Dynamic microbial and metabolic changes during Apulian Caciocavallo cheese-making and ripening produced according to a standardized protocol.

The cheese microbiota plays a critical role in influencing its sensory and physicochemical properties. In this study, traditional Apulian Caciocavallo cheese coming from 4 different dairies in the same area and produced following standardized procedures have been examined, as well as the different bulk milks and natural whey starter cultures used. Moreover, considering the cheese wheels as the blocks of Caciocavallo cheeses as whole, these were characterized at different layers (i.e., core, under-rind, and rind) of the block using a multi-omics approach. In addition to physical-chemical characterization, culturomics, quantitative PCR, metagenomics, and metabolomics analysis, have been carried out post-salting and throughout ripening time (2 mo) to investigate the major shifts in the succession of the microbiota and flavor development. Culture-dependent and 16S rRNA metataxonomics results clearly clustered samples based on the microbiota biodiversity related to the production dairy plant as the result of the use of different NWS or intrinsic conditions of each production site. At the beginning of the ripening, cheeses were dominated by the Lactobacillus and, in 2 dairies (Art and SdC), Streptococcus genera associated with the NWS. The analysis allowed us to show that, although the diversity of identified genera did not change significantly between the rind, under-rind and core fractions of the same samples, there was an evolution in the relative abundance and absolute quantification, modifying and differentiating profiles during ripening. The qPCR mainly differentiated the temporal adaptation of those species originating from bulk milks and those provided by NWSs. The primary starter detected in NWS and cheese reassured the high relative concentration of 1-butanol, 2-butanol, 2-heptanol, 2-butanone, acetoin, delta-dodecalactone, hexanoic acid ethyl ester, octanoic acid ethyl ester, and VFFA during ripening, while cheeses displaying low abundances of Streptococcus and Lactococcus (dairy Del) have a lower total concentration of acetoin compared with Art and SdC. However, the sub-dominant strains and NSLAB present in cheeses are responsible for the production of secondary metabolites belonging to the chemical classes of ketones, alcohols, and organic acids, reaffirming the importance and relevance of autochthonous strains of each dairy plant although considering a delimited production area.

In vivo evaluation of an innovative synbiotics on stage IIIb-IV chronic kidney disease patients.

Background: Microbiota unbalance has been proven to affect chronic kidney disease (CKD) patients and, noteworthy, microbiota composition and activity are implicated in CKD worsening. The progression of kidney failure implies an exceeding accumulation of waste compounds deriving from the nitrogenous metabolism in the intestinal milieu. Therefore, in the presence of an altered intestinal permeability, gut-derived uremic toxins, i.e., indoxyl sulfate (IS) and p-cresyl sulfate (PCS), can accumulate in the blood. Methods: In a scenario facing the nutritional management as adjuvant therapy, the present study assessed the effectiveness of an innovative synbiotics for its ability to modulate the patient gut microbiota and metabolome by setting a randomized, single-blind, placebo-controlled, pilot trial accounting for IIIb-IV stage CKD patients and healthy controls. Metataxonomic fecal microbiota and fecal volatilome were analyzed at the run-in, after 2 months of treatment, and after 1 month of wash out. Results: Significant changes in microbiota profile, as well as an increase of the saccharolytic metabolism, in feces were found for those CKD patients that were allocated in the synbiotics arm. Conclusions: Noteworthy, the here analyzed data emphasized a selective efficacy of the present synbiotics on a stage IIIb-IV CKD patients. Nonetheless, a further validation of this trial accounting for an increased patient number should be considered. Clinical trial registration: https://clinicaltrials.gov/, identifier NCT03815786.

Gut Microbiota, Metabolome, and Body Composition Signatures of Response to Therapy in Patients with Advanced Melanoma.

