Genome-scale metabolic reconstruction of 7,302 human microorganisms for personalized medicine.

The human microbiome influences the efficacy and safety of a wide variety of commonly prescribed drugs. Designing precision medicine approaches that incorporate microbial metabolism would require strain- and molecule-resolved, scalable computational modeling. Here, we extend our previous resource of genome-scale metabolic reconstructions of human gut microorganisms with a greatly expanded version. AGORA2 (assembly of gut organisms through reconstruction and analysis, version 2) accounts for 7,302 strains, includes strain-resolved drug degradation and biotransformation capabilities for 98 drugs, and was extensively curated based on comparative genomics and literature searches. The microbial reconstructions performed very well against three independently assembled experimental datasets with an accuracy of 0.72 to 0.84, surpassing other reconstruction resources and predicted known microbial drug transformations with an accuracy of 0.81. We demonstrate that AGORA2 enables personalized, strain-resolved modeling by predicting the drug conversion potential of the gut microbiomes from 616 patients with colorectal cancer and controls, which greatly varied between individuals and correlated with age, sex, body mass index and disease stages. AGORA2 serves as a knowledge base for the human microbiome and paves the way to personalized, predictive analysis of host-microbiome metabolic interactions.

Early-Life Adversity Leaves Its Imprint on the Oral Microbiome for More Than 20 Years and Is Associated with Long-Term Immune Changes.

The early-life microbiome (ELM) interacts with the psychosocial environment, in particular during early-life adversity (ELA), defining life-long health trajectories. The ELM also plays a significant role in the maturation of the immune system. We hypothesised that, in this context, the resilience of the oral microbiomes, despite being composed of diverse and distinct communities, allows them to retain an imprint of the early environment. Using 16S amplicon sequencing on the EpiPath cohort, we demonstrate that ELA leaves an imprint on both the salivary and buccal oral microbiome 24 years after exposure to adversity. Furthermore, the changes in both communities were associated with increased activation, maturation, and senescence of both innate and adaptive immune cells, although the interaction was partly dependent on prior herpesviridae exposure and current smoking. Our data suggest the presence of multiple links between ELA, Immunosenescence, and cytotoxicity that occur through long-term changes in the microbiome.

Genome-Scale Metabolic Modeling of the Human Microbiome in the Era of Personalized Medicine.

The human microbiome plays an important role in human health and disease. Meta-omics analyses provide indispensable data for linking changes in microbiome composition and function to disease etiology. Yet, the lack of a mechanistic understanding of, e.g., microbiome-metabolome links hampers the translation of these findings into effective, novel therapeutics. Here, we propose metabolic modeling of microbial communities through constraint-based reconstruction and analysis (COBRA) as a complementary approach to meta-omics analyses. First, we highlight the importance of microbial metabolism in cardiometabolic diseases, inflammatory bowel disease, colorectal cancer, Alzheimer disease, and Parkinson disease. Next, we demonstrate that microbial community modeling can stratify patients and controls, mechanistically link microbes with fecal metabolites altered in disease, and identify host pathways affected by the microbiome. Finally, we outline our vision for COBRA modeling combined with meta-omics analyses and multivariate statistical analyses to inform and guide clinical trials, yield testable hypotheses, and ultimately propose novel dietary and therapeutic interventions.

Integration of constraint-based modeling with fecal metabolomics reveals large deleterious effects of Fusobacterium spp. on community butyrate production.

