Adherence to the Mediterranean diet is associated with decreased fecal calprotectin in patients with ulcerative colitis after pouch surgery.

BACKGROUND: Mediterranean diet (MED) is associated with health benefits, yet scarce data exist regarding the role of MED in inflammatory bowel diseases (IBD). Herein, we aimed to evaluate the association between MED and inflammatory markers in patients with IBD after pouch surgery. METHODS: Consecutive patients after pouch surgery due to ulcerative colitis (UC) were recruited at a comprehensive pouch clinic. Adherence to MED was calculated according to MED score, ranging from 0 (low adherence) to 9 (high adherence), based on food-frequency questionnaires. Pouch behavior was defined as normal pouch (NP) or pouchitis based on Pouchitis Disease Activity Index (PDAI) and disease activity was defined as active or inactive. C-reactive protein (CRP) and fecal calprotectin were assessed. RESULTS: Overall 153 patients were enrolled (male gender 47%; mean age 46 ± 14 years; mean pouch age 9.5 ± 7 years). MED scores were higher in patients with normal vs. elevated CRP and calprotectin levels (4.6 ± 1.8 vs. 4.4 ± 1.6, p = 0.28; 4.8 ± 1.8 vs. 4.07 ± 1.7, p < 0.05, respectively). In a multivariate regression, MED score was associated with decreased calprotectin levels (OR = 0.74 [0.56-0.99]). Adherence to MED was associated with dietary fiber and antioxidants intake. Finally, in a subgroup of patients with NP followed up for 8 years, higher adherence to MED trended to be inversely associated with the onset of pouchitis (log rank = 0.17). CONCLUSIONS: In patients with UC after pouch surgery, adherence to MED is associated with decreased calprotectin levels. Thus, MED may have a role in modifying intestinal inflammation in IBD.

An electrode-assisted anaerobic digestion process for the production of high-quality biogas.

Biogas is a sustainable, renewable energy source generated from organic waste degradation during anaerobic digestion (AD). AD is applied for treating different types of wastewater, mostly containing high organic load. However, AD practice is still limited due to the low quality of the produced biogas. Upgrading biogas to natural gas quality (>90% CH4) is essential for broad applications. Here, an innovative bio-electrochemically assisted AD process was developed, combining wastewater treatment and biogas upgrading. This process was based on a microbial electrolysis cell (MEC) that produced hydrogen from wastewater at a relatively high efficiency, followed by high-rate anaerobic systems for completing biodegradation of organic matter and an in situ bio-methanation process. Results showed that CH4 production yield was substantially improved upon coupling of the MEC with the AD system. Interestingly, CH4 production yield increase was most notable once circulation between AD and MEC was applied, while current density was not markedly affected by the circulation rates. The microbial community analysis confirmed that the MEC enhanced hydrogen production, leading to the enrichment of hydrogenotrophic methanogens. Thus, directing soluble hydrogen from the MEC to AD is plausible, and has great potential for biogas upgrading, avoiding the need for direct hydrogen harvesting.

Increasing adherence to the Mediterranean diet and lifestyle is associated with reduced fecal calprotectin and intra-individual changes in microbial composition of healthy subjects.

The Mediterranean diet (MED) is associated with the modification of gut microbial composition. In this pilot study, we investigate the feasibility of a microbiota-targeted MED-based lifestyle intervention in healthy subjects. MED intervention integrating dietary counseling, a supporting mobile application, and daily physical activity measurement using step trackers was prospectively applied for 4 weeks. Blood and fecal samples were collected at baseline, after the 4-week intervention, and at 6 and 12 months. Blood counts, inflammatory markers, microbial and eukaryotic composition were analyzed. Dietary adherence was assessed using daily questionnaires. All 20 healthy participants (females 65%, median age 37), completed the 4-week intervention. Adherence to MED increased from 15.6 ± 4.1 (baseline) to 23.2 ± 3.6 points (4 weeks), p < .01, reflected by increased dietary fiber and decreased saturated fat intake (both p < .05). MED intervention modestly reduced fecal calprotectin, white blood cell, neutrophil, and lymphocyte counts, within the normal ranges (P < .05). Levels of butyrate producers including Faecalibacterium and Lachnospira were positively correlated with adherence to MED and the number of daily steps. Bacterial composition was associated with plant-based food intake, while fungal composition with animal-based food as well as olive oil and sweets. Increasing adherence to MED correlated with increased absolute abundances of multiple beneficial gut symbionts. Therefore, increasing adherence to MED is associated with reduction of fecal calprotectin and beneficial microbial alterations in healthy subjects. Microbiota targeted lifestyle interventions may be used to modify the intestinal ecosystem with potential implications for microbiome-mediated diseases.

