AI powered quantification of nuclear morphology in cancers enables prediction of genome instability and prognosis.

While alterations in nucleus size, shape, and color are ubiquitous in cancer, comprehensive quantification of nuclear morphology across a whole-slide histologic image remains a challenge. Here, we describe the development of a pan-tissue, deep learning-based digital pathology pipeline for exhaustive nucleus detection, segmentation, and classification and the utility of this pipeline for nuclear morphologic biomarker discovery. Manually-collected nucleus annotations were used to train an object detection and segmentation model for identifying nuclei, which was deployed to segment nuclei in H&E-stained slides from the BRCA, LUAD, and PRAD TCGA cohorts. Interpretable features describing the shape, size, color, and texture of each nucleus were extracted from segmented nuclei and compared to measurements of genomic instability, gene expression, and prognosis. The nuclear segmentation and classification model trained herein performed comparably to previously reported models. Features extracted from the model revealed differences sufficient to distinguish between BRCA, LUAD, and PRAD. Furthermore, cancer cell nuclear area was associated with increased aneuploidy score and homologous recombination deficiency. In BRCA, increased fibroblast nuclear area was indicative of poor progression-free and overall survival and was associated with gene expression signatures related to extracellular matrix remodeling and anti-tumor immunity. Thus, we developed a powerful pan-tissue approach for nucleus segmentation and featurization, enabling the construction of predictive models and the identification of features linking nuclear morphology with clinically-relevant prognostic biomarkers across multiple cancer types.

Transferable Immunoglobulin A-Coated Odoribacter splanchnicus in Responders to Fecal Microbiota Transplantation for Ulcerative Colitis Limits Colonic Inflammation.

BACKGROUND & AIMS: Fecal microbiota transplantation (FMT) is an emerging treatment modality for ulcerative colitis (UC). Several randomized controlled trials have shown efficacy for FMT in the treatment of UC, but a better understanding of the transferable microbiota and their immune impact is needed to develop more efficient microbiome-based therapies for UC. METHODS: Metagenomic analysis and strain tracking was performed on 60 donor and recipient samples receiving FMT for active UC. Sorting and sequencing of immunoglobulin (Ig) A-coated microbiota (called IgA-seq) was used to define immune-reactive microbiota. Colonization of germ-free or genetically engineered mice with patient-derived strains was performed to determine the mechanism of microbial impact on intestinal immunity. RESULTS: Metagenomic analysis defined a core set of donor-derived transferable bacterial strains in UC subjects achieving clinical response, which predicted response in an independent trial of FMT for UC. IgA-seq of FMT recipient samples and gnotobiotic mice colonized with donor microbiota identified Odoribacter splanchnicus as a transferable strain shaping mucosal immunity, which correlated with clinical response and the induction of mucosal regulatory T cells. Colonization of mice with O splanchnicus led to an increase in Foxp3+/RORγt+ regulatory T cells, induction of interleukin (IL) 10, and production of short chain fatty acids, all of which were required for O splanchnicus to limit colitis in mouse models. CONCLUSIONS: This work provides the first evidence of transferable, donor-derived strains that correlate with clinical response to FMT in UC and reveals O splanchnicus as a key component promoting both metabolic and immune cell protection from colitis. These mechanistic features will help enable strategies to enhance the efficacy of microbial therapy for UC. Clinicaltrials.gov ID NCT02516384.

Beyond Fecal Microbiota Transplantation: Developing Drugs from the Microbiome.

The transfer of live gut microbes may transform patient care across a range of autoimmune, metabolic, hepatic, and infectious diseases. One early approach, fecal microbiota transplantation, has shown promise in Clostridiodes difficile infection and the potential for improving clinical and public health outcomes for other antibiotic-resistant bacteria. These clinical successes have motivated the development of microbiome drugs, which will need to address challenges in safety, uniformity, and delivery while seeking to preserve the benefits of using whole microbiome communities as novel therapeutics and an innovative platform for drug discovery.

