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ABSTRACTIntratumoral bacteria flexibly contribute to cellular and molecular tumor heterogeneity for supporting cancer recurrence through poorly understood mechanisms. Using spatial metabolomic profiling technologies and 16SrRNA sequencing, we herein report that right-sided colorectal tumors are predominantly populated with Colibactin-producing Escherichia coli (CoPEC) that are locally establishing a high-glycerophospholipid microenvironment with lowered immunogenicity. It coincided with a reduced infiltration of CD8+ T lymphocytes that produce the cytotoxic cytokines IFN-γ where invading bacteria have been geolocated. Mechanistically, the accumulation of lipid droplets in infected cancer cells relied on the production of colibactin as a measure to limit genotoxic stress to some extent. Such heightened phosphatidylcholine remodeling by the enzyme of the Land’s cycle supplied CoPEC-infected cancer cells with sufficient energy for sustaining cell survival in response to chemotherapies. This accords with the lowered overall survival of colorectal patients at stage III-IV who were colonized by CoPEC when compared to patients at stage I-II. Accordingly, the sensitivity of CoPEC-infected cancer cells to chemotherapies was restored upon treatment with an acyl-CoA synthetase inhibitor. By contrast, such metabolic dysregulation leading to chemoresistance was not observed in human colon cancer cells that were infected with the mutant strain that did not produce colibactin (11G5∆ClbQ). This work revealed that CoPEC locally supports an energy trade-off lipid overload within tumors for lowering tumor immunogenicity. This may pave the way for improving chemoresistance and subsequently outcome of CRC patients who are colonized by CoPEC.

Background Studies have demonstrated the efficacy of Palbociclib (CDK 4/6 inhibitor), Gedatolisib (PI3K/mTOR dual inhibitor) and PD0325901 (MEK1/2 inhibitor) in colorectal cancer (CRC), however single agent therapeutics are often limited by the development of resistance. Methods We compared the anti-proliferative effects of the combination of Gedatolisib and Palbociclib and Gedatolisib and PD0325901 in five CRC cell lines with varying mutational background and tested their combinations on total and phosphoprotein levels of signaling pathway proteins. Results The combination of Palbociclib and Gedatolisib was superior to the combination of Palbociclib and PD0325901. The combination of Palbociclib and Gedatolisib had synergistic anti-proliferative effects in all cell lines tested [CI range: 0.11–0.69] and resulted in the suppression of S6rp (S240/244), without AKT reactivation. The combination of Palbociclib and Gedatolisib increased BAX and Bcl−2 levels in PIK3CA mutated cell lines. The combination of Palbociclib and Gedatolisib caused MAPK/ERK reactivation, as seen by an increase in expression of total EGFR, regardless of the mutational status of the cells. Conclusion This study shows that the combination of Palbociclib and Gedatolisib has synergistic anti-proliferative effects in both wild-type and mutated CRC cell lines. Separately, the phosphorylation of S6rp may be a promising biomarker of responsiveness to this combination.

The surface zone of articular cartilage is the first area impacted by cartilage defects, commonly resulting in osteoarthritis. Chondrocytes in the surface zone of articular cartilage synthesize and secrete lubricin, a proteoglycan that functions as a lubricant protecting the deeper layers from shear stress. Notably, 3D bioprinting is a tissue engineering technique that uses cells encapsulated in biomaterials to fabricate 3D constructs. Gelatin methacrylate (GelMA) is a frequently used biomaterial for 3D bioprinting cartilage. Oxidized methacrylated alginate (OMA) is a chemically modified alginate designed for its tunable degradation rate and mechanical properties. To determine an optimal combination of GelMA and OMA for lubricin expression, we used our novel high-throughput human articular chondrocyte reporter system. Primary human chondrocytes were transduced with PRG4 (lubricin) promoter-driven Gaussia luciferase, allowing for temporal assessment of lubricin expression. A lubricin expression-driven Design of Experiment screen and subsequent validation identified 14% GelMA/2% OMA for further study. Therefore, DoE optimized 14% GelMA/2% OMA, 14% GelMA control, and 16% GelMA (total solid content control) were 3D bioprinted. The combination of lubricin protein expression and shape retention over the 22 days in culture, successfully determined the 14% GelMA/2%OMA to be the optimal formulation for lubricin secretion. This strategy allows for rapid analysis of the role(s) of biomaterial composition, stiffness or other cell manipulations on lubricin expression by chondrocytes, which may improve therapeutic strategies for cartilage regeneration.