Fruit Consumption is Associated with Alterations in Microbial Composition and Lower Rates of Pouchitis.

BACKGROUND: Patients with ulcerative colitis [UC] who undergo proctocolectomy with an ileal pouch-anal anastomosis commonly develop pouch inflammation [pouchitis]. Pouchitis develops in a previously normal small intestine and may involve environmental factors. We explored whether diet and microbiota alterations contributed to the pathogenesis of pouchitis. METHODS: Patients were recruited and prospectively followed at a comprehensive pouch clinic. Pouch behaviour was clinically defined as a normal pouch [NP] or pouchitis. Patients completed Food Frequency Questionnaires [FFQs]. Faecal samples were analysed for microbial composition [16S rRNA gene pyrosequencing]. RESULTS: Nutritional evaluation was performed in 172 patients [59% females], and of these, faecal microbial analysis was performed in 75 patients (microbiota cohort: NP [n = 22], pouchitis [n = 53]). Of the entire cohort, a subgroup of 39 [22.6%] patients had NP at recruitment [NP cohort]. Of these, 5 [12.8%] developed pouchitis within a year. Patients at the lowest tertile of fruit consumption [<1.45 servings/day] had higher rates of pouchitis compared with those with higher consumption [30.8% vs 3.8%, log rank, p = 0.03]. Fruit consumption was correlated with microbial diversity [r = 0.35, p = 0.002] and with the abundance of several microbial genera, including Faecalibacterium [r = 0.29, p = 0.01], Lachnospira [r = 0.38, p = 0.001], and a previously uncharacterized genus from the Ruminococcaceae family [r = 0.25, p = 0.05]. Reduction in fruit consumption over time was associated with disease recurrence and with reduced microbial diversity [Δ = -0.8 ± 0.3, p = 0.008]. CONCLUSIONS: Fruit consumption is associated with modification of microbial composition, and lower consumption was correlated with the development of pouchitis. Thus, fruit consumption may protect against intestinal inflammation via alteration of microbial composition.

Microbiome associated with denture malodour.

In the past, our inability to cultivate most of the oral microorganisms has limited our view of this complex ecosystem. In the present study, we utilized next generation deep sequencing techniques to revisit the microbiome associated with denture malodour, a growing field with the rise in life expectancy. The study population comprised 26 full dentures patients (mean age 71 ± 6.4, 10 males, 16 females) who visited the Tel Aviv University dental geriatric clinic. Denture malodour was rated organoleptically by a single odour judge, and dentures scoring 2 and above were considered malodour positive. DNA was extracted from the swab samples and analysed using next generation deep sequencing 16 s rDNA technology. Taxa identified could be classified into nine phyla, 29 genera and 117 species. Malodour positive samples showed a higher abundance of the phyla Firmicutes and Fusobacteria and the genera Leptotrichia, Atopobium, Megasphaera, Oribacterium and Campylobacter. Microbiome analysis demonstrated higher bacterial diversity within the malodourous samples and a significant difference in the microbial profile within the two groups. Taken together these results suggest a difference between the microbial populations of malodourous and non-malodourous dentures both in composition and diversity.

Developmentally regulated interactions of liver nuclear factors with the rat phosphoenolpyruvate carboxykinase promoter.

A sequential pattern of interactions of trans-acting factors in rat liver with the phosphoenolpyruvate carboxykinase promoter during late development was observed. A liver-enriched factor, possibly AF1, interacted with the promoter in fetal liver, whereas a factor with the characteristics of C/EBP bound the promoter after birth with the onset of the gene expression.

Transcriptional regulation of the phosphoenolpyruvate carboxykinase gene by cooperation between hepatic nuclear factors.

