Pharmacological activation of constitutive androstane receptor induces female-specific modulation of hepatic metabolism.

Background & Aims: The constitutive androstane receptor (CAR) is a nuclear receptor that binds diverse xenobiotics and whose activation leads to the modulation of the expression of target genes involved in xenobiotic detoxification and energy metabolism. Although CAR hepatic activity is considered to be higher in women than in men, its sex-dependent response to an acute pharmacological activation has seldom been investigated. Methods: The hepatic transcriptome, plasma markers, and hepatic metabolome, were analysed in Car+/+ and Car-/- male and female mice treated either with the CAR-specific agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) or with vehicle. Results: Although 90% of TCPOBOP-sensitive genes were modulated in a sex-independent manner, the remaining 10% showed almost exclusive female liver specificity. These female-specific CAR-sensitive genes were mainly involved in xenobiotic metabolism, inflammation, and extracellular matrix organisation. CAR activation also induced higher hepatic oxidative stress and hepatocyte cytolysis in females than in males. Hepatic expression of flavin monooxygenase 3 (Fmo3) was almost abolished and was associated with a decrease in hepatic trimethylamine-N-oxide (TMAO) concentration in TCPOBOP-treated females. In line with a potential role in the control of TMAO homeostasis, CAR activation decreased platelet hyper-responsiveness in female mice supplemented with dietary choline. Conclusions: More than 10% of CAR-sensitive genes are sex-specific and influence hepatic and systemic responses such as platelet aggregation. CAR activation may be an important mechanism of sexually-dimorphic drug-induced liver injury. Impact and implications: CAR is activated by many drugs and pollutants. Its pharmacological activation had a stronger impact on hepatic gene expression and metabolism in females than in males, and had a specific impact on liver toxicity and trimethylamine metabolism. Sexual dimorphism should be considered when testing and/or prescribing xenobiotics known to activate CAR.

A polyphenol-rich green Mediterranean diet enhances epigenetic regulatory potential: the DIRECT PLUS randomized controlled trial.

BACKGROUND: The capacity of a polyphenol-enriched diet to modulate the epigenome in vivo is partly unknown. Given the beneficial metabolic effects of a Mediterranean (MED) diet enriched in polyphenols and reduced in red/processed meat (green-MED), as previously been proven by the 18-month DIRECT PLUS randomized controlled trial, we analyzed the effects of the green-MED diet on methylome and transcriptome levels to highlight molecular mechanisms underlying the observed metabolic improvements. METHODS: Our study included 260 participants (baseline BMI = 31.2 kg/m2, age = 5 years) of the DIRECT PLUS trial, initially randomized to one of the intervention arms: A. healthy dietary guidelines (HDG), B. MED (440 mg polyphenols additionally provided by walnuts), C. green-MED (1240 mg polyphenols additionally provided by walnuts, green tea, and Mankai: green duckweed shake). Blood methylome and transcriptome of all study subjects were analyzed at baseline and after completing the 18-month intervention using Illumina EPIC and RNA sequencing technologies. RESULTS: A total of 1573 differentially methylated regions (DMRs; false discovery rate (FDR) < 5 %) were found in the green-MED compared to the MED (177) and HDG (377) diet participants. This corresponded to 1753 differentially expressed genes (DEGs; FDR < 5 %) in the green-MED intervention compared to MED (7) and HDG (738). Consistently, the highest number (6 %) of epigenetic modulating genes was transcriptionally changed in subjects participating in the green-MED intervention. Weighted cluster network analysis relating transcriptional and phenotype changes among participants subjected to the green-MED intervention identified candidate genes associated with serum-folic acid change (all P < 1 × 10-3) and highlighted one module including the KIR3DS1 locus, being negatively associated with the polyphenol changes (e.g. P < 1 × 10-4), but positively associated with the MRI-assessed superficial subcutaneous adipose area-, weight- and waist circumference- 18-month change (all P < 0.05). Among others, this module included the DMR gene Cystathionine Beta-Synthase, playing a major role in homocysteine reduction. CONCLUSIONS: The green-MED high polyphenol diet, rich in green tea and Mankai, renders a high capacity to regulate an individual's epigenome. Our findings suggest epigenetic key drivers such as folate and green diet marker to mediate this capacity and indicate a direct effect of dietary polyphenols on the one­carbon metabolism.

Cocoa seeds and chocolate products interaction with gut microbiota; mining microbial and functional biomarkers from mechanistic studies, clinical trials and 16S rRNA amplicon sequencing.

