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As screening strategies employ better biomarkers and genetics to identify individuals at an increased risk of prostate cancer, there are currently no chemotherapeutic prevention strategies. With any chemoprevention strategy, the population will be younger and healthier; therefore, they will be less tolerant of side effects. This study translated findings from screening a natural product library and pre-clinical evaluation of curcumin (CURC) in combination with ursolic acid (UA) in prostate cancer models. After manufacturing capsules for each compound, 18 subjects were enrolled. The study used a 3 × 3 phase 1 clinical trial to evaluate CURC (1200 mg/day) and UA (300 mg/day) alone and in combination over a 2-week period with endpoints of safety, bioavailability, and microbiome alterations. After enrolling six subjects in each arm, we found no grade 3 or 4 events and only minor changes in the safety laboratory values. In the pooled analysis of groups, we noted a statistically significant difference between median serum levels of UA when administered alone vs administered in the combination (2.7 ng/mL vs 43.8 ng/mL, p = 0.03). Individuals receiving the combination also had a favorable impact on gut microbiome status and a reduction in “microbiome score” predictive of prostate cancer risk.

Here, the authors present ConQuR, a conditional quantile regression method that removes microbiome batch effects through non-parametric modeling of complex microbial read counts, while preserving the signals of interest.

Detection of mutational signatures in cell-free DNA (cfDNA) is challenging due to low sequence coverage and low mutant allele fractions. Here, the authors identify mutational signatures in plasma whole genome sequencing of cancer patients and use machine learning to distinguish them from healthy individuals.

The genomic profiling of tumours has not been widely adopted in the clinic due to technical and practical hurdles. Here, the authors develop PGDx elio tissue complete, a scalable, standardised and FDA-cleared test comprising a targeted gene panel and automated machine-learning analysis, which detects clinically relevant sequence biomarkers in cancer samples.

The Fall Midwater Trawl (FMWT) Survey has been conducted near continuously since 1967 to assess the abundance and distribution of pelagic fish species throughout the San Francisco Bay/Sacramento-San Joaquin estuary (Bay Delta). For most of this period, sampling 100 core stations provided data for abundance and distribution analyses. Another 22 (non-core) stations were added to the FMWT 8 to 28 years ago to supplement the original 100 (core) stations. However, relative abundance indices are published annually from only the data collected at the core stations. Here we incorporate data from non-core stations along with core station data to calculate an alternative index that also integrates modern estimates of water volume within the Bay Delta into an index calculation. The use of data from non-core stations in calculating the alternative index was particularly useful for American Shad (Alosa sapidissima) and Threadfin Shad (Dorosoma petenense). Consistently high catches at non-core stations for a couple species and modest catches for a couple additional species highlight the value of these additional catch data for our understanding of how fishes are distributed in the estuary.

Recent studies in cancer patients and animal models demonstrate that intestinal microbiota influence the therapeutic efficacy of cancer treatments, including immune checkpoint inhibition. However, no studies to-date have investigated relationships between gastrointestinal microbiota composition and response to checkpoint inhibition in advanced metastatic castrate resistant prostate cancer (mCRPC). We performed 16S rRNA gene sequencing of fecal DNA from 23 individuals with mCRPC progressing on enzalutamide and just prior to treatment with anti-PD-1 (pembrolizumab) to determine whether certain features of the microbiome are associated with treatment response (defined as serum PSA decrease >50% at any time on treatment or radiographic response per RECIST V.1.1). Global bacterial composition was similar between responders and non-responders, as assessed by multiple alpha and beta diversity metrics. However, certain bacterial taxa identified by sequencing across multiple 16S rRNA hypervariable regions were consistently associated with response, including the archetypal oral bacterium Streptococcus salivarius. Quantitative PCR (qPCR) of DNA extracts from fecal samples confirmed increased Streptococcus salivarius fecal levels in responders, whereas qPCR of oral swish DNA extracts showed no relationship between oral Streptococcus salivarius levels and response status. Contrary to previous reports in other cancer types, Akkermansia muciniphila levels were reduced in responder samples as assessed by both 16S rRNA sequencing and qPCR. We further analyzed our data in the context of a previously published “integrated index” describing bacteria associated with response and non-response to checkpoint inhibition. We found that the index was not reflective of response status in our cohort. Lastly, we demonstrate little change in the microbiome over time, and with pembrolizumab treatment. Our results suggest that the association between fecal microbiota and treatment response to immunotherapy may be unique to cancer type and/or previous treatment history.

DNA from tumour cells can be detected in the blood of cancer patients. Here, the authors show that cell free DNA fragmentation patterns can identify lung cancer patients and when this information is further interrogated it can be used to predict lung cancer histological subtype.

Abstract Prosthetically driven workflows using CBCT, digital optical scanning, 3D‐printed molds and frameworks, and dental implant component attachments to osseointegrated fixtures can produce anatomically accurate, esthetic, durable silicone ear replacements.

Abstract Integration of CBCT imaging with dental implant treatment planning software and 3D‐printed surgical guides can facilitate surgical planning for extraoral implant placement. In the current case, this combined planning strategy enabled navigation of challenging osseous anatomy, avoided critical structures, and optimized osseointegration for prosthesis retention.