Abstract Background Comparing the composition of microbial communities among groups of interest (e.g., patients vs healthy individuals) is a central aspect in microbiome research. It typically involves sequencing, data processing, statistical analysis and graphical display. Such an analysis is normally obtained by using a set of different applications that require specific expertise for installation, data processing and in some cases, programming skills. Results Here, we present SHAMAN, an interactive web application we developed in order to facilitate the use of (i) a bioinformatic workflow for metataxonomic analysis, (ii) a reliable statistical modelling and (iii) to provide the largest panel of interactive visualizations among the applications that are currently available. SHAMAN is specifically designed for non-expert users. A strong benefit is to use an integrated version of the different analytic steps underlying a proper metagenomic analysis. The application is freely accessible at http://shaman.pasteur.fr/ , and may also work as a standalone application with a Docker container (aghozlane/shaman), conda and R. The source code is written in R and is available at https://github.com/aghozlane/shaman . Using two different datasets (a mock community sequencing and a published 16S rRNA metagenomic data), we illustrate the strengths of SHAMAN in quickly performing a complete metataxonomic analysis. Conclusions With SHAMAN, we aim at providing the scientific community with a platform that simplifies reproducible quantitative analysis of metagenomic data.

Purpose: To assess the frequency of intraoperative intravenous sedation administration during routine resident-performed cataract surgery among patients receiving pre-operative oral sedation at a Veterans Affairs Medical Center and its impact on patient safety and system cost. Methods: Retrospective review of all resident-performed cataract surgeries performed at the Iowa City Veterans Affairs Medical Center in 2013 and 2017. Cases monitored by a registered nurse were included. Combined cases and cases monitored by an anesthesia provider were excluded. Pre-operative placement of an intravenous (IV) catheter, administration of intra-operative IV sedation, oral diazepam administration, anesthesia type, conditions for administering intraoperative IV medication, and cost of IV catheter placement were recorded. Results: Of 1025 patient cases included for analysis, 972 received pre-operative diazepam (94.9%) and 1017 (99.3%) had IV catheters placed. One patient received a planned dose of IV methylprednisolone. Zero patients received supplemental intraoperative IV sedation. The estimated materials cost of unused IV catheters was $10,668 over 2 years. Conclusions: Pre-operative IV catheter placement may not be necessary in patients undergoing routine resident cataract surgery with pre-operative oral sedation. Discontinuation of routine IV placement may improve patient satisfaction and decrease health care costs without compromising patient safety.

