Human gut microbiota and their production of endocannabinoid-like mediators are directly affected by a dietary oil.

Dietary omega-3 polyunsaturated fatty acids (n-3 PUFAs) and the gut microbiome affect each other. We investigated the impact of supplementation with Buglossoides arvensis oil (BO), rich in stearidonic acid (SDA), on the human gut microbiome. Employing the Mucosal Simulator of the Human Intestinal Microbial Ecosystem (M-SHIME), we simulated the ileal and ascending colon microbiomes of four donors. Our results reveal two distinct microbiota clusters influenced by BO, exhibiting shared and contrasting shifts. Notably, Bacteroides and Clostridia abundance underwent similar changes in both clusters, accompanied by increased propionate production in the colon. However, in the ileum, cluster 2 displayed a higher metabolic activity in terms of BO-induced propionate levels. Accordingly, a triad of bacterial members involved in propionate production through the succinate pathway, namely Bacteroides, Parabacteroides, and Phascolarctobacterium, was identified particularly in this cluster, which also showed a surge of second-generation probiotics, such as Akkermansia, in the colon. Finally, we describe for the first time the capability of gut bacteria to produce N-acyl-ethanolamines, and particularly the SDA-derived N-stearidonoyl-ethanolamine, following BO supplementation, which also stimulated the production of another bioactive endocannabinoid-like molecule, commendamide, in both cases with variations across individuals. Spearman correlations enabled the identification of bacterial genera potentially involved in endocannabinoid-like molecule production, such as, in agreement with previous reports, Bacteroides in the case of commendamide. This study suggests that the potential health benefits on the human microbiome of certain dietary oils may be amenable to stratified nutrition strategies and extend beyond n-3 PUFAs to include microbiota-derived endocannabinoid-like mediators.

Curcumin attenuates replicative senescence in human dental follicle cells and restores their osteogenic differentiation.

OBJECTIVE: This study aimed to examine the therapeutic effects of curcumin against replicative senescence in dental follicle cells (DFCs). METHODS: Human DFCs were cultured in Dulbecco's Modified Eagle Medium with growth supplements. Replicative senescence in DFCs at different passages was assessed using ß-galactosidase activity assay. Cell proliferation and size of DFCs at different passages were determined by CCK-8 kit and microscopy method, respectively. In addition, curcumin's effect on replicative senescence, cell proliferation, and size of DFCs at different passages was analyzed. Using western-blot analysis and siRNA-mediated gene silencing, we determined the molecular mechanisms involved in curcumin's effect against replicative senescence and osteogenic differentiation in DFCs at different passages. RESULTS: We observed decreased proliferation and increased cell size and replicative senescence in cultured human DFCs at higher passages. Intriguingly, despite not showing any effect on cell size, curcumin (50 µM) significantly restored proliferation ability in DFCs and inhibited their replicative senescence. Concerning mechanisms, we found that curcumin inhibits replicative senescence in DFCs via down-regulation of senescence markers (P16 & P21) and restoration of proliferation markers (E2F1 & P53). Additionally, curcumin also rescued the osteogenic differentiation potential in higher-passage DFCs via restoration of osteogenic markers RUNX2 and OPN. CONCLUSION: Our findings reveal for the first time that curcumin could act as a potential anti-senescence therapeutic for DFCs via regulation of proliferation, senescence, and osteogenic differentiation markers.

Spontaneous and Naloxone-Precipitated Withdrawal Behaviors From Chronic Opiates are Accompanied by Changes in N-Oleoylglycine and N-Oleoylalanine Levels in the Brain and Ameliorated by Treatment With These Mediators.

