Longitudinal analysis reveals transition barriers between dominant ecological states in the gut microbiome.

The Pioneer 100 Wellness Project involved quantitatively profiling 108 participants\u27 molecular physiology over time, including genomes, gut microbiomes, blood metabolomes, blood proteomes, clinical chemistries, and data from wearable devices. Here, we present a longitudinal analysis focused specifically around the Pioneer 100 gut microbiomes. We distinguished a subpopulation of individuals with reduced gut diversity, elevated relative abundance of the genus Prevotella, and reduced levels of the genus Bacteroides We found that the relative abundances of Bacteroides and Prevotella were significantly correlated with certain serum metabolites, including omega-6 fatty acids. Primary dimensions in distance-based redundancy analysis of clinical chemistries explained 18.5% of the variance in bacterial community composition, and revealed a Bacteroides/Prevotella dichotomy aligned with inflammation and dietary markers. Finally, longitudinal analysis of gut microbiome dynamics within individuals showed that direct transitions between Bacteroides-dominated and Prevotella-dominated communities were rare, suggesting the presence of a barrier between these states. One implication is that interventions seeking to transition between Bacteroides- and Prevotella-dominated communities will need to identify permissible paths through ecological state-space that circumvent this apparent barrier

Inhibition of Breast Cancer Metastasis and Regulation of Lipid Metabolism by the Active Vitamin D Metabolite 1α,25-Dihydroxyvitamin D3

Breast cancer continues to be one of the most commonly diagnosed cancers in the United States, with 1 in 8 women expected to develop invasive breast cancer during her lifetime. Metastatic breast cancer in particular is a debilitating disease accounting for over 40,000 deaths each year. Therefore, it is imperative to identify effective preventive compounds against breast cancer metastasis that may prolong survival of breast cancer patients. Epidemiological evidence suggests that vitamin D may protect against metastatic breast cancer. However, the mechanisms orchestrating vitamin D’s antimetastatic effect are unclear and necessitate futher investigation. In the present studies, the effect of 1α,25 dihydroxyvitamin D (1,25(OH)2D), the bioactive form of vitamin D, on metastatic breast cancer capability was investigated using an in vitro (rMET) model that accurately recapitulates multiple steps of breast to bone metastasis, the most common metastatic site in breast cancer patients. Treatment with 1,25(OH)2D (10 nM) in the rMET model significantly inhibited the metastatic potential of MCF10CA1a and MDA-MB-231 breast epithelial cells. 1,25(OH)2D further inhibited the ability of MCF10CA1a epithelial cells to adapt to the drastically different bone environment recapitulated in the rMET model, suggesting 1,25(OH)2D may inhibit both the migration of cells away from the primary tumor as well as their ability to colonize secondary organs. Epithelial-mesenchymal transition is an essential feature of metastatic breast cancer cells characterized by loss of epithelial cell-cell junction proteins (E-cadherin) and acquisition of mesenchymal markers (N-cadherin). Invesitigation into the effect of 1,25(OH)2D E- and N-cadherin expression in MCF10CA1a breast epithelial cells demonstrated an inhibitory effect of 1,25(OH)2D on EMT. E-cadherin mRNA and protein expression were both upregulated while N-cadherin mRNA was downregulated with 1,25(OH)2D treatment in this cell line. breast cancer cells undergo multiple genetic and phenotypic changes towards their progression to a metastatic phenotype. Lipid metabolic reprograming characterized by upregulated de novo fatty acid synthesis as well as increased neutral lipid accumulation is a comon characteristic observed in metastatic breast cancer cells, making it a promising therapeutic target in metastatic breast cancer prevention. Therefore, the effect of 1,25(OH)2D on metastatic breast cancer lipid metabolism was investigated using MCF10CA1a breast epithelial cells. 1,25(OH)2D treatment drastically inhibited de novo fatty acid synthesis, as measured by [U]-13C-glucose incorporation into palmitate and stearate through liquid chromatography coupled mass spectrometry. Furthermore, measurement of neutral lipid accumulation using a triacylglycerol colorimetric assay as well as confocal microscopy demonstrated that treatment with 1,25(OH)2D inhibited neutral lipid levels in metastatic breast cancer cells in a time dependent manner. The anaplerotic enzyme pyruvate carboxylase (PC) emerged as a target of 1,25(OH)2D-mediated regulation. PC expression was decreased with 1,25(OH)2D treatment and its overexpression reversed the effect of 1,25(OH)2D on de novo fatty acid synthesis in MCF10CA1a cells. PC overexpression further promoted a metastatic phenotype as measured by a Boyden Chamber migration assay in MCF10CA1a and MCF10A-Harvey ras oncogene transfected cells, a model of early breast cancer progression. Together these studies suggest that PC is an essential enzyme in breast cancer metastasis and that 1,25(OH)2D may potentially inhibit metastasis through downregulation of PC expression. PC was shown to play an important role in maintaining redox balance in pancreatic β-cells. We therefore hypothesized that PC downregulation by 1,25(OH)2D may promote oxidative stress. To test this hypothesis, the effect of 1,25(OH)2D on oxidative stress was investigated in MCF10A-Harvey ras oncogene transfected breast epithelial cells. 1,25(OH)2D increased reactive oxygen species as well as cell susceptibility to hydrogen peroxide induced cell death. Treatment with 1,25(OH)2D further depleted reduced glutathione levels, demonstrating a pro-oxidative effect of vitamin D in early breast cancer progression. Treatment with the mitochondrial pyruvate carrier inhibitor UK-5099 mimicked the effect of 1,25(OH)2D on GSH levels, with 1,25(OH)2D having no further effect. Administration of oxaloacetate (2 mM), the product of pyruvate metabolism in the mitochondria by PC, rescued 1,25(OH)2D treated cells from hydrogen peroxide induced cell death. Collectively, the present studies provide novel insight into vitamin D mediated mechanisms in early and later breast cancer prevention, potentially through the downregulation of the mitochondrial enzyme PC. Results of the present investigation provide additional evidence for a protective role of vitamin D against breast cancer development and metastasis