Inflammatory Bowel Disease Outcomes Following Fecal Microbiota Transplantation for Recurrent C. difficile Infection.

BACKGROUND: Recurrent Clostridioides difficile infection (CDI) in patients with inflammatory bowel disease (IBD) is a clinical challenge. Fecal microbiota transplantation (FMT) has emerged as a recurrent CDI therapy. Anecdotal concerns exist regarding worsening of IBD activity; however, prospective data among IBD patients are limited. METHODS: Secondary analysis from an open-label, prospective, multicenter cohort study among IBD patients with 2 or more CDI episodes was performed. Participants underwent a single FMT by colonoscopy (250 mL, healthy universal donor). Secondary IBD-related outcomes included rate of de novo IBD flares, worsening IBD, and IBD improvement-all based on Mayo or Harvey-Bradshaw index (HBI) scores. Stool samples were collected for microbiome and targeted metabolomic profiling. RESULTS: Fifty patients enrolled in the study, among which 15 had Crohn's disease (mean HBI, 5.8 ± 3.4) and 35 had ulcerative colitis (mean partial Mayo score, 4.2 ± 2.1). Overall, 49 patients received treatment. Among the Crohn's disease cohort, 73.3% (11 of 15) had IBD improvement, and 4 (26.6%) had no disease activity change. Among the ulcerative colitis cohort, 62% (22 of 34) had IBD improvement, 29.4% (11 of 34) had no change, and 4% (1 of 34) experienced a de novo flare. Alpha diversity significantly increased post-FMT, and ulcerative colitis patients became more similar to the donor than Crohn's disease patients (P = 0.04). CONCLUSION: This prospective trial assessing FMT in IBD-CDI patients suggests IBD outcomes are better than reported in retrospective studies.

Re-Evaluating the Evidence for Faecal Microbiota Transplantation 'Super-Donors' in Inflammatory Bowel Disease.

BACKGROUND: Faecal microbiota transplantation [FMT] is a recommended treatment for recurrent Clostridioides difficile infection, and there is promise that FMT may be effective for conditions such as inflammatory bowel disease [IBD]. Previous FMT clinical trials have considered the possibility of a 'donor effect', that is, that FMT material from different donors has different clinical efficacies. METHODS: Here we re-evaluate evidence for donor effects in published FMT clinical trials for IBD. RESULTS: In ten of 12 published studies, no statistically significant donor effect was detected when rigorously re-evaluating the original analyses. One study showed statistically significant separation of microbiota composition of pools of donor stool when stratified by patient outcome. One study reported a significant effect but did not have underlying data available for re-evaluation. When quantifying the uncertainty on the magnitude of the donor effect, confidence intervals were large, including both zero donor effects and very substantial donor effects. CONCLUSION: Although we found very little evidence for donor effects, the existing data cannot rule out the possibility that donor effects are clinically important. Large clinical trials prospectively designed to detect donor effects are probably needed to determine if donor effects are clinically relevant for IBD.

Effects of Fecal Microbiota Transplantation With Oral Capsules in Obese Patients.