Rationale: The endocannabinoidome mediators, N-Oleoylglycine (OlGly) and N-Oleoylalanine (OlAla), have been shown to reduce acute naloxone-precipitated morphine withdrawal affective and somatic responses. Objectives: To determine the role and mechanism of action of OlGly and OlAla in withdrawal responses from chronic exposure to opiates in male Sprague-Dawley rats. Methods: Opiate withdrawal was produced: 1) spontaneously 24 h following chronic exposure to escalating doses of morphine over 14 days (Experiments 1 and 2) and steady-state exposure to heroin by minipumps for 12 days (Experiment 3), 2) by naloxone injection during steady-state heroin exposure (Experiment 4), 3) by naloxone injection during operant heroin self-administration (Experiment 5). Results: In Experiment 1, spontaneous morphine withdrawal produced somatic withdrawal reactions. The behavioral withdrawal reactions were accompanied by suppressed endogenous levels of OlGly in the nucleus accumbens, amygdala, and prefrontal cortex, N-Arachidonylglycerol and OlAla in the amygdala, 2-arachidonoylglycerol in the nucleus accumbens, amygdala and interoceptive insular cortex, and by changes in colonic microbiota composition. In Experiment 2, treatment with OlAla, but not OlGly, reduced spontaneous morphine withdrawal responses. In Experiment 3, OlAla attenuated spontaneous steady-state heroin withdrawal responses at both 5 and 20 mg/kg; OlGly only reduced withdrawal responses at the higher dose of 20 mg/kg. Experiment 4 demonstrated that naloxone-precipitated heroin withdrawal from steady-state exposure to heroin (7 mg/kg/day for 12 days) is accompanied by tissue-specific changes in brain or gut endocannabinoidome mediator, including OlGly and OlAla, levels and colonic microbiota composition, and that OlAla (5 mg/kg) attenuated behavioural withdrawal reactions, while also reversing some of the changes in brain and gut endocannabinoidome and gut microbiota induced by naloxone. Experiment 5 demonstrated that although OlAla (5 mg/kg) did not interfere with operant heroin self-administration on its own, it blocked naloxone-precipitated elevation of heroin self-administration behavior. Conclusion: These results suggest that OlAla and OlGly are two endogenous mediators whose brain concentrations respond to chronic opiate treatment and withdrawal concomitantly with changes in colon microbiota composition, and that OlAla may be more effective than OlGly in suppressing chronic opiate withdrawal responses.

Three of a Kind: Control of the Expression of Liver-Expressed Antimicrobial Peptide 2 (LEAP2) by the Endocannabinoidome and the Gut Microbiome.

The endocannabinoidome (expanded endocannabinoid system, eCBome)-gut microbiome (mBIome) axis plays a fundamental role in the control of energy intake and processing. The liver-expressed antimicrobial peptide 2 (LEAP2) is a recently identified molecule acting as an antagonist of the ghrelin receptor and hence a potential effector of energy metabolism, also at the level of the gastrointestinal system. Here we investigated the role of the eCBome-gut mBIome axis in the control of the expression of LEAP2 in the liver and, particularly, the intestine. We confirm that the small intestine is a strong contributor to the circulating levels of LEAP2 in mice, and show that: (1) intestinal Leap2 expression is profoundly altered in the liver and small intestine of 13 week-old germ-free (GF) male mice, which also exhibit strong alterations in eCBome signaling; fecal microbiota transfer (FMT) from conventionally raised to GF mice completely restored normal Leap2 expression after 7 days from this procedure; in 13 week-old female GF mice no significant change was observed; (2) Leap2 expression in organoids prepared from the mouse duodenum is elevated by the endocannabinoid noladin ether, whereas in human Caco-2/15 epithelial intestinal cells it is elevated by PPARγ activation by rosiglitazone; (3) Leap2 expression is elevated in the ileum of mice with either high-fat diet-or genetic leptin signaling deficiency-(i.e., ob/ob and db/db mice) induced obesity. Based on these results, we propose that LEAP2 originating from the small intestine may represent a player in eCBome- and/or gut mBIome-mediated effects on food intake and energy metabolism.

Butyrate induced Tregs are capable of migration from the GALT to the pancreas to restore immunological tolerance during type-1 diabetes.

Type-1 diabetes (T1D) is an autoimmune disease caused by progressive loss of insulin-producing beta cells in the pancreas. Butyrate is a commensal microbial-derived metabolite, implicated in intestinal homeostasis and immune regulation. Here, we investigated the mechanism of diabetes remission in non-obese diabetic (NOD) mice following butyrate administration. Sodium butyrate (150 mM) was administered to female NOD mice in drinking water after the onset of hyperglycemia (15-25 weeks age) and at 4 weeks of age (early-intervention group). Butyrate administration reduced the progression of hyperglycemia in diabetic mice and delayed onset of diabetes in the early-intervention group with a reduction in insulitis. Butyrate administration increased regulatory T cells (Tregs) in the colon, mesenteric lymph nodes, Peyer's patches, and its protective effects diminished upon depletion of Tregs. Further, an increase in α4ß7, CCR9, and GPR15 expressing Tregs in the pancreatic lymph nodes (PLN) and pancreas in butyrate-treated mice suggested migration of gut-primed Tregs towards the pancreas. Finally, the adoptive transfer experiments demonstrated that induced Tregs from gut-associated lymphoid tissue can migrate towards the pancreas and PLN and delay the onset of diabetes. Our results thus suggest that early administration of butyrate can restore immunological tolerance during T1D via induction of Tregs with migratory capabilities.

In vitro evidence of NLRP3 inflammasome regulation by histone demethylase LSD2 in renal cancer: a pilot study.