In recent years, gut microbiome has evolved as a focal point of interest with growing recognition that a well-balanced gut microbiota is highly relevant to an individual's health status. The present review provides a mechanistic insight on the effects of cocoa chemicals on the gut microbiome and further reveals in silico biomarkers, taxonomic and functional features that distinguish gut microbiome of cocoa consumers and controls by using 16S rRNA gene sequencing data. The polyphenols in cocoa can change the gut microbiota either by inhibiting the growth of pathogenic bacteria in the gut such as Clostridium perfringens or by increasing the growth of beneficial microbiota in the gut such as Lactobacillus and Bifidobacterium. This paper demonstrates the holistic effect of gut microbiota on cocoa chemicals and how it impacts human health. We present herein the first comprehensive review and analysis of how raw and roasted cocoa and its products can specifically influence gut homeostasis, and likewise, how microbiota metabolizes cocoa chemicals. In addition to that, our 16S rRNA amplicon sequencing analysis revealed that the flavone and flavonols metabolism, aminobenzoate degradation and fatty acid elongation pathways represent the three most important signatures of microbial functions associated with cocoa consumption.

The effect of high-polyphenol Mediterranean diet on visceral adiposity: the DIRECT PLUS randomized controlled trial.

BACKGROUND: Mediterranean (MED) diet is a rich source of polyphenols, which benefit adiposity by several mechanisms. We explored the effect of the green-MED diet, twice fortified in dietary polyphenols and lower in red/processed meat, on visceral adipose tissue (VAT). METHODS: In the 18-month Dietary Intervention Randomized Controlled Trial PoLyphenols UnproceSsed (DIRECT-PLUS) weight-loss trial, 294 participants were randomized to (A) healthy dietary guidelines (HDG), (B) MED, or (C) green-MED diets, all combined with physical activity. Both isocaloric MED groups consumed 28 g/day of walnuts (+ 440 mg/day polyphenols). The green-MED group further consumed green tea (3-4 cups/day) and Wolffia globosa (duckweed strain) plant green shake (100 g frozen cubes/day) (+ 800mg/day polyphenols) and reduced red meat intake. We used magnetic resonance imaging (MRI) to quantify the abdominal adipose tissues. RESULTS: Participants (age = 51 years; 88% men; body mass index = 31.2 kg/m2; 29% VAT) had an 89.8% retention rate and 79.3% completed eligible MRIs. While both MED diets reached similar moderate weight (MED: - 2.7%, green-MED: - 3.9%) and waist circumference (MED: - 4.7%, green-MED: - 5.7%) loss, the green-MED dieters doubled the VAT loss (HDG: - 4.2%, MED: - 6.0%, green-MED: - 14.1%; p < 0.05, independent of age, sex, waist circumference, or weight loss). Higher dietary consumption of green tea, walnuts, and Wolffia globosa; lower red meat intake; higher total plasma polyphenols (mainly hippuric acid), and elevated urine urolithin A polyphenol were significantly related to greater VAT loss (p < 0.05, multivariate models). CONCLUSIONS: A green-MED diet, enriched with plant-based polyphenols and lower in red/processed meat, may be a potent intervention to promote visceral adiposity regression. TRIAL REGISTRATION: ClinicalTrials.gov , NCT03020186.

The effect of a high-polyphenol Mediterranean diet (Green-MED) combined with physical activity on age-related brain atrophy: the Dietary Intervention Randomized Controlled Trial Polyphenols Unprocessed Study (DIRECT PLUS).

