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Abstract Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disorder characterized by the progressive loss of motor neurons, with genetic and environmental factors contributing to its complex pathogenesis. Dysregulated immune responses and altered energetic metabolism are key features, with emerging evidence implicating the gut microbiota (GM) in disease progression. We investigated the interplay among genetic background, GM composition, metabolism, and immune response in two distinct ALS mouse models: 129Sv_G93A and C57Ola_G93A, representing rapid and slow disease progression, respectively. Using 16 S rRNA sequencing and fecal metabolite analysis, we characterized the GM composition and metabolite profiles in non-transgenic (Ntg) and SOD1G93A mutant mice of both strains. Our results revealed strain-specific differences in GM composition and functions, particularly in the abundance of taxa belonging to Erysipelotrichaceae and the levels of short and medium-chain fatty acids in fecal samples. The SOD1 mutation induces significant shifts in GM colonization in both strains, with C57Ola_G93A mice showing changes resembling those in 129 Sv mice, potentially affecting disease pathogenesis. ALS symptom progression does not significantly alter microbiota composition, suggesting stability. Additionally, we assessed systemic immunity and inflammatory responses revealing strain-specific differences in immune cell populations and cytokine levels. Our findings underscore the substantial influence of genetic background on GM composition, metabolism, and immune response in ALS mouse models. These strain-specific variations may contribute to differences in disease susceptibility and progression rates. Further elucidating the mechanisms underlying these interactions could offer novel insights into ALS pathogenesis and potential therapeutic targets.

Background & Aims: Among the reprogrammed metabolic pathways described in cancer stem cells, aberrant lipid metabolism has recently drawn increasing attention. Our study explored the contribution of fatty acids (FA) in the regulation of stem-like features in intrahepatic cholangiocarcinoma (iCCA). Methods: We previously identified a functional stem-like subset in human iCCA by using a three-dimensional sphere (SPH) model in comparison to parental cells grown as monolayers (MON). In this study, quantification of intracellular free FA and lipidomic analysis (triacylglycerol [TAG] composition, de novo synthesis products) was performed by Liquid chromatography–mass spectrometry (LC–MS); quadrupole time-of-flight liquid chromatography/mass spectrometry (Q-TOF LC/MS), respectively, in both SPH and MON cultures. Results: Stem-like SPH showed a superior content of free FA (citric, palmitic, stearic, and oleic acids) and unsaturated TAG. Molecularly, SPH showed upregulation of key metabolic enzymes involved in de novo FA biosynthesis (AceCS1, ACLY, ACAC, FASN, ACSL1) and the mTOR signalling pathway. In patients with iCCA (n = 68), tissue expression of FASN, a key gene involved in FA synthesis, correlated with 5-year overall survival. Interference with FASN activity in SPH cells through both specific gene silencing (siRNA) or pharmacological inhibition (orlistat) decreased sphere-forming ability and expression of stem-like markers. In a murine xenograft model obtained by injection of iCCA-SPH cells, FASN inhibition by orlistat or injection of FASN-silenced cells significantly reduced tumour growth and expression of stem-like genes. Conclusion: Altered FA metabolism contributes to the maintenance of a stem-like phenotype in iCCA. FASN inhibition may represent a new approach to interfere with the progression of this deadly disease. Impact and implications: Recent evidence indicates that metabolic disorders correlate with an increased susceptibility to intrahepatic cholangiocarcinoma (iCCA). Our investigation emphasises the pivotal involvement of lipid metabolism in the tumour stem cell biology of iCCA, facilitated by the upregulation of crucial enzymes and the mTOR signalling pathway. From a clinical perspective, this underscores the dual role of FASN as both a prognostic indicator and a therapeutic target, suggesting that FASN inhibitors could enhance patient outcomes by diminishing stemness and tumour aggressiveness. These findings pave the way for novel therapeutic strategies for iCCA and shed light on its relationship with metabolic disorders such as diabetes, obesity, metabolic syndrome, and metabolic dysfunction-associated steatotic liver disease.

Abstract Systemic inflammation and neuroinflammation affect the natural course of the sporadic form of Alzheimer’s disease (AD), as supported by epidemiological and preclinical data, and several epidemiological studies indicate a higher prevalence of AD in patients with inflammatory bowel disease. In this study, we explored whether colitis induced by dextran sulfate sodium (DSS) in young, presymptomatic/preplaque mice worsens and/or anticipates age-dependent cognitive impairment in Tg2576, a widely used mouse model of AD. We demonstrated that DSS colitis induced in young Tg2576 mice anticipates the onset age of learning and memory deficit in the Morris water maze test. To explore potential mechanisms behind the acceleration of cognitive decline in Tg2576 mice by DSS colitis, we focused on gut microbiota, systemic inflammation and neuroinflammation markers. We observed a Firmicutes/Bacteroidetes ratio change in Tg2576 DSS animals comparable to that of elderly Tg2576 mice, suggesting accelerated microbiota aging in Tg2576 DSS mice, a change not observed in C57BL6 DSS mice. We also observed substantial differences between Tg2576 and WT mice in several inflammation and neuroinflammation-related parameters as early as 3 months of age, well before plaque deposition, a picture which evolved rapidly (between 3 and 5.5 months of age) in contrast to Tg2576 and WT littermates not treated with DSS. In detail, following induction of DSS colitis, WT and Tg2576 mice exhibited contrasting features in the expression level of inflammation-evoked astrocyte-associated genes in the hippocampus. No changes in microglial features occurred in the hippocampus between the experimental groups, whereas a reduced glial fibrillary acidic protein immunoreactivity was observed in Tg2576 vs. WT mice. This finding may reflect an atrophic, “loss-of-function” profile, further exacerbated by DSS where a decreased of GFAP mRNA expression level was detected. In conclusion, we suggest that as-yet unidentified peripheral mediators evoked by DSS colitis and involving the gut-brain axis emphasize an astrocyte “loss-of-function” profile present in young Tg2576 mice, leading to impaired synaptic morphological and functional integrity as a very early sign of AD.