In the intestine, the Na+/H+ exchanger 3 (NHE3) plays a critical role for Na+ and fluid absorption. NHE3 deficiency predisposes patients to inflammatory bowel disease (IBD). In mice, selective deletion of intestinal NHE3 causes various local and systemic pathologies due to dramatic changes in the intestinal environment, which can influence microbiota colonization. By using metagenome shotgun sequencing, we determined the effect of inducible intestinal epithelial cell-specific deletion of NHE3 (NHE3IEC-KO) in adulthood on the gut microbiome in mice. Compared with control mice, NHE3IEC-KO mice show a significantly different gut microbiome signature, with an unexpected greater diversity. At the phylum level, NHE3IEC-KO mice showed a significant expansion in Proteobacteria and a tendency for lower Firmicutes/Bacteroidetes (F/B) ratio, an indicator of dysbiosis. At the family level, NHE3IEC-KO mice showed significant expansions in Bacteroidaceae, Rikenellaceae, Tannerellaceae, Flavobacteriaceae and Erysipelotrichaceae, but had contractions in Lachnospiraceae, Prevotellaceae and Eubacteriaceae. At the species level, after removing those with lowest occurrence and abundance, we identified 23 species that were significantly expanded (several of which are established pro-inflammatory pathobionts); whereas another 23 species were found to be contracted (some of which are potential anti-inflammatory probiotics) in NHE3IEC-KO mice. These results reveal that intestinal NHE3 deletion creates an intestinal environment favoring the competitive advantage of inflammophilic over anti-inflammatory species, which is commonly featured in conventional NHE3 knockout mice and patients with IBD. In conclusion, our study emphasizes the importance of intestinal NHE3 for gut microbiota homeostasis, and provides a deeper understanding regarding interactions between NHE3, dysbiosis, and IBD.

Short read 16 S rRNA amplicon sequencing is a common technique used in microbiome research. However, inaccuracies in estimated bacterial community composition can occur due to amplification bias of the targeted hypervariable region. A potential solution is to sequence and assess multiple hypervariable regions in tandem, yet there is currently no consensus as to the appropriate method for analyzing this data. Additionally, there are many sequence analysis resources for data produced from the Illumina platform, but fewer open-source options available for data from the Ion Torrent platform. Herein, we present an analysis pipeline using open-source analysis platforms that integrates data from multiple hypervariable regions and is compatible with data produced from the Ion Torrent platform. We used the ThermoFisher Ion 16 S Metagenomics Kit and a mock community of twenty bacterial strains to assess taxonomic classification of six amplicons from separate hypervariable regions (V2, V3, V4, V6-7, V8, V9) using our analysis pipeline. We report that different amplicons have different specificities for taxonomic classification, which also has implications for global level analyses such as alpha and beta diversity. Finally, we utilize a generalized linear modeling approach to statistically integrate the results from multiple hypervariable regions and apply this methodology to data from a representative clinical cohort. We conclude that examining sequencing results across multiple hypervariable regions provides more taxonomic information than sequencing across a single region. The data across multiple hypervariable regions can be combined using generalized linear models to enhance the statistical evaluation of overall differences in community structure and relatedness among sample groups.

Neoplastic pancreatic cysts are associated with invasive pancreatic cancer, but their origins and evolutionary relationships are unclear. Here, the authors present the evolutionary analysis of neoplastic cysts and report them as precursors of invasive pancreatic cancer, and that SMAD4/TGFBR2 alterations are likely drivers of invasion in a subset of cases.

Background & Aims: In short-bowel syndrome (SBS), inadequate intestinal adaptation is responsible for the majority of complications, including sepsis, liver failure, and death. In this study, we sought to further delineate the adaptive response to identify potential therapeutic targets. Methods: We performed a 75% small-bowel resection (SBR) or sham operation on C57Bl/6J wild-type (WT), lipocalin-2 (LCN2)-/-, and interleukin 22 (IL22)-/- mice. Exogenous IL22 was administered to SBR WT mice. Cecal fecal matter from SBR WT and SBR LCN2-/- mice were transplanted into germ-free mice. Intestinal permeability, inflammation, proliferation, and the microbiome were evaluated 1 week after surgery. CD4+IL22+ laminal propria lymphocytes were sorted by flow cytometry. Naïve T cells were polarized to T-helper cells with or without LCN2. Results: A 75% SBR in a mouse re-creates the increased intestinal permeability, enterocyte proliferation, and intestinal dysbiosis seen in SBS. LCN2 expression increases after 75% SBR, and this increase can be abrogated with broad-spectrum antibiotic treatment. LCN2-/- mice have less intestinal inflammation, increased IL22 expression, and greater adaptation as evidenced by less intestinal permeability, increased carbohydrate enzyme expression, less weight loss, and less dysbiosis after 75% SBR than WT mice. The proinflammatory and anti-adaptive effects of LCN2 can be transferred to germ-free mice via a fecal transplant. Administration of exogenous IL22 improves adaptation and restores the normal microbiome after 75% SBR in WT mice. Conclusions: LCN2 promotes inflammation and slows intestinal adaptation through changes in the microbiome and IL22 inhibition in a mouse SBS model. Strategies to reduce LCN2 may offer novel therapeutic approaches to enhance adaptation in SBS.