BACKGROUND: The effect of diet on age-related brain atrophy is largely unproven. OBJECTIVES: We aimed to explore the effect of a Mediterranean diet (MED) higher in polyphenols and lower in red/processed meat (Green-MED diet) on age-related brain atrophy. METHODS: This 18-mo clinical trial longitudinally measured brain structure volumes by MRI using hippocampal occupancy score (HOC) and lateral ventricle volume (LVV) expansion score as neurodegeneration markers. Abdominally obese/dyslipidemic participants were randomly assigned to follow 1) healthy dietary guidelines (HDG), 2) MED, or 3) Green-MED diet. All subjects received free gym memberships and physical activity guidance. Both MED groups consumed 28 g walnuts/d (+440 mg/d polyphenols). The Green-MED group consumed green tea (3-4 cups/d) and Mankai (Wolffia-globosa strain, 100 g frozen cubes/d) green shake (+800 mg/d polyphenols). RESULTS: Among 284 participants (88% men; mean age: 51 y; BMI: 31.2 kg/m2; APOE-ε4 genotype = 15.7%), 224 (79%) completed the trial with eligible whole-brain MRIs. The pallidum (-4.2%), third ventricle (+3.9%), and LVV (+2.2%) disclosed the largest volume changes. Compared with younger participants, atrophy was accelerated among those ≥50 y old (HOC change: -1.0% ± 1.4% compared with -0.06% ± 1.1%; 95% CI: 0.6%, 1.3%; P < 0.001; LVV change: 3.2% ± 4.5% compared with 1.3% ± 4.1%; 95% CI: -3.1%, -0.8%; P = 0.001). In subjects ≥ 50 y old, HOC decline and LVV expansion were attenuated in both MED groups, with the best outcomes among Green-MED diet participants, as compared with HDG (HOC: -0.8% ± 1.6% compared with -1.3% ± 1.4%; 95% CI: -1.5%, -0.02%; P = 0.042; LVV: 2.3% ± 4.7% compared with 4.3% ± 4.5%; 95% CI: 0.3%, 5.2%; P = 0.021). Similar patterns were observed among younger subjects. Improved insulin sensitivity over the trial was the parameter most strongly associated with brain atrophy attenuation (P < 0.05). Greater Mankai, green tea, and walnut intake and less red and processed meat were significantly and independently associated with reduced HOC decline (P < 0.05). Elevated urinary concentrations of the polyphenols urolithin-A (r = 0.24; P = 0.013) and tyrosol (r = 0.26; P = 0.007) were significantly associated with lower HOC decline. CONCLUSIONS: A Green-MED (high-polyphenol) diet, rich in Mankai, green tea, and walnuts and low in red/processed meat, is potentially neuroprotective for age-related brain atrophy.This trial was registered at clinicaltrials.gov as NCT03020186.

The Metabolomic-Gut-Clinical Axis of Mankai Plant-Derived Dietary Polyphenols.

BACKGROUND: Polyphenols are secondary metabolites produced by plants to defend themselves from environmental stressors. We explored the effect of Wolffia globosa 'Mankai', a novel cultivated strain of a polyphenol-rich aquatic plant, on the metabolomic-gut clinical axis in vitro, in-vivo and in a clinical trial. METHODS: We used mass-spectrometry-based metabolomics methods from three laboratories to detect Mankai phenolic metabolites and examined predicted functional pathways in a Mankai artificial-gut bioreactor. Plasma and urine polyphenols were assessed among the 294 DIRECT-PLUS 18-month trial participants, comparing the effect of a polyphenol-rich green-Mediterranean diet (+1240 mg/polyphenols/day, provided by Mankai, green tea and walnuts) to a walnuts-enriched (+440 mg/polyphenols/day) Mediterranean diet and a healthy controlled diet. RESULTS: Approximately 200 different phenolic compounds were specifically detected in the Mankai plant. The Mankai-supplemented bioreactor artificial gut displayed a significantly higher relative-abundance of 16S-rRNA bacterial gene sequences encoding for enzymes involved in phenolic compound degradation. In humans, several Mankai-related plasma and urine polyphenols were differentially elevated in the green Mediterranean group compared with the other groups (p < 0.05) after six and 18 months of intervention (e.g., urine hydroxy-phenyl-acetic-acid and urolithin-A; plasma Naringenin and 2,5-diOH-benzoic-acid). Specific polyphenols, such as urolithin-A and 4-ethylphenol, were directly involved with clinical weight-related changes. CONCLUSIONS: The Mankai new plant is rich in various unique potent polyphenols, potentially affecting the metabolomic-gut-clinical axis.

Roux-en-Y gastric bypass contributes to weight loss-independent improvement in hypothalamic inflammation and leptin sensitivity through gut-microglia-neuron-crosstalk.

OBJECTIVE: Hypothalamic inflammation and endoplasmic reticulum (ER) stress are extensively linked to leptin resistance and overnutrition-related diseases. Surgical intervention remains the most efficient long-term weight-loss strategy for morbid obesity, but mechanisms underlying sustained feeding suppression remain largely elusive. This study investigated whether Roux-en-Y gastric bypass (RYGB) interacts with obesity-associated hypothalamic inflammation to restore central leptin signaling as a mechanistic account for post-operative appetite suppression. METHODS: RYGB or sham surgery was performed in high-fat diet-induced obese Wistar rats. Sham-operated rats were fed ad libitum or by weight matching to RYGB via calorie restriction (CR) before hypothalamic leptin signaling, microglia reactivity, and the inflammatory pathways were examined to be under the control of gut microbiota-derived circulating signaling. RESULTS: RYGB, other than CR-induced adiposity reduction, ameliorates hypothalamic gliosis, inflammatory signaling, and ER stress, which are linked to enhanced hypothalamic leptin signaling and responsiveness. Mechanistically, we demonstrate that RYGB interferes with hypothalamic ER stress and toll-like receptor 4 (TLR4) signaling to restore the anorexigenic action of leptin, which most likely results from modulation of a circulating factor derived from the altered gut microbial environment upon RYGB surgery. CONCLUSIONS: Our data demonstrate that RYGB interferes with hypothalamic TLR4 signaling to restore the anorexigenic action of leptin, which most likely results from modulation of a circulating factor derived from the post-surgical altered gut microbial environment.