Healthy aging and the human gut microbiome: why we cannot just turn back the clock.

The aging research field has largely focused on reversing aging-related changes in the body. However, emerging evidence about the gut microbiome indicates that it may not be optimal to just turn back the clock. Here, we advocate for a more tailored and function-focused approach promoting health across the lifespan

D3T acts as a pro-oxidant in a cell culture model of diabetes-induced peripheral neuropathy

Diabetes mellitus is one of the most common chronic diseases in the United States and peripheral neuropathy (PN) affects at least 50% of diabetic patients. Medications available for patients ameliorate symptoms (pain), but do not protect against cellular damage and come with severe side effects, leading to discontinued use. Our research group uses differentiated SH-SY5Y cells treated with advanced glycation end products (AGE) as a model to mimic diabetic conditions and to study the mechanisms of oxidative stress mediated cell damage and antioxidant protection. N-acetylcysteine (NAC), a common antioxidant supplement, was previously shown by our group to fully protect against AGE-induced damage. We have also shown that 3H-1,2-dithiole-3-thione (D3T), a cruciferous vegetable constituent and potent inducer of nuclear factor (erythroid-derived 2)- like 2 (Nrf2), can significantly increase cellular GSH concentrations and protect against oxidant species–induced cell death. Paradoxically, D3T conferred no protection against AGE-induced cell death or neurite degeneration. In the present study we establish a mechanism for this paradox by showing that D3T in combination with AGE increased oxidant species generation and depleted GSH via inhibition of glutathione reductase (GR) activity and increased expression of the NADPH generating enzyme glucose-6-phosphate dehydrogenase (G6PD). Blocking NADPH generation with the G6PD inhibitor dehydroepiandrosterone was found to protect against AGE-induced oxidant species generation, loss of viability, and neurite degeneration. It further reversed the D3T potentiation effect under AGE-treated conditions. Collectively, these results suggest that strategies aimed at combating oxidative stress that rely on upregulation of the endogenous antioxidant defense system via Nrf2 may backfire and promote further damage in diabetic PN. Keywords: Peripheral neuropathy, Diabetes, D3T, NAC, Redox balance, Oxidative stres