NLRP3 pathway plays a vital role in the pathogenesis of different human cancers but still the regulation of NLRP3 pathway largely unknown. Therefore, we examined the levels of NLRP3 and its downstream components (caspase-1 and IL-1ß) and its relationship with histone modifiers in renal cancer pathogenesis. Total 30 cases of clear cell renal cell carcinoma (ccRCC), were studied for NLRP3, caspase-1 and IL-1ß expression using real-time PCR, which showed the augmented levels of all the three components of NLRP3 inflammasome pathway in ccRCC. Next, role of the FAD dependent monoamine oxidases (LSD2) and jumonji C (JmjC)-domain-containing, iron-dependent dioxygenases (KDM5A) histone demethylases were evaluated in regulation of NLRP3 inflammasome pathway in-vitro using RCC cell line. It was observed that silencing of KDM5A didn't alter the levels of neither of the NLRP3 component but inhibition of LSD2 showed significant effect on NLRP3 expression while no change in caspase-1 and IL-1ß levels. This study suggests that rather LSD2 not KDM5A lysine demethylase family might be involved in the regulation of NLRP3 inflammasome in cancer cells which could be useful for deciphering the future therapeutic targets for the disease.

Expression profile of H3K4 demethylases with their clinical and pathological correlation in patients with clear cell renal cell carcinoma.

OBJECTIVE: Clear cell renal cell carcinoma (ccRCC) is one of the deadly diseases with poor metastatic disease prognosis. There is an urgent need to explore the potential molecular markers which can improve the prognosis of the disease. Histone demethylases have emerged as a powerful tool for cancer prognosis and therapeutics during the last decade. The implications of demethylases of histone 3 lysine 4 (H3K4) in ccRCC are however unrevealed. We therefore evaluated the expression of H3K4 demethylases in ccRCC, with emphasis on their clinical significance as a prognostic marker. METHODS: Total 50 histopathological confirmed cases of ccRCC were enrolled in the study. The expression of seven H3K4 demethylases was determined by Real-Time PCR using gene specific primers and correlated with tumor stage, grade and metastasis. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the prognostic significance of H3K4 demethylases. RESULTS: The median age of the patients was 54 years with predominance of male patients by 2.6-fold. Among seven genes viz FBXL10, LSD1, LSD2, KDM5A, KDM5B, KDM5C and KDM5D analyzed, LSD2 was found to be significantly associated with tumor stage and metastasis. The optimal cut-off value for LSD2 was 3.2 as calculated from ROC curve analysis for metastasis as well as TNM stage with area under curve of 0.74 and 0.78 respectively. In addition, LSD2 expression showed significant positive correlation with LSD1 expression in tumor metastasis. CONCLUSION: The expression of LSD2 was associated with higher TNM stage and metastasis of the tumor and thus, might serve as a useful marker for ccRCC progression.

Pathogenic and Therapeutic Role of H3K4 Family of Methylases and Demethylases in Cancers.

Histone modifications occupy an essential position in the epigenetic landscape of the cell, and their alterations have been linked to cancers. Histone 3 lysine 4 (H3K4) methylation has emerged as a critical epigenetic cue for the regulation of gene transcription through dynamic modulation by several H3K4 methyltransferases (writers) and demethylases (erasers). Any disturbance in the delicate balance of writers and erasers can result in the mis-regulation of H3K4 methylation, which has been demonstrated in several human cancers. Therefore, H3K4 methylation has been recognized as a putative therapeutic or prognostic tool and drug trials of different inhibitors of this process have demonstrated promising results. Henceforth, more detailed knowledge of H3K4 methylation is utmost important for elucidating the complex cellular processes, which might help in improving the disease outcome. The primary focus of this review will be directed on deciphering the role of H3K4 methylation along with its writers/erasers in different cancers.

Influence of autocrine growth hormone on NF-κB activation leading to epithelial-mesenchymal transition of mammary carcinoma.

Progression of breast cancers often depends on hormones among which human growth hormone is prominently involved in breast cancer progression. Earlier studies have reported constitutive activation of nuclear factor-κB, a key regulator of growth hormone receptor-mediated signaling pathway in breast carcinoma, but the precise molecular mechanisms are still elusive. In this study, we investigated the effect of human growth hormone on nuclear factor-κB activation and epithelial-mesenchymal transition in breast carcinoma. Our results explored that autocrine production of human growth hormone enhances cellular proliferation by the activation of nuclear factor-κB (65 kDa) and downregulation of E-cadherin expression. Furthermore, enhanced nuclear factor-κB expression significantly increases cell proliferation and diminishes apoptosis in MCF-7 cell line. Increased expression of nuclear factor-κB significantly enhances mammary carcinoma cell migration and invasion stimulated by autocrine human growth hormone, which results in epithelial-mesenchymal transition of MCF-7 cells. In conclusion, our study revealed the influence of human growth hormone on nuclear factor-κB activity and epithelial-mesenchymal transition in mammary carcinoma. Our findings will help to understand molecular role of "growth hormone-nuclear factor-κB axis" in mammary carcinogenesis which may facilitate the discovery of suitable pathway inhibitors for disease treatment.