Purpose: Fluoroquinolone-resistant (FQR) Escherichia coli causes transrectal prostate biopsy infections. In order to reduce colonization of these bacteria in carriers, we would like to understand the surrounding microbiome to determine targets for decolonization. Materials and Methods: We perform an observational study to investigate the microbiome differences in men with and without FQR organisms found on rectal culture. A rectal swab with two culturettes was performed on men before an upcoming prostate biopsy procedure as standard of care to perform “targeted prophylaxis.” Detection of FQR was performed by the standard microbiology lab inoculates the swab onto MacConkey agar containing ciprofloxacin. The extra swab was sent for 16S rRNA amplicon sequencing (MiSeq paired-end) using the V1V2 primer. Alpha and beta-diversity analysis were performed using QIIME. We used PERMANOVA to evaluate the statistical significance of beta-diversity distances within and between groups of interest. Results: We collected 116 rectal swab samples before biopsy for 16S rRNA amplicon sequencing. We identified 18 isolates (15.5%, 18/116) that were positive and had relative reduced diversity profiles (p

Objective. To perform a proof of concept microbiome evaluation and PD-L1 expression profiling in clear-cell renal cell carcinoma (cc-RCC) with associated tumor thrombus (TT). Methods. After IRB approval, six patients underwent radical nephrectomy (RN) with venous tumor thrombectomy (VTT). We collected fresh tissue specimens from normal adjacent, tumor, and thrombus tissues. We utilized RNA sequencing to obtain PD-L1 expression profiles and perform microbiome analysis. Statistical assessment was performed using Student’s t-test, chi-square, and spearman rank correlations using SPSS v25. Results. We noted the tumor thrombus to be mostly devoid of diverse microbiota. A large proportion of Staphylococcus epidermidus was detected and unknown if this is a surgical or postsurgical contaminant; however, it was noted more in the thrombus than other tissues. Microbiome diversity profiles were most abundant in the primary tumor compared to the thrombus or normal adjacent tissue. Differential expression of PD-L1 was examined in the tumor thrombus to the normal background tissue and noted three of the six subjects had a threshold above 2-fold. These three similar subjects had foreign microbiota that are typical residents of the oral microbiome. Conclusion. Renal tumors have more diverse microbiomes than normal adjacent tissue. Identification of resident oral microbiome profiles in clear-cell renal cancer with tumor thrombus provides a potential biomarker for thrombus response to PD-L1 inhibition.

Abstract Background Lung cancer is the leading cancer diagnosis worldwide and the number one cause of cancer deaths. Exposure to cigarette smoke, the primary risk factor in lung cancer, reduces epithelial barrier integrity and increases susceptibility to infections. Herein, we hypothesize that somatic mutations together with cigarette smoke generate a dysbiotic microbiota that is associated with lung carcinogenesis. Using lung tissue from 33 controls and 143 cancer cases, we conduct 16S ribosomal RNA (rRNA) bacterial gene sequencing, with RNA-sequencing data from lung cancer cases in The Cancer Genome Atlas serving as the validation cohort. Results Overall, we demonstrate a lower alpha diversity in normal lung as compared to non-tumor adjacent or tumor tissue. In squamous cell carcinoma specifically, a separate group of taxa are identified, in which Acidovorax is enriched in smokers. Acidovorax temporans is identified within tumor sections by fluorescent in situ hybridization and confirmed by two separate 16S rRNA strategies. Further, these taxa, including Acidovorax, exhibit higher abundance among the subset of squamous cell carcinoma cases with TP53 mutations, an association not seen in adenocarcinomas. Conclusions The results of this comprehensive study show both microbiome-gene and microbiome-exposure interactions in squamous cell carcinoma lung cancer tissue. Specifically, tumors harboring TP53 mutations, which can impair epithelial function, have a unique bacterial consortium that is higher in relative abundance in smoking-associated tumors of this type. Given the significant need for clinical diagnostic tools in lung cancer, this study may provide novel biomarkers for early detection.

Following publication of the original paper [1], the authors submitted a new Additional file 5 to replace the one containing formatting issues. The updated Additional file 5 is published in this correction.

Food microbiome composition impacts food safety and quality. The resident microbiota of many food products is influenced throughout the farm to fork continuum by farming practices, environmental factors, and food manufacturing and processing procedures. Currently, most food microbiology studies rely on culture-dependent methods to identify bacteria. However, advances in high-throughput DNA sequencing technologies have enabled the use of targeted 16S rRNA gene sequencing to profile complex microbial communities including non-culturable members. In this study we used 16S rRNA gene sequencing to assess the microbiome profiles of plant and animal derived foods collected at two points in the manufacturing process; post-harvest/pre-retail (cilantro) and retail (cilantro, masala spice mixes, cucumbers, mung bean sprouts, and smoked salmon). Our findings revealed microbiome profiles, unique to each food, that were influenced by the moisture content (dry spices, fresh produce), packaging methods, such as modified atmospheric packaging (mung bean sprouts and smoked salmon), and manufacturing stage (cilantro prior to retail and at retail). The masala spice mixes and cucumbers were comprised mainly of Proteobacteria, Firmicutes, and Actinobacteria. Cilantro microbiome profiles consisted mainly of Proteobacteria, followed by Bacteroidetes, and low levels of Firmicutes and Actinobacteria. The two brands of mung bean sprouts and the three smoked salmon samples differed from one another in their microbiome composition, each predominated by either by Firmicutes or Proteobacteria. These data demonstrate diverse and highly variable resident microbial communities across food products, which is informative in the context of food safety, and spoilage where indigenous bacteria could hamper pathogen detection, and limit shelf life.