Characterizing the metabolic functions of the gut microbiome in health and disease is pivotal for translating alterations in microbial composition into clinical insights. Two major analysis paradigms have been used to explore the metabolic functions of the microbiome but not systematically integrated with each other: statistical screening approaches, such as metabolome-microbiome association studies, and computational approaches, such as constraint-based metabolic modeling. To combine the strengths of the two analysis paradigms, we herein introduce a set of theoretical concepts allowing for the population statistical treatment of constraint-based microbial community models. To demonstrate the utility of the theoretical framework, we applied it to a public metagenomic dataset consisting of 365 colorectal cancer (CRC) cases and 251 healthy controls, shining a light on the metabolic role of Fusobacterium spp. in CRC. We found that (1) glutarate production capability was significantly enriched in CRC microbiomes and mechanistically linked to lysine fermentation in Fusobacterium spp., (2) acetate and butyrate production potentials were lowered in CRC, and (3) Fusobacterium spp. presence had large negative ecological effects on community butyrate production in CRC cases and healthy controls. Validating the model predictions against fecal metabolomics, the in silico frameworks correctly predicted in vivo species metabolite correlations with high accuracy. In conclusion, highlighting the value of combining statistical association studies with in silico modeling, this study provides insights into the metabolic role of Fusobacterium spp. in the gut, while providing a proof of concept for the validity of constraint-based microbial community modeling.

Metabolic modelling reveals broad changes in gut microbial metabolism in inflammatory bowel disease patients with dysbiosis.

Inflammatory bowel diseases, such as Crohn's Disease, are characterised by an altered blood and faecal metabolome, and changes in gut microbiome composition. Here, we present an efficient, scalable, tractable systems biology framework to mechanistically link microbial strains and faecal metabolites. We retrieve strain-level relative abundances from metagenomics data from a cohort of paediatric Crohn's Disease patients with and without dysbiosis and healthy control children and construct and interrogate a personalised microbiome model for each sample. Predicted faecal secretion profiles and strain-level contributions to each metabolite vary broadly between healthy, dysbiotic, and non-dysbiotic microbiomes. The reduced microbial diversity in IBD results in reduced numbers of secreted metabolites, especially in sulfur metabolism. We demonstrate that increased potential to synthesise amino acids is linked to Proteobacteria contributions, in agreement with experimental observations. The established modelling framework yields testable hypotheses that may result in novel therapeutic and dietary interventions targeting the host-gut microbiome-diet axis.

Parkinson's disease-associated alterations of the gut microbiome predict disease-relevant changes in metabolic functions.

BACKGROUND: Parkinson's disease (PD) is a systemic disease clinically defined by the degeneration of dopaminergic neurons in the brain. While alterations in the gut microbiome composition have been reported in PD, their functional consequences remain unclear. Herein, we addressed this question by an analysis of stool samples from the Luxembourg Parkinson's Study (n = 147 typical PD cases, n = 162 controls). RESULTS: All individuals underwent detailed clinical assessment, including neurological examinations and neuropsychological tests followed by self-reporting questionnaires. Stool samples from these individuals were first analysed by 16S rRNA gene sequencing. Second, we predicted the potential secretion for 129 microbial metabolites through personalised metabolic modelling using the microbiome data and genome-scale metabolic reconstructions of human gut microbes. Our key results include the following. Eight genera and seven species changed significantly in their relative abundances between PD patients and healthy controls. PD-associated microbial patterns statistically depended on sex, age, BMI, and constipation. Particularly, the relative abundances of Bilophila and Paraprevotella were significantly associated with the Hoehn and Yahr staging after controlling for the disease duration. Furthermore, personalised metabolic modelling of the gut microbiomes revealed PD-associated metabolic patterns in the predicted secretion potential of nine microbial metabolites in PD, including increased methionine and cysteinylglycine. The predicted microbial pantothenic acid production potential was linked to the presence of specific non-motor symptoms. CONCLUSION: Our results suggest that PD-associated alterations of the gut microbiome can translate into substantial functional differences affecting host metabolism and disease phenotype.

Integrated Analyses of Microbiome and Longitudinal Metabolome Data Reveal Microbial-Host Interactions on Sulfur Metabolism in Parkinson's Disease.

Parkinson's disease (PD) exhibits systemic effects on the human metabolism, with emerging roles for the gut microbiome. Here, we integrate longitudinal metabolome data from 30 drug-naive, de novo PD patients and 30 matched controls with constraint-based modeling of gut microbial communities derived from an independent, drug-naive PD cohort, and prospective data from the general population. Our key results are (1) longitudinal trajectory of metabolites associated with the interconversion of methionine and cysteine via cystathionine differed between PD patients and controls; (2) dopaminergic medication showed strong lipidomic signatures; (3) taurine-conjugated bile acids correlated with the severity of motor symptoms, while low levels of sulfated taurolithocholate were associated with PD incidence in the general population; and (4) computational modeling predicted changes in sulfur metabolism, driven by A. muciniphila and B. wadsworthia, which is consistent with the changed metabolome. The multi-omics integration reveals PD-specific patterns in microbial-host sulfur co-metabolism that may contribute to PD severity.