Despite the recent breakthroughs in targeted and immunotherapy for melanoma, the overall survival rate remains low. In recent years, considerable attention has been paid to the gut microbiota and other modifiable patient factors (e.g., diet and body composition), though their role in influencing therapeutic responses has yet to be defined. Here, we characterized a cohort of 31 patients with unresectable IIIC-IV-stage cutaneous melanoma prior to initiation of targeted or first-line immunotherapy via the following methods: (i) fecal microbiome and metabolome via 16S rRNA amplicon sequencing and gas chromatography/mass spectrometry, respectively, and (ii) anthropometry, body composition, nutritional status, physical activity, biochemical parameters, and immunoprofiling. According to our data, patients subsequently classified as responders were obese (i.e., with high body mass index and high levels of total, visceral, subcutaneous, and intramuscular adipose tissue), non-sarcopenic, and enriched in certain fecal taxa (e.g., Phascolarctobacterium) and metabolites (e.g., anethole), which were potentially endowed with immunostimulatory and oncoprotective activities. On the other hand, non-response was associated with increased proportions of Streptococcus, Actinomyces, Veillonella, Dorea, Fusobacterium, higher neutrophil levels (and a higher neutrophil-to-lymphocyte ratio), and higher fecal levels of butyric acid and its esters, which also correlated with decreased survival. This exploratory study provides an integrated list of potential early prognostic biomarkers that could improve the clinical management of patients with advanced melanoma, in particular by guiding the design of adjuvant therapeutic strategies to improve treatment response and support long-term health improvement.

Adipocyte-hepatocyte crosstalk in cellular models of obesity: Role of soluble factors.

Hepatic steatosis is often a consequence of obesity. Adipose tissue is an important endocrine regulator of metabolic homeostasis in the body. In obesity, adipocytes become hypertrophic and develop an inflammatory phenotype, altering the panel of secreted adipokines. Moreover, excess fatty acids are, in part, released by adipocytes and delivered to the liver. These multiple pathways of adipose-liver crosstalk contribute to the development and progression of liver disease: TNFα induces hepatocyte dysfunction, excess of circulating fatty acids promotes hepatic steatosis and inflammation, whilst adipokines mediate and exacerbate liver injury. In this study, we investigated in vitro the effects and mechanisms of the crosstalk between adipocytes and hepatocytes, as a function of the different adipocyte status (mature vs hypertrophic) being mediated by soluble factors. We employed the conditioned medium method to test how mature and hypertrophic adipocytes distinctively affect the liver, leading to metabolic dysfunction. The media collected from adipocytes were characterized by high triglyceride content and led to lipid accumulation and fat-dependent dysfunction in hepatocytes. The present findings seem to suggest that, in addition to triglycerides, other soluble mediators, cytokines, are released by mature and hypertrophic adipocytes and influence the metabolic status of liver cells. Understanding the precise factors involved in the pathogenesis and pathophysiology of NAFLD in obesity will provide important insights into the mechanisms responsible for the metabolic complications of obesity, paving the way for new possible approaches.

Clinical and Metabolomic Effects of Lactiplantibacillus plantarum and Pediococcus acidilactici in Fructose Intolerant Patients

Fructose intolerance (FI) is a widespread non-genetic condition in which the incomplete absorption of fructose leads to gastro-intestinal disorders. The crucial role of microbial dysbiosis on the onset of these intolerance symptoms together with their persistence under free fructose diets are driving the scientific community towards the use of probiotics as a novel therapeutic approach. In this study, we evaluated the prevalence of FI in a cohort composed of Romanian adults with Functional Grastrointestinal Disorders (FGIDs) and the effectiveness of treatment based on the probiotic formulation EQBIOTA® (Lactiplantibacillus plantarum CECT 7484 and 7485 and Pediococcus acidilactici CECT 7483). We evaluated the impact of a 30-day treatment both on FI subjects and healthy volunteers. The gastrointestinal symptoms and fecal volatile metabolome were evaluated. A statistically significant improvement of symptoms (i.e., bloating, and abdominal pain) was reported in FI patient after treatment. On the other hand, at the baseline, the content of volatile metabolites was heterogeneously distributed between the two study arms, whereas the treatment led differences to decrease. From our analysis, how some metabolomics compounds were correlated with the improvement and worsening of clinical symptoms clearly emerged. Preliminary observations suggested how the improvement of gastrointestinal symptoms could be induced by the increase of anti-inflammatory and protective substrates. A deeper investigation in a larger patient cohort subjected to a prolonged treatment would allow a more comprehensive evaluation of the probiotic treatment effects.

