Search

Abstract Perennial grains, which remain productive for multiple years, rather than growing for only one season before harvest, have deep, dense root systems that can support a richness of beneficial microorganisms, which are mostly underexplored. In this work we isolated forty-three bacterial strains associated with the rhizosphere of the OK72 perennial wheat line, developed from a cross between winter common wheat and Thinopyrum ponticum. Identified using 16S rDNA sequencing, these bacteria were assessed for plant growth-promoting traits such as indole-3-acetic acid, siderophores and ACC-deaminase acid production, biofilm formation, and the ability to solubilize phosphate and proteins. Twenty-five strains exhibiting in vitro significant plant growth promoting traits, belong to wheat keystone genera Pseudomonas, Microbacterium, Variovorax, Pedobacter, Dyadobacter, Plantibacter, and Flavobacterium. Seven strains, including Aeromicrobium and Okibacterium genera, were able to promote root growth in a commercial annual wheat cultivar while strains from Pseudomonas genus inhibited the growth of Aspergillus flavus and Fusarium species, using direct antagonism assays. The same strains produced a high amount of 1-undecanol a volatile organic compound, which may aid in suppressing fungal growth. The study highlights the potential of these bacteria to form new commercial consortia, enhancing the health and productivity of annual wheat crops within sustainable agricultural practices.

Abstract Background Intrahepatic cholangiocarcinoma (iCCA) is a lethal primary liver tumor characterized by clinical aggressiveness, poor prognosis, and scarce therapeutic possibilities. Therefore, new treatments are urgently needed to render this disease curable. Since cumulating evidence supports the oncogenic properties of the Heat Shock Factor 1 (HSF1) transcription factor in various cancer types, we investigated its pathogenetic and therapeutic relevance in iCCA. Methods Levels of HSF1 were evaluated in a vast collection of iCCA specimens. The effects of HSF1 inactivation on iCCA development in vivo were investigated using three established oncogene-driven iCCA mouse models. In addition, the impact of HSF1 suppression on tumor cells and tumor stroma was assessed in iCCA cell lines, human iCCA cancer-associated fibroblasts (hCAFs), and patient-derived organoids. Results Human preinvasive, invasive, and metastatic iCCAs displayed widespread HSF1 upregulation, which was associated with a dismal prognosis of the patients. In addition, hydrodynamic injection of a dominant-negative form of HSF1 (HSF1dn), which suppresses HSF1 activity, significantly delayed cholangiocarcinogenesis in AKT/NICD, AKT/YAP, and AKT/TAZ mice. In iCCA cell lines, iCCA hCAFs, and patient-derived organoids, administration of the HSF1 inhibitor KRIBB-11 significantly reduced proliferation and induced apoptosis. Cell death was profoundly augmented by concomitant administration of the Bcl-xL/Bcl2/Bcl-w inhibitor ABT-263. Furthermore, KRIBB-11 reduced mitochondrial bioenergetics and glycolysis of iCCA cells. Conclusions The present data underscore the critical pathogenetic, prognostic, and therapeutic role of HSF1 in cholangiocarcinogenesis.

Soil salinity is considered one of the most limiting factors in large agricultural areas, and tomato (Solanum lycopersicum L.) is highly sensitive to this abiotic stress, which exacerbates under drought. The use of halotolerant PGPR is a promising strategy to enhance tomato tolerance and productivity in saline soils. The present study tested the capability of the recently discovered bacterial strain PVr_9 to increase tolerance to salinity in tomato plants in vitro, greenhouse and open field conditions. When inoculated with PVr_9, in vitro seedlings showed a significant increase in primary root length, number of secondary roots and fresh weight both in absence of stress and when exposed to 150 mM NaCl. In plants exposed to salt treatment, PVr_9 upregulated the salt tolerance genes SOS1 and NHX1, and the enzyme prolyl aminopeptidase involved in proline metabolism. Proteins associated with resistance against pathogens were also upregulated by PVr_9 in absence of salt stress. In greenhouse, PVr_9-inoculated tomato plants treated for 1-week with 150 mM NaCl showed a significant increase in shoot fresh biomass, chlorophyll, proline content, and APX activity. Tomato fruits from PVr_9 inoculated plants, exposed in the field to salt stress during the flowering stage, showed higher levels of carotenoids, lycopene, and l-ascorbic acid, as well as an increased antioxidant capacity compared to fruits of uninoculated plants. These results indicate PVr_9 as a potential biostimulant in tomato for better tackling soil salinization in a context of climate change and expansion of coastal salinity, while there is large scope for the improvement of the nutritional characteristics of tomato fruits.

Background: Consumption of flavonoid-rich orange juice has been shown to reduce adiposity and liver steatosis in murine models of diet-induced obesity. However, little is known about the effects of whole orange intake, independent of body weight changes, on liver function and steatosis in individuals with metabolic dysfunction-associated steatotic liver disease (MASLD). The goal is to understand the direct impact of orange consumption on metabolic health. Methods: Sixty-two men and women aged 30–65 with MASLD (Controlled Attenuation Parameter, (CAP) > 275 dB/m) were randomly assigned to consume either 400 g of whole oranges or non-citrus fruits daily for 4 weeks. Baseline evaluations included medical assessments, blood tests, and body composition. Liver health was assessed using transient elastography (FibroScan®) for steatosis and fibrosis, conducted by blinded personnel. This clinical trial was registered at ClinicalTrials.gov (NCT05558592). Results: After 4 weeks of orange supplementation, liver steatosis decreased in the treatment group, with 70.9% showing steatosis compared to 100% in controls (p < 0.004), indicating a 30% reduction in liver disease prevalence. There were no significant changes in fibrosis or plasma liver enzymes, though plasma gamma glutaril transferase (GGT) levels decreased significantly. Body weight, waist circumference, body composition, lipid profile, fasting glucose, insulin, and C-reactive protein levels remained unchanged. Dietary analysis revealed no change in caloric intake, but vitamins C, A, thiamine, and riboflavin increased in the orange group. Conclusions: Our findings suggest that phytochemical-rich foods, especially whole fruits like oranges, may enhance liver function as an adjunct treatment for MASLD. The notable reduction in liver steatosis prevalence occurred independently of body weight changes. Further studies are needed to investigate the long-term effects of orange supplementation on steatosis and fibrosis progression and to identify the specific bioactive compounds and mechanisms involved.

