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Pancreatic ductal adenocarcinoma (PDAC) is expected to be the second most deadly cancer by 2040, owing to the high incidence of metastatic disease and limited responses to treatment1,2. Less than half of all patients respond to the primary treatment for PDAC, chemotherapy3,4, and genetic alterations alone cannot explain this5. Diet is an environmental factor that can influence the response to therapies, but its role in PDAC is unclear. Here, using shotgun metagenomic sequencing and metabolomic screening, we show that the microbiota-derived tryptophan metabolite indole-3-acetic acid (3-IAA) is enriched in patients who respond to treatment. Faecal microbiota transplantation, short-term dietary manipulation of tryptophan and oral 3-IAA administration increase the efficacy of chemotherapy in humanized gnotobiotic mouse models of PDAC. Using a combination of loss- and gain-of-function experiments, we show that the efficacy of 3-IAA and chemotherapy is licensed by neutrophil-derived myeloperoxidase. Myeloperoxidase oxidizes 3-IAA, which in combination with chemotherapy induces a downregulation of the reactive oxygen species (ROS)-degrading enzymes glutathione peroxidase 3 and glutathione peroxidase 7. All of this results in the accumulation of ROS and the downregulation of autophagy in cancer cells, which compromises their metabolic fitness and, ultimately, their proliferation. In humans, we observed a significant correlation between the levels of 3-IAA and the efficacy of therapy in two independent PDAC cohorts. In summary, we identify a microbiota-derived metabolite that has clinical implications in the treatment of PDAC, and provide a motivation for considering nutritional interventions during the treatment of patients with cancer.

Probiotic microbes such as Lactobacillus may reduce serum total cholesterol (TC) and low-density lipoprotein (LDL) cholesterol. The objective of this study was to assess the effect of Lactobacillus plantarum strains CECT7527, CECT7528 and CECT7529 (LP) on the serum lipids, cardiovascular parameters and fecal gut microbiota composition in patients with mild hypercholesterolemia. A randomized, double-blinded, placebo-controlled clinical trial with 86 healthy adult participants with untreated elevated LDL cholesterol ≥ 160 mg/dL was conducted. Participants were randomly allocated to either placebo or LP (1.2 x109 CFU/d) for 12 weeks. LDL, HDL, TC and triglycerides (TG), cardiovascular parameters (blood pressure, arterial stiffness) and fecal gut microbiota composition (16S rRNA gene sequencing) were assessed at baseline and after 12 weeks. Both groups were comparable regarding age, sex and LDL-C at baseline. LDL-C decreased (mean decrease − 6.6 mg/dl ± -14.0 mg/dl, Ptime*intervention = 0.006) in the LP group but not in the placebo group. No effects were observed on HDL, TG or cardiovascular parameters or overall gut microbiota composition. Responders to LP intervention (> 5% LDL-C reduction) were characterized by higher BMI, pronounced TC reduction, higher abundance of fecal Roseburia and lower abundance of Oscillibacter. In conclusion, 12-week of L. plantarum intake moderately reduced LDL-C and TC as compared to placebo. LDL-C lowering efficacy of L. plantarum strains may potentially be dependent on individual difference in the gut microbiota. Trial registration: DRKS00006189, dated 17/12/2019.

Prevotella copri is a prevalent inhabitant of the human gut and has been associated with plant‐rich diet consumption and diverse health states. The underlying genetic basis of these associations remains enigmatic due to the lack of genetic tools. Here, we developed a novel versatile genetic toolbox for rapid and efficient genetic insertion and allelic exchange applicable to P. copri strains from multiple clades. Enabled by the genetic platform, we systematically investigated the specificity of polysaccharide utilization loci (PULs) and identified four highly conserved PULs for utilizing arabinan, pectic galactan, arabinoxylan, and inulin, respectively. Further genetic and functional analysis of arabinan utilization systems illustrate that P. copri has evolved two distinct types of arabinan‐processing PULs (PULAra) and that the type‐II PULAra is significantly enriched in individuals consuming a vegan diet compared to other diets. In summary, this genetic toolbox will enable functional genetic studies for P. copri in future., A novel conjugation‐based gene insertion/excision system allowing genetic manipulation of the previously intractable bacterium Prevotella copri provides insight into human diet effects on commensal bacterial metabolism.