BACKGROUND & AIMS: Studies in mice have shown that the intestinal microbiota can contribute to obesity via the anorexigenic gut hormone glucagon-like peptide 1 (GLP1) and bile acids, which affect lipid metabolism. We performed a randomized, placebo-controlled, pilot study of the effects of fecal microbiota transplantation (FMT) in obese, metabolically uncompromised patients. METHODS: We performed a double-blind study of 22 obese patients (body mass index [BMI] ≥5 kg/m2) without a diagnosis of diabetes, nonalcoholic steatohepatitis, or metabolic syndrome. Participants were assigned randomly (1:1) to groups that received FMT by capsules (induction dose of 30 capsules at week 4 and maintenance dose of 12 capsules at week 8) or placebo capsules. FMT capsules were derived from a single lean donor (BMI, 17.5 kg/m2). Patients were followed up through week 26; the primary outcome was safety. Stool and serum samples were collected from patients at baseline and at weeks 1, 4, 6, 8, and 12 after administration of the first dose of FMT or placebo and analyzed by 16S RNA gene sequencing. Stool and serum samples were analyzed for metabolomics by liquid chromatography-mass spectrometry. Additional outcomes were the change in area under the curve for GLP1 at week 12. RESULTS: We observed no significant differences in adverse events between patients who received FMT vs placebo. There was no increase in the area under the curve of GLP1 in either group. Patients who received FMT had sustained shifts in microbiomes associated with obesity toward those of the donor (P < .001). Patients who received FMT had a sustained decrease in stool levels of taurocholic acid (P < .05) compared with baseline; bile acid profiles began to resemble those of the donor more closely. We did not observe significant changes in mean BMI at week 12 in either group. CONCLUSIONS: In a placebo-controlled pilot study, we found that FMT capsules (derived from a lean donor) were safe but did not reduce BMI in obese metabolically uncompromised patients. The FMT capsules were well tolerated and led to sustained changes in the intestinal microbiome and bile acid profiles that were similar to those of the lean donor. ClinicalTrials.gov number: NCT02741518.

Combined Endoscopic and Oral Fecal Microbiota Transplantation in Patients with Antibiotic-Dependent Pouchitis: Low Clinical Efficacy due to Low Donor Microbial Engraftment.

BACKGROUND AND OBJECTIVE: A significant number of pouch patients develop antibiotic-dependent pouchitis (ADP). Microbial dysbiosis is thought to be a major driver of clinical symptoms in ADP. The objective of this proof of concept study was to evaluate safety, efficacy, and donor microbial engraftment of an intensified fecal microbiota transplant (FMT) consisting of a single endoscopic FMT followed by daily oral FMT for 2 weeks in patients with ADP. METHODS: We performed a prospective placebo-controlled double-blind FMT trial in patents with established ADP and planned to enroll 20 patients in this proof of concept study. In case of non-response, patients were offered an optional open label active FMT treatment. The endpoints were safety, clinical remission without need for antibiotics during 16 weeks of follow-up, quantitative changes of fecal calprotectin (FCP), and engraftment of donor FMT as determined by metagenomic sequencing of the V4 region of the 16S rRNA gene. RESULTS: Due to a lower than expected clinical remission rate and low FMT engraftment, enrollment in the study was stopped prematurely after 6 patients were included. All 6 patients enrolled in the placebo-controlled portion failed to respond and needed antibiotic rescue therapy shortly after FMT. FCP increased in the majority of patients in the setting of relapse after FMT. In the active open label FMT extension study 1 out of 5 patients achieved antibiotic-free clinical remission. FMT engraftment after active FMT was observed only in this single patient, whereas engraftment of donor FMT occurred in none of the other patients receiving active FMT, paralleling the lack of clinical response. CONCLUSIONS: Low donor FMT engraftment resulted in low clinical efficacy of FMT in patients with ADP. Before embarking on larger clinical trials with FMT in patients with ADP or other forms of pouchitis, it is mandatory to explore approaches for superior FMT engraftment.

Fecal Microbiota Transplantation in Patients With Primary Sclerosing Cholangitis: A Pilot Clinical Trial.

BACKGROUND: Primary sclerosing cholangitis (PSC) is a cholestatic liver disease with no effective medical therapies. A perturbation of the gut microbiota has been described in association with PSC, and fecal microbiota transplantation (FMT) has been reported to restore the microbiome in other disease states. Accordingly, we aimed at evaluating the safety, change in liver enzymes, microbiota, and metabolomic profiles in patients with PSC after FMT. METHODS: An open-label pilot study of patients with PSC with concurrent inflammatory bowel disease and alkaline phosphatase (ALP) > 1.5× the upper limit of normal was conducted. The patients underwent a single FMT by colonoscopy. Liver enzyme profiles and stool microbiome and metabolomic analysis were conducted at baseline and weeks 1, 4, 8, 12, and 24 post-FMT. The primary outcome was safety, and the secondary outcome was a decrease in ALP levels ≥50% from baseline by week 24 post-FMT; stool microbiota (by 16S rRNA gene profiling) and metabonomic dynamics were assessed. RESULTS: Ten patients underwent FMT. Nine patients had ulcerative colitis, and 1 had Crohn's colitis. The mean baseline ALP level was 489 U/L. There were no related adverse events. Overall, 30% (3/10) experienced a ≥50% decrease in ALP levels. The diversity increased in all patients post-FMT, as early as week 1 (P < 0.01). Importantly, abundance of engrafter operational taxonomic units in patients post-FMT correlated with decreased ALP levels (P = 0.02). DISCUSSION: To our knowledge, this is the first study to demonstrate that FMT in PSC is safe. In addition, increases in bacterial diversity and engraftment may correlate with an improvement in ALP among patients with PSC.

