhenium(i) tricarbonyl tetrazolato complex using epifluorescence microscopy in conjunction with X-ray fluorescence imaging.

Optical epifluorescence microscopy was used in conjunction with X-ray fluorescence imaging to monitor the stability and intracellular distribution of the luminescent rhenium(i) complex fac-[Re(CO)3(phen)L], where phen = 1,10-phenathroline and L = 5-(4-iodophenyl)tetrazolato, in 22Rv1 cells. The rhenium complex showed no signs of ancillary ligand dissociation, a conclusion based on data obtained via X-ray fluorescence imaging aligning iodine and rhenium distributions. A diffuse reticular localisation was detected for the complex in the nuclear/perinuclear region of cells, by either optical or X-ray fluorescence imaging techniques. X-ray fluorescence also showed that the rhenium complex disrupted the homeostasis of some biologically relevant elements, such as chlorine, potassium and zinc.