Objective Protein disulfide isomerases (PDIs) are oxidoreductases that are involved in catalyzing the formation and rearrangement of disulfide bonds during protein folding. One of the PDI members is the PDI-associated 6 (PDIA6) protein, which has been shown to play a vital role in β-cell dysfunction and diabetes. However, very little is known about the function of this protein in β-cells in vivo. This study aimed to describe the consequences of a point mutation in Pdia6 on β-cell development and function. Methods We generated an ENU mouse model carrying a missense mutation (Phe175Ser) in the second thioredoxin domain of the Pdia6 gene. Using biochemical and molecular tools, we determined the effects of the mutation on the β-cell development at embryonic day (E)18.5 and β-cell identity as well as function at postnatal stages. Results Mice homozygous for the Phe175Ser (F175S) mutation were mildly hyperglycemic at weaning and subsequently became hypoinsulinemic and overtly diabetic at the adult stage. Although no developmental phenotype was detected during embryogenesis, mutant mice displayed reduced insulin-expressing β-cells at P14 and P21 without any changes in the rate of cell death and proliferation. Further analysis revealed an increase in BiP and the PDI family member PDIA4, but without any concomitant apoptosis and cell death. Instead, the expression of prominent markers of β-cell maturation and function, such as Ins2, Mafa, and Slc2a2, along with increased expression of α-cell markers, Mafb, and glucagon was observed in adult mice, suggesting loss of β-cell identity. Conclusions The results demonstrate that a global Pdia6 mutation renders mice hypoinsulinemic and hyperglycemic. This occurs due to the loss of pancreatic β-cell function and identity, suggesting a critical role of PDIA6 specifically for β-cells., Highlights • Mutation in the second thioredoxin domain (F175S) of PDIA6 does not affect pancreatic islet development. • F175S mutation leads to development of neonatal diabetes in mice. • Diabetic phenotype is due to severe lack of insulin and loss of β-cell identity.

OBJECTIVES: The effects of fermented foods on the gut microbiome are of great interest, yet evidence regarding its potential to increase gut microbial diversity, a measure likely associated with health, is lacking. Therefore, we analyzed the microbial composition (bacteria and yeasts) of commercially available fermented vegetables. Furthermore, we conducted a pilot study to assess the feasibility of studying effects of regular consumption of fermented vegetables on the gut microbiome. METHODS: Six healthy male volunteers (age: 25.5 ± 2.9yrs, BMI: 24.3 ± 1.2kg/m(2)) participated in a randomized crossover trial, with two 2-week intervention phases each of which was preceded by a 2-week washout phase. Participants consumed 150g/d of either sauerkraut (intervention 1) or a variety of six different fermented vegetables (intervention 2). We used 16S rRNA sequencing to assess the effects of each dietary regime on the composition, diversity and dynamics of the gut microbiome, as well as the composition and diversity of the fermented vegetable microbiome. RESULTS: Lactobacillus was the dominant genus in all fermented vegetables; still, the alpha diversity, richness and evenness of the microbiota differed substantially among the different products. Among our study participants, we observed an increase in alpha diversity (Shannon index) after both, consumption of sauerkraut (pre intervention: 3.31 ± 0.74, post intervention: 3.58 ± 0.68) and the selection of fermented vegetables (pre: 3.60 ± 0.93, post: 3.84 ± 0.81). However, the results did not reach statistical significance, due to the high inter- and intra-individual variability as evaluated by beta diversity of the gut microbial communities. CONCLUSIONS: A longer-term intervention study with fermented vegetables and/or sauerkraut seems feasible. Consumption of fermented vegetables appears to increase the diversity of the gut microbiome, even after a relatively short period of time. However, further studies with a larger sample size are warranted to verify our observations. FUNDING SOURCES: Institutional budget.