Abstract Background Emerging evidence suggests that breast microbiota dysbiosis contributes to cancer initiation, progression, prognosis and treatment efficacy. Anyway, available data are referred only to female patients, and studies on males are completely missing. Male breast cancer (MBC) is 70–100 times less frequent, but the mortality rate adjusted to incidence is higher in men than in females. Currently, MBC diagnostic approaches and treatments have generally been extrapolated from the clinical experience gained in women, while few studies focus on characterizing male cancer biology. Taking into account the rising importance of the oncobiome field and the need of MBC targeted studies, we explored the breast cancer oncobiome of male and female patients. Methods 16S rRNA gene sequencing was performed in 20 tumor and 20 non-pathological adjacent FFPE breast tissues from male and female patients. Results We documented, for the first time, the presence of a sexually dimorphic breast-associated microbiota, here defined as “breast microgenderome”. Moreover, the paired analysis of tumor and non-pathological adjacent tissues suggests the presence of a cancer-associated dysbiosis in male patients, with surrounding tissue conserving a healthier microbiome, whereas in female patients, the entire breast tissue is predisposed to cancer development. Finally, the phylum Tenericutes, especially the genera Mesoplasma and Mycobacterium, could to be involved in breast carcinogenesis, in both sexes, deserving further investigation, not only for its role in cancer development but even as potential prognostic biomarker. Conclusions Breast microbiota characterization can enhance the understanding of male breast cancer pathogenesis, being useful for detection of new prognostic biomarkers and development of innovative personalized therapies, remarking the relevant gender differences.

In the early stages of carcinogenesis, the transformed cells become “invisible” to the immune system. From this moment on, the evolution of the tumor depends essentially on the genotype of the primitive cancer cells and their subsequent genetic drift. The role of the immune system in blocking tumor progression from the earliest stages is largely underestimated because by the time tumors are clinically detectable, the immune system has already completely failed its task. Therefore, a clinical treatment capable of restoring the natural anti-tumor role of the immune system could prove to be the “ultimate weapon” against cancer. Herein, we propose a novel therapeutic approach for the treatment of solid cancer that exploits the capability of activated monocytes to transfer major histocompatibility complex I (MHC-I) molecules bound to antigenic peptides to cancer cells using microvesicles as cargo, making tumor cells target of a “natural” CD8+ T lymphocyte cytotoxic response.

Introduction: Calcific aortic valve disease (CAVD) is the most common heart valve disorder, defined by a remodeling multistep process: namely, valve fibrosis with its area narrowing, impaired blood flow, and final calcification phase. Nowadays, the only treatment is the surgical valve replacement. As for other cardiovascular diseases, growing evidence suggest an active role of the immune system in the calcification process that could be modulated by the microbiota. To address this point, we aimed to investigate and characterize, for the first time, the presence of a valve microbiota and associated immune response in human CAVD. Method: Calcified aortic valve (CAV) samples from twenty patients (11 from Germany and 9 from Italy) with diagnosis of severe symptomatic CAVD were used to assess the presence of infiltrating T cells, by cloning approach, and to characterize the valve microbiota, by 16S rRNA gene sequencing (NGS). Results: We documented the presence of infiltrating T lymphocytes, especially the T helper subset, in CAV samples. Moreover, we found a tissue-associated microbiota in freshly collected CAV samples, which was significantly different in Italian and German patients, suggesting potential correlation with other cardiovascular risk factors. Conclusion: The presence of microbiota in inflamed CAV samples represents the right trigger point to explain the valve calcification process, encouraging further studies to explore the potential link between bacteria and adaptive immune response and to define the critical role of local microbiota-immunity axis on CAVD development.

Background: Approximately 95% of Colorectal cancers (CRC) consist of adenocarcinomas originating from colonic Adenomatous polyps (AP). Increasing importance in CRC occurrence and progression has been attributed to the gut microbiota; however, a huge proportion of microorganisms inhabit the human digestive system. So, to comprehensively study the microbial spatial variations and their role in CRC progression, from AP to the different CRC phases, a holistic vision is imperative, including the simultaneous evaluation of multiple niches from the gastrointestinal system. Through an integrated approach, we identified potential microbial and metabolic biomarkers, able to discriminate human CRC from AP and/or also the different Tumor node metastasis (TNM) staging. In addition, as the microbiota contributes to the production of essential metabolic products detectable in fecal samples, we analysed and compared metabolites obtained from CRC and AP patients by using a Nuclear magnetic resonance (NMR) approach. Methods: In this observational study, saliva, tissue and stool samples from 61 patients, have been collected, including 46 CRC and 15 AP patients, age and sex-matched, undergoing surgery in 2018 at the Careggi University Hospital (Florence, Italy). First, the microbiota in the three-district between CRC and AP patients has been characterized, as well as in different CRC TNM stages. Subsequently, proton NMR spectroscopy has been used in combination with multivariate and univariate statistical approaches, to define the fecal metabolic profile of a restricted group of CRC and AP patients. Results: CRC patients display a different profile of tissue and fecal microbiota with respect to AP patients. Significant differences have been observed in CRC tissue microbial clades, with a rise of the Fusobacterium genus. In addition, significant taxa increase at the genus level has been observed in stool samples of CRC patients. Furthermore, Fusobacterium found in intestinal tissue has been positively correlated with fecal Parvimonas, for the first time. Moreover, as predicted by metagenomics pathway analysis, a significant increase of lactate (p=0.037) has been observed in the CRC fecal metabolic profiles, and positively correlated with Bifidobacterium (p=0.036). Finally, minor bacterial differences in CRC patients at stage T2 (TNM classification) have been detected, with a raise of the Spirochaetota phylum in CRC samples, with a slight increase of the Alphaproteobacteria class in fecal samples. Conclusion: Our results suggest the importance of microbiota communities and oncometabolites in CRC development. Further studies on CRC/AP management with a focus on CRC assessment are needed to investigate novel microbial-related diagnostic tools aimed to improve therapeutic interventions.