Abstract Background Chronic kidney disease (CKD) is a devastating illness associated with increased mortality, reduced quality of life, impaired growth, neurocognitive impairment and psychosocial maladjustment in children. There is growing evidence of socioeconomic disparities in health outcomes among children with CKD. Patient navigators are trained non-medical personnel who assist patients with chronic conditions journey through the continuum of care and transit across different care settings. They help vulnerable and underserved populations to better understand their diagnosis, treatment options, and available resources, guide them through complex medical systems, and help them to overcome barriers to health care access. Given the complexity and chronicity of the disease process and concerns that current models of care may not adequately support the provision of high-level care in children with CKD from socioeconomically disadvantaged backgrounds, a patient navigator program may improve the provision of care and overall health of children with CKD. Methods The NAV-KIDS2 trial is a multi-centre, staggered entry, waitlisted randomised controlled trial assessing the health benefits and costs of a patient navigator program in children with CKD (stages 3–5, on dialysis, and with kidney transplants), who are of low socioeconomic backgrounds. Across 5 sites, 210 patients aged from 3 to 17 years will be randomised to immediate receipt of a patient navigator intervention for 24 weeks or waitlisting with standard care until receipt of a patient navigator at 24 weeks. The primary outcome is child self-rated health (SRH) 6-months after completion of the intervention. Other outcomes include utility-based quality of life, caregiver SRH, satisfaction with healthcare, progression of kidney dysfunction, other biomarkers, missed school days, hospitalisations and mortality. The trial also includes an economic evaluation and process evaluation, which will assess the cost-effectiveness, fidelity and barriers and enablers of implementing a patient navigator program in this setting. Discussion This study will provide clear evidence on the effectiveness and cost-effectiveness of a new intervention aiming to improve overall health and well-being for children with CKD from socioeconomically disadvantaged backgrounds, through a high quality, well-powered clinical trial. Trial registration Prospectively registered (12/07/2018) on the Australian New Zealand Clinical Trials Registry (ACTRN12618001152213).

Background Prematurity disrupts the perinatal maturation of the microvasculature and macrovasculature and confers high risk of vascular dysfunction later in life. No previous studies have investigated the crosstalk between the microvasculature and macrovasculature in childhood. Methods and Results In a case‐control study, we enrolled 55 children aged 11 years weighing

Abstract Background Objective Structured Clinical Exams are used to increase reliability and validity, yet they only achieve a modest level of reliability. This low reliability is due in part to examiner variance which is greater than the variance of students. This variance often represents indecisiveness at the cut score with apparent confusion over terms such as “borderline pass”. It is amplified by a well reported failure to fail. Methods A borderline grade (meaning performance is neither a clear pass nor a clear fail) was introduced in a high stakes undergraduate medical clinical skills exam to replace a borderline pass grade (which was historically resolved as 50%) in a 4 point scale (distinction, pass, borderline, fail). Each Borderline grade was then resolved into a Pass or Fail grade by a formula referencing the difficulty of the station and the performance in the same domain by the student in other stations. Raw pass or fail grades were unaltered. Mean scores and 95%CI were calculated per station and per domain for the unmodified and the modified scores/grades (results are presented on error bars). To estimate the defensibility of these modifications, similar analysis took place for the P and the F grades which resulted from the modification of the B grades. Results Of 14,634 observations 4.69% were Borderline. Application of the formula did not impact the mean scores in each domain but the failure rate for the exam increased from 0.7 to 4.1%. Examiners and students expressed satisfaction with the Borderline grade, resolution formula and outcomes. Mean scores (by stations and by domains respectively) of students whose B grades were modified to P were significantly higher than their counterparts whose B grades were modified to F. Conclusions This study provides a feasible and defensible resolution to situations where the examinee’s performance is neither a clear pass nor a clear fail, demonstrating the application of the resolution of borderline formula in a high stakes exam. It does not create a new performance standard but utilises real data to make judgements about these small number of candidates. This is perceived as a fair approach to Pass/Fail decisions.

The field of Dual use research of concern (DURC) has been rapidly expanding in the last decade. Though this has led to unprecedented breakthroughs in Life sciences and Technology, it is also associated with significant risks. These risks are enormous, transcend local and national boundaries and have the potential of catastrophic effects. Some of these include the release of highly infectious pathogens capable of sparking unnatural outbreaks, with resultant widespread illness and deaths. Yet Clinicians who are significant first responders involved in the management of cases in outbreaks, often lack relevant training on DURC and its associated risks. We propose that the World Health Organization (WHO) should develop global guidelines on the integration of relevant DURC training into undergraduate and postgraduate medical curricula. In addition, country-level national policy should be developed by each member country in line with their specific context.