Prospective associations of androgens and sex hormone-binding globulin with 12-month, lifetime and incident anxiety and depressive disorders in men and women from the general population.

BACKGROUND: Findings on associations of androgens and sex hormone-binding globulin (SHBG) with anxiety and depressive disorders in the general population remain inconclusive. METHODS: We used data of n = 993 men and n = 980 women from the Study of Health in Pomerania (SHIP, a prospective-longitudinal general population study from northeastern Germany). Immunoassay-measured serum concentrations of total testosterone, androstenedione and SHBG were assessed when participants were aged 20-80. 12-month, lifetime and incident DSM-IV anxiety and depressive disorders were assessed with the DIA-X/M-CIDI at 10-year follow-up, when participants were aged 29-89. Logistic regressions were adjusted for age, smoking, alcohol consumption, physical activity, waist circumference, hypertension and oral contraceptive use (women only) at baseline and follow-up interval. RESULTS: In men and women, androgens and SHBG were not associated significantly with incident anxiety and depressive disorders. In men, higher total testosterone predicted any 12-month (OR = 1.46) and lifetime (OR = 1.34) anxiety disorder, lifetime social phobia (OR = 2.15), and 12-month (OR = 1.48) and lifetime (OR = 1.39) specific phobia, but neither 12-month nor lifetime depression. Moreover, androstenedione in men interacted with age in predicting lifetime anxiety disorders (OR = 0.98): Higher androstenedione more strongly predicted lifetime anxiety in younger vs. older men. These findings, however, did not survive correction for multiple testing. In women, androgens and SHBG were not associated significantly with 12-month and lifetime anxiety and depressive disorders. LIMITATIONS: The follow-up period was relatively long and other factors might have affected the examined associations. CONCLUSIONS: Higher serum total testosterone in men and androstenedione in younger men may relate to an increased risk of anxiety disorders.

Microglia ablation alleviates myelin-associated catatonic signs in mice.

The underlying cellular mechanisms of catatonia, an executive "psychomotor" syndrome that is observed across neuropsychiatric diseases, have remained obscure. In humans and mice, reduced expression of the structural myelin protein CNP is associated with catatonic signs in an age-dependent manner, pointing to the involvement of myelin-producing oligodendrocytes. Here, we showed that the underlying cause of catatonic signs is the low-grade inflammation of white matter tracts, which marks a final common pathway in Cnp-deficient and other mutant mice with minor myelin abnormalities. The inhibitor of CSF1 receptor kinase signaling PLX5622 depleted microglia and alleviated the catatonic symptoms of Cnp mutants. Thus, microglia and low-grade inflammation of myelinated tracts emerged as the trigger of a previously unexplained mental condition. We observed a very high (25%) prevalence of individuals with catatonic signs in a deeply phenotyped schizophrenia sample (n = 1095). Additionally, we found the loss-of-function allele of a myelin-specific gene (CNP rs2070106-AA) associated with catatonia in 2 independent schizophrenia cohorts and also associated with white matter hyperintensities in a general population sample. Since the catatonic syndrome is likely a surrogate marker for other executive function defects, we suggest that microglia-directed therapies may be considered in psychiatric disorders associated with myelin abnormalities.

Visceral adipose tissue but not subcutaneous adipose tissue is associated with urine and serum metabolites.