(1) Background: Metabolic dysfunction-associated liver disease (MASLD) is one of the most important causes of liver disease worldwide. Meat consumption is a growing trend and white meat has been shown to have beneficial effects on cardiometabolic risk factors. The aim of this study was to investigate the dose–response relationship between white meat intake and MASLD at survey level in a Southern Italy setting. (2) Methods: This cross-sectional study encompassed 1192 subjects (509 males, 42.7%) without missing data from the second wave of the NUTRIHEP cohort (2014–2016). Adjusted dose–response modeling was employed for statistical analysis; (3) Results: There were 587 subjects with MASLD (49.2%), i.e., 278 males (54.6%) and 309 females (45.2%). By increasing the intake, an unfavorable influence of white meat on MASLD was significantly revealed in females, whereas a protective effect of white meat was detectable in males. Male sex was shown to be involved in other associations in this study, such as influencing the preference for specific foods such as poultry and chicken skin. (4) Conclusions: Our data suggest that white meat does not have a clear-cut independent dose–response effect on MASLD, but sex may be a trigger moderator for age and BMI, with an increasing unfavorable effect of white meat in women, and a favorable effect in men.

Background: The minor G-allele of FOXO3 rs2802292 is associated with human longevity. The aim of this study was to test the protective effect of the variant against the association with type 2 Diabetes and NAFLD. Methods: rs2802292 was genotyped in a large population of middle-aged subjects (n = 650) from a small city in Southern Italy. All participants were interviewed to collect information about lifestyle and dietary habits; clinical characteristics were recorded, and blood samples were collected from all subjects. The association between rs2802292 and NAFLD or diabetes was tested using a logistic model and mediation analysis adjusted for covariates. Results: Overall, the results indicated a statistical association between diabetes and rs2802292, especially for the TT genotype (OR = 2.14, 1.01 to 4.53 95% C.I., p = 0.05) or in any case for those who possess the G-allele (OR = 0.45, 0.25 to 0.81 95% C.I., p = 0.008). Furthermore, we found a mediation effect of rs2802292 on diabetes (as mediator) and NAFLD. There is no direct relationship between rs2802292 and NAFLD, but the effect is direct (β = 0.10, −0.003 to 0.12 95% C.I., p = 0.04) on diabetes, but only in TT genotypes. Conclusions: The data on our cohort indicate that the longevity-associated FOXO3 variant may have protective effects against diabetes and NAFLD.

Crohn’s disease (CD) is a type of inflammatory bowel disease (IBD) affecting the gastrointestinal tract that can also cause extra-intestinal complications. Following exposure to the mRNA vaccine BNT162b2 (Pfizer-BioNTech) encoding the SARS-CoV-2 Spike (S) protein, some patients experienced a lack of response to the biological drug Adalimumab and a recrudescence of the disease. In CD patients in progression, resistant to considered biological therapy, an abnormal increase in intestinal permeability was observed, more often with a modulated expression of different proteins such as Aquaporin 8 (AQP8) and in tight junctions (e.g., ZO-1, Claudin1, Claudin2, Occludin), especially during disease flares. The aim of this study is to investigate how the SARS-CoV-2 vaccine could interfere with IBD therapy and contribute to disease exacerbation. We investigated the role of the SARS-CoV-2 Spike protein, transported by extracellular vesicles (EVs), and the impact of various EVs components, namely, exosomes (EXOs) and microvesicles (MVs), in modulating the expression of molecules involved in the exacerbation of CD, which remains unknown.

Cyclic nucleotide phosphodiesterases (PDEs) consist of a family of enzymes expressed in several types of cells, including inflammatory cells, that play a pivotal role in inflammation. Several studies have demonstrated that the inhibition of PDE4 results in a reduced inflammatory response via PKA and CREB signaling. Hence, PDE4 suppression improves the inflammatory feedback typical of several diseases, such as inflammatory bowel disease (IBD). In our previous studies, we have demonstrated that miR-369-3p regulates inflammatory responses, modulating different aspects of the inflammatory process. The aim of this study was to demonstrate an additional anti-inflammatory effect of miR-369-3p targeting PDE4B, one of the widely expressed isoforms in immune cells. We found that miR-369-3p was able to reduce the expression of PDE4B, elevating the intracellular levels of cAMP. This accumulation increased the expression of PKA and pCREB, mitigating the release of pro-inflammatory cytokines and promoting the release of anti-inflammatory cytokines. To prove that PDE4B is a good therapeutic target in IBD, we also demonstrate that the expression of PDE4B was increased in UC patients compared to healthy controls, affecting the immune infiltrate. PDE4B is considered an important player in inflammatory progression; hence, our results show the ability of miR-369-3p to ameliorate inflammation by targeting PDE4B, supporting its future application as a new therapeutic approach in IBD.

Abstract The NLRP3 inflammasome is an intracellular sensor and an essential component of the innate immune system involved in danger recognition. An important hallmark of inflammasome activation is the formation of a single supramolecular punctum, known as a speck, per cell, which is the site where the pro-inflammatory cytokines IL-1β and IL-18 are converted into their bioactive form. Speck also provides the platform for gasdermin D protein activation, whose N-terminus domain perforates the plasma membrane, allowing the release of mature cytokines alongside with a highly inflammatory form of cell death, namely pyroptosis. Although controlled NLRP3 inflammasome-pyroptosis pathway activation preserves mucosal immunity homeostasis and contributes to host defense, a prolonged trigger is deleterious and could lead, in genetically predisposed subjects, to the onset of inflammatory bowel disease, including Crohn's disease and ulcerative colitis, as well as to gastrointestinal cancer. Experimental evidence shows that the NLRP3 inflammasome has both protective and pathogenic abilities. In this review we highlight the impact of the NLRP3-pyroptosis axis on the pathophysiology of the gastrointestinal tract at molecular level, focusing on newly discovered features bearing pro- and anti-inflammatory and neoplastic activity, and on targeted therapies tested in preclinical and clinical trials.