Abstract Background Antimicrobial resistance is a major global health challenge. Metagenomics allows analyzing the presence and dynamics of “resistomes” (the ensemble of genes encoding antimicrobial resistance in a given microbiome) in disparate microbial ecosystems. However, the low sensitivity and specificity of available metagenomic methods preclude the detection of minority populations (often present below their detection threshold) and/or the identification of allelic variants that differ in the resulting phenotype. Here, we describe a novel strategy that combines targeted metagenomics using last generation in-solution capture platforms, with novel bioinformatics tools to establish a standardized framework that allows both quantitative and qualitative analyses of resistomes. Methods We developed ResCap, a targeted sequence capture platform based on SeqCapEZ (NimbleGene) technology, which includes probes for 8667 canonical resistance genes (7963 antibiotic resistance genes and 704 genes conferring resistance to metals or biocides), and 2517 relaxase genes (plasmid markers) and 78,600 genes homologous to the previous identified targets (47,806 for antibiotics and 30,794 for biocides or metals). Its performance was compared with metagenomic shotgun sequencing (MSS) for 17 fecal samples (9 humans, 8 swine). ResCap significantly improves MSS to detect “gene abundance” (from 2.0 to 83.2%) and “gene diversity” (26 versus 14.9 genes unequivocally detected per sample per million of reads; the number of reads unequivocally mapped increasing up to 300-fold by using ResCap), which were calculated using novel bioinformatic tools. ResCap also facilitated the analysis of novel genes potentially involved in the resistance to antibiotics, metals, biocides, or any combination thereof. Conclusions ResCap, the first targeted sequence capture, specifically developed to analyze resistomes, greatly enhances the sensitivity and specificity of available metagenomic methods and offers the possibility to analyze genes related to the selection and transfer of antimicrobial resistance (biocides, heavy metals, plasmids). The model opens the possibility to study other complex microbial systems in which minority populations play a relevant role.

Abstract Background Most assessments in health professions education consist of knowledge-based examinations as well as practical and clinical examinations. Among the most challenging aspects of clinical assessments is decision making related to borderline grades assigned by examiners. Borderline grades are commonly used by examiners when they do not have sufficient information to make clear pass/fail decisions. The interpretation of these borderline grades is rarely discussed in the literature. This study reports the application of the Objective Borderline Method (version 2, henceforth: OBM2) to a high stakes Objective Structured Clinical Examination undertaken at the end of the final year of a Medicine program in Australia. Methods The OBM2 uses all examination data to reclassify borderline grades as either pass or fail. Factor analysis was used to estimate the suitability of data for application of OBM2. Student’s t-tests, utilising bootstrapping, were used to compare the OBM2 with ‘traditional’ results. Interclass correlations were used to estimate the association between the grade reclassification and all other grades in this examination. Results The correlations between scores for each station and pass/fail outcomes increased significantly after the mark reclassification, yet the reclassification did not significantly impact on students’ total scores. Examiners, students and program leaders expressed high levels of satisfaction and the Faculty’s Curriculum Development Committee has decided that the OBM2 will be used for all future clinical examinations. Implications of the OBM2 are discussed. Conclusions The OBM2 provides a feasible, defensible and acceptable solution for classification of borderline grades as either pass or fail.