Maternal paraben exposure triggers childhood overweight development.

Parabens are preservatives widely used in consumer products including cosmetics and food. Whether low-dose paraben exposure may cause adverse health effects has been discussed controversially in recent years. Here we investigate the effect of prenatal paraben exposure on childhood overweight by combining epidemiological data from a mother-child cohort with experimental approaches. Mothers reporting the use of paraben-containing cosmetic products have elevated urinary paraben concentrations. For butyl paraben (BuP) a positive association is observed to overweight within the first eight years of life with a stronger trend in girls. Consistently, maternal BuP exposure of mice induces a higher food intake and weight gain in female offspring. The effect is accompanied by an epigenetic modification in the neuronal Pro-opiomelanocortin (POMC) enhancer 1 leading to a reduced hypothalamic POMC expression. Here we report that maternal paraben exposure may contribute to childhood overweight development by altered POMC-mediated neuronal appetite regulation.

Ethnomedicinal survey and in vitro confirmation of anti-inflammatory and antispasmodic properties of the termite strain Macrotermes bellicosus used in traditional medicine in the Republic of Benin.

ETHNOPHARMACOLOGICAL RELEVANCE: Insects and insect-derived products play a vital role in traditional medicine in many parts of the world since ancient times. Among these insects, fungus-growing termites like Macrotermes bellicosus (M. bellicosus) are widely used in nutrition and traditional medicine in various societies of sub-Saharan Africa. AIM OF THE STUDY: Aim of the present study was to explore the traditional applications of M. bellicosus and subsequently investigate the anti-inflammatory and spasmolytic activity of samples collected in Benin. MATERIAL AND METHODS: An ethnomedicinal survey with thirty active healers in Benin was conducted and the anti-inflammatory activity of an ethanolic extract of M. bellicosus was investigated. Thus, LPS-induced TNFα release from differentiated human macrophages (THP-1) and IL-8 release from cytokine (IL-1ß/TNFα/IFNγ)-challenged human intestinal epithelial cells (Caco-2) was measured by enzyme-linked immunosorbent assay. Furthermore, the influence of M. bellicosus extract on basal tone and induced contractions in isolated rat small intestinal preparations was determined to examine the influence on intestinal motility. RESULTS: The survey of 30 active healers demonstrated that M. bellicosus and its products (termites' mound and fungus comb) are used in Benin for therapeutic purposes mainly to treat infectious and inflammatory diseases including digestive disorders, snake bites and diarrhea. It was found that M. bellicosus extract inhibited both LPS-induced TNFα release from human macrophages and cytokine-induced IL-8 release from intestinal epithelial cells comparable to budesonide. In addition, isometric contraction measurement with isolated rat small intestinal preparations demonstrated a mild spasmolytic effect of the termite extract in higher concentrations with a suppression of induced contractions and relaxation of basal tone. CONCLUSION: M. bellicosus which is used in traditional medicine in Benin to treat infectious and inflammatory diseases showed anti-inflammatory activity by inhibiting pro-inflammatory cytokine release and a moderate influence on intestinal motility.

The Effect of Wolffia globosa Mankai, a Green Aquatic Plant, on Postprandial Glycemic Response: A Randomized Crossover Controlled Trial.