Toxoplasma gondii is an obligate intracellular parasite with worldwide distribution. Felines are the definitive hosts supporting the complete life cycle of T. gondii. However, other warm-blooded animals such as rodents and humans can also be infected. Infection of such secondary hosts results in long-term infection characterized by the presence of tissue cysts in the brain and other organs. While it is known that T. gondii infection in rodents is associated with behavioral changes, the mechanisms behind these changes remain unclear. Alterations of the host intestinal microflora are recognized as a prominent role player in shaping host behavior and cognition. It has been shown that acute T. gondii infection of mice results in microflora changes as a result of gastrointestinal inflammation in inbred mouse models. The long-term effects of chronic T. gondii infection on microbial communities, however, are unknown. In this study, after we verified using our model in terms of measuring microflora changes during an acute episode of toxoplasmosis, we assessed the microbiome changes that occur during a long-term infection; then we further investigated these changes in a follow-up study of chronic infection. These analyses were performed by constructing and sequencing 16S rRNA amplicon DNA libraries from small intestine fecal specimens. We found that acute infection with the GT1 strain of T. gondii caused an enrichment of Bacteroidetes compared with controls in CD1 mice. Strikingly, this enrichment upheld throughout long-term chronic infection. The potential biological consequences of this alteration in rodents and humans should be subjected to further exploration.

16S rRNA community profiling continues to be a useful tool to study microbiome composition and dynamics, in part due to advances in next generation sequencing technology that translate into reductions in cost. Reliable taxonomic identification to the species-level, however, remains difficult, especially for short-read sequencing platforms, due to incomplete coverage of the 16S rRNA gene. This is especially true for Salmonella enterica, which is often found as a low abundant member of the microbial community, and is often found in combination with several other closely related enteric species. Here, we report on the evaluation and application of Resphera Insight, an ultra-high resolution taxonomic assignment algorithm for 16S rRNA sequences to the species level. The analytical pipeline achieved 99.7% sensitivity to correctly identify S. enterica from WGS datasets extracted from the FDA GenomeTrakr Bioproject, while demonstrating 99.9% specificity over other Enterobacteriaceae members. From low-diversity and low-complexity samples, namely ice cream, the algorithm achieved 100% specificity and sensitivity for Salmonella detection. As demonstrated using cilantro and chili powder, for highly complex and diverse samples, especially those that contain closely related species, the detection threshold will likely have to be adjusted higher to account for misidentifications. We also demonstrate the utility of this approach to detect Salmonella in the clinical setting, in this case, bloodborne infections.

Flexibility in behavior is advantageous for organisms that transition between stages of a complex life history. However, various constraints can set limits on plasticity, giving rise to the existence of personalities that have associated costs and benefits. Here, we document a field and laboratory experiment that examines the consistency of measures of boldness, activity, and aggressive behavior in the young of a tropical reef fish, Pomacentrus amboinensis (Pomacentridae) immediately following their transition between pelagic larval and benthic juvenile habitats. Newly-settled fish were observed in aquaria and in the field on replicated patches of natural habitat cleared of resident fishes. Seven behavioral traits representing aspects of boldness, activity and aggression were monitored directly and via video camera over short (minutes), medium (hours), and long (3 days) time scales. With the exception of aggression, these behaviors were found to be moderately or highly consistent over all time scales in both laboratory and field settings, implying that these fish show stable personalities within various settings. Our study is the first to examine the temporal constancy of behaviors in both field and laboratory settings in over various time scales at a critically important phase during the life cycle of a reef fish.

The syndecans are the major family of transmembrane proteoglycans, usually bearing multiple heparan sulfate chains. They are present on virtually all nucleated cells of vertebrates and are also present in invertebrates, indicative of a long evolutionary history. Genetic models in both vertebrates and invertebrates have shown that syndecans link to the actin cytoskeleton and can fine-tune cell adhesion, migration, junction formation, polarity and differentiation. Although often associated as co-receptors with other classes of receptors (e.g. integrins, growth factor and morphogen receptors), syndecans can nonetheless signal to the cytoplasm in discrete ways. Syndecan expression levels are upregulated in development, tissue repair and an array of human diseases, which has led to the increased appreciation that they may be important in pathogenesis not only as diagnostic or prognostic agents, but also as potential targets. Here, their functions in development and inflammatory diseases are summarized, including their potential roles as conduits for viral pathogen entry into cells.

Syndecans are a four member multifunctional family of cell surface molecules with diverse biological roles. Syndecan-3 (SDC3) is the largest of these, but in comparison to the other family members relatively little is known about this molecule. SDC3 null mice grow and develop normally, all be it with subtle anatomical phenotypes in the brain. Roles for this molecule in both neuronal and brain tissue have been identified, and is associated with altered satiety responses. Recent studies suggest that SDC3 expression is not restricted to neuronal tissues and has important roles in inflammatory disorders such as rheumatoid arthritis, disease associated processes such as angiogenesis and in the facilitation of infection of dendritic cells by HIV. The purpose of this review article is to explore these new biological insights into SDC3 functions in inflammatory disease.

The potential of induced pluripotent stem cells (iPSCs) in disease modeling and regenerative medicine is vast, but current methodologies remain inefficient. Understanding the cellular mechanisms underlying iPSC reprogramming, such as the metabolic shift from oxidative to glycolytic energy production, is key to improving its efficiency. We have developed a lentiviral reporter system to assay longitudinal changes in cell signaling and transcription factor activity in living cells throughout iPSC reprogramming of human dermal fibroblasts. We reveal early NF-κB, AP-1, and NRF2 transcription factor activation prior to a temporal peak in hypoxia inducible factor α (HIFα) activity. Mechanistically, we show that an early burst in oxidative phosphorylation and elevated reactive oxygen species generation mediates increased NRF2 activity, which in turn initiates the HIFα-mediated glycolytic shift and may modulate glucose redistribution to the pentose phosphate pathway. Critically, inhibition of NRF2 by KEAP1 overexpression compromises metabolic reprogramming and results in reduced efficiency of iPSC colony formation.