Abstract Excessive toxic lipid accumulation in hepatocytes underlies the development of non-alcoholic fatty liver disease (NAFLD), phenotypically characterized by necrosis and steato-fibrosis, whose molecular mechanism is not yet fully understood. Patients with NAFLD display an imbalanced palmitic (PA) to oleic acid (OA) ratio. Moreover, increasing experimental evidence points out a relevant involvement of the exosomal content in disease progression. Aim of the study was to highlight the PA/OA imbalance within circulating exosomes, the subsequent intracellular alterations, and the impact on NALFD. Liver cells were challenged with exosomes isolated from both healthy subjects and NAFLD patients. The exosomal PA/OA ratio was artificially modified, and biological effects were evaluated. A NAFLD-derived exosomal PA/OA imbalance impacts liver cell cycle and cell viability. OA-modified NAFLD-derived exosomes restored cellular viability and proliferation, whereas the inclusion of PA into healthy subjects-derived exosomes negatively affected cell viability. Moreover, while OA reduced the phosphorylation and activation of the necroptosis marker, Receptor-interacting protein 1 (phospho-RIP-1), PA induced the opposite outcome, alongside increased levels of stress fibers, such as vimentin and fibronectin. Administration of NAFLD-derived exosomes led to increased expression of Elongase 6 (ELOVL6), Stearoyl-CoA desaturase 1 (SCD1), Tumor necrosis factor α (TNF-α), Mixed-lineage-kinase-domain-like-protein (MLKL) and RIP-1 in the hepatocytes, comparable to mRNA levels in the hepatocytes of NAFLD patients reported in the Gene Expression Omnibus (GEO) database. Genetic and pharmacological abrogation of ELOVL6 elicited a reduced expression of downstream molecules TNF-α, phospho-RIP-1, and phospho-MLKL upon administration of NAFLD-derived exosomes. Lastly, mice fed with high-fat diet exhibited higher phospho-RIP-1 than mice fed with control diet. Targeting the Elongase 6–RIP-1 signaling pathway offers a novel therapeutic approach for the treatment of the NALFD-induced exosomal PA/OA imbalance.

Abstract Background Autotaxin (ATX) is a secreted enzyme that converts lysophosphatidylcholine to lysophosphatidic acid (LPA). LPA stimulates cell proliferation and migration and promotes wound repair following tissue damage. ATX levels are directly correlated with stage and grade in several human cancers. Several small molecule ATX inhibitors have been developed in recent years. IOA-289 is a potent ATX inhibitor, developed to treat cancers containing fibrosis. In this study, we tested IOA-289 treatment on different gastrointestinal tract tumor cell lines, in order to evaluate its effects on viability and motility. Methods To determine the effects on cell viability and proliferation of treatment with increasing concentrations of IOA-289, we used the crystal violet assay, a clonogenic assay in matrigel, and we evaluated the inhibitor’s effect on formation of 3D spheroids in an in vitro model. The effect of IOA-289 on cell cycle phases was analysed with a redox dye reagent. Cell migration capacity was evaluated by wound healing assay and transwell migration assay. To evaluate the pro-apoptotic effect of the inhibitor, cells were stained with Annexin V and immunofluorescence and flow cytometry analysis were performed. An antibody array was also used, to discriminate, in various samples, the differential expression of 43 proteins involved in the apoptosis pathway. Results We found that IOA-289 is able to inhibit both growth and migration of gastrointestinal tract tumor cell lines, both in 2D (crystal violet assay) and 3D in vitro models (spheroid formation and clonogenic assay in matrigel). This effect is dose-dependent, and the drug is most effective when administered in FBS-free culture medium. The inhibitory effect on cell growth is due to a pro-apoptotic effect of IOA-289. Staining with FITC-conjugated Annexin V showed that IOA-289 induced a dose-dependent increase in fluorescence following incubation for 24 h, and apoptotic cells were also distinguished in flow cytometry using Annexin/PI staining. The antibody array shows that treatment with IOA-289 causes the increased expression of several pro-apoptotic proteins in all tested cell lines. Conclusions These results indicate that IOA-289 may be an effective drug for the treatment of tumors of the gastrointestinal tract, particularly those characterized by a high degree of fibrosis.

The VLCKD is a diet recognized to promote rapid fat mobilization and reduce inflammation, hepatic steatosis, and liver fibrosis. Extracellular vesicles (EVs) mediate cell-to-cell communication. The aim of the study is to investigate the role of circulating EVs in cell proliferation, ketone bodies, and ROS production in patients on an 8-week VLCKD regimen. Participants were classified as responders (R) or non-responders (NR) to VLCKD treatment based on their fibroscan results. In vitro experiments with the hepatic cell lines HEPA-RG (normal hepatocytes) and LX-2 (stellate cells) were conducted to investigate the effects of circulating EVs on cell viability, ROS production, and ketone body presence. The findings reveal a notable reduction in cell viability in both cell lines when treated with exosomes (EXOs). In contrast, treatment with microvesicles (MVs) did not appear to affect cell viability, which remained unchanged. Additionally, the levels of ketone bodies measured in urine were not consistently correlated with the reduction of fibrosis in responders (R). Similarly, an increase in ketone bodies was observed in non-responders (NR), which was also not aligned with the expected reduction in fibrosis. This inconsistency stands in stark contrast to the levels of Reactive Oxygen Species (ROS), which exhibited a clear and consistent pattern in accordance with the dietary intervention. Finally, in this preliminary study, ROS has been identified as a potential diet adherence marker for VLCKD patients; the ROS levels reliably follow the progression of the fibrosis response, providing a more accurate reflection of the therapeutic effects.

This greenhouse study evaluated the effects of soil enrichment with Pteris vittata rhizosphere bacteria on the growth and accumulation of arsenic in P. vittata grown on a naturally As-rich soil. Inoculations were performed with a consortium of six bacteria resistant to 100 mM arsenate and effects were compared to those obtained on the sterilized soil. Selected bacteria from the consortium were also utilized individually: PVr_9 homologous to Agrobacterium radiobacter that produces IAA and siderophores and shows ACC deaminase activity, PVr_15 homologous to Acinetobacter schindleri that contains the arsenate reductase gene, and PVr_5 homologous to Paenarthrobacter ureafaciens that possesses all traits from both PVr_9 and PVr_15. Frond and root biomass significantly increased in ferns inoculated with the consortium only on non-sterilized soil. A greater increase was obtained with PVr_9 alone, while only an increased root length was found in those inoculated with either PVr_5 or PVr_15. Arsenic content significantly decreased only in ferns inoculated with PVr_9 while it increased in those inoculated with PVr_5 and PVr_15. In conclusion, inoculations with the consortium and PVr_9 alone increase plant biomass, but no increase in As phytoextraction occurs with the consortium and even a reduction is seen with PVr_9 alone. Conversely, inoculations with PVr_5 and PVr_15 have the capacity of increasing As phytoextraction.