OBJECTIVE: To compare the postprandial and overnight glycemic response using a novel green aquatic plant thought to provide a dietary source for high-quality protein, with an iso-carbohydrate/protein/caloric dairy shake. RESEARCH DESIGN AND METHODS: This is a randomized controlled crossover trial among 20 abdominally obese participants (age 51.4 years; fasting plasma glucose 110.9 mg/dL), who were allocated to replace dinner with either, first, a green shake containing Wolffia globosa duckweed (Mankai: specific-strain) or an iso-carbohydrate/protein/calorie yogurt shake. A 2-week flash glucose-monitoring system was used to assess postmeal glucose dynamics (6 net administration days; 97 observation days in total). We further obtained from each participant dietary/daily activity/satiety scale/sleep logs. Participants were recruited from the green-Mediterranean diet arm of the 18-month Dietary Intervention Randomized Controlled Trial-Polyphenols Unprocessed (DIRECT-PLUS) study. RESULTS: Wolffia globosa Mankai elicited a lower postprandial glucose peak compared with yogurt (∆peak = 13.4 ± 9.2 vs. 19.3 ± 15.1 mg/dL; P = 0.044), which occurred later (77.5 ± 29.2 vs. 59.2 ± 28.4 min; P = 0.037) and returned faster to baseline glucose levels (135.8 ± 53.1 vs. 197.5 ± 70.2 min; P = 0.012). The mean post-net incremental area under the curve (netAUC) was lower with Wolffia globosa up to 60 and 180 min (netAUC 60 min: 185.1 ± 340.1 vs. 441.4 ± 336.5 mg/dL/min, P = 0.005; netAUC 180 min: 707.9 ± 1,428.5 vs. 1,576.6 ± 1,810.1 mg/dL/min, P = 0.037). A Wolffia globosa-based shake replacing dinner resulted in lower next-morning fasting glucose levels (83.2 ± 0.8 vs. 86.6 ± 13 mg/dL; P = 0.041). Overall, postprandial glucose levels from the shake administration until the next morning were lower in the Wolffia globosa Mankai green shake compared with the yogurt shake (P < 0.001). Overnight sleep duration was similar (378.2 ± 22.4 vs. 375.9 ± 28.4 min; P = 0.72), and satiety rank was slightly higher for the Wolffia globosa shake compared with the yogurt shake (7.5 vs. 6.5; P = 0.035). CONCLUSIONS: Wolffia globosa Mankai duckweed may serve as an emerging alternative plant protein source with potential beneficial postprandial glycemic effects.

Assessment of maternal drug intake by urinary bio monitoring during pregnancy and postpartally until the third perinatal year.

PURPOSE: Although sales of prescribed and over-the-counter (OTC) medication are rising, little is known about individual drug intake. This study was aimed to obtain complementary information about drug intake. METHOD: Information on drug utilization was obtained in a female cohort for five different time points (TP): 36th week of pregnancy (n = 622), 7th perinatal week (n = 533), 3rd perinatal month (n = 340), and 1st perinatal (n = 534) and 3rd perinatal year (n = 324) by a validated urine screening method. RESULTS: Drugs were detected 807 times among all analyzed samples (n = 2353) with less drug intake for early TP compared with later TP (~24.4%, n = 152; ~33.8%, n = 180; ~23.2%, n = 79; ~42.5%, n = 227; and ~52.2%, n = 169). The diversity of drugs increased from 25 up to 40 different drugs for the investigated period. OTC drugs were detected most frequently reflected by the top three drugs: acetaminophen (~37%, n = 292), ibuprofen (~23%, n = 183), and xylometazoline (~12%, n = 98). Mainly guideline-orientated drug therapy was observed. However, contraindicated ibuprofen intake during third trimester urine samples (n = 26) and a repeated usage of acetaminophen and/or ibuprofen (n = 9), as well as xylometazoline (n = 7), reveal missing information about drug safety. CONCLUSION: Bio monitoring was applied for detection of drug intake revealing a lack of information about OTC products and their health risks. Hence, information about health risks for certain drugs and patient groups must be improved for and by pharmacists, to avoid (i) usage of contraindicated drugs and (ii) abuse of OTC drugs.

Effects of ceftiofur treatment on the susceptibility of commensal porcine E.coli--comparison between treated and untreated animals housed in the same stable.

BACKGROUND: Healthy farm animals have been found to act as a reservoir of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (E. coli). Therefore, the objective of the study was to determine the input of antimicrobial active ceftiofur metabolites in the stable via faeces and urine after intramuscular administration of the drug to pigs and the elucidation of the Escherichia coli ESBL resistance pattern of treated and untreated pigs housed in the same barn during therapy. METHODS: For determination of the minimal inhibitory concentration (MIC) the method of microdilutionaccording to the recommended procedure of the Clinical and Laboratory Standards Institute was used. Inaddition to that, a qualitative determination was performed by agar dilution. Unsusceptible E. coli speciesselected via agar dilution with cefotaxime were confirmed by MALDI-TOF and ESBL encoding genes wereidentified by PCR. The amounts of ceftiofur measured as desfuroylceftiofur (DFC) in the different probes (plasma, urine, faeces and dust) were analysed by UPLC-MS/MS. RESULTS: In a first experiment two groups of pigs (6 animals per group) were housed in the same barn in two separated boxes. One group (group B) were treated with ceftiofur according to the licence (3 mg/kg administered intramuscularly (i.m.) on three consecutive days, day 1-3). During a second treatment period (day 29-31) an increased rate of ESBL resistant E. coli was detectable in these treated pigs and in the air of the stable. Moreover, the second group of animals (group A) formerly untreated but housed for the whole period in the same stable as the treated animals revealed increased resistance rates during their first treatment (day 45-47) with ceftiofur. In order to investigate the environmental input of ceftiofur during therapy and to simulate oral uptake of ceftiofur residues from the air of the stable a second set of experiments were performed. Pigs (6 animals) were treated with an interval of 2 weeks for 3 days with different doses of ceftiofur (3 mg/kg, 1 mg/kg and 0.3 mg/kg i.m.) as well as with 3 mg/kg per os) and the renal and biliary excretion of ceftiofur as its active metabolite were measured in comparison to the plasma levels. In addition to that, probes of the sedimentation dust and the air of the stable were analysed for drug residues. CONCLUSION: The present study shows that treatment of several animals in a stable with ceftiofur influences the resistance pattern of intestinal Escherichia coli of the treated as well as untreated animals housed in the same stable. During therapy with the drug which was administered by injection according to the licence we detected nameable amounts of ceftiofur and its active metabolites in the dust and air of the stable.