Obesity is a complex multifactorial phenotype that influences several metabolic pathways. Yet, few studies have examined the relations of different body fat compartments to urinary and serum metabolites. Anthropometric phenotypes (visceral adipose tissue (VAT), subcutaneous adipose tissue (SAT), the ratio between VAT and SAT (VSR), body mass index (BMI), waist circumference (WC)) and urinary and serum metabolite concentrations measured by nuclear magnetic resonance spectroscopy were measured in a population-based sample of 228 healthy adults. Multivariable linear and logistic regression models, corrected for multiple testing using the false discovery rate, were used to associate anthropometric phenotypes with metabolites. We adjusted for potential confounding variables: age, sex, smoking, physical activity, menopausal status, estimated glomerular filtration rate (eGFR), urinary glucose, and fasting status. In a fully adjusted logistic regression model dichotomized for the absence or presence of quantifiable metabolite amounts, VAT, BMI and WC were inversely related to urinary choline (ß = -0.18, p = 2.73*10-3), glycolic acid (ß = -0.20, 0.02), and guanidinoacetic acid (ß = -0.12, p = 0.04), and positively related to ethanolamine (ß = 0.18, p = 0.02) and dimethylamine (ß = 0.32, p = 0.02). BMI and WC were additionally inversely related to urinary glutamine and lactic acid. Moreover, WC was inversely associated with the detection of serine. VAT, but none of the other anthropometric parameters, was related to serum essential amino acids, such as valine, isoleucine, and phenylalanine among men. Compared to other adiposity measures, VAT demonstrated the strongest and most significant relations to urinary and serum metabolites. The distinct relations of VAT, SAT, VSR, BMI, and WC to metabolites emphasize the importance of accurately differentiating between body fat compartments when evaluating the potential role of metabolic regulation in the development of obesity-related diseases, such as insulin resistance, type 2 diabetes, and cardiovascular disease.

Community-Dwelling People Screened Positive for Dementia in Primary Care: A Comprehensive, Multivariate Descriptive Analysis Using Data from the DelpHi-Study.

BACKGROUND: Efficient help and care for people with dementia (PWD) is dependent on knowledge about PWD in primary care. OBJECTIVE: This analysis comprehensively describes community-dwelling PWD in primary care with respect to various dementia care specific variables. METHODS: The analyses are based on baseline data of the ongoing general practitioner-based, randomized, controlled intervention trial DelpHi-MV (Dementia: life- and person-centered help). 6,838 patients were screened for dementia in 136 GP practices; 17.1% were screened positive, 54.4% of those agreed to participate and data could be assessed in n = 516 subjects. We assessed age, sex, living situation, cognitive status, functional status, level of impairment, comorbidities, formal diagnosis of dementia, depression, neuropsychiatric symptoms, quality of life, utilization of medical support, and pharmacological therapy. RESULTS: Concerning clinical-, dementia-, and health-related variables, the sample under examination was on average mildly cognitively and functionally impaired (MMSE, m = 22.2; BADL, m = 3.7). A level of care was assigned in 38.0%. Depression was identified in 15.4% and other frequent comorbidities were high blood pressure (83.3%), coronary heart diseases (37.1%), cerebrovascular diseases (22.3%), among others. In 48.6%, neuropsychiatric symptoms were present in a clinically relevant severity. Pharmacological treatment with antidementia medication was received by 25.8% and antidepressant medication by 14.0%. Utilization of services was generally low. CONCLUSION: The comprehensive description of people screened positive for dementia in primary care reveals a complex and unique population of patients. They are considerably underdiagnosed and in their majority mildly to moderately affected. More in-depth analyses are needed to study relations, associations and interactions between different variables.

Measuring Biological Age via Metabonomics: The Metabolic Age Score.

Chronological age is one of the most important risk factors for adverse clinical outcome. Still, two individuals at the same chronological age could have different biological aging states, leading to different individual risk profiles. Capturing this individual variance could constitute an even more powerful predictor enhancing prediction in age-related morbidity. Applying a nonlinear regression technique, we constructed a metabonomic measurement for biological age, the metabolic age score, based on urine data measured via (1)H NMR spectroscopy. We validated the score in two large independent population-based samples by revealing its significant associations with chronological age and age-related clinical phenotypes as well as its independent predictive value for survival over approximately 13 years of follow-up. Furthermore, the metabolic age score was prognostic for weight loss in a sample of individuals who underwent bariatric surgery. We conclude that the metabolic age score is an informative measurement of biological age with possible applications in personalized medicine.