Autotaxin (ATX) is a member of the ectonucleotide pyrophosphate/phosphodiesterase (ENPP) family; it is encoded by the ENPP2 gene. ATX is a secreted glycoprotein and catalyzes the hydrolysis of lysophosphatidylcholine to lysophosphatidic acid (LPA). LPA is responsible for the transduction of various signal pathways through the interaction with at least six G protein-coupled receptors, LPA Receptors 1 to 6 (LPAR1–6). The ATX–LPA axis is involved in various physiological and pathological processes, such as angiogenesis, embryonic development, inflammation, fibrosis, and obesity. However, significant research also reported its connection to carcinogenesis, immune escape, metastasis, tumor microenvironment, cancer stem cells, and therapeutic resistance. Moreover, several studies suggested ATX and LPA as relevant biomarkers and/or therapeutic targets. In this review of the literature, we aimed to deepen knowledge about the role of the ATX–LPA axis as a promoter of cancer development, progression and invasion, and therapeutic resistance. Finally, we explored its potential application as a prognostic/predictive biomarker and therapeutic target for tumor treatment.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide. Curative treatments are available to a minority of patients, as HCC is often diagnosed at an advanced stage. For patients with unresectable and multifocal HCC, tyrosine kinase inhibitor drugs (TKIs) are the only potential treatment option. Despite extensive research, predictors of response to these therapies remain elusive. This study aimed to analyze the biological and histopathological characteristics of HCC patients treated with TKIs, focusing on angiogenesis and lymphangiogenesis. Immunohistochemistry quantified the expression of angiopoietin-2 (Ang2), lymphatic endothelial cells (LEC) podoplanin, and C-type Lectin Domain Family 2 (CLEC-2), key factors in neoangiogenesis and lymphangiogenesis. An increased expression of endothelial Ang2 and LEC podoplanin predicted a lower risk of metastasis. Female patients had significantly longer overall survival and survival on TKIs, associated with higher tumor expression of endothelial Ang2 and LEC podoplanin. Moreover, LEC podoplanin expression and a longer time on TKIs were independently correlated with improved survival on TKI therapy at Cox regression analysis. These findings suggest that endothelial Ang2 and LEC podoplanin could be potential biomarkers for predicting treatment outcomes and guiding therapeutic strategies in HCC patients treated with TKIs.

Background: Ultra-Processed Foods (UPFs) are increasingly consumed worldwide, even in regions with strong dietary traditions like the Mediterranean and can play a crucial role in the development of chronic diseases, including cancer. This population-based prospective cohort study investigates the association between UPF consumption and gastrointestinal cancers and other causes of mortality in Southern Italy. Methods: Data were collected from 4870 participants in the MICOL and NUTRIHEP cohorts. The EPIC questionnaire was used to elicit information on food and drink consumption and UPFs were categorized by degree of processing according to the NOVA classification. Cox proportional hazards regression and competing risk models were employed for statistical analysis. Results: UPF consumption was positively associated with all-cause mortality: participants in the 3rd UFP quartile, as compared to the lowest, had a 27% higher risk of death (SHR 1.27 95% CI, 1.03; 1.57), while in the highest quartile as compared to the lowest, the risk was 34% higher (SHR 1.34 95% CI, 1.00; 1.79). Higher UPFs intake was also correlated with an increased gastrointestinal cancers mortality risk, especially the 2nd (SHR 1.65, 95% CI: 1.01; 2.71) and 4th quartile (SHR 3.14 95% CI: 1.56; 6.32), with a dose-dependent effect. For the other cancers, a SHR 1.61 (95% CI 1.03; 2.54) was observed for the 3rd quartile. Conclusions: Our results reinforce the link between UPF consumption and cancer risk, emphasizing the urgent need for interventions targeting dietary patterns.

A very low calorie ketogenic diet (VLCKD) impacts host metabolism in people marked by an excess of visceral adiposity, and it affects the microbiota composition in terms of taxa presence and relative abundances. As a matter of fact, there is little available literature dealing with microbiota differences in obese patients marked by altered intestinal permeability. With the aim of inspecting consortium members and their related metabolic pathways, we inspected the microbial community profile, together with the set of volatile organic compounds (VOCs) from untargeted fecal and urine metabolomics, in a cohort made of obese patients, stratified based on both normal and altered intestinal permeability, before and after VLCKD administration. Based on the taxa relative abundances, we predicted microbiota-derived metabolic pathways whose variations were explained in light of our cohort symptom picture. A totally different number of statistically significant pathways marked samples with altered permeability, reflecting an important shift in microbiota taxa. A combined analysis of taxa, metabolic pathways, and metabolomic compounds delineates a set of markers that is useful in describing obesity dysfunctions and comorbidities.

Pinin (PNN) is a desmosome-associated protein that reinforces the organization of keratin intermediate filaments and stabilizes the anchoring of the cytoskeleton network to the lateral surface of the plasma membrane. The aberrant expression of PNN affects the strength of cell adhesion as well as modifies the intracellular signal transduction pathways leading to the onset of CRC. In our previous studies, we characterized the role of miR-195-5p in the regulation of desmosome junctions and in CRC progression. Here, with the aim of investigating additional mechanisms related to the desmosome complex, we identified PNN as a miR-195-5p putative target. Using a public data repository, we found that PNN was a negative prognostic factor and was overexpressed in colon cancer tissues from stage 1 of the disease. Then, we assessed PNN expression in CRC tissue specimens, confirming the overexpression of PNN in tumor sections. The increase in intracellular levels of miR-195-5p revealed a significant decrease in PNN at the mRNA and protein levels. As a consequence of PNN regulation by miR-195-5p, the expression of KRT8 and KRT19, closely connected to PNN, was affected. Finally, we investigated the in vivo effect of miR-195-5p on PNN expression in the colon of AOM/DSS-treated mice. In conclusion, we have revealed a new mechanism driven by miR-195-5p in the regulation of desmosome components, suggesting a potential pharmacological target for CRC therapy.