To study the transcriptional regulation of the liver gluconeogenic phenotype, the underdifferentiated mouse Hepa-1c1c7 (Hepa) hepatoma cell line was used. These cells mimicked the fetal liver by appreciably expressing the alpha-fetoprotein and albumin genes but not the phosphoenolpyruvate carboxykinase (PEPCK) gene. Unlike the fetal liver, however, Hepa cells failed to express the early-expressed factors hepatocyte nuclear factor 1 alpha (HNF-1 alpha) and HNF-4 and the late-expressed factor C/EBP alpha, thereby providing a suitable system for examining possible cooperation between these factors in the transcriptional regulation of the PEPCK gene. Transient transfection assays of a chimeric PEPCK-chloramphenicol acetyltransferase construct showed a residual PEPCK promoter activity in the Hepa cell line, which was slightly stimulated by cotransfection with a single transcription factor from either the C/EBP family or HNF-1 alpha but not at all affected by cotransfection of HNF-4. In contrast, cotransfection of the PEPCK construct with members from the C/EBP family plus HNF-1 alpha resulted in a synergistic stimulation of the PEPCK promoter activity. This synergistic effect depended on the presence in the PEPCK promoter region of the HNF-1 recognition sequence and on the presence of two C/EBP recognition sequences. The results demonstrate a requirement for coexistence and cooperation between early and late liver-enriched transcription factors in the transcriptional regulation of the PEPCK gene. In addition, the results suggest redundancy between members of the C/EBP family of transcription factors in the regulation of PEPCK gene expression.

trans activation of rat phosphoenolpyruvate carboxykinase (GTP) gene expression by micro-coinjection of rat liver mRNA in Xenopus laevis oocytes.

To study the liver-specific trans activation of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene, the PEPCK promoter was linked to a reporter gene and was microinjected into Xenopus laevis oocytes alone or in conjunction with rat liver poly(A)+ RNA. The rat liver mRNA markedly enhanced the expression of the PEPCK-chimeric construct. This effect appeared to be sequence specific, as it was dependent on the presence of the intact promoter. Moreover, the RNA effect was limited to mRNA preparations from PEPCK-expressing tissues only. Finally, microinjection of size-fractionated liver mRNA revealed that the trans-acting factor(s) is encoded by RNA of 1,600 to 2,000 nucleotides, providing a direct bioassay for the gene(s) involved in this tissue-specific trans-activation process.

Differential regulation of the rat phosphoenolpyruvate carboxykinase gene expression in several tissues of transgenic mice.

The selective expression of a unique copy gene in several mammalian tissues has been approached by studying the regulatory sequences needed to control expression of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene in transgenic mice. A transgene containing the entire PEPCK gene, including 2.2 kb of the 5'-flanking region and 0.5 kb of the 3'-flanking region, exhibits tissue-specific expression in the liver, kidney, and adipose tissue, as well as the hormonal and developmental regulation inherent to endogenous gene expression. Deletions of the 5'-flanking region of the gene have shown the need for sequences downstream of position -540 of the PEPCK gene for expression in the liver and sequences downstream of position -362 for expression in the kidney. Additional sequences upstream of position -540 (up to -2200) are required for expression in adipose tissue. In addition, the region containing the glucocorticoid-responsive elements of the gene used by the kidney was identified. This same sequence was found to be needed specifically for developmental regulation of gene expression in the kidney and, together with upstream sequences, in the intestine. The apparently distinct sequence requirements in the various tissues indicate that the tissues use different mechanisms for expression of the same gene.

Cis-regulatory elements that confer differential expression upon the rat gene encoding phosphoenolpyruvate carboxykinase in kidney and liver.

The PCK gene, encoding cytosolic phosphoenolpyruvate carboxykinase, is specifically expressed in gluconeogenic tissues, liver and kidney. Hence it serves as a model of a class of single-copy genes whose transcription is restricted to a few tissues, rather than a unique tissue. To begin delineating the mechanisms that govern this pattern of expression, cis-regulatory elements of PCK were examined using transient transfection assays in PCK-expressing kidney and hepatoma cell lines. The analyses enabled us to identify a proximal element, between nucleotide (nt) positions -121 and -98, relative to the transcription start point that is sufficient for specific expression in kidney cells, but is just one of the elements required for expression in hepatoma cells. A distal element (between nt -487 and -417), which is essential for hepatoma-specific expression, is not needed in kidney cells. We suggest that the differential regulation of PCK expression in the liver and kidney results from an interplay between different cis-regulatory elements and trans-acting factors.