Beneficial Effects of Carvacrol on In Vitro Models of Metabolically-Associated Liver Steatosis and Endothelial Dysfunction: A Role for Fatty Acids in Interfering with Carvacrol Binding to Serum Albumin.

BACKGROUND: Carvacrol, a plant phenolic monoterpene, is largely employed as food additive and phytochemical. OBJECTIVE: We aimed to assess the lipid lowering and protective effects of carvacrol in vitro using cellular models of hepatic steatosis and endothelial dysfunction. We also investigated if and how the binding of carvacrol to albumin, the physiological transporter for small compounds in the blood, might be altered by the presence of high levels of fatty acids (FAs). METHODS: Hepatic FaO cells treated with exogenous FAs mimic hepatosteatosis; endothelial HECV cells exposed to hydrogen peroxide are a model of endothelial dysfunction. In these models, we measured spectrophotometrically lipid accumulation and release, lipoperoxidation, free radical production, and nitric oxide release before and after treatment with carvacrol. The carvacrol binding to albumin in the presence or absence of high levels of FAs was assessed by absorption and emission spectroscopies. RESULTS: Carvacrol counteracted lipid accumulation and oxidative stress in hepatocytes and protected endothelial cells from oxidative stress and dysfunction. Moreover, high levels of FAs reduced the binding of carvacrol to albumin. CONCLUSION: The results suggest the good potential of carvacrol in ameliorating dysfunction of hepatic and endothelial cells in vitro. High levels of circulating FAs might compete with carvacrol for binding to albumin thus influencing its transport and bio-distribution.

Extent and features of liver steatosis in vitro pave the way to endothelial dysfunction without physical cell-to-cell contact.

BACKGROUND AND AIMS: Several chronic multifactorial diseases originate from energy unbalance between food intake and body energy expenditure, including non-alcoholic fatty liver disease (NAFLD), diabetes, and cardiovascular disorders. Vascular endothelium plays a central role in body homeostasis, and NAFLD is often associated with endothelial dysfunction (ED), the first step in atherosclerosis. Both sugars and fatty acids (FAs) are fuel sources for energy production, but their excess leads to liver steatosis which may trigger ED through a network of mechanisms which need to be clarified. Here, we investigated the crosstalk pathways between in vitro cultured steatotic hepatocytes (FaO) and endothelial cells (HECV) being mediated by soluble factors. METHODS AND RESULTS: We employed the conditioned medium approach to test how different extent and features of hepatic steatosis distinctively affect endothelium leading to ED. The steatogenic media collected from steatotic hepatocytes were characterized by high triglyceride content and led to lipid accumulation and fat-dependent dysfunction in HECV cells. We found a parallelism between (i) extent of hepatocyte steatosis and level of lipid accumulation in HECV cells; (ii) type of hepatocyte steatosis (with macro- or microvesicular LDs) and extent of oxidative stress, lipid peroxidation, nitric oxide release and expression of ED markers in HECV cells. CONCLUSIONS: The present findings seem to suggest that, in addition to triglycerides, other soluble mediators should be released by steatotic hepatocytes and may influence lipid accumulation and function of HECV cells. Further studies need to depict the exact profile of soluble factors involved in steatotic hepatocyte-endothelium crosstalk.

Antitumor Activity of Ethanolic Extract from Thymbra Spicata L. aerial Parts: Effects on Cell Viability and Proliferation, Apoptosis Induction, STAT3, and NF-kB Signaling.