Many exercisers listen to their favorite music during thei r workouts, and this has been shown to increase exercise enjoyment and decrease perceptions of exercise exertion. This study investigated whether suggestion of increased enjoyment from using music would create an expectancy effect that would, in turn, influence actual reported enjoyment of an exercise session. Participants (N=69)intending to voluntarily exercise at a college wellness center while listening to self-chosen music were invited to participate in the study. All participants were asked to volunteer to fill in an exercise enjoyment scale after their session, but using a simplerandomized experimental design in a field setting, expectancy of exercise enjoyment was manipulated by telling half of them that their choice of music was already known to improve exercise enjoyment before they began their workouts. There was a significant effect (p < 0.05) on reported exercise enjoyment with the experimental group scoring higher (ES = 0.48) on the modified PACES scale. This preliminary study indicates creating expectancy can influence the effect of music on reported exercise enjoyment. This effect might have measurement implications for future research and possible practical implications for the promotion of physicalactivity for health and wellness reasons.

We analyzed data from two independent samples to assess the predictive relationships between eating disorder risk (RISK) and physical self-esteem and social physique anxiety. Female college athletes completed the Physical Self-Perception Profile (PSPP), the Social Physique Anxiety Scale (SPAS), and the Drive For Thinness (THIN), Bulimia (BUL), and Body Dissatisfaction (BDIS) subscales of the Eating Disorder Inventory. They also completed socially desirability (SD) scales. Our analyses assessed the unique contribution of BDIS to risk prediction (THIN and BUL) after SD, SPAS and PSPP had been statistically controlled for. The associations between variables, and the predictive contributions of PSPP, SPAS, and BDIS to eating disorder risk as measured by THIN were similar in both studies. However, we found dissimilar results with regard to prediction of BUL. The R2 shrinkage between studies suggests satisfactory cross validation with respect to predicting THIN, but not for the prediction of BUL. No scales were unduly prone to distortion by SD responding. These data support the hypothesis that PSPP and SPAS may contribute to predicting aspects of eating disorder risk, and further study of the contributions of PSPP and SPAS to eating disorder risk seems to be warranted.

Syndecan-3 is one of the four members of the syndecan family of heparan sulphate proteoglycans and has been shown to interact with numerous growth factors via its heparan sulphate chains. The extracellular core proteins of syndecan-1,-2 and -4 all possess adhesion regulatory motifs and we hypothesized that syndecan-3 may also possess such characteristics. Here we show that a bacterially expressed GST fusion protein consisting of the entire mature syndecan-3 ectodomain has anti-angiogenic properties and acts via modulating endothelial cell migration. This work identifies syndecan-3 as a possible therapeutic target for anti-angiogenic therapy.

Background We conducted the first trial of neoadjuvant PD-1 blockade in resectable non-small cell lung cancer (NSCLC), finding nivolumab monotherapy to be safe and feasible with an encouraging rate of pathologic response. Building on these results, and promising data for nivolumab plus ipilimumab (anti-CTLA-4) in advanced NSCLC, we expanded our study to include an arm investigating neoadjuvant nivolumab plus ipilimumab.Methods Patients with resectable stage IB (≥4 cm)–IIIA (American Joint Committee on Cancer Tumor Node Metastases seventh edition), histologically confirmed, treatment-naïve NSCLC received nivolumab 3 mg/kg intravenously plus ipilimumab 1 mg/kg intravenously 6 weeks prior to planned resection. Nivolumab 3 mg/kg was given again approximately 4 and 2 weeks preoperatively. Primary endpoints were safety and feasibility with a planned enrollment of 15 patients. Pathologic response was a key secondary endpoint.Results While the treatment regimen was feasible per protocol, due to toxicity, the study arm was terminated early by investigator consensus after 9 of 15 patients were enrolled. All patients received every scheduled dose of therapy and were fit for planned surgery; however, 6 of 9 (67%) experienced treatment-related adverse events (TRAEs) and 3 (33%) experienced grade ≥3 TRAEs. Three of 9 patients (33%) had biopsy-confirmed tumor progression precluding definitive surgery. Of the 6 patients who underwent resection, 3 are alive and disease-free, 2 experienced recurrence and are actively receiving systemic treatment, and one died postoperatively due to acute respiratory distress syndrome. Two patients who underwent resection had tumor pathologic complete responses (pCRs) and continue to remain disease-free over 24 months since surgery. Pathologic response correlated with pre-treatment tumor PD-L1 expression, but not tumor mutation burden. Tumor KRAS/STK11 co-mutations were identified in 5 of 9 patients (59%), of whom two with disease progression precluding surgery had tumor KRAS/STK11/KEAP1 co-mutations.Conclusions Though treatment was feasible, due to toxicity the study arm was terminated early by investigator consensus. In light of this, and while the long-term disease-free status of patients who achieved pCR is encouraging, further investigation of neoadjuvant nivolumab plus ipilimumab in patients with resectable NSCLC requires the identification of predictive biomarkers that enrich for response.