Obesity and metabolic syndrome are linked to steatotic liver disease (SLD), the most common form of chronic liver disease. Lifestyle modifications and dieting are strategies that can prevent metabolic dysfunction-associated steatotic liver disease (MASLD). The very low-calorie ketogenic diet (VLCKD) is a helpful treatment for MASLD and has been recommended for people affected by obesity; we evaluated the effect of gender on steatosis and fibrosis in a cohort of 112 overweight or obese patients undergoing an eight-week treatment with a VLCKD. Differences between the genders in terms of anthropometric measures, body composition, and metabolic indicators were examined before, during, and after the nutritional intervention. At baseline, there were significant differences between men and women in terms of anthropometric parameters, blood pressure, Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), fasting insulin, hepatic markers, and lipid profile. Men had considerably higher levels of liver steatosis (measured by CAP) and liver stiffness (measured by E) under basal conditions than women. After the VLCKD, there were reductions in both genders of controlled attenuation parameter (CAP), body weight, body mass index (BMI), waist circumference, systolic and diastolic blood pressure, insulin resistance, fat mass (FM), free fat mass (FFM), and fasting blood glucose, insulin, glycated hemoglobin (HbA1c), triglycerides, total cholesterol, low-density lipoprotein (LDL) cholesterol, alanine transaminase (ALT), gamma-glutamyl transferase (γGT), and uric acid levels. Only in men, liver stiffness, aspartate aminotransferase (AST), creatinine, and C-reactive protein (CRP) levels significantly decreased. Moreover, men had significantly greater levels of liver steatosis: the male gender featured an increase of 23.96 points of the Fibroscan CAP. Men exhibited higher levels of steatosis and fibrosis than women, and these differences persist despite VLCKD. These gender-specific variations in steatosis and fibrosis levels could be caused by hormonal and metabolic factors, suggesting that different therapeutic strategies might be required depending on the gender.

Liver cancer is one of the leading causes of cancer-related mortality. Hepatocellular carcinoma and cholangiocarcinoma are the most common types, and despite numerous advances, therapeutic options still remain poor for these cancer patients. Tumor development and progression strictly depend on a supportive tumor microenvironment (TME). Tumor-associated macrophages (TAMs) are the most abundant immune cells population within a tumorigenic liver; they sustain cancer cells’ growth and invasiveness, and their presence is correlated with a poor prognosis. Furthermore, TAM cross-talk with cells and components of the TME promotes immunosuppression, a desmoplastic response, and angiogenesis. In this review, we summarize the latest advances in understanding TAM heterogeneity and function, with a particular focus on TAM modulation of the TME. We also discuss the potential of targeting macrophage subpopulations and how this is now being exploited in current clinical trials for the treatment of liver cancer.

Background: Dietary guidelines recommend limiting red meat intake because it has been amply associated with increased cancer mortality, particularly in patients with liver conditions, such as metabolic dysfunction-associated fatty liver disease (MASLD). MASLD is the leading cause of liver dysfunction in the world today, and no specific treatment other than lifestyle correction has yet been established. The aim of this study was to explore the protective role of leafy vegetables when associated with high red meat consumption. Methods: The study cohort included 1646 participants assessed during the fourth recall of the MICOL study, subdivided into two groups based on red meat intake (≤50 g/die vs. >50 g/die), in order to conduct a cancer mortality analysis. The prevalence of subjects that consumed >50 g/die was only 15.73%. Leafy vegetable intake was categorized based on median g/die consumption, and it was combined with red meat intake. Conclusions: This is the first study to demonstrate that the consumption of about 30 g/die of leafy vegetables reduces the risk of mortality. A strong association with mortality was observed in subjects with MASLD, and the protective role of vegetables was demonstrated.

Extracellular vesicles (EVs), acting as inherent nanocarriers adept at transporting a range of different biological molecules such as proteins, lipids, and genetic material, exhibit diverse functions within the gastroenteric tract. In states of normal health, they participate in the upkeep of systemic and organ homeostasis. Conversely, in pathological conditions, they significantly contribute to the pathogenesis of gastrointestinal diseases (GIDs). Isolating EVs from patients’ biofluids facilitates the discovery of new biomarkers that have the potential to offer a rapid, cost-effective, and non-invasive method for diagnosing and prognosing specific GIDs. Furthermore, EVs demonstrate considerable therapeutic potential as naturally targeted physiological carriers for the intercellular delivery of therapeutic cargo molecules or as nanoscale tools engineered specifically to regulate physio-pathological conditions or disease progression. Their attributes including safety, high permeability, stability, biocompatibility, low immunogenicity, and homing/tropism capabilities contribute to their promising clinical therapeutic applications. This review will delve into various examples of EVs serving as biomarkers or nanocarriers for therapeutic cargo in the context of GIDs, highlighting their clinical potential for both functional and structural gastrointestinal conditions. The versatile and advantageous properties of EVs position them as promising candidates for innovative therapeutic strategies in advancing personalized medicine approaches tailored to the gastroenteric tract, addressing both functional and structural GIDs.

Natural compounds like flavonoids preserve intestinal mucosal integrity through their antioxidant, anti-inflammatory, and antimicrobial properties. Additionally, some flavonoids show prebiotic abilities, promoting the growth and activity of beneficial gut bacteria. This study investigates the protective impact of Lens culinaris extract (LE), which is abundant in flavonoids, on intestinal mucosal integrity during LPS-induced inflammation. Using Caco-2 cells as a model for the intestinal barrier, the study found that LE did not affect cell viability but played a cytoprotective role in the presence of LPS. LE improved transepithelial electrical resistance (TEER) and tight junction (TJ) protein levels, which are crucial for barrier integrity. It also countered the upregulation of pro-inflammatory genes TRPA1 and TRPV1 induced by LPS and reduced pro-inflammatory markers like TNF-α, NF-κB, IL-1β, and IL-8. Moreover, LE reversed the LPS-induced upregulation of AQP8 and TLR-4 expression. These findings emphasize the potential of natural compounds like LE to regulate the intestinal barrier and reduce inflammation’s harmful effects on intestinal cells. More research is required to understand their mechanisms and explore therapeutic applications, especially for gastrointestinal inflammatory conditions.

The most common form of chronic liver disease, recently defined as MASLD, is strongly linked to obesity and metabolic syndrome. Lifestyle changes are part of MASLD prevention. The very low-calorie ketogenic diet (VLCKD) is a useful option for treating MASLD and reducing liver steatosis in patients with obesity. We assessed whether a greater degree of steatosis could have a positive or negative impact on how well 8 weeks of using the VLCKD improve steatosis and fibrosis in a patient population of overweight and obese individuals. Anthropometric parameters, along with changes in hormone and metabolic biomarkers, were also assessed both before and after the dietary change. The study population included 111 overweight (14.41%) or obese subjects (85.59%) aged between 18 and 64 years; the 75 women and 36 men involved were not taking any medicine. In both the raw (0.37 95% CI 0.21; 0.52) and the multivariate models (model a: 0.439 95% CI 0.26; 0.62; model b: 0.437 95% CI 0.25; 0.63), there was a positive and statistically significant correlation between the CAP delta value and the CAP before using the VLCKD. Additionally, the liver stiffness delta was found to be positively and statistically significantly correlated with liver stiffness before the use of the VLCKD in both models: the multivariate model (model a: 0.560 95% CI 0.40; 0.71; model b: 0.498 95% CI 0.34; 0.65) and the raw model (0.52 95% CI 0.39; 0.65). Systolic and diastolic blood pressure, insulin resistance (measured by HOMA-IR), insulin, HbA1c, fasting blood glucose, total cholesterol, LDL cholesterol, HDL cholesterol and triglycerides, BMI, waist circumference, and fat mass, were all decreased (p < 0.001) following the use of the VLCKD. However, following the use of the VLCKD, there was an increase in vitamin D levels. (p < 0.001). We found that using the VLCKD for 8 weeks has a greater effect on improving steatosis and fibrosis in subjects who initially have more severe forms of these conditions.