Until recently the in utero environment of pregnant women was considered sterile. Recent high-sensitivity molecular techniques and high-throughput sequencing lead to some evidence for a low-biomass microbiome associated with the healthy placenta. Other studies failed to reveal evidence for a consistent presence of bacteria using either culture or molecular based techniques. Comparing conflicting "placental microbiome" studies is complicated by the use of varied and inconsistent protocols. Given this situation, we undertook an evaluation of the in utero environment sterility using several controlled methods, in the same study, to evaluate the presence or absence of bacteria and to explain contradictions present in the literature. Healthy pregnant women (n = 38) were recruited in three maternity wards. Placenta were collected after cesarean section with or without Alexis® and vaginal delivery births. For this study we sampled fetal membranes, umbilical cord and chorionic villi. Bacterial presence was analyzed using bacterial culture and qPCR on 34 fetal membranes, umbilical cord and chorionic villi samples. Shotgun metagenomics was performed on seven chorionic villi samples. We showed that the isolation of meaningful quantities of viable bacteria or bacterial DNA was possible only outside the placenta (fetal membranes and umbilical cords) highlighting the importance of sampling methods in studying the in utero environment. Bacterial communities described by metagenomics analysis were similar in chorionic villi samples and in negative controls and were dependent on the database chosen for the analysis. We conclude that the placenta does not harbor a specific, consistent and functional microbiota.

The bacterial pathway for fatty acid biosynthesis, FASII, is a target for development of new anti-staphylococcal drugs. Here, Morvan et al. show that exogenous fatty acids can favour the emergence of staphylococcal strains displaying resistance to multiple FASII inhibitors.

Background: Somatic mutations in the ERBB genes (epidermal growth factor receptor: EGFR, ERBB2, ERBB3, ERBB4 ) promote oncogenesis and lapatinib resistance in metastatic HER2+ (human epidermal growth factor-like receptor 2) breast cancer in vitro . Our study aimed to determine the frequency of mutations in four genes: EGFR, ERBB2, ERBB3 and ERBB4 and to investigate whether these mutations affect cellular behaviour and therapy response in vitro and outcomes after adjuvant trastuzumab-based therapy in clinical samples. Methods: We performed Agena MassArray analysis of 227 HER2+ breast cancer samples to identify the type and frequency of ERBB family mutations. Of these, two mutations, the somatic mutations ERBB4 -V721I and ERBB4 -S303F, were stably transfected into HCC1954 (PIK3CA mutant), HCC1569 (PIK3CA wildtype) and BT474 (PIK3CA mutant, ER positive) HER2+ breast cancer cell lines for functional in vitro experiments. Results: A total of 12 somatic, likely deleterious mutations in the kinase and furin-like domains of the ERBB genes (3 EGFR , 1 ERBB2 , 3 ERBB3 , 5 ERBB4 ) were identified in 7% of HER2+ breast cancers, with ERBB4 the most frequently mutated gene. The ERBB4 -V721I kinase domain mutation significantly increased 3D-colony formation in 3/3 cell lines, whereas ERBB4 -S303F did not increase growth rate or 3D colony formation in vitro . ERBB4-V721I sensitized HCC1569 cells (PIK3CA wildtype) to the pan class I PI3K inhibitor copanlisib but increased resistance to the pan-HER family inhibitor afatinib. The combinations of copanlisib with trastuzumab, lapatinib, or afatinib remained synergistic regardless of ERBB4 -V721I or ERBB4 -S303F mutation status. Conclusions: ERBB gene family mutations, which are present in 7% of our HER2+ breast cancer cohort, may have the potential to alter cellular behaviour and the efficacy of HER- and PI3K-inhibition.

Semantic Web Services (SWS) are Web Service (WS) descriptions augmented with semantic information. SWS enable intelligent reasoning and automation in areas such as service discovery, composition, mediation, ranking and invocation. This paper applies SWS to a previous protocol adapter which, operating within clearly defined constraints, maps SOAP Web Services to RESTful HTTP format. However, in the previous adapter, the configuration element is manual and the latency implications are locally based. This paper applies SWS technologies to automate the configuration element and the latency tests are conducted in a more realistic Internet based setting.