Background Tumor mutational burden (TMB), defined as the number of somatic mutations per megabase of interrogated genomic sequence, demonstrates predictive biomarker potential for the identification of patients with cancer most likely to respond to immune checkpoint inhibitors. TMB is optimally calculated by whole exome sequencing (WES), but next-generation sequencing targeted panels provide TMB estimates in a time-effective and cost-effective manner. However, differences in panel size and gene coverage, in addition to the underlying bioinformatics pipelines, are known drivers of variability in TMB estimates across laboratories. By directly comparing panel-based TMB estimates from participating laboratories, this study aims to characterize the theoretical variability of panel-based TMB estimates, and provides guidelines on TMB reporting, analytic validation requirements and reference standard alignment in order to maintain consistency of TMB estimation across platforms.Methods Eleven laboratories used WES data from The Cancer Genome Atlas Multi-Center Mutation calling in Multiple Cancers (MC3) samples and calculated TMB from the subset of the exome restricted to the genes covered by their targeted panel using their own bioinformatics pipeline (panel TMB). A reference TMB value was calculated from the entire exome using a uniform bioinformatics pipeline all members agreed on (WES TMB). Linear regression analyses were performed to investigate the relationship between WES and panel TMB for all 32 cancer types combined and separately. Variability in panel TMB values at various WES TMB values was also quantified using 95% prediction limits.Results Study results demonstrated that variability within and between panel TMB values increases as the WES TMB values increase. For each panel, prediction limits based on linear regression analyses that modeled panel TMB as a function of WES TMB were calculated and found to approximately capture the intended 95% of observed panel TMB values. Certain cancer types, such as uterine, bladder and colon cancers exhibited greater variability in panel TMB values, compared with lung and head and neck cancers.Conclusions Increasing uptake of TMB as a predictive biomarker in the clinic creates an urgent need to bring stakeholders together to agree on the harmonization of key aspects of panel-based TMB estimation, such as the standardization of TMB reporting, standardization of analytical validation studies and the alignment of panel-based TMB values with a reference standard. These harmonization efforts should improve consistency and reliability of panel TMB estimates and aid in clinical decision-making.

Background Immune checkpoint inhibitors (ICIs) have expanded treatment options for metastatic renal cell carcinoma (mRCC); however, there are limited predictive biomarkers for response to ICIs in this indication, with programmed death-ligand 1 (PD-L1) status demonstrating little predictive utility in mRCC. While predictive of ICI response in other tumor types, the utility of tumor mutation burden (TMB) in mRCC is unclear. Here, we assess TMB, loss of antigen presentation genes and PD-L1 status correlated with outcomes to ICI treatment in mRCC.Methods Tumor samples from 34 patients with mRCC treated with ICI therapy at Duke Cancer Institute were retrospectively evaluated using Personal Genome Diagnostics elio tissue complete (RUO version), a tumor genomic profiling assay for somatic variants, TMB, microsatellite status and genomic status of antigen presentation genes. Tumor samples were also analyzed with the Dako 28-8 PD-L1 immunohistochemistry assay. Deidentified clinical information was extracted from the medical record, and tumor response was evaluated based on the Response Evaluation Criteria In Solid Tumors (RECIST) V.1.1 criteria.Results Patients were stratified by overall response following ICI therapy and designated as progressive disease (PD; n=18) or disease control groups (DC; n=16). TMB scores ranged from 0.36 to 12.24 mutations/Mb (mean 2.83 mutations/Mb) with no significant difference between the PD and DC groups (3.01 vs 2.63 mutations/Mb, respectively; p=0.7682). Interestingly, 33% of PD patients displayed loss of heterozygosity of major histocompatibility complex class I genes (LOH-MHC) vs 6% of DC patients. Nine of 34 samples were PD-L1-positive (4 in the PD group; 5 in the DC group), suggesting no correlation between PD-L1 expression and response to ICI therapy. Notably, the DC group displayed an enrichment of mutations in DNA repair genes (p=0.04), with 68.8% exhibiting at least one mutated homologous recombination repair (HRR)-related gene compared with only 38.9% of the PD group (p=0.03).Conclusions Overall, neither TMB nor PD-L1 correlated with ICI response and TMB was not significantly associated with PD-L1 expression. The higher incidence of LOH-MHC in PD group suggests that loss of antigen presentation may restrict response to ICIs. Separately, enrichment of HRR gene mutations in the DC group suggests potential utility in predicting ICI response and a potential therapeutic target, warranting future studies.

There is an extensive literature establishing, validating, and quantifying a wide range of responses of fishes to fasting. Our study complements this work by comparing fed and unfed treatments of hatchery-raised Delta Smelt (Hypomesus transpacificus)-an imperiled fish that is endemic to the San Francisco Estuary and its tributaries in California, USA-across a diverse suite of endpoints over a two-month time series. The experiment was conducted at 15.9°C, and individuals were sampled at 12 time points as starvation became increasingly severe. We found that hepatosomatic index and condition factor were relatively sensitive to starvation, becoming significantly depressed at Day 4 and 7, respectively. Histological analysis of liver showed elevated cytoplasmic inclusion bodies at Day 7, followed by increased glycogen depletion, single cell necrosis, and hydropic vacuolar degeneration at Day 14, 21, and 28, respectively. Of four antioxidants measured, glutathione decreased at Day 4, superoxide dismutase increased at Day 14, catalase increased at Day 56, and glutathione peroxidase was not affected by starvation. The net result was a ~2-fold increase in lipid peroxidation (malondialdehyde) in fasted fish that was highly inconsistent through time. RNA to DNA ratio and triglycerides in muscle were relatively insensitive to starvation, only consistently decreasing with fasting after mortality began increasing in the 'No Feeding' treatment, at Day 21. Together, these results suggest that Delta Smelt mobilize hepatic energy stores far more rapidly than lipids in muscle when subjected to fasting, leading to rapid atrophy of liver and the development of cytoplasmic inclusion bodies-possibly autophagosomes-in hepatocytes.