Background: Liver diseases are constantly increasing throughout the world and are often associated with other diseases, but above all they are caused by improper diet. Adherence to a diet with abundant vegetables has now been widely demonstrated to be important in combating this pathological condition. The aim of this study was to explore the protective role of lycopene (LYC) extracts from cooked and fresh tomato. Methods: The study cohort included 969 participants assessed in the NUTRIHEP cohort (2005–2006) and the associated follow-up (2014–2016), divided into two groups, based on liver condition: NAFLD, or AFLD and FLD. Results: The results indicated a statistical significance of LYC consumption, showing a protective role against liver disease, the best concentration being 9.50 mg/die, with an RR value of 0.59, p = 0.01, 0.39 to 0.90 at 95% C.I., and RRR = 0.40, p = 0.002, 0.22 to 0.71 at 95% C.I. Conclusions: The protective role of LYC extracts from tomato has not been amply demonstrated in humans. We conclude that this is one of the few papers in the literature to evaluate the protective effect of LYC against liver disease, as well as how this molecule could be used in future possible treatments. Utilizing lycopene as a supplement alone or in combination with other foods could be useful for developing treatments with reduced contraindications.

Background & Aims: Fatty liver disease with metabolic dysfunction (MAFLD) is a new concept proposed to replace the previous concept of Non-Alcoholic Hepatic Steatosis (NAFLD). We developed and internally validated a prognostic model to predict the likelihood of death in a cohort of subjects with MAFLD. Methods: Our work involved two steps: the first was the construction of a bootstrapped multivariable Cox model for mortality risk prognosis and the second was its validation. Results: The study cohort included 1506 subjects, of which 907 were used for internal validation. Discriminant measures for the final model were R2D 0.6845 and Harrell’s C 0.8422 in the development and R2D 0.6930 and Harrell’s C 0.8465 in the validation. We used the nine independent prognostic factors selected by the LASSO Cox procedure and fitted by the bootstrap Cox survival model, and observed β were: Gender 0.356 1.42 (p < 0.008), Age 0.146 (p < 0.001), Glycemia 0.004 (p < 0.002), Total Cholesterol −0.0040 (p < 0.009), Gamma Glutamyl Transpeptidase 0.009 (p < 0.001), SBP 0.009 (p < 0.036), DBP −0.016 (p < 0.041), ALP 0.008 (p < 0.071) and Widowhood 0.550 (p < 0.001). Conclusions: We produced and validated a model to estimate the probability of death in subjects with MAFLD. The instruments we used showed satisfactory predictive capabilities.

The NADPH oxidase NOX4 has been proposed as necessary for the apoptosis induced by the Transforming Growth Factor-beta (TGF-β) in hepatocytes and hepatocellular carcinoma (HCC) cells. However, whether NOX4 is required for TGF-β-induced canonical (SMADs) or non-canonical signals is not fully understood yet, neither its potential involvement in other parallel actions induced by TGF-β. In this work we have used CRISPR Cas9 technology to stable attenuate NOX4 expression in HCC cells. Results have indicated that NOX4 is required for an efficient SMAD2/3 phosphorylation in response to TGF-β, whereas non-canonical signals, such as the phosphorylation of the Epidermal Growth Receptor or AKT, are higher in NOX4 silenced cells. TGF-β-mediated inhibition of cell proliferation and viability is attenuated in NOX4 silenced cells, correlating with decreased response in terms of apoptosis, and maintenance of high expression of MYC and CYCLIN D1. These results would indicate that NOX4 is required for all the tumor suppressor actions of TGF-β in HCC. However, analysis in human HCC tumors has revealed a worse prognosis for patients showing high expression of TGF-β1-related genes concomitant with high expression of NOX4. Deepening into other tumorigenic actions of TGF-β that may contribute to tumor progression, we found that NOX4 is also required for TGF-β-induced migratory effects. The Epithelial-Mesenchymal transition (EMT) program does not appear to be affected by attenuation of NOX4 levels. However, TGF-β-mediated regulation of cytoskeleton dynamics and focal adhesions require NOX4, which is necessary for TGF-β-induced increase in the chaperone Hsp27 and correct subcellular localization of Hic-5 within focal adhesions, as well for upregulation of the metalloprotease MMP9. All these results together point to NOX4 as a key element in the whole TGF-β signaling in HCC cells, revealing an unknown role for NOX4 as tumor promoter in HCC patients presenting activation of the TGF-β pathway.

Abstract Background Intrahepatic cholangiocarcinoma (iCCA) is a highly malignant tumor characterized by an intensive desmoplastic reaction due to the exaggerated presence of the extracellular (ECM) matrix components. Liver fibroblasts close to the tumor, activated by transforming growth factor (TGF)-β1 and expressing high levels of α-smooth muscle actin (α-SMA), become cancer-associated fibroblasts (CAFs). CAFs are deputed to produce and secrete ECM components and crosstalk with cancer cells favoring tumor progression and resistance to therapy. Overexpression of Notch signaling is implicated in CCA development and growth. The study aimed to determine the effectiveness of the Notch inhibitor, Crenigacestat, on the surrounding microenvironment of iCCA. Methods We investigated Crenigacestat’s effectiveness in a PDX model of iCCA and human primary culture of CAFs isolated from patients with iCCA. Results In silico analysis of transcriptomic profiling from PDX iCCA tissues treated with Crenigacestat highlighted “liver fibrosis” as one of the most modulated pathways. In the iCCA PDX model, Crenigacestat treatment significantly (p