Correction to: Fecal Microbiota Transplantation Capsules with Targeted Colonic Versus Gastric Delivery in Recurrent Clostridium difficile Infection: A Comparative Cohort Analysis of High and Low Dose.

The original version of the article unfortunately contained an error in article title. The corrected title is 'Fecal Microbiota Transplantation Capsules with Targeted Colonic Versus Gastric Delivery in Recurrent Clostridium difficile Infection: A Comparative Cohort Analysis of High and Low Dose'.

Fecal Microbiota Transplantation Capsules with Targeted Colonic Versus Gastric Delivery in Recurrent Clostridium difficile Infection: A Comparative Cohort Analysis of High and Lose Dose.

BACKGROUND: Fecal microbiota transplantation (FMT) is an effective therapy for recurrent Clostridium. difficile infection (rCDI). FMT capsules have emerged, and it is unknown if delivery location and dose impact efficacy. METHODS: We compared two cohorts of patients receiving two capsule formulations: gastric release (FMTgr) and targeted colonic release (FMTcr) at two different sites. Cohort A received FMTgr at (1) high dose: 60 capsules and low dose: 30 capsules. Patients in Cohort B received FMTcr at (1) high dose: 30 capsules (2) low dose: 10 capsules. Clinical cure rates and adverse events were monitored through week 8. Paired t-tests were used to compare diversity pre- and post-FMT. RESULTS: 51 rCDI patients were enrolled. Cohort A contained n = 20 and Cohort B contained n = 31. Overall cure at week 8 for FMTgr was 75% (15/20) compared to 80.6% for FMTcr, (25/31), p = 0.63. Both formulations were safe with no serious adverse events. FMTcr was superior at increasing gut microbial diversity. DISCUSSION: To our knowledge, this is the first study to compare targeted delivery of FMT capsules. While both capsules were safe and efficacious, microbial engraftment patterns were superior in FMTcr.

Single Delivery of High-Diversity Fecal Microbiota Preparation by Colonoscopy Is Safe and Effective in Increasing Microbial Diversity in Active Ulcerative Colitis.

BACKGROUND: Recent trials suggest fecal microbiota transplantation (FMT) with repeated enemas and high-diversity FMT donors is a promising treatment to induce remission in ulcerative colitis. METHODS: We designed a prospective, open-label pilot study to assess the safety, clinical efficacy, and microbial engraftment of single FMT delivery by colonoscopy for active ulcerative colitis using a 2-donor fecal microbiota preparation (FMP). Safety and clinical endpoints of response, remission, and mucosal healing at week 4 were assessed. Fecal DNA and rectal biopsies were used to characterize the microbiome and mucosal CD4 T cells, respectively, before and after FMT. RESULTS: Of the 20 patients enrolled in this study, 7 patients (35%) achieved a clinical response by week 4. Three patients (15%) were in remission at week 4 and 2 of these patients (10%) achieved mucosal healing. Three patients (15%) required escalation of care. No serious adverse events were observed. Microbiome analysis revealed that restricted diversity of recipients pre-FMT was significantly increased by high-diversity 2-donor FMP. The microbiome of recipients post-transplant was more similar to the donor FMP than the pretransplant recipient sample in both responders and nonresponders. Notably, donor composition correlated with clinical response. Mucosal CD4 T-cell analysis revealed a reduction in both Th1 and regulatory T-cells post-FMT. CONCLUSIONS: High-diversity, 2-donor FMP delivery by colonoscopy seems safe and effective in increasing fecal microbial diversity in patients with active ulcerative colitis. Donor composition correlated with clinical response and further characterization of immunological parameters may provide insight into factors influencing clinical outcome.