Background and aimIn recent decades, obesity prevalence has reached epidemic proportions and considering the pivotal role of gut microbiota (GM) in the regulation of energy balance, alternative non-pharmacological approaches involving probiotics’ administration have been proposed. The aim of the present study was to evaluate the effect of Lactiplantibacillus plantarum IMC 510® supplementation on anthropometric and biochemical parameters, GM composition and functionality, and gastrointestinal and general symptoms of overweight/obese subjects.MethodsForty overweight/obese subjects were randomly assigned to daily consume the probiotic Lactiplantibacillus plantarum IMC 510® or placebo for 3 months. Before and after the administration period, anthropometric and biochemical parameters, self-administered questionnaires, and plasma and stool samples were obtained from each participant. The GM characterization was performed with 16S rRNA sequencing, while fecal short (SCFAs) and medium (MCFAs) chain fatty acids were analyzed with a gas chromatography–mass spectrometry protocol.ResultsCompared to placebo, probiotic supplementation determined a significant decrease in body weight, BMI, waist circumference, waist-to-height ratio, and blood glucose. Moreover, probiotic administration produced a significant decrease of the genera Hafnia-Obesumbacterium and Romboutsia and an increase of Succiniclasticum spp.; conversely, placebo administration resulted in the decrease of Actinomycetaceae and an increase of both Alloprevotella spp. and of the levels of pro-inflammatory hexanoic and heptanoic acids.ConclusionThanks to its effect in increasing some beneficial gut bacteria and lowering effects on waist circumference, fasting glucose levels and gastrointestinal symptoms of obese subjects, Lactiplantibacillus plantarum IMC 510® supplementation could represent a future and encouraging strategy for the prevention or treatment of obesity.

Pathway analysis is ubiquitous in biological data analysis due to the ability to integrate small simultaneous changes in functionally related components. While pathways are often defined based on either manual curation or network topological properties, an attractive alternative is to generate pathways around specific functions, in which metabolism can be defined as the production and consumption of specific metabolites. In this work, we present an algorithm, termed MetPath, that calculates pathways for condition-specific production and consumption of specific metabolites. We demonstrate that these pathways have several useful properties. Pathways calculated in this manner (1) take into account the condition-specific metabolic role of a gene product, (2) are localized around defined metabolic functions, and (3) quantitatively weigh the importance of expression to a function based on the flux contribution of the gene product. We demonstrate how these pathways elucidate network interactions between genes across different growth conditions and between cell types. Furthermore, the calculated pathways compare favorably to manually curated pathways in predicting the expression correlation between genes. To facilitate the use of these pathways, we have generated a large compendium of pathways under different growth conditions for E. coli. The MetPath algorithm provides a useful tool for metabolic network-based statistical analyses of high-throughput data.

IntroductionPotential celiac disease (pCD) is characterized by genetic predisposition, positive anti-endomysial and anti-tissue transglutaminase antibodies, but a normal or almost normal jejunal mucosa (e.g., minor histological abnormalities without villous atrophy). To gain further insights into basic mechanisms involved in the development of intestinal villous atrophy, we evaluated and compared the microbial, lipid, and immunological signatures of pCD and atrophic CD (aCD).Materials and methodsThis study included 17 aCD patients, 10 pCD patients, and 12 healthy controls (HC). Serum samples from all participants were collected to analyze free fatty acids (FFAs). Duodenal mucosa samples of aCD and pCD patients were taken to evaluate histology, tissue microbiota composition, and mucosal immune response.ResultsWe found no significant differences in the mucosa-associated microbiota composition of pCD and aCD patients. On the other hand, in pCD patients, the overall abundance of serum FFAs showed relevant and significant differences in comparison with aCD patients and HC. In detail, compared to HC, pCD patients displayed higher levels of propionic, butyric, valeric, 2-ethylhexanoic, tetradecanoic, hexadecanoic, and octadecanoic acids. Instead, aCD patients showed increased levels of propionic, isohexanoic, and 2-ethylhexanoic acids, and a lower abundance of isovaleric and 2-methylbutyricacids when compared to HC. In addition, compared to aCD patients, pCD patients showed a higher abundance of isobutyric and octadecanoic acid. Finally, the immunological analysis of duodenal biopsy revealed a lower percentage of CD4+ T lymphocytes in pCD infiltrate compared to that observed in aCD patients. The functional characterization of T cells documented a pro-inflammatory immune response in both aCD and pCD patients, but the pCD patients showed a higher percentage of Th0/Th17 and a lower percentage of Th1/Th17.ConclusionThe results of the present study show, for the first time, that the duodenal microbiota of patients with pCD does not differ substantially from that of aCD; however, serum FFAs and local T cells displayed a distinctive profile between pCD, aCD, and HC. In conclusion, our result may help to shed new light on the “gut microbiota-immunity axis,” lipid metabolites, and duodenal immune response in overt CD and pCD patients, opening new paradigms in understanding the pathogenesis behind CD progression.

BackgroundMerTK is a tyrosine kinase receptor that belongs to the TAM (Tyro3/Axl/Mer) receptor family. It is involved in different processes including cellular proliferation/survival, cellular adhesion/migration, and release of the inflammatory/anti-inflammatory cytokines. Although it is reported that MERTK polymorphisms affect the severity of viral and metabolic liver diseases, being able to influence fibrosis progression and hepatocellular carcinoma development, the mechanisms remain unknown. Methods: using a microarray approach, we evaluated the liver expression of genes involved in fibrogenesis and hepatocarcinogenesis in patient with chronic hepatitis C (CHC), stratified for MERTK genotype and MERTK expression. Results: we found that the rs 4374383 AA homozygosity is associated with lower MERTK expression in CHC patients and that, depending on MERTK genotype, Matrix Metallopeptidase 9 (MMP9), Matrix Metallopeptidase 7 (MMP7), Secreted Frizzled Related Protein 1 (SFRP1) and WNT gene family 11(WNT11) show differential expression in patients with CHC with or without neoplastic progression. Conclusions: our results confirm that MERTK represents a genetic biomarker for progression of liver disease and are suggestive of translational relevance for the study of downstream pathways involved in fibrogenesis and hepatocarcinogenesis.