Fate of polyoma origin of replication after its direct introduction into mice.

Recently we have developed a method for direct introduction of calcium phosphate-precipitated DNA into newborn rats. To examine whether the foreign DNA can replicate, a plasmid containing a polyoma origin of replication was injected into newborn mice. The plasmid was found intact in liver and spleen and able to transform bacteria. The foreign DNA had disappeared by the seventh day after injection. Yet, the plasmid DNA containing the polyoma origin of replication had undergone replication in both the liver and the spleen.

Repression and activation of transcription of phosphoenolpyruvate carboxykinase gene during liver development.

Transcriptional activation of the hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene at birth is critical since PEPCK appearance initiates hepatic gluconeogenesis. A delayed appearance results in hypoglycemia, while a premature appearance results in neonatal diabetes, both are incompatible with sustaining life. Experiments using transgenic mice and transfected hepatoma cells suggest that both repression and activation underlie the correct onset of hepatic PEPCK gene transcription. In transgenic mice, transgenes driven by the proximal PEPCK promoter are prematurely expressed in the fetal liver and over-expressed in the neonatal liver, indicating that sequences upstream of the proximal promoter restrain perinatal expression. In Hepa1c1c7 cells, which mimic the fetal liver, the proximal PEPCK promoter (597 bp) exhibited a 3. 5-10-fold higher activity than longer promoters. Repression of the longer promoter (2000 bp) was diminished upon deletion of the sequence spanning positions(-840) to(- 1116) which contains a PPAR/RXR recognition element. The intact 2000 bp PEPCK promoter could be markedly activated by co-transfecting the transcription factor HNF-1 together with C/EBP. It could be repressed by co-transfection with RXRalpha and adding PPARalpha relieved this inhibition.

Involvement of HNF-1 in the regulation of phosphoenolpyruvate carboxykinase gene expression in the kidney.

The cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (PEPCK) gene is differentially expressed in several tissues. A specific set of regulatory elements in the promoter are responsible for the control of PEPCK gene transcription and, in turn, determine its distinct metabolic role in each tissue. DNase I footprinting analysis of the PEPCK promoter, using nuclear proteins from tissues which express the gene for PEPCK, and transient expression assays in renal cell lines have demonstrated that the HNF-1 recognition motif (P2) in the PEPCK promoter characterizes kidney-specific expression. This site is required also for the response to acidosis. Since the P2 site is not involved in the expression of the PEPCK gene in the liver, we propose that its critical role in the kidney stems from a combination of abundance of HNF-1 together with low concentrations of members of the C/EBP family in this tissue.

Conservation from rat to human of cytosolic phosphoenolpyruvate carboxykinase and the control of its gene expression.

Structural conservation of cytosolic phosphoenolpyruvate carboxykinase protein and mRNA sequence was found in all species examined from rodents to human. The mitochondrial isoenzyme, in all species tested, represents a distinct protein. Moreover, irrespective of the ratio of cytosolic to mitochondrial isoenzyme, cytosolic phosphoenolpyruvate carboxykinase activity in the human as in the rat is controlled at the level of gene expression and through the same multiple hormonal stimulation. This evolutionary conservation of the cytosolic phosphoenolpyruvate carboxykinase structure and mode of regulation supports the enzymes' physiological importance in mammals.

Regulation of tissue- and development-specific gene expression in the liver.

The liver is equipped with a repertoire of enzymatic activities essential for executing its specialized role in metabolism, the expression of which is regulated during development. The liver-specific phenotype is the consequence of a developmental tissue-specific program of gene expression. Sequences close to many characterized structural liver-specific genes (cis-regulatory elements) regulate their transcription. Identification of such cis-regulatory elements, capable of conferring a hepatocyte-specific gene expression, has been achieved by the introduction of chimeric genes into germ lines, producing transgenic animals, into differentiated cultured cells and into a cell-free transcription system. Such cis-elements in the DNA are recognized by specific DNA-binding nuclear proteins (trans-acting factors) which are liver-enriched and developmentally controlled. The interaction of defined cis-acting elements, near liver-specific genes, with liver-specific trans-acting factors might result in the differentiation of cells of the endoderm lineage into hepatocyte cells.