A competitive trade-off limits the selective advantage of increased antibiotic production.

In structured environments, antibiotic-producing microorganisms can gain a selective advantage by inhibiting nearby competing species1. However, despite their genetic potential2,3, natural isolates often make only small amounts of antibiotics, and laboratory evolution can lead to loss rather than enhancement of antibiotic production4. Here, we show that, due to competition with antibiotic-resistant cheater cells, increased levels of antibiotic production can actually decrease the selective advantage to producers. Competing fluorescently labelled Escherichia coli colicin producers with non-producing resistant and sensitive strains on solid media, we found that although producer colonies can greatly benefit from the inhibition of nearby sensitive colonies, this benefit is shared with resistant colonies growing in their vicinity. A simple model, which accounts for such local competitive and inhibitory interactions, suggests that the advantage of producers varies non-monotonically with the amount of production. Indeed, experimentally varying the amount of production shows a peak in selection for producers, reflecting a trade-off between benefit gained by inhibiting sensitive competitors and loss due to an increased contribution to resistant cheater colonies. These results help explain the low level of antibiotic production observed for natural species and can help direct laboratory evolution experiments selecting for increased or novel production of antibiotics.

Isolated cell behavior drives the evolution of antibiotic resistance.

Bacterial antibiotic resistance is typically quantified by the minimum inhibitory concentration (MIC), which is defined as the minimal concentration of antibiotic that inhibits bacterial growth starting from a standard cell density. However, when antibiotic resistance is mediated by degradation, the collective inactivation of antibiotic by the bacterial population can cause the measured MIC to depend strongly on the initial cell density. In cases where this inoculum effect is strong, the relationship between MIC and bacterial fitness in the antibiotic is not well defined. Here, we demonstrate that the resistance of a single, isolated cell-which we call the single-cell MIC (scMIC)-provides a superior metric for quantifying antibiotic resistance. Unlike the MIC, we find that the scMIC predicts the direction of selection and also specifies the antibiotic concentration at which selection begins to favor new mutants. Understanding the cooperative nature of bacterial growth in antibiotics is therefore essential in predicting the evolution of antibiotic resistance.

The Arabidopsis ROCK-N-ROLLERS gene encodes a homolog of the yeast ATP-dependent DNA helicase MER3 and is required for normal meiotic crossover formation.

Recent studies in Saccharomyces cerevisiae have unveiled that meiotic recombination crossovers are formed by two genetically distinct pathways: a major interference-sensitive pathway and a minor interference-insensitive pathway. Several proteins, including the MSH4/MSH5 heterodimer and the MER3 DNA helicase, are indispensable for the interference-sensitive pathway. MSH4 homologs have been identified in mice and Arabidopsis and shown to be required for normal levels of crossovers, suggesting that the function of MSH4 may be conserved among major eukaryotic kingdoms. However, it is not known whether an MER3-like function is also required for meiosis in animals and plants. We have identified an Arabidopsis gene that encodes a putative MER3 homolog and is preferentially expressed in meiocytes. T-DNA insertional mutants of this gene exhibit defects in fertility and meiosis. Detailed cytological studies indicate that the mutants are defective in homolog synapsis and crossover formation, resulting in a reduction of bivalents and in the formation of univalents at late prophase I. We have named this gene ROCK-N-ROLLERS (RCK) to reflect the mutant phenotype of chromosomes undergoing the meiotic 'dance' either in pairs or individually. Our results demonstrate that an MER3-like function is required for meiotic crossover in plants and provide further support for the idea that Arabidopsis, like the budding yeast, possesses both interference-sensitive and insensitive pathways for crossover formation.