Metaproteomics and metabolomics analyses of chronically petroleum-polluted sites reveal the importance of general anaerobic processes uncoupled with degradation.

Crude oil is one of the most important natural assets for humankind, yet it is a major environmental pollutant, notably in marine environments. One of the largest crude oil polluted areas in the word is the semi-enclosed Mediterranean Sea, in which the metabolic potential of indigenous microbial populations towards the large-scale chronic pollution is yet to be defined, particularly in anaerobic and micro-aerophilic sites. Here, we provide an insight into the microbial metabolism in sediments from three chronically polluted marine sites along the coastline of Italy: the Priolo oil terminal/refinery site (near Siracuse, Sicily), harbour of Messina (Sicily) and shipwreck of MT Haven (near Genoa). Using shotgun metaproteomics and community metabolomics approaches, the presence of 651 microbial proteins and 4776 metabolite mass features have been detected in these three environments, revealing a high metabolic heterogeneity between the investigated sites. The proteomes displayed the prevalence of anaerobic metabolisms that were not directly related with petroleum biodegradation, indicating that in the absence of oxygen, biodegradation is significantly suppressed. This suppression was also suggested by examining the metabolome patterns. The proteome analysis further highlighted the metabolic coupling between methylotrophs and sulphate reducers in oxygen-depleted petroleum-polluted sediments.

High metabolic in vivo stability and bioavailability of a palmitoylated ghrelin receptor ligand assessed by mass spectrometry.

The constitutive activity of the ghrelin receptor is of high physiological and pathophysiological relevance. In-depth structure-activity relationship studies revealed a palmitoylated ghrelin receptor ligand that displays an in vitro binding affinity in the low nanomolar range. Activity studies revealed inverse agonistic as well as antagonistic properties and in vitro metabolic analysis indicated a high stability in blood serum and liver homogenate. For metabolic testing in vivo, a combined approach of stable isotopic labeling and mass spectrometry-based analysis was established. Therefore, a heavy isotopic version of the peptide containing a (13)C-labeled palmitic acid was synthesized and a 1:1 ratio of a (12)C/(13)C-peptide mixture was injected into rats. Biological samples were analyzed by multiple reaction monitoring allowing simultaneous peptide detection and quantification. Measurements revealed a suitable bioavailability over 24h in rat serum and subsequent high-resolution mass spectrometry investigations showed only negligible degradation and slow body clearance. Hence, this method combination allowed the identification and evaluation of a highly potent and metabolically stable ghrelin receptor ligand in vivo.

Diverse sulfate-reducing bacteria of the Desulfosarcina/Desulfococcus clade are the key alkane degraders at marine seeps.

Biogeochemical and microbiological data indicate that the anaerobic oxidation of non-methane hydrocarbons by sulfate-reducing bacteria (SRB) has an important role in carbon and sulfur cycling at marine seeps. Yet, little is known about the bacterial hydrocarbon degraders active in situ. Here, we provide the link between previous biogeochemical measurements and the cultivation of degraders by direct identification of SRB responsible for butane and dodecane degradation in complex on-site microbiota. Two contrasting seep sediments from Mediterranean Amon mud volcano and Guaymas Basin (Gulf of California) were incubated with (13)C-labeled butane or dodecane under sulfate-reducing conditions and analyzed via complementary stable isotope probing (SIP) techniques. Using DNA- and rRNA-SIP, we identified four specialized clades of alkane oxidizers within Desulfobacteraceae to be distinctively active in oxidation of short- and long-chain alkanes. All clades belong to the Desulfosarcina/Desulfococcus (DSS) clade, substantiating the crucial role of these bacteria in anaerobic hydrocarbon degradation at marine seeps. The identification of key enzymes of anaerobic alkane degradation, subsequent ß-oxidation and the reverse Wood-Ljungdahl pathway for complete substrate oxidation by protein-SIP further corroborated the importance of the DSS clade and indicated that biochemical pathways, analog to those discovered in the laboratory, are of great relevance for natural settings. The high diversity within identified subclades together with their capability to initiate alkane degradation and growth within days to weeks after substrate amendment suggest an overlooked potential of marine benthic microbiota to react to natural changes in seepage, as well as to massive hydrocarbon input, for example, as encountered during anthropogenic oil spills.