Metabolic dysfunction-associated steatotic liver disease (MASLD) is closely related to some metabolic disorders, such as central obesity and type 2 diabetes (T2D). Glucagon-like peptide 1 receptor agonists (GLP-1RAs), such as semaglutide, may have therapeutic roles in MASLD associated with T2D. This study aims to investigate the molecular mechanisms underlying the effectiveness of semaglutide on MASLD in terms of progression from liver steatosis to fibrosis. We characterized exosomes from ten patients with type 2 diabetes (T2D) before (T0) and after 12 months (T12) of treatment with once-weekly subcutaneous semaglutide. Six of ten patients were considered responders to therapy (R) based on MASLD severity downgrading by at least one class according to a validated ultrasonographic (US) score. Normal hepatocytes (HEPA-RG) and stellate (LX-2) cells were challenged with exosomes from R and NR patients, isolated before and after 12 months of therapy. Exosomes from both R and NR patients isolated at T0 significantly affected LX-2 viability. After 12 months of treatment, only those isolated from R patients restored cell viability, whereas those from NR patients did not. No effects were observed on HEPA-RG cells. Exosomes at T12 from R but not from NR patients significantly decreased the production of α-SMA, a marker of LX-2 activation, a liver stellate cell model, and ph-SMAD2 and CTGF, involved in fibrosis processes. TGF-β1 was not modulated by the exosomes of R and NR patients. As a downstream effect, Vimentin, Collagen 1A1, and Fibronectin extracellular matrix components were also downregulated, as measured by droplets digital PCR. In conclusion, these results shed light on the potential effectiveness of semaglutide in improving liver fibrosis in MASLD.

Background: Steatotic liver disease (SLD) is defined as a fat accumulation in more than 5% of hepatocytes; it can progress to non-alcoholic steatohepatitis (NASH), associated with an increased state of inflammation. The aim of this study was to explore the protective effects of eating eggs and any association with SLD and hypertension (HTN). Methods: The study cohort included 908 participants assessed in the fourth recall of the MICOL study, grouped into four groups, based on NALFD and/or HTN. Results: The prevalence of HTN and SLD among participants was 31.61%. Overall, the results indicated a statistical significance of egg consumption, showing a protective role against the two disease conditions, in both the raw and adjusted models (RRR = 0.34, p = 0.009, 0.15 to 0.76 95% C.I.). Conclusions: Many differences were found among the groups, and the protective role of eating eggs was amply demonstrated. We can conclude that it is unwise to demonize the intake of this food and its nutritional properties, in contrast with previous reports in the literature.

A healthy intestinal permeability facilitates the selective transport of nutrients, metabolites, water, and bacterial products, involving cellular, neural, hormonal, and immune factors. An altered intestinal permeability indicates pathologic phenotypes and is associated with the exacerbation of obesity and related comorbidities. To investigate the impact of altered permeability in obese patients undergoing a calorie-restrictive dietary regimen (VLCKD), we collected urinary and fecal samples from obese patients with both normal and altered permeability (determined based on the lactulose/mannitol ratio) before and after treatment. The analysis of volatile organic compounds (VOCs) aids in understanding the metabolites produced by the intestinal microbiota in this unique ecological niche. Furthermore, we examined clinical and anthropometric variables from the cohort and compared them to significant VOC panels. Consequently, we identified specific markers in the metabolomics data that differentiated between normal and altered profiles before and after the diet. These markers indicated how the variable contribution specifically accounted for interleukins and lipopolysaccharides (LPS). The targeted metabolomics experiment detected no differences in measured short-chain fatty acids (SCFA). In summary, our study evaluated metabolomic markers capable of distinguishing low-grade inflammation conditions, exacerbated in more advanced stages of obesity with altered intestinal permeability.

Desmosomes are essential structures for ensuring tissue functions, and their deregulation is involved in the development of colorectal cancer (CRC). JUP (γ-catenin) is a desmosome adhesion component that also acts as a signaling hub, suggesting its potential involvement in CRC progression. In this context, we recently demonstrated that miR-195-5p regulated JUP and desmosome cadherins expression. In addition, miR-195-5p gain of function indirectly modulated the expression of key effectors of the Wnt pathway involved in JUP-dependent signaling. Here, our purpose was to demonstrate the aberrant expression of miR-195-5p and JUP in CRC patients and to functionally characterize the role of miR-195-5p in the regulation of desmosome function. First, we showed that miR-195-5p was downregulated in CRC tumors compared to adjacent normal tissue. Then, we demonstrated that JUP expression was significantly increased in CRC tissues compared to adjacent normal tissues. The effects of miR-195-5p on CRC progression were assessed using in vitro transient transfection experiments and in vivo miRNA administration. Increased miR-195-5p in colonic epithelial cells strongly inhibits cell proliferation, viability, and invasion via JUP. In vivo gain of function of miR-195-5p reduced the numbers and sizes of tumors and significantly ameliorated the histopathological changes typical of CRC. In conclusion, our findings indicate a potential pharmacological target based on miR-195-5p replacement as a new therapeutic approach in CRC.

Background: Alcohol is a psychoactive substance with deleterious effects on human health and mortality. This study aims to investigate the joint associations between the Mediterranean Diet (MedDiet), alcohol- consumption patterns and mortality from the following: all causes, cardiovascular, neoplastic, the digestive system, and other causes. Methods: A sample of 3411 alcohol consumers aged ≥18 years was selected from two prospective cohort studies: the MICOL and NUTRIHEP Study. Cohorts were enrolled in 2005–2006, and followed up until December 2022, capturing data on alcohol consumption, diet, and mortality. Adherence to the MedDiet was measured by the relative Mediterranean score (rMED), and alcohol consumption by the Mediterranean Alcohol-drinking Pattern index (MADP). Statistical analyses included flexible parametric survival models and subdistribution hazard ratios, to consider different causes of death. Results: a significant increase in digestive-system (SHR 2.77, 95% CI 1.16; 63) and cancer mortality risk (SHR 2.25, 95% CI 1.08; 4.70) was observed among individuals with low adherence to the MADP. Low adherence to the Mediterranean pattern of alcohol consumption, combined with low adherence to the MedDiet, was associated with higher overall mortality (HR 2.29, 95% CI 1.04, 5.04), and, in particular, with higher mortality from digestive system diseases (SHR 4.38, 95% CI 1.22, 15.8). Conclusions: This study suggests that deleterious effects of alcohol on mortality vary, depending on alcohol consumption patterns and dietary context. Higher adherence to the MedDiet appears to mitigate the adverse effects of moderate alcohol consumption, particularly for wine drinkers.