Este trabalho analisa alguns aspectos da fotodegradação heterogênea do polipropileno (PP) e compósitos de PP com talco. Amostras moldadas por compressão e por injeção foram expostas à radiação ultravioleta em laboratório por períodos de até 48 semanas. Procedimentos sistemáticos de exposição e coleta de material para análise foram adotados visando-se minimizar as variabilidades inerentes deste estudo. As amostras foram caracterizadas por cromatografia de permeação em gel, espectroscopia de infravermelho, calorimetria diferencial de varredura, difração de raios-X, comportamento mecânico e microscopia ótica e eletrônica. As heterogeneidades na fotodegradação do PP podem estar relacionadas com três principais aspectos: (i) natureza semi-cristalina do polímero; (ii) perfil de degradação; (iii) variações estruturais em diferentes posições nos corpos de prova. As consequências da fotodegradação heterogênea foram discutidas, enfatizando-se os efeitos nas propriedades físicas.In this paper some characteristics of the heterogeneous photodegradation of polypropylene (PP) and PP containing talc were analyzed. Samples produced by injection and compression moulding were exposed to ultraviolet radiation in the laboratory for periods of up to 48 weeks. Systematic procedures for exposure and sampling were adopted in an attempt to minimize the inherent variabilities of this type of study. The specimens were characterized by gel permeation chromatography, infrared spectroscopy, differential scanning calorimetry, X-ray diffraction, mechanical properties and light and electron microscopy. The heterogeneities in PP photodegradation can be related to three main aspects: (i) the semi-crystalline nature of PP; (ii) the depth-profile of degradation; (iii) the structural variations in different positions along the test bar. The consequences of these heterogeneities were discussed with emphasis on the effects on physical properties.

Boldness is the propensity of an animal to engage in risky behavior. Many variations of novel-object or novel-environment tests have been used to quantify the boldness of animals, although the relationship between test outcomes has rarely been investigated. Furthermore, the relationship of outcomes to any ecological aspect of fitness is generally assumed, rather than measured directly. Our study is the first to compare how the outcomes of the same test of boldness differ among observers and how different tests of boldness relate to the survival of individuals in the field. Newly-metamorphosed lemon damselfish, Pomacentrus moluccensis, were placed onto replicate patches of natural habitat. Individual behavior was quantified using four tests (composed of a total of 12 different measures of behavior): latency to enter a novel environment, activity in a novel environment, and reactions to threatening and benign novel objects. After behavior was quantified, survival was monitored for two days during which time fish were exposed to natural predators. Variation among observers was low for most of the 12 measures, except distance moved and the threat test (reaction to probe thrust), which displayed unacceptable amounts of inter-observer variation. Overall, the results of the behavioral tests suggested that novel environment and novel object tests quantified similar behaviors, yet these behavioral measures were not interchangeable. Multiple measures of behavior within the context of novel environment or object tests were the most robust way to assess boldness and these measures have a complex relationship with survivorship of young fish in the field. Body size and distance ventured from shelter were the only variables that had a direct and positive relationship with survival.

The theory of behavioral syndromes focuses on quantifying variation in behavior within and among individual organisms and attempts to account for the maintenance of differences in behavior that occur in a consistent manner among individuals. Behavioral syndromes have potentially important ecological consequences (e.g. survivorship tradeoffs) and can be shaped by population dynamics through selective mortality. Here, we search for any evidence for consistency of behavior across situations in juveniles of a common damselfish, Pomacentrus amboinensis (Pomacentridae) at the transition between larval habitats in the plankton and juvenile habitats on the reef. Naïve fish leaving the pelagic phase to settle on reefs were caught by light traps and their behaviors observed using similar methods across three different situations (small aquaria, large aquaria, field setting); all of which represent low risk and well-sheltered environments. Seven behavioral traits were compared within and among individuals across situations to determine if consistent behavioral syndromes existed. No consistency was found in any single or combination of behavioral traits for individuals across all situations. We suggest that high behavioral flexibility is likely beneficial for newly-settled fish at this ontogenetic transition and it is possible that consistent behavioral syndromes are unlikely to emerge in juveniles until environmental experience is gained or certain combinations of behaviors are favored by selective mortality.

Celiac disease (CD) is a unique autoimmune disorder in which the genetic factors (DQ2/DQ8) and the environmental trigger (gluten) are known and necessary but not sufficient for its development. Other environmental components contributing to CD are poorly understood. Studies suggest that aspects of gluten intake might influence the risk of CD occurrence and timing of its onset, i.e., the amount and quality of ingested gluten, together with the pattern of infant feeding and the age at which gluten is introduced in the diet. In this study, we hypothesize that the intestinal microbiota as a whole rather than specific infections dictates the switch from tolerance to immune response in genetically susceptible individuals. Using a sample of infants genetically at risk of CD, we characterized the longitudinal changes in the microbial communities that colonize infants from birth to 24 months and the impact of two patterns of gluten introduction (early vs. late) on the gut microbiota and metabolome, and the switch from gluten tolerance to immune response, including onset of CD autoimmunity. We show that infants genetically susceptible to CD who are exposed to gluten early mount an immune response against gluten and develop CD autoimmunity more frequently than at-risk infants in which gluten exposure is delayed until 12 months of age. The data, while derived from a relatively small number of subjects, suggest differences between the developing microbiota of infants with genetic predisposition for CD and the microbiota from infants with a non-selected genetic background, with an overall lack of bacteria of the phylum Bacteriodetes along with a high abundance of Firmicutes and microbiota that do not resemble that of adults even at 2 years of age. Furthermore, metabolomics analysis reveals potential biomarkers for the prediction of CD. This study constitutes a definite proof-of-principle that these combined genomic and metabolomic approaches will be key to deciphering the role of the gut microbiota on CD onset.