Twin and family studies have established the contribution of genetic factors to variation in metabolic, hematologic and immunological parameters. The majority of these studies analyzed single or combined traits into pre-defined syndromes. In the present study, we explore an alternative multivariate approach in which a broad range of metabolic, hematologic, and immunological traits are analyzed simultaneously to determine the resemblance of monozygotic (MZ) twin pairs, twin-spouse pairs and unrelated, non-cohabiting individuals. A total of 517 participants from the Netherlands Twin Register, including 210 MZ twin pairs and 64 twin-spouse pairs, took part in the study. Data were collected on body composition, blood pressure, heart rate, and multiple biomarkers assessed in fasting blood samples, including lipid levels, glucose, insulin, liver enzymes, hematological measurements and cytokine levels. For all 51 measured traits, pair-wise Pearson correlations, correcting for family relatedness, were calculated across all the individuals in the cohort. Hierarchical clustering techniques were applied to group the measured traits into sub-clusters based on similarity. Sub-clusters were observed among metabolic traits and among inflammatory markers. We defined a phenotypic profile as the collection of all the traits measured for a given individual. Average within-pair similarity of phenotypic profiles was determined for the groups of MZ twin pairs, spouse pairs and pairs of unrelated individuals. The average similarity across the full phenotypic profile was higher for MZ twin pairs than for spouse pairs, and lowest for pairs of unrelated individuals. Cohabiting MZ twins were more similar in their phenotypic profile compared to MZ twins who no longer lived together. The correspondence in the phenotypic profile is therefore determined to a large degree by familial, mostly genetic, factors, while household factors contribute to a lesser degree to profile similarity.

Cheese ripening is a complex biochemical process driven by microbial communities composed of both eukaryotes and prokaryotes. Surface-ripened cheeses are widely consumed all over the world and are appreciated for their characteristic flavor. Microbial community composition has been studied for a long time on surface-ripened cheeses, but only limited knowledge has been acquired about its in situ metabolic activities. We applied metagenomic, metatranscriptomic and biochemical analyses to an experimental surface-ripened cheese composed of nine microbial species during four weeks of ripening. By combining all of the data, we were able to obtain an overview of the cheese maturation process and to better understand the metabolic activities of the different community members and their possible interactions. Furthermore, differential expression analysis was used to select a set of biomarker genes, providing a valuable tool that can be used to monitor the cheese-making process.

CD8(+) T cells infiltrate the brain during an anti-viral immune response. Within the brain CD8(+) T cells recognize cells expressing target antigens, become activated, and secrete IFNγ. However, there are no methods to recognize individual cells that respond to IFNγ. Using a model that studies the effects of the systemic anti-adenoviral immune response upon brain cells infected with an adenoviral vector in mice, we describe a method that identifies individual cells that respond to IFNγ. To identify individual mouse brain cells that respond to IFNγ we constructed a series of adenoviral vectors that contain a transcriptional response element that is selectively activated by IFNγ signaling, the gamma-activated site (GAS) promoter element; the GAS element drives expression of a transgene, Cre recombinase (Ad-GAS-Cre). Upon binding of IFNγ to its receptor, the intracellular signaling cascade activates the GAS promoter, which drives expression of the transgene Cre recombinase. We demonstrate that upon activation of a systemic immune response against adenovirus, CD8(+) T cells infiltrate the brain, interact with target cells, and cause an increase in the number of cells expressing Cre recombinase. This method can be used to identify, study, and eventually determine the long term fate of infected brain cells that are specifically targeted by IFNγ. The significance of this method is that it will allow to characterize the networks in the brain that respond to the specific secretion of IFNγ by anti-viral CD8(+) T cells that infiltrate the brain. This will allow novel insights into the cellular and molecular responses underlying brain immune responses.

Parasitological analyses of the third Pavilion du Levant and the Royal Pavilion are in continuity with those carried out by Françoise Bouchet on the latrines of the second Pavilion du Levant, published in 1998. The initial objective is to confirm the excremental nature of these different deposits and then to reconstitute the health status and microbial population of the intestinal flora of the subjects of Louis XIV’s court. To answer this question, a large multidisciplinary scientific team intervened to carry out the various parasitological and molecular analyses. This first report sets out the means put in place as well as the results relating to optical microscopy, toxicological and metagenomic data.