Identification of new protein coding sequences and signal peptidase cleavage sites of Helicobacter pylori strain 26695 by proteogenomics.

Correct annotation of protein coding genes is the basis of conventional data analysis in proteomic studies. Nevertheless, most protein sequence databases almost exclusively rely on gene finding software and inevitably also miss protein annotations or possess errors. Proteogenomics tries to overcome these issues by matching MS data directly against a genome sequence database. Here we report an in-depth proteogenomics study of Helicobacter pylori strain 26695. MS data was searched against a combined database of the NCBI annotations and a six-frame translation of the genome. Database searches with Mascot and X! Tandem revealed 1115 proteins identified by at least two peptides with a peptide false discovery rate below 1%. This represents 71% of the predicted proteome. So far this is the most extensive proteome study of Helicobacter pylori. Our proteogenomic approach unambiguously identified four previously missed annotations and furthermore allowed us to correct sequences of six annotated proteins. Since secreted proteins are often involved in pathogenic processes we further investigated signal peptidase cleavage sites. By applying a database search that accommodates the identification of semi-specific cleaved peptides, 63 previously unknown signal peptides were detected. The motif LXA showed to be the predominant recognition sequence for signal peptidases. BIOLOGICAL SIGNIFICANCE: The results of MS-based proteomic studies highly rely on correct annotation of protein coding genes which is the basis of conventional data analysis. However, the annotation of protein coding sequences in genomic data is usually based on gene finding software. These tools are limited in their prediction accuracy such as the problematic determination of exact gene boundaries. Thus, protein databases own partly erroneous or incomplete sequences. Additionally, some protein sequences might also be missing in the databases. Proteogenomics, a combination of proteomic and genomic data analyses, is well suited to detect previously not annotated proteins and to correct erroneous sequences. For this purpose, the existing database of the investigated species is typically supplemented with a six-frame translation of the genome. Here, we studied the proteome of the major human pathogen Helicobacter pylori that is responsible for many gastric diseases such as duodenal ulcers and gastric cancer. Our in-depth proteomic study highly reliably identified 1115 proteins (FDR<0.01%) by at least two peptides (FDR<1%) which represent 71% of the predicted proteome deposited at NCBI. The proteogenomic data analysis of our data set resulted in the unambiguous identification of four previously missed annotations, the correction of six annotated proteins as well as the detection of 63 previously unknown signal peptides. We have annotated proteins of particular biological interest like the ferrous iron transport protein A, the coiled-coil-rich protein HP0058 and the lipopolysaccharide biosynthesis protein HP0619. For instance, the protein HP0619 could be a drug target for the inhibition of the LPS synthesis pathway. Furthermore it has been proven that the motif "LXA" is the predominant recognition sequence for the signal peptidase I of H. pylori. Signal peptidases are essential enzymes for the viability of bacterial cells and are involved in pathogenesis. Therefore signal peptidases could be novel targets for antibiotics. The inclusion of the corrected and new annotated proteins as well as the information of signal peptide cleavage sites will help in the study of biological pathways involved in pathogenesis or drug response of H. pylori.

Microbiota from the distal guts of lean and obese adolescents exhibit partial functional redundancy besides clear differences in community structure.

Recent research has disclosed a tight connection between obesity, metabolic gut microbial activities and host health. Obtaining a complete understanding of this relationship remains a major goal. Here, we conducted a comparative metagenomic and metaproteomic investigation of gut microbial communities in faecal samples taken from an obese and a lean adolescent. By analysing the diversity of 16S rDNA amplicons (10% operational phylogenetic units being common), 22 Mbp of consensus metagenome sequences (~70% common) and the expression profiles of 613 distinct proteins (82% common), we found that in the obese gut, the total microbiota was more abundant on the phylum Firmicutes (94.6%) as compared with Bacteroidetes (3.2%), although the metabolically active microbiota clearly behaves in a more homogeneous manner with both contributing equally. The lean gut showed a remarkable shift towards Bacteroidetes (18.9% total 16S rDNA), which become the most active fraction (81% proteins). Although the two gut communities maintained largely similar gene repertoires and functional profiles, improved pili- and flagella-mediated host colonization and improved capacity for both complementary aerobic and anaerobic de novo B(12) synthesis, 1,2-propanediol catabolism (most likely participating in de novo B(12) synthesis) and butyrate production were observed in the obese gut, whereas bacteria from lean gut seem to be more engaged in vitamin B(6) synthesis. Furthermore, this study provides functional evidence that variable combinations of species from different phyla could 'presumptively' fulfil overlapping and/or complementary functional roles required by the host, a scenario where minor bacterial taxa seem to be significant active contributors.