BACKGROUND: Marine sponge species are of significant interest to many scientific fields including marine ecology, conservation biology, genetics, host-microbe symbiosis and pharmacology. One of the most intriguing aspects of the sponge "holobiont" system is the unique physiology, interaction with microbes from the marine environment and the development of a complex commensal microbial community. However, intraspecific variability and temporal stability of sponge-associated bacterial symbionts remain relatively unknown. METHODOLOGY/PRINCIPAL FINDINGS: We have characterized the bacterial symbiont community biodiversity of seven different individuals of the Caribbean reef sponge Axinella corrugata, from two different Florida reef locations during variable seasons using multiplex 454 pyrosequencing of 16 S rRNA amplicons. Over 265,512 high-quality 16 S rRNA sequences were generated and analyzed. Utilizing versatile bioinformatics methods and analytical software such as the QIIME and CloVR packages, we have identified 9,444 distinct bacterial operational taxonomic units (OTUs). Approximately 65,550 rRNA sequences (24%) could not be matched to bacteria at the class level, and may therefore represent novel taxa. Differentially abundant classes between seasonal Axinella communities included Gammaproteobacteria, Flavobacteria, Alphaproteobacteria, Cyanobacteria, Acidobacter and Nitrospira. Comparisons with a proximal outgroup sponge species (Amphimedon compressa), and the growing sponge symbiont literature, indicate that this study has identified approximately 330 A. corrugata-specific symbiotic OTUs, many of which are related to the sulfur-oxidizing Ectothiorhodospiraceae. This family appeared exclusively within A. corrugata, comprising >34.5% of all sequenced amplicons. Other A. corrugata symbionts such as Deltaproteobacteria, Bdellovibrio, and Thiocystis among many others are described. CONCLUSIONS/SIGNIFICANCE: Slight shifts in several bacterial taxa were observed between communities sampled during spring and fall seasons. New 16 S rDNA sequences and concomitant identifications greatly expand the microbial community profile for this model reef sponge, and will likely be useful as a baseline for any future comparisons regarding sponge microbial community dynamics.

BackgroundThe widespread popularity of genomic applications is threatened by the "bioinformatics bottleneck" resulting from uncertainty about the cost and infrastructure needed to meet increasing demands for next-generation sequence analysis. Cloud computing services have been discussed as potential new bioinformatics support systems but have not been evaluated thoroughly.ResultsWe present benchmark costs and runtimes for common microbial genomics applications, including 16S rRNA analysis, microbial whole-genome shotgun (WGS) sequence assembly and annotation, WGS metagenomics and large-scale BLAST. Sequence dataset types and sizes were selected to correspond to outputs typically generated by small- to midsize facilities equipped with 454 and Illumina platforms, except for WGS metagenomics where sampling of Illumina data was used. Automated analysis pipelines, as implemented in the CloVR virtual machine, were used in order to guarantee transparency, reproducibility and portability across different operating systems, including the commercial Amazon Elastic Compute Cloud (EC2), which was used to attach real dollar costs to each analysis type. We found considerable differences in computational requirements, runtimes and costs associated with different microbial genomics applications. While all 16S analyses completed on a single-CPU desktop in under three hours, microbial genome and metagenome analyses utilized multi-CPU support of up to 120 CPUs on Amazon EC2, where each analysis completed in under 24 hours for less than $60. Representative datasets were used to estimate maximum data throughput on different cluster sizes and to compare costs between EC2 and comparable local grid servers.ConclusionsAlthough bioinformatics requirements for microbial genomics depend on dataset characteristics and the analysis protocols applied, our results suggests that smaller sequencing facilities (up to three Roche/454 or one Illumina GAIIx sequencer) invested in 16S rRNA amplicon sequencing, microbial single-genome and metagenomics WGS projects can achieve cost-efficient bioinformatics support using CloVR in combination with Amazon EC2 as an alternative to local computing centers.

The assembly methods used for whole-genome shotgun (WGS) data have a major impact on the quality of resulting draft genomes. We present a novel algorithm to generate a set of "reliable" overlaps based on identifying repeat k-mers. To demonstrate the benefits of using reliable overlaps, we have created a version of the Phrap assembly program that uses only overlaps from a specific list. We call this version PhrapUMD. Integrating PhrapUMD and our "reliable-overlap" algorithm with the Baylor College of Medicine assembler, Atlas, we assemble the BACs from the Rattus norvegicus genome project. Starting with the same data as the Nov. 2002 Atlas assembly, we compare our results and the Atlas assembly to the 4.3 Mb of rat sequence in the 21 BACs that have been finished. Our version of the draft assembly of the 21 BACs increases the coverage of finished sequence from 93.4% to 96.3%, while simultaneously reducing the base error rate from 4.5 to 1.1 errors per 10,000 bases. There are a number of ways of assessing the relative merits of assemblies when the finished sequence is available. If one views the overall quality of an assembly as proportional to the inverse of the product of the error rate and sequence missed, then the assembly presented here is seven times better. The UMD Overlapper with options for reliable overlaps is available from the authors at http://www.genome.umd.edu. We also provide the changes to the Phrap source code enabling it to use only the reliable overlaps.