Biochemical and genome sequence analyses of Megasphaera sp. strain DISK18 from dental plaque of a healthy individual reveals commensal lifestyle.

Much of the work in periodontal microbiology in recent years has focused on identifying and understanding periodontal pathogens. As the majority of oral microbes have not yet been isolated in pure form, it is essential to understand the phenotypic characteristics of microbes to decipher their role in oral environment. In this study, strain DISK18 was isolated from gingival sulcus and identified as a Megasphaera species. Although metagenomics studies revealed Megasphaera species as a major group within the oral habitat, they have never been isolated in cultivable form to date. Therefore, we have characterized the DISK18 strain to better understand its role in the periodontal ecosystem. Strain Megasphaera sp. DISK18 displayed the ability to adhere and self-aggregate, which are essential requisite features for inhabiting and persisting in oral cavity. It also coaggregated with other pioneer oral colonizers like Streptococcus and Lactobacillus species but not with Veillonella. This behaviour points towards its role in the ecologic succession of a multispecies biofilm as an early colonizer. The absence of virulence determining genes as observed in whole genome sequence analysis coupled with an inability to degrade collagen reveals that Megasphaera sp. strain DISK18 is likely not a pathogenic species and emphasizes its commensal lifestyle.

Laterosporulin10: a novel defensin like Class IId bacteriocin from Brevibacillus sp. strain SKDU10 with inhibitory activity against microbial pathogens.

Bacteriocins are antimicrobial peptides (AMPs) produced by bacteria to acquire survival benefits during competitive inter- and intra-species interactions in complex ecosystems. In this study, an AMP-producing soil bacterial strain designated SKDU10 was isolated and identified as a member of the genus Brevibacillus. The AMP produced by strain SKDU10 identified as a class IId bacteriocin with 57.6 % homology to laterosporulin, a defensin-like class IId bacteriocin. However, substantial differences were observed in the antimicrobial activity spectrum of this bacteriocin named laterosporulin10 when compared to laterosporulin. Laterosporulin10 effectively inhibited the growth of Staphylococcus aureus and Mycobacterium tuberculosis (Mtb H37Rv) with LD50 values of 4.0 µM and 0.5 µM, respectively. Furthermore, laterosporulin10 inhibited the growth of Mtb H37Rv strain at about 20 times lower MIC value compared to S. aureus MTCC 1430 or M. smegmatis MC2 155 in vitro and ex vivo. Electron micrographs along with membrane permeabilization studies using FACS analysis revealed that laterosporulin10 is a membrane-permeabilizing peptide. Interestingly, laterosporulin10 was able to efficiently kill Mtb H37Rv strain residing inside the macrophages and did not show haemolysis up to 40 µM concentration.

Contribution of cyclin D1 (CCND1) and E-cadherin (CDH1) alterations to colorectal cancer susceptibility: a case-control study.

Cyclin D1 (CCND1) and E-cadherin (CDH1) are two important genes of the ß-catenin/LEF pathway that is involved in tumorigenesis of various cancers including colorectal cancer (CRC). However, studies of the association between genetic variants of these two genes and CRC have shown conflicting results. We conducted a genetic association study in South Indian population (cases, 103; controls, 107) to assess the association of CCND1 870G/A and CDH1 -160C/A single nucleotide polymorphisms (SNPs) with CRC risk. Genotyping of SNPs was performed by PCR sequencing analysis. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D') for pair-wise linkage disequilibrium (LD) were assessed by Haploview Software. In addition, to better understand the role of CCND1 and CDH1 in the pathophysiology of CRC, the expression pattern was evaluated in analogous tumor and adjacent normal tissues from 23 CRC patients by Western blot analysis. The frequencies of CCND1 870A/A (P = 0.045) genotype, CDH1 -160A allele (P = 0.042), and 870A/-160A haplotype (P = 0.002) were significantly higher in patients as compared with controls. Strong LD was observed between 870G/A and -160C/A SNPs in cases (D' = 0.76) as compared to controls (D' = 0.32). Furthermore, elevated CCND1 and diminished CDH1 expression was observed in tumor tissue as compared with analogous normal tissue of CRC patients. Interestingly, advanced-stage tumors showed wider expression alterations than in early-stage tumors. In conclusion, CCND1 870G/A and CDH1 -160C/A SNPs may modify the risk of CRC susceptibility in South Indian population. In addition, elevated CCND1 and diminished CDH1 expression appears to be useful prognostic markers for CRC.