Thyme-like plants including Thymbra spicata L. are widely used as food and folk medicinal remedies in the Mediterranean area. This study aimed to explore the in vitro antitumor potential of polyphenol-enriched extracts from aerial parts of T. spicata. The ethanolic extract significantly inhibited proliferation of different human tumor cell lines, without significant effects on non-neoplastic cells. A deeper investigation of the molecular mechanism sustaining the in vitro antitumor activity of the extract was carried on the human breast cancer cells MCF-7 in comparison with the normal breast cells MCF-10A. The effects on MCF-7 cells were associated with the following: (i) production of reactive oxygen species (ROS) and release of nitric oxide; (ii) apoptosis induction; and (iii) reduction in STAT3 and NF-kB phosphorylation. The ethanolic extract from T. spicata leaves might represent a novel therapeutic tool in combination with conventional chemotherapy to reduce the adverse side effects and drug resistance.

Protective effects of extracts from Ephedra foeminea Forssk fruits against oxidative injury in human endothelial cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Ephedra foeminea is a member of the Ephedraceae family which is widespread in the eastern Mediterranean area. In Lebanon, Ephedra is a popular remedy in traditional medicine to prevent and/or counteract many stress oxidative-related diseases like inflammation and bacterial infections. AIM OF THE STUDY: Oxidative stress leads to endothelial cell dysfunction, and is a major factor contributing to etiology of atherosclerosis and related diseases. This study aims to investigate the antioxidant and cytoprotective potential of extracts from E. foeminea fruits on human endothelial cells exposed to hydrogen peroxide (H2O2) to mimic in vitro vascular endothelium dysfunction. MATERIALS AND METHODS: Different extracts of E. foeminea fruits were prepared using pure ethanol (EE), methanol/water (EMW), pure hexane (Ehex) or ethyl acetate/water (Epoly) as extraction solvents. The phenolome profile of each extract was characterized using high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS/MS). Total phenolic and flavonoid content, and radical scavenging properties of the extracts were assessed spectrophotometrically. Then, the effects on human endothelial cells HECV were evaluated. RESULTS: Epoly extract showed the highest phenol and flavonoid content, and the highest radical scavenging capacity. On H2O2-insulted HECV cells Epoly was able: (i) to counteract the ROS/RNS production and lipid peroxidation; (ii) to rescue the ROS-dependent decrease in the mitochondrial membrane potential; (iii) to counteract the apoptosis induction; (iv) to restore endothelial cell viability and migration. CONCLUSIONS: The findings indicated that the polyphenol-enriched extract Epoly from E. foeminea fruits is endowed with in vitro anti-oxidant and anti-apoptotic effects and might be used as nutraceutical for treating ROS-related endothelium dysfunction and inflammation.

Aquaporin-9 is involved in the lipid-lowering activity of the nutraceutical silybin on hepatocytes through modulation of autophagy and lipid droplets composition.

Hepatic steatosis is the hallmark of non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome and insulin resistance with potential evolution towards non-alcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma. Key roles of autophagy and oxidative stress in hepatic lipid accumulation and NAFLD progression are recognized. Here, we employed a rat hepatoma cell model of NAFLD progression made of FaO cells exposed to oleate/palmitate followed or not by TNFα treatment to investigate the molecular mechanisms through which silybin, a lipid-lowering nutraceutical, may improve hepatic lipid dyshomeostasis. The beneficial effect of silybin was found to involve amelioration of the fatty acids profile of lipid droplets, stimulation of the mitochondrial oxidation and upregulation of a microRNA of pivotal relevance in hepatic fat metabolism, miR-122. Silybin was also found to restore the levels of Aquaporin-9 (AQP9) and glycerol permeability while reducing the activation of the oxidative stress-dependent transcription factor NF-κB, and autophagy turnover. In conclusion, silybin was shown to have molecular effects on signaling pathways that were previously unknown and potentially protect the hepatocyte. These actions intersect TG metabolism, fat-induced autophagy and AQP9-mediated glycerol transport in hepatocytes.