Prevotella copriis a prevalent inhabitant of the human gut and has been associated with plant-rich diet consumption and diverse health states. The underlying genetic basis of these associations remains enigmatic due to the lack of genetic tools. Here, we developed a novel versatile genetic toolbox for rapid and efficient genetic insertion and allelic exchange applicable toP. copristrains from multiple clades. Enabled by the genetic platform, we systematically investigated the specificity of polysaccharide utilization loci (PULs), and identified four highly conserved PULs for utilizing arabinan, pectic galactan, arabinoxylan and inulin, respectively. Further genetic and functional analysis of arabinan utilization systems illustrate thatP. coprihas evolved two distinct types of arabinan-processing PULs (PULAra) and that the type-II PULArais significantly enriched in individuals consuming a vegan diet compared to other diets. In summary, this genetic toolbox will enable functional genetic studies forP. copriin the future.

The transcription factor PAX6 is involved in the development of the eye and pancreatic islets, besides being associated with sleep–wake cycles. Here, we investigated a point mutation in the RED subdomain of PAX6, previously described in a human patient, to present a comprehensive study of a homozygous Pax6 mutation in the context of adult mammalian metabolism and circadian rhythm. Pax6Leca2 mice lack appropriate retinal structures for light perception and do not display normal daily rhythmic changes in energy metabolism. Despite β cell dysfunction and decreased insulin secretion, mutant mice have normal glucose tolerance. This is associated with reduced hepatic glucose production possibly due to altered circadian variation in expression of clock and metabolic genes, thereby evading hyperglycemia. Hence, our findings show that while the RED subdomain is important for β cell functional maturity, the Leca2 mutation impacts peripheral metabolism via loss of circadian rhythm, thus revealing pleiotropic effects of PAX6., Nirav Chhabra et al. characterize adult mice carrying a homozygous mutation in Pax6 that was identified in a patient with foveal hypoplasia. They find that the Pax6 point mutation has pleiotropic effects, including defects in the mouse retinal structures, loss of the optic nerve, changes in energy metabolism and circadian rhythms, and dysregulation of genes expressed in the pancreas.

CD81 plays a role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus. Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81-cholesterol association, but had disparate effects on HCV, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified an allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol unbound) or closed (cholesterol bound) conformation. The open mutant of CD81 exhibited reduced receptor activity whereas the closed mutant was enhanced. These data are consistent with cholesterol switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81-partner networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry and CD81's function as a molecular scaffold; these insights are relevant to CD81's varied roles in health and disease.

Diverse microbial signatures within the intestinal microbiota have been associated with intestinal and systemic inflammatory diseases, but whether these candidate microbes actively modulate host phenotypes or passively expand within the altered microbial ecosystem is frequently not known. Here we demonstrate that colonization of mice with a member of the genus Prevotella, which has been previously associated to colitis in mice, exacerbates intestinal inflammation. Our analysis revealed that Prevotella intestinalis alters composition and function of the ecosystem resulting in a reduction of short-chain fatty acids, specifically acetate, and consequently a decrease in intestinal IL-18 levels during steady state. Supplementation of IL-18 to Prevotella-colonized mice was sufficient to reduce intestinal inflammation. Hence, we conclude that intestinal Prevotella colonization results in metabolic changes in the microbiota, which reduce IL-18 production and consequently exacerbate intestinal inflammation, and potential systemic autoimmunity.

Prevotella spp. form one of the most dominant bacterial genera in the human gut. Multiple Prevotella spp. can co-exist in some but not all individuals consuming plant-based diets. To functionally investigate the relationship between Prevotella competition and diet, we isolated several novel Prevotella spp. from the mouse gut, performed genome and metatranscriptome analysis as well as a series of competition experiments under different dietary conditions. These experiments revealed that diverse dominant Prevotella species compete for similar metabolic niches in vivo, which is linked to the upregulation of specific Polysaccharide Utilization Loci (PULs). Complex plant-derived polysaccharides are required, and particularly arabinoxylan is sufficient, for the expansion of Prevotella spp. The most dominant Prevotella species encodes a specific tandem-repeat trsusC/D PUL, which is conserved in highly abundant Prevotella copri strains further expanded in humans consuming a vegan diet. Our data suggest efficient (arabino)xylan-utilization as a factor contributing to Prevotella dominance.