Burden of Behavioral and Psychiatric Symptoms in People Screened Positive for Dementia in Primary Care: Results of the DelpHi-Study.

BACKGROUND: There is limited knowledge about the range and effects of neuropsychiatric symptoms shown by persons with dementia (PWD) living in the community and their related caregiver burden. OBJECTIVE: To examine neuropsychiatric symptoms in PWD in primary care with regard to frequency, severity, and burden to caregiver; to compare PWD with and without symptoms with regard to sociodemographics, care-related, and disease-related variables; and to identify variables associated with symptoms. METHODS: A general physician-based epidemiological cohort of 248 people screened positive for dementia over the age of 70 (living at home) and their caregivers, was assessed using the Neuropsychiatric Interview (NPI), sociodemographics, and disease-related variables. RESULTS: In preliminary analyses, neuropsychiatric symptoms were frequent in PWD. Prevalence numbers ofdysphoria/depression, apathy, and agitation/aggression were each more than 30% . The severity of neuropsychiatric symptoms in people screened positive for dementia in primary care is moderate with a mean NPI score of m = 11.91 (SD = 16.0). Overall, caregiver distress is low, indicated by a total distress score of m = 5.94 (SD = 7.2, range 0-39). Common or frequent symptoms are not necessarily the most distressing symptoms. CONCLUSIONS: Neuropsychiatric symptoms are common in people screened positive for dementia in primary care. While frequency, severity, and perceived distress might be low in the total sample, we identified the dimensions delusions, aggression, anxiety, disinhibition, and depression to be perceived "severely" to "extremely" distressing in more than 30% of the caregivers affected. The association between activities of daily living and symptoms needs further attention.

Transcription of the vascular endothelial growth factor receptor-3 (VEGFR3) gene is regulated by the zinc finger proteins Sp1 and Sp3 and is under epigenetic control: transcription of vascular endothelial growth factor receptor 3.

BACKGROUND: In the past, the vascular endothelial growth factor receptor-3 (VEGFR-3) has been linked to the regulation of lymphangiogenesis and the lymphatic spread of solid malignancies. The molecular mechanisms controlling VEGFR3 gene expression have, however, remained poorly understood. Here, we aimed at assessing these mechanisms through VEGFR3 gene promoter analysis and the identification of transcription factors binding to it. In addition, we focussed on epigenetic modifications underlying VEGFR3 transcription regulation. METHODS: 5' Deletion analyses for the identification of functional promoter elements, electrophoretic mobility shift assays, chromatin immunoprecipitations, methylation-specific PCRs, and Trichostatin A (TSA) and 5-Aza desoxycytidine (5-Aza dC) treatments were performed in this study. RESULTS: Following the isolation of a 2 kb stretch of 5'-flanking DNA of VEGFR3, we identified a novel GC-rich element (GRE) spanning -101/-66 sufficient for VEGFR3 transcription and activated by Sp1 and Sp3, respectively. Histone de-acetylase inhibition by TSA led to the accumulation of acetylated histones H3/H4 at the VEGFR3 gene promoter, up-regulation of its mRNA levels, and transactivation of promoter reporter constructs in endothelial cell lines. Similarly, methylation inhibition by 5-Aza dC triggered up-regulation of VEGFR3 mRNA levels and increased promoter activity. TSA and 5-Aza-dC did not influence Sp1/Sp3 binding, but increased the transactivating capacity of both transcription factors, suggesting epigenetic modification as an underlying mechanism. CONCLUSIONS: Here we describe the identification of regulatory elements controlling human VEGFR3 gene expression and show that histone acetylation and CpG methylation are important determinants of VEGFR3 transcription regulation. These findings may facilitate the development of intervention strategies aimed at targeting VEGFR3-based tumor lymphangiogenesis and/or lymphatic tumor spread.