Aggressive hepatocellular carcinoma (HCC) overexpressing Angiopoietin-2 (ANG-2) (a protein linked with angiogenesis, proliferation, and epithelial–mesenchymal transition (EMT)), shares 95% of up-regulated genes and a similar poor prognosis with the proliferative subgroup of intrahepatic cholangiocarcinoma (iCCA). We analyzed the pro-invasive effect of ANG-2 and its regulator vascular endothelial growth factor (VEGF) on HCC and CCA spheroids to uncover posUsible common ways of response. Four cell lines were used: Hep3B and HepG2 (HCC), HuCC-T1 (iCCA), and EGI-1 (extrahepatic CCA). We treated the spheroids with recombinant human (rh) ANG-2 and/or VEGF and then observed the changes at the baseline, after 24 h, and again after 48 h. Proangiogenic stimuli increased migration and invasion capability in HCC- and iCCA-derived spheroids and were associated with a modification in EMT phenotypic markers (a decrease in E-cadherin and an increase in N-cadherin and Vimentin), especially at the migration front. Inhibitors targeting ANG-2 (Trebananib) and the VEGF (Bevacizumab) effectively blocked the migration ability of spheroids that had been stimulated with rh-ANG-2 and rh-VEGF. Overall, our findings highlight the critical role played by ANG-2 and the VEGF in enhancing the ability of HCC- and iCCA-derived spheroids to migrate and invade, which are key processes in cancer progression.

Desmosomes play a key role in the regulation of cell adhesion and signaling. Dysregulation of the desmosome complex is associated with the loss of epithelial cell polarity and disorganized tissue architecture typical of colorectal cancer (CRC). The aim of this study was to investigate and characterize the effect of miR-195-5p on desmosomal junction regulation in CRC. In detail, we proposed to investigate the deregulation of miR-195-5p and JUP, a gene target that encodes a desmosome component in CRC patients. JUP closely interacts with desmosomal cadherins, and downstream, it regulates several intracellular transduction factors. We restored the miR-195-5p levels by transient transfection in colonic epithelial cells to examine the effects of miR-195-5p on JUP mRNA and protein expression. The JUP regulation by miR-195-5p, in turn, determined a modulation of desmosome cadherins (Desmoglein 2 and Desmocollin 2). Furthermore, we focused on whether the miR-195-5p gain of function was also able to modulate the expression of key components of Wnt signaling, such as NLK, LEF1 and Cyclin D1. In conclusion, we have identified a novel mechanism controlled by miR-195-5p in the regulation of adhesive junctions, suggesting its potential clinical relevance for future miRNA-based therapy in CRC.

Extra virgin olive oil (EVOO) has healthy benefits for noncommunicable diseases (NCDs). However, limited evidence is available about the effects of liver disease and non-alcoholic fatty liver disease (NAFLD). We evaluate whether dose-increased consumption of EVOO is associated with a lower prevalence of NAFLD and if these effects vary based on body weight. The study included 2436 subjects with a 33% NAFLD prevalence. Daily EVOO was categorized into tertiles: low (0–24 g/day), moderate (25–37 g/day), and high consumption (>37 g/day). Subjects were also classified by body mass index (BMI) as normo-weight (18.5–24.9), overweight (25–29.9), and obese (≥30). Logistic regression analysis was applied to calculate odds ratios (ORs) for NAFLD, considering a 20-gram increment in EVOO intake and accounting for EVOO categories combined with BMI classes. The ORs were 0.83 (0.74;0.93) C.I. p = 0.0018 for continuous EVOO, 0.89 (0.69;1.15) C.I. p = 0.37, and 0.73 (0.55;0.97) C.I. p = 0.03 for moderate and high consumption, respectively, when compared to low consumption. Overall, the percent relative risk reductions (RRR) for NAFLD from low to high EVOO consumption were 18% (16.4%;19.2%) C.I. and 26% (25%;27.4%) C.I. in overweight and obese subjects. High EVOO consumption is associated with a reduced risk of NAFLD. This effect is amplified in overweight subjects and even more in obese subjects.

Overweight and obesity have been suggested as significant factors in irritable bowel syndrome (IBS) development. However, the relationship between overweight/obesity and IBS is unclear. It is known that a modified intestinal barrier, especially the permeability of the small intestine (s-IP), can play a significant role in the pathogenesis of both obesity and IBS. Moreover, dietary interventions are essential for treating both pathologies. We evaluated the gastrointestinal (GI) symptoms and the urinary and circulating markers of GI barrier function and integrity, the markers of intestinal dysbiosis and bacterial translocation, in 40 IBS patients with predominant diarrhea (IBS-D) (32 females and 8 males; mean age = 43.5 ± 1.4 years), categorized using their Body Mass Index levels as normal (NW) and overweight (OW). Evaluations were performed before and after 12 weeks of a Low FODMAP Diet (LFD). At the baseline, OW patients showed a significantly higher s-IP than NW. After an LFD, a significant improvement of s-IP in OW patients occurred, along with a significant decrease in markers of epithelial integrity and bacterial translocation. Our findings highlight the close relationship between overweight and the intestinal barrier and support their involvement in IBS-D pathophysiology. Furthermore, the positive role of an LFD in managing overweight IBS-D was highlighted.

Colorectal cancer (CRC) is the third leading cause of cancer-related death worldwide. Metastasis is the prime driver of CRC-related mortality, and the liver is the organ most frequently involved. Despite the overall success of current treatments, colorectal liver metastasis (CRLM) is associated with poor prognoses and a survival rate of only 14%. Recent studies have highlighted the importance of the tumor microenvironment (TME) and the crosstalk within it in determining the invasion of distant organs by circulating cancer cells. In the TME, cellular communication is mediated via soluble molecules, among which cytokines have recently emerged as key regulators, involved in every aspect of tumor progression and the metastatic cascade. Indeed, in the serum of CRC patients elevated levels of several cytokines are associated with cancer development and progression. The current review evaluates the role of different cytokines during CRLM development. Additionally, considering the increasing amount of data concerning the importance of cytokine complex networks, we outline the potential of combination treatments using targeted cytokines together with other well-established therapies, such as immune checkpoint blockades, chemotherapy, or gene therapy, to improve therapeutic outcomes.

Background: Hepatocellular carcinoma (HCC) accounts for more than 75% of primary liver cancers, which are the second leading cause of cancer-related deaths. The GALAD (gender, age, AFP-L3, AFP, and des-carboxy-prothrombin) score is a