OBJECTIVES: Caloric restriction has been associated with beneficial effects on metabolic diseases such as type 2 diabetes, cardiovascular diseases, and cancer but the mechanisms are diverse and not fully understood. Interactions between the human host and its gut microbiota might play a crucial role in this context, but these have not yet been analysed in detail. Therefore, we conducted a pilot intervention study to investigate how the microbial community in the human gut adapts to caloric restriction. Additionally, we investigated whether the compliance improved if the participants during fasting consumed prebiotics, which are supposed to reduce sensations of hunger while not providing calories through digestion in the small intestine. METHODS: We recruited six healthy volunteers (age: 20–50 years, BMI: 20–25 kg/m(2)) for a pilot intervention study using a randomized crossover design. The study consisted of two sequential fasting periods of which one included additional prebiotics. Participants were fasting for three consecutive days consuming a total of 300 kcal daily provided by vegetable juices, framed by two days with a total daily calorie intake of 800 kcal, respectively. During one of the fasting periods, participants consumed additionally 24 g of inulin daily. Stool samples were collected for the analysis of the microbial composition by 16S rRNA sequencing. RESULTS: We observed no clear change in alpha diversity during the period of caloric reduction. However, quantitative changes in the composition of the microbiome was visible in mainly all participants. Additional intake of the prebiotic inulin did not influence compliance for the fasting intervention. However, we observed a clear reduction in alpha diversity in the gut microbiome after inulin ingestion during the fasting period. CONCLUSIONS: Caloric restriction appears to have no significant influence on the gut microbiome itself but inulin consumption during fasting seems to selectively promote the growth of some bacterial families leading to an overall decrease of alpha diversity in the gut microbiome. Further studies with a larger sample size are warranted to verify our observations. FUNDING SOURCES: Institutional budget, no external funding.

Numerous studies have associated alterations in the gut microbiota composition with almost every known inflammatory disease. However, proving the biological relevance of distinct microbial signatures and linking specific microorganisms to host phenotypes, remains a considerable challenge. Correspondingly, increased abundance of members of Prevotella genus within microbial communities colonizing distinct mucosal surfaces has been found in individuals diagnosed with rheumatoid arthritis, periodontitis, metabolic disorders, and intestinal and vaginal dysbiosis. Still, the role of Prevotella spp. in the incidence of these diseases continues to be debated. For many years, poor understanding of Prevotella biology could be in large part attributed to the lack of experimental tools. However, in the recent years significant advances have been made towards overcoming these limitations, including increased number of isolates and improved understanding of genetic diversity. Besides discussing the most relevant associations between Prevotella spp. and inflammatory disorders, in the present review we examine the recent efforts to expand the Prevotella experimental “toolbox” and we highlight remaining experimental challenges that should advance future research and our understanding of Prevotella-host interplay.

Correct innervation of the main respiratory muscle in mammals, namely the thoracic diaphragm, is a crucial pre-requisite for the functionality of this muscle and the viability of the entire organism. Systemic impairment of Sema3A–Npn-1 (Npn-1 is also known as NRP1) signaling causes excessive branching of phrenic nerves in the diaphragm and into the central tendon region, where the majority of misguided axons innervate ectopic musculature. To elucidate whether these ectopic muscles are a result of misguidance of myoblast precursors due to the loss of Sema3A–Npn-1 signaling, we conditionally ablated Npn-1 in somatic motor neurons, which led to a similar phenotype of phrenic nerve defasciculation and, intriguingly, also formation of innervated ectopic muscles. We therefore hypothesize that ectopic myocyte fusion is caused by additional factors released by misprojecting growth cones. Slit2 and its Robo receptors are expressed by phrenic motor axons and migrating myoblasts, respectively, during innervation of the diaphragm. In vitro analyses revealed a chemoattractant effect of Slit2 on primary diaphragm myoblasts. Thus, we postulate that factors released by motor neuron growth cones have an influence on the migration properties of myoblasts during establishment of the diaphragm., Highlighted Article: Sema3A–Npn-1 signaling cell autonomously influences phrenic nerve branching in the diaphragm and induces ectopic muscle formation through secondary mechanisms, possibly by Slit–Robo signaling.