Evaluation of the tolerability and immunogenicity of ultraviolet C-irradiated autologous platelets in a dog model.

BACKGROUND: The THERAFLEX ultraviolet (UV) platelets (PLTs) pathogen reduction system for PLT concentrates (PCs) operates using ultraviolet C (UVC) light at a wavelength of 254 nm. UVC treatment can potentially alter proteins, which may affect drug tolerance in humans and influence the immunogenicity of blood products. This preclinical study in beagle dogs was designed to evaluate the safety pharmacology of UVC-irradiated PCs after intravenous administration and to determine whether they are capable of eliciting humoral responses to PLTs and plasma proteins. STUDY DESIGN AND METHODS: Six beagle dogs each were transfused once every other week for 10 weeks with UVC-irradiated or nonirradiated PCs. All PCs were autologous canine single-donor products prepared from whole blood. Safety pharmacology variables were regularly assessed. The impact of UVC irradiation on PLT and plasma proteomes was analyzed by one- and two-dimensional gel electrophoresis. Serum samples were tested for UVC-induced antibodies by Western blot and flow cytometry. RESULTS: Dogs transfused with UVC-irradiated PCs showed no signs of local or systemic intolerance. Few but significant changes in PLT protein integrity were observed after UVC irradiation. Even after repeated administration of UVC-irradiated PCs, no antibodies against UVC-exposed plasma or PLT proteins were detected. CONCLUSIONS: Repeated transfusions of autologous UVC-treated PCs were well tolerated in all dogs studied. UVC irradiation did not cause significant plasma or PLT protein modifications capable of inducing specific antibody responses in the dogs. High-resolution proteomics combined with antibody analysis introduces a comprehensive and sensitive method for screening of protein modifications and antibodies specific for pathogen reduction treatment.

Peptide NMHRYPNQ of the cellular prion protein (PrP(C)) inhibits aggregation and is a potential key for understanding prion-prion interactions.

Pathogenesis of transmissible spongiform encephalopathies is correlated with a conversion of the normal cellular form of the prion protein (PrP(C)) into the abnormal isoform (scrapie form of PrP). Contact of the normal PrP with its abnormal isoform, the scrapie form of PrP, induces the transformation. Knowledge of molecules that inhibit such contacts leads to an understanding of the mechanism of the aggregation, and these molecules may serve as leads for drugs against transmissible spongiform encephalopathies. Therefore, we screened a synthetic octapeptide library of the globular domain of the human PrP(C) for binding affinity to PrP(C). Two fragments with binding affinity, 149YYRENMHR156 and 153NMHRYPNQ160, were identified with K(d) values of 21 and 25 microM, respectively. A 10-fold excess of peptide 153NMHRYPNQ160 inhibits aggregation of the PrP by 99%. NMR and mass spectrometry showed that the binding region of the peptide 153NMHRYPNQ160 is located at helix 3 of the PrP.

Screening for inhibitors of tau polymerization.

The histopathological diagnosis of Alzheimer's disease relies on two kinds of proteinaceous aggregates: the extracellular plaques built from filaments of the Abeta-peptide and the intracellular tangles consisting of tau polymerized into Paired Helical Filaments (PHFs). The order of aggregation events is still under debate, but it is well accepted that tau-related changes have an important impact on the viability of neurons. In neurons, early morphological changes are seen in axons which begin to loose and retract synapses. This process is accompanied by an increase of aggregated tau protein. Thus the prevention of tau aggregation seems to be a valuable target for therapy of Alzheimer's disease. Here we present a screening procedure by which we identified inhibitors of tau polymerization. In the primary screen we used a thioflavin-S based assay which detects PHF formation in solution. These initial hits were further analyzed for their capacity to depolymerize preformed PHFs. These results were confirmed by several secondary assays (tryptophan fluorescence, pelleting, filter trapping and electron microscopy). By this approach it is possible to identify small molecule compounds which prevent or reverse the aggregation of tau and thereby might improve the viability of neurons in a therapeutic approach.