Regulation of vascular endothelial growth factor D by orphan receptors hepatocyte nuclear factor-4 alpha and chicken ovalbumin upstream promoter transcription factors 1 and 2.

Vascular endothelial growth factor D has recently been linked to the control of lymphangiogenesis and lymphatic metastasis. The molecular determinants regulating vegf-D gene transcription, however, have not yet been identified. After isolation of 2 kb of 5'-flanking DNA of the human vegf-D gene, we identified a novel, atypical direct repeat (DR) element consisting of a consensus half-site (AGGTCA) at -125/-119 and a degenerated DR half-site (ATGTTA) at -99/-94 as sufficient and necessary for vegf-D transcription. The vegf-D DR element is bound and activated by the orphan receptors hepatocyte nuclear factor 4 alpha (HNF-4 alpha) and chicken ovalbumin upstream promoter transcription factor (COUP-TF)-1/COUP-TF2. Additionally, chromatin immunoprecipitation assays identified transcriptional coactivators cyclic AMP-responsive element binding protein-binding protein and glucocorticoid receptor interacting protein 1 at the vegf-D DR element and functional assays confirmed their stimulatory effect on the vegf-D promoter. Histone deacetylase inhibition by trichostatin A led to accumulation of acetylated histones H3/H4 at the vegf-D promoter, up-regulation of vegf-D mRNA levels, and transactivation of vegf-D promoter reporter gene constructs in cancer cell lines. This study for the first time describes the molecular determinants in cis and trans controlling vegf-D gene transcription and identifies interaction of HNF-4 alpha and COUP-TF1/COUP-TF2 with a proximal, atypical DR element as indispensable for vegf-D transcription. Moreover, our findings suggest that epigenetic control of histone acetylation represents an important determinant of vegf-D gene expression in cancer cells. These results provide novel insights into the molecular machinery controlling vegf-D gene expression and may add to a better understanding of the regulation of lymphangiogenesis in vascular development and cancer.

Vascular endothelial growth factor-D and its receptor VEGFR-3: two novel independent prognostic markers in gastric adenocarcinoma.

PURPOSE: Vascular endothelial growth factor (VEGF)-D and its homolog VEGF-C influence lymphangiogenesis through activation of VEGF receptor 3 (VEGFR-3), and have been implicated in lymphatic tumor spread. Nodal dissemination of gastric adenocarcinomas critically determines clinical outcome and therapeutic options of affected patients. Therefore, we analyzed expression and prognostic significance of VEGF-D along with VEGF-C, and VEGFR-3 in gastric adenocarcinomas. MATERIALS AND METHODS: VEGF-C, VEGF-D, and VEGFR-3 were analyzed in 91 R(0)-resected primary gastric adenocarcinomas, corresponding noncancerous gastric mucosa, and lymph node metastases employing immunohistochemistry and/or in situ hybridization. Blood and lymph vessel densities were assessed after staining with CD31 and LYVE-1-specific antibodies. RESULTS: VEGF-D and VEGF-C were detected in 67.0% and 50.5% of gastric cancers, respectively. Healthy gastric mucosa was negative for VEGF-C and in 12.5% positive for VEGF-D. Presence of VEGF-D (P = .005) or VEGF-C (P = .006) was correlated with lymphatic metastases and decreased survival (VEGF-D, P < .05; VEGF-C, P < .05). VEGFR-3 was correlated with reduced carcinoma-specific survival (P < .05), and Cox multivariate regression analysis qualified VEGF-D and VEGFR-3, but not VEGF-C, as independent prognostic parameters. In lymph node-positive gastric cancers, presence of VEGF-D/VEGFR-3 was associated with poor survival, whereas absence of VEGF-D/VEGFR-3 defined a subgroup of patients with clearly favorable prognosis. CONCLUSION: VEGF-D and VEGFR-3 are novel independent prognostic marker molecules aiding to identify patients with poor prognosis after curative resection of gastric adenocarcinomas. Combined analysis of the VEGF-C/VEGF-D/VEGFR-3 system can be useful to identify patients with unfavorable clinical outcome and thereby may help to refine therapeutic decisions in gastric cancer.