Background and aimIn recent years, many studies have suggested that ancient wheat products might have beneficial effects on cardiometabolic risk profile, but little is known about their effect on gut microbiota (GM). The aim of the present study was to evaluate whether a replacement diet with pasta made from ancient wheat (AD) could influence the GM composition and its metabolites’ production compared to a replacement diet with pasta made from modern wheat (CD).MethodsA randomized, double-blinded crossover trial with two intervention phases was conducted on 20 clinically healthy adults (9 females; 11 males; mean age 43.1 ± 12.5 years). Study participants were assigned to consume pasta made using semi-whole flour from organic wheat that was either from ancient or modern control wheat for 8 weeks in a random order. An 8-week washout period was implemented between the interventions. Stool samples were collected from all subjects at the beginning and at the end of each intervention period. GM composition, and short- (SCFAs) and medium- chain fatty acids (MCFAs) production was evaluated.ResultsDietary interventions did not produce significant diversity in the GM composition at higher ranks (phylum, class, order and family), but only at genus level. In detail, the AD significantly (adj. p < 0.05) changed the abundance of Erysipelatoclostridium spp., Bacteroides_pectinophilus_group spp., CAG-873 spp., and Holdemanella spp. The CD significantly affected the abundance of Akkermansia spp., CAG-873 spp., Hungatella spp., Lachnospiraceae_UCG-008 spp., NK4A214_group spp., Frisingicoccus spp., Megasphaera spp., Synergistes spp., and Tyzzerella spp. Regarding the production of SCFAs and MCFAs, AD resulted in a significant increase of fecal acetic (+0.7%), isobutyric (+30.1%), 2-methylbutyric (+64.2%), and isovaleric (+22.5%) acids. On the other hand, CD resulted in increased levels of isobutyric (+71.4%), 2-methylbutyric (+116.2%), isovaleric (+99%), and valeric (+21.4%) acids, and a reduction of butyric (-31.6%) and hexanoic (-66.4%) acids.ConclusionA short-term replacement diet with both ancient and modern wheat pasta determined significant changes in GM composition at the genus level but notably the AD resulted in a greater beneficial impact on anti-inflammatory SCFAs.

Background and aimsCrohn’s disease (CD) pathogenesis is still unclear. Remodeling in mucosal microbiota and systemic immunoregulation may represent an important component in tissue injury. Here, we aim to characterize the ileal microbiota in both pathological and healthy settings and to evaluate the correlated systemic microbial-associated inflammatory markers comparing first-time surgery and relapse clinical conditions.MethodsWe enrolled 28 CD patients at surgery; we collected inflamed and non-inflamed mucosa tissues and blood samples from each patient. Bacterial wall adherence was observed histologically, while its composition was assessed through amplicon sequencing of the 16S rRNA gene. In addition, we evaluated the systemic microRNA (miRNA) using quantitative real-time PCR amplification and free fatty acids (FFAs) using gas chromatography–mass spectroscopy.ResultsThe total number of mucosal adherent microbiota was enriched in healthy compared to inflamed mucosa. In contrast, the phylum Tenericutes, the family Ruminococcaceae, and the genera Mesoplasma and Mycoplasma were significantly enriched in the pathological setting. Significant microbiota differences were observed between the relapse and first surgery patients regarding the families Bacillaceae 2 and Brucellaceae and the genera Escherichia/Shigella, Finegoldia, Antrobacter, Gemmatimonas, Moraxella, Anoxibacillus, and Proteus. At the systemic level, we observed a significant downregulation of circulating miR-155 and miR-223, as well as 2-methyl butyric, isobutyric, and hexanoic (caproic) acids in recurrence compared to the first surgery patients. In addition, the level of hexanoic acid seems to act as a predictor of recurrence risk in CD patients (OR 18; 95% confidence interval 1.24–261.81; p = 0.006).ConclusionsWe describe a dissimilarity of ileal microbiota composition comparing CD and healthy settings, as well as systemic microbial-associated inflammatory factors between first surgery and surgical relapse. We suggest that patterns of microbiota, associated with healthy ileal tissue, could be involved in triggering CD recurrence. Our findings may provide insight into the dynamics of the gut microbiota–immunity axis in CD surgical recurrence, paving the way for new diagnostics and therapeutics aimed not only at reducing inflammation but also at maintaining a general state of eubiosis in healthy tissue.

Food contamination can be a serious concern for public health because it can be related to the severe spreading of pathogens. This is a main issue, especially in the case of fresh fruits and vegetables; indeed, they have often been associated with gastrointestinal outbreak events, due to contamination with pathogenic bacteria. However, little is known about the physiological adaptation and bacterial response to stresses encountered in the host plant. Thus, this work aimed to investigate the adaptation of a commensal E. coli strain while growing in tomato pericarp. Pre-adapted and non-adapted cells were compared and used to contaminate tomatoes, demonstrating that pre-adaptation boosted cell proliferation. DNA extracted from pre-adapted and non-adapted cells was sequenced, and their methylation profiles were compared. Hence, genes involved in cell adhesion and resistance against toxic compounds were identified as genes involved in adaptation, and their expression was compared in these two experimental conditions. Finally, pre-adapted and non-adapted E. coli were tested for their ability to resist the presence of toxic compounds, demonstrating that adaptation exerted a protective effect. In conclusion, this work provides new information about the physiological adaptation of bacteria colonizing the tomato fruit pericarp.

In this exploratory study, we investigate the variation in the facial skin microbiome architecture through aging and their functional association with host genetic factors in a cohort of healthy women, living in the same area and without cutaneous diseases. Notably, facial skin microbiota (SM) samples were collected from a cohort of 15 healthy Caucasian females, firstly divided into three age groups (younger women aged 20–35 years old; middle aged women of 36–52 years old; and older women aged 53–68 years old). Then, the recruited cohort was divided into two groups based on their facial hydration level (dry and normal skin). The facial SM revealed a different composition in the three analyzed aging groups and between normal and dry skins. The middle-aged women also revealed functional variations associated with collagen biosynthesis and oxidative stress damage repair. Otherwise, the association between selected host SNPs (single nucleotide polymorphisms) and the facial SM profile showed significant associations, suggesting a negative correlation with collagen metabolism and ROS damage protection. Finally, the composition and functionality of the facial SM seemed to affect the aging process through the two aging-correlated pathways of host ROS damage repair and collagen metabolism. Our exploratory data could be useful for future studies characterizing the structure, function, and dynamics of the SM in the aging process to design personalized therapeutic agents focusing on potential genomic targets, microbes, and their metabolites.

Background and aimGut microbiota (GM) can support colorectal cancer (CRC) progression by modulating immune responses through the production of both immunostimulatory and/or immunosuppressive cytokines. The role of IL-9 is paradigmatic because it can either promote tumor progression in hematological malignancies or inhibit tumorigenesis in solid cancers. Therefore, we investigate the microbiota–immunity axis in healthy and tumor mucosa, focusing on the correlation between cytokine profile and GM signature.MethodsIn this observational study, we collected tumor (CRC) and healthy (CRC-S) mucosa samples from 45 CRC patients, who were undergoing surgery in 2018 at the Careggi University Hospital (Florence, Italy). First, we characterized the tissue infiltrating lymphocyte subset profile and the GM composition. Subsequently, we evaluated the CRC and CRC-S molecular inflammatory response and correlated this profile with GM composition, using Dirichlet multinomial regression.ResultsCRC samples displayed higher percentages of Th17, Th2, and Tregs. Moreover, CRC tissues showed significantly higher levels of MIP-1α, IL-1α, IL-1β, IL-2, IP-10, IL-6, IL-8, IL-17A, IFN-γ, TNF-α, MCP-1, P-selectin, and IL-9. Compared to CRC-S, CRC samples also showed significantly higher levels of the following genera: Fusobacteria, Proteobacteria, Fusobacterium, Ruminococcus2, and Ruminococcus. Finally, the abundance of Prevotella spp. in CRC samples negatively correlated with IL-17A and positively with IL-9. On the contrary, Bacteroides spp. presence negatively correlated with IL-9.ConclusionsOur data consolidate antitumor immunity impairment and the presence of a distinct microbiota profile in the tumor microenvironment compared with the healthy mucosa counterpart. Relating the CRC cytokine profile with GM composition, we confirm the presence of bidirectional crosstalk between the immune response and the host’s commensal microorganisms. Indeed, we document, for the first time, that Prevotella spp. and Bacteroides spp. are, respectively, positively and negatively correlated with IL-9, whose role in CRC development is still under debate.

Eosinophilic granulomatosis with polyangiitis (EGPA) is an anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis. A genome-wide association study showed a correlation between ANCA-negative EGPA and variants of genes encoding proteins with intestinal barrier functions, suggesting that modifications of the mucosal layer and consequent gut dysbiosis might be involved in EGPA pathogenesis. Here, we characterized the gut microbiota (GM) composition and the intestinal immune response in a cohort of EGPA patients. Faeces from 29 patients and 9 unrelated healthy cohabitants were collected, and GM and derived metabolites’ composition were compared. Seven intestinal biopsies from EGPA patients with gastrointestinal manifestations were analysed to assess the T-cell distribution and its correlation with GM and EGPA clinical and laboratory features. No significant differences in GM composition, nor in the total amount of faecal metabolites, emerged between patients and controls. Nevertheless, differences in bacterial taxa abundances and compositional GM-derived metabolites profile were observed. Notably, an enrichment of potential pathobionts (Enterobacteriacee and Streptococcaceae) was found in EGPA, particularly in patients with active disease, while lower levels were found in patients on immunosuppression, compared with non-immunosuppressed ones. Significantly lower amounts of hexanoic acid were found in patients, compared to controls. The analysis of the immune response in the gut mucosa revealed a high frequency of IFN-γ/IL-17-producing T lymphocytes, and a positive correlation between EGPA disease activity and intestinal T-cell levels. Our data suggest that an enrichment in potential intestinal pathobionts might drive an imbalanced inflammatory response in EGPA.

Congenital Central Hypoventilation Syndrome (CCHS) is a rare genetic disorder of the autonomic nervous system and in particular of the respiratory control during sleep. No drug therapy is, to date, available; therefore, the survival of these patients depends on lifelong ventilatory support during sleep. Reactive oxygen species (ROS)-induced oxidative stress is a recognized risk factor involved in the pathogenesis of several chronic diseases. Therefore, monitoring systemic oxidative stress could provide important insights into CCHS outcomes. Because ROS-induced oxidative products are excreted as stable metabolites in urine, we performed an HPLC-MS/MS analysis for the quantitative determination of the three main representative oxidative biomarkers (i.e., diY, MDA, and 8-OHdG) in the urine of CCHS patients. Higher levels of urinary MDA were found in CCHS patients compared with age-matched control subjects. The noteworthy finding is the identification of urinary MDA as relevant biomarker of systemic oxidative status in CCHS patients. This study is a concise and smart communication about the impact that oxidative stress has in CCHS, and suggests the monitoring of urinary MDA levels as a useful tool for the management of these patients.

Investigation of the fungal communities in animal models of Inflammatory Bowel Diseases (IBD) showed a controversial role of Saccharomyces cerevisiae and Candida spp. In health and disease. These conflicting observations could be ascribed to immunogenic differences among co-specific strains. To assess the relevance of intra-strains differences on yeast immunogenicity and impact on the microbiota, we screened S. cerevisiae and Candida spp. Strains isolated from fecal samples of IBD patients. We compared the cytokine profiles, obtained upon stimulation of Peripheral Blood Mononuclear Cells (PBMCs) and Dendritic Cells with different yeast strains, and evaluated the relationship between strain’s cell wall sugar amount and immune response. Moreover, the gut microbiota composition was explored in relation to fungal isolation from fecal samples by metabarcoding analysis. The comparison of cytokine profiles showed strain dependent rather than species-dependent differences in immune responses. Differences in immunogenicity correlated with the cell wall composition of S. cerevisiae intestinal strains. Stimulation of human healthy PBMCs with different strains showed a pro-inflammatory IL-6 response counterbalanced by IL-10 production. Interestingly, Crohn’s (CD) patients responded differently to “self” and “non-self” strains, eliciting pure Th1 or Th17 cytokine patterns. The differences observed in vitro were recapitulated in vivo, where different strains contributed in dramatically different ways to local epithelial activity and to the inflammation of wild type and Interleukin-deficient mice. Furthermore, we observed that the gut microbiota profiles significantly differentiated according to the presence of Saccharomyces or Candida spp. or the absence of fungal isolates in fecal samples. Our results show the importance to deepen metagenomics and immunophenotyping analyses to the strain level, to elucidate the role of fungal and bacterial communities in health and disease.

The release of monomers from the homotetrameric protein transthyretin (TTR) is the first event of a cascade, eventually leading to sporadic or familial TTR amyloidoses. Thus, ligands able to stabilize TTR and inhibit monomer release are subject of intense scrutiny as potential treatments against these pathologies. Here, we investigated the interaction between TTR and a non-glycated derivative of the main olive polyphenol, oleuropein (OleA), known to interfere with TTR aggregation. We coupled fluorescence studies with molecular docking to investigate the OleA/TTR interaction using wild-type TTR, a monomeric variant, and the L55P cardiotoxic mutant. We characterized a fluorescence band emitted by OleA upon formation of the OleA/TTR complex. Exploiting this signal, we found that a poorly specific non-stoichiometric interaction occurs on the surface of the protein and a more specific stabilizing interaction takes place in the ligand binding pocket of TTR, exhibiting a KD of 3.23 ± 0.32 µM, with two distinct binding sites. OleA interacts with TTR in different modes, stabilizing it and preventing its dissociation into monomers, with subsequent misfolding. This result paves the way to the possible use of OleA to prevent degenerative diseases associated with TTR misfolding.

Summary: Endocrine therapy (ET) is the standard of care for estrogen receptor-positive (ER+) breast cancers. Despite its efficacy, ∼40% of women relapse with ET-resistant (ETR) disease. A global transcription analysis in ETR cells reveals a downregulation of the neutral and basic amino acid transporter SLC6A14 governed by enhanced miR-23b-3p expression, resulting in impaired amino acid metabolism. This altered amino acid metabolism in ETR cells is supported by the activation of autophagy and the enhanced import of acidic amino acids (aspartate and glutamate) mediated by the SLC1A2 transporter. The clinical significance of these findings is validated by multiple orthogonal approaches in a large cohort of ET-treated patients, in patient-derived xenografts, and in in vivo experiments. Targeting these amino acid metabolic dependencies resensitizes ETR cells to therapy and impairs the aggressive features of ETR cells, offering predictive biomarkers and potential targetable pathways to be exploited to combat or delay ETR in ER+ breast cancers. : Bacci et al. find that endocrine-resistant ER+ breast cancers are characterized by enhanced miR-23b-3p, autophagy activation, and import of aspartate and glutamate that fuel catabolic and anabolic pathways, which are essential for their aggressive features. The molecular players involved in this metabolic scenario are of clinical significance and have prognostic and predictive value. Keywords: endocrine therapy, resistance, metabolic reprogramming, estrogen receptor, amino acid transporters, aspartate, glutamate, miRNA, SLCs

Diabetes is a chronic metabolic disease with a strong social impact worldwide. Under chronic hyperglycemia, protein glycation strongly contributes to diabetes-related complications onset. Anti-glycation agents and inhibitors of α-glucosidase are often therapeutically used to control postprandial glycemia in order to prevent development of long-term diabetic complications. Given drug resistance and adverse effects of conventional antidiabetic therapies, the discovery of new effective and non-toxic naturally occurring compounds is needed to prevent and/or to manage life-threatening diabetic complications. Annona cherimola Miller fruit has been used in Mexican traditional medicine as natural remedy against diabetes. In this work, the in vitro anti-glycation and anti-α-glucosidase roles of Annona cherimola Miller pulp extract (CE) were investigated. Moreover, healthy and diabetic subjects were enrolled in a cross-over design intervention study aimed at investigating the effects of pulp intake on postprandial glycemia. This work shows that CE was able to inhibit albumin glycation in vitro and to inhibit α-glucosidase enzyme. Furthermore, the pulp intake did not contribute to an increase in postprandial glycemia, making it a suitable source of health-promoting phytonutrients and a potential functional food in diabetics and pre-diabetics diet.

Neurodegenerative diseases are generally characterized by the presence of neurotoxic amyloid aggregates underlying progressive neuronal death. Since ancient times, natural compounds have been used as curative agents for human health. Amyloid research is constantly looking for safe natural molecules capable of blocking toxic amyloid aggregates’ formation. From the marine environment, seaweeds are recognized as rich reservoirs of molecules with multiple bioactivities, including the anti-amyloidogenic activity. Here, hydroalcoholic extracts of two seasonal samples of the Mediterranean red seaweed Halophytis incurva (HIEs) were characterized by the HPLC-DAD-MS analysis. The H. incurva anti-amyloidogenic role was explored by incubating both HIEs with hen egg white lysozyme (HEWL), a well-known protein model widely used in amyloid aggregation experiments. The aggregation kinetics and morphological analysis of amyloid aggregates were performed by ThT and AFM analysis, respectively, while their cytotoxicity on SH-SY5Y human neuroblastoma cells was examined by MTT assay. HIEs showed a different efficacy, probably dependent on their metabolic composition, both in inhibiting amyloid fibrillation and in obtaining short and less toxic pre-fibrillary aggregates. Overall, this work sheds light, for the first time, on a Mediterranean red seaweed as a promising renewable resource of bioactive compounds, potentially useful in preventing the formation of toxic amyloid aggregates.

Xylella fastidiosa causing disease on different plant species has been reported in several European countries, since 2013. Based on multilocus sequence typing (MLST) results, there is evidence of repeated introductions of the pathogen in Spain and France. In contrast, in the Salento area of Apulia (Puglia) in Southern Italy, the existence of a unique Apulian MLST genotype of X. fastidiosa, causing the olive quick decline syndrome (OQDS; also referred to as “CoDiRO” or “ST53”) was proven, and this was tentatively ascribed to X. fastidiosa subsp. pauca. In order to acquire information on intra population diversity European Food Safety Authority (EFSA) has strongly called for the characterization of X. fastidiosa isolates from Apulia to produce the necessary data to better understand strain diversity and evolution. In this work, for the first time the existence of sub-variants within a set of 14 “ST53” isolates of X. fastidiosa collected from different locations was searched using DNA typing methods targeting the whole pathogen genome. Invariably, VNTR, RAPD and rep-PCR (ERIC and BOX motifs) analyses indicated that all tested isolates possessed the same genomic fingerprint, supporting the existence of predominant epidemiological strain in Apulia. To further explore the degree of clonality within this population, two isolates from two different Salento areas (Taviano and Ugento) were completely sequenced using PacBio SMRT technology. The whole genome map and sequence comparisons revealed that both isolates are nearly identical, showing less than 0.001% nucleotide diversity. However, the complete and circularized Salento-1 and Salento-2 genome sequences were different, in genome and plasmid size, from the reference strain 9a5c of X. fastidiosa subsp. pauca (from citrus), and showed a PCR-proved large genome inversion of about 1.7 Mb. Genome-wide indices ANIm and dDDH indicated that the three isolates of X. fastidiosa from Salento (Apulia, Italy), namely Salento-1, Salento-2, and De Donno, whose complete genome sequence has been recently released, share a very recent common ancestor. This highlights the importance of continuous and extensive monitoring of molecular variation of this invasive pathogen to understand evolution of adaptive traits, and the necessity for adoption of all possible measures to reduce the risk of new introductions that may augment pathogen diversity.

Diet is one of the main factors that affects the composition of gut microbiota. When people move from a rural environment to urban areas, and experience improved socio-economic conditions, they are often exposed to a “globalized” Western type diet. Here, we present preliminary observations on the metagenomic scale of microbial changes in small groups of African children belonging to the same ethnicity and living in different environments, compared to children living on the urban area of Florence (Italy). We analyzed dietary habits and, by pyrosequencing of the 16S rRNA gene, gut microbiota profiles from fecal samples of children living in a rural village of Burkina Faso (n = 11), of two groups of children living in different urban settings (Nanoro town, n = 8; Ouagadougou, the capital city, n = 5) and of a group of Italian children (n = 13). We observed that when foods of animal origin, those rich in fat and simple sugars are introduced into a traditional African diet, composed of cereals, legumes and vegetables, the gut microbiota profiles changes. Microbiota of rural children retain a geographically unique bacterial reservoir (Prevotella, Treponema, and Succinivibrio), assigned to ferment fiber and polysaccharides from vegetables. Independently of geography and ethnicity, in children living in urban areas these bacterial genera were progressively outcompeted by bacteria more suited to the metabolism of animal protein, fat and sugar rich foods, similarly to Italian children, as resulted by PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States), a predictive functional profiling of microbial communities using 16S rRNA marker gene. Consequently, we observed a progressive reduction of SCFAs measured by gas chromatography–mass spectrometry, in urban populations, especially in Italian children, respect to rural ones. Our results even if in a limited number of individuals point out that dietary habit modifications in the course of urbanization play a role in shaping gut microbiota, and that ancient microorganisms, such as fiber-degrading bacteria, are at risk of being eliminated by the fast paced globalization of foods and by the advent of westernized lifestyle.

Posidonia oceanica (L.) Delile is a marine plant with interesting biological properties potentially ascribed to the synergistic combination of bioactive compounds. Our previously described extract, obtained from the leaves of P. oceanica, showed the ability to impair HT1080 cell migration by targeting both expression and activity of gelatinases. Commonly, the lack of knowledge about the mechanism of action of phytocomplexes may be an obstacle regarding their therapeutic use and development. The aim of this study was to gain insight into the molecular signaling through which such bioactive compounds impact on malignant cell migration and gelatinolytic activity. The increase in autophagic vacuoles detected by confocal microscopy suggested an enhancement of autophagy in a time and dose dependent manner. This autophagy activation was further confirmed by monitoring pivotal markers of autophagy signaling as well as by evidencing an increase in IGF-1R accumulation on cell membranes. Taken together, our results confirm that the P. oceanica phytocomplex is a promising reservoir of potent and cell safe molecules able to defend against malignancies and other diseases in which gelatinases play a major role in progression. In conclusion, the attractive properties of this phytocomplex may be of industrial interest in regard to the development of novel health-promoting and pharmacological products for the treatment or prevention of several diseases.

The study of compounds able to interfere in various ways with amyloid aggregation is of paramount importance in amyloid research. Molecules characterized by a 4-thiaflavane skeleton have received great attention in chemical, medicinal, and pharmaceutical research. Such molecules, especially polyhydroxylated 4-thiaflavanes, can be considered as structural mimickers of several natural polyphenols that have been previously demonstrated to bind and impair amyloid fibril formation. In this work, we tested five different 4-thiaflavanes on the hen egg-white lysozyme (HEWL) amyloid model for their potential anti-amyloid properties. By combining a thioflavin T assay, atomic force microscopy, and a cell toxicity assay, we demonstrated that such compounds can impair the formation of high-order amyloid aggregates and mature fibrils. Despite this, the tested 4-thiaflavanes, although non-toxic per se, are not able to prevent amyloid toxicity on human neuroblastoma cells. Rather, they proved to block early aggregates in a stable, toxic conformation. Accordingly, 4-thiaflavanes can be proposed for further studies aimed at identifying blocking agents for the study of toxicity mechanisms of amyloid aggregation.

Among the enzymes involved in NAD homeostasis, nicotinamide mononucleotide adenylyltransferases (NMNAT1-3) are central to intracellular NAD formation. Although NMNAT3 is postulated to be a mitochondrial enzyme contributing to NAD-dependent organelle functioning, information on endogenous proteins is lacking. We report that in human cells a single gene nmnat3 localized on chromosome 3 codes for two mRNA splice variants NMNATv1 and FKSG76, whereas the previously reported NMNAT3v2 transcript is not present. However, NMNAT3v1 and FKSG76 proteins are not detectable, consistent with the finding that an upstream ORF in their mRNAs negatively regulates translation. NMNAT3v1 transfection demonstrates that the protein is cytosolic and inactive, whereas FKSG76 is mitochondrial but operates NAD cleavage rather than synthesis. In keeping with the lack of NMNAT3, we show that extracellular NAD, but not its metabolic precursors, sustains mitochondrial NAD pool in an ATP-independent manner. Data of the present study modify the scenario of the origin of mitochondrial NAD by showing that, in human cells, NMNAT3 is absent in mitochondria, and, akin to plants and yeast, cytosolic NAD maintains the mitochondrial NAD pool.

Nine human neurodegenerative diseases, including Huntington's disease and several spinocerebellar ataxia, are associated to the aggregation of proteins comprising an extended tract of consecutive glutamine residues (polyQs) once it exceeds a certain length threshold. This event is believed to be the consequence of the expansion of polyCAG codons during the replication process. This is in apparent contradiction with the fact that many polyQs-containing proteins remain soluble and are encoded by invariant genes in a number of eukaryotes. The latter suggests that polyQs expansion and/or aggregation might be counter-selected through a genetic and/or protein context. To identify this context, we designed a software that scrutinize entire proteomes in search for imperfect polyQs. The nature of residues flanking the polyQs and that of residues other than Gln within polyQs (insertions) were assessed. We discovered strong amino acid residue biases robustly associated to polyQs in the 15 eukaryotic proteomes we examined, with an over-representation of Pro, Leu and His and an under-representation of Asp, Cys and Gly amino acid residues. These biases are conserved amongst unrelated proteins and are independent of specific functional classes. Our findings suggest that specific residues have been co-selected with polyQs during evolution. We discuss the possible selective pressures responsible of the observed biases.

BackgroundAmyloid fibril formation is the hallmark of many human diseases, including Alzheimer's disease, type II diabetes and amyloidosis. Amyloid fibrils deposit in the extracellular space and generally co-localize with the glycosaminoglycans (GAGs) of the basement membrane. GAGs have been shown to accelerate the formation of amyloid fibrils in vitro for a number of protein systems. The high number of data accumulated so far has created the grounds for the construction of a database on the effects of a number of GAGs on different proteins.Methodology/principal findingsIn this study, we have constructed such a database and have used a computational approach that uses a combination of single parameter and multivariate analyses to identify the main chemical factors that determine the GAG-induced acceleration of amyloid formation. We show that the GAG accelerating effect is mainly governed by three parameters that account for three-fourths of the observed experimental variability: the GAG sulfation state, the solute molarity, and the ratio of protein and GAG molar concentrations. We then combined these three parameters into a single equation that predicts, with reasonable accuracy, the acceleration provided by a given GAG in a given condition.Conclusions/significanceIn addition to shedding light on the chemical determinants of the protein:GAG interaction and to providing a novel mathematical predictive tool, our findings highlight the possibility that GAGs may not have such an accelerating effect on protein aggregation under the conditions existing in the basement membrane, given the values of salt molarity and protein:GAG molar ratio existing under such conditions.

Formation of amyloid-like fibrils is involved in numerous human protein deposition diseases, but is also an intrinsic property of polypeptide chains in general. Progress achieved recently now allows the aggregation propensity of proteins to be analyzed over large scales. In this work we used a previously developed predictive algorithm to analyze the propensity of the 34,180 protein sequences of the human proteome to form amyloid-like fibrils. We show that long proteins have, on average, less intense aggregation peaks than short ones. Human proteins involved in protein deposition diseases do not differ extensively from the rest of the proteome, further demonstrating the generality of protein aggregation. We were also able to reproduce some of the results obtained with other algorithms, demonstrating that they do not depend on the type of computational tool employed. For example, proteins with different subcellular localizations were found to have different aggregation propensities, in relation to the various efficiencies of quality control mechanisms. Membrane proteins, intrinsically disordered proteins, and folded proteins were confirmed to have very different aggregation propensities, as a consequence of their different structures and cellular microenvironments. In addition, gatekeeper residues at strategic positions of the sequences were found to protect human proteins from aggregation. The results of these comparative analyses highlight the existence of intimate links between the propensity of proteins to form aggregates with beta-structure and their biology. In particular, they emphasize the existence of a negative selection pressure that finely modulates protein sequences in order to adapt their aggregation propensity to their biological context.