Pyruvate carboxylase supports the pulmonary tropism of metastatic breast cancer

Abstract Background Overcoming systemic dormancy and initiating secondary tumor grow under unique microenvironmental conditions is a major rate-limiting step in metastatic progression. Disseminated tumor cells encounter major changes in nutrient supplies and oxidative stresses compared to the primary tumor and must demonstrate significant metabolic plasticity to adapt to specific metastatic sites. Recent studies suggest that differential utilization of pyruvate sits as a critical node in determining the organotropism of metastatic breast cancer. Pyruvate carboxylase (PC) is key enzyme that converts pyruvate into oxaloacetate for utilization in gluconeogenesis and replenishment of the TCA cycle. Methods Patient survival was analyzed with respect to gene copy number alterations and differential mRNA expression levels of PC. Expression of PC was analyzed in the MCF-10A, D2-HAN and the 4 T1 breast cancer progression series under in vitro and in vivo growth conditions. PC expression was depleted via shRNAs and the impact on in vitro cell growth, mammary fat pad tumor growth, and pulmonary and non-pulmonary metastasis was assessed by bioluminescent imaging. Changes in glycolytic capacity, oxygen consumption, and response to oxidative stress were quantified upon PC depletion. Results Genomic copy number increases in PC were observed in 16–30% of metastatic breast cancer patients. High expression of PC mRNA was associated with decreased patient survival in the MCTI and METABRIC patient datasets. Enhanced expression of PC was not recapitulated in breast cancer progression models when analyzed under glucose-rich in vitro culture conditions. In contrast, PC expression was dramatically enhanced upon glucose deprivation and in vivo in pulmonary metastases. Depletion of PC led to a dramatic decrease in 4 T1 pulmonary metastasis, but did not affect orthotopic primary tumor growth. Tail vein inoculations confirmed the role of PC in facilitating pulmonary, but not extrapulmonary tumor initiation. PC-depleted cells demonstrated a decrease in glycolytic capacity and oxygen consumption rates and an enhanced sensitivity to oxidative stress. Conclusions Our studies indicate that PC is specifically required for the growth of breast cancer that has disseminated to the lungs. Overall, these findings point to the potential of targeting PC for the treatment of pulmonary metastatic breast cancer

From taxonomy to metabolic output: what factors define gut microbiome health?

Many studies link the composition of the human gut microbiome to aberrant health states. However, our understanding of what constitutes a ‘healthy’ gut ecosystem, and how to effectively monitor and maintain it, are only now emerging. Here, we review current approaches to defining and monitoring gut microbiome health, and outline directions for developing targeted ecological therapeutics. We emphasize the importance of identifying which ecological features of the gut microbiome are most resonant with host molecular phenotypes, and highlight certain gut microbial metabolites as potential biomarkers of gut microbiome health. We further discuss how multi-omic measurements of host phenotypes, dietary information, and gut microbiome profiles can be integrated into increasingly sophisticated host-microbiome mechanistic models that can be leveraged to design personalized interventions. Overall, we summarize current progress on defining microbiome health and highlight a number of paths forward for engineering the ecology of the gut to promote wellness

Additional file 3: of Pyruvate carboxylase supports the pulmonary tropism of metastatic breast cancer

Figure S3. Transient induction of EMT does not induce expression of PC. (A) Photomicrographs showing EMT-like changes in cellular morphology of the control (scram) and PC-depleted (shPC25 and shPC28) 4 T1 cells upon treatment with exogenous TGF-β1 (5 ng/ml) for 4 days. (B) Immunoblot analyses for PC in cells shown in Panel A. β-tubulin served as a loading control. (C) The RAS transformed MCF-10A-T1 k cells were constructed to express a doxycycline-inducible vector encoding the EMT transcription factor Snail. Expression of PC was analyzed upon addition of doxycycline (Dox) at the indicated concentrations. Expression of Snail and β-tubulin (β-Tub) served as loading controls. (PDF 1401 kb

Additional file 1: of Pyruvate carboxylase supports the pulmonary tropism of metastatic breast cancer

Figure S1. PC expression is not correlated with primary tumor size. Patient samples within the METABRIC dataset were analyzed for PC expression in relation to primary tumor size. Data are analyzed by a nonparametric Spearman correlation resulting in the indicated r and P values. The linear regression for these data is also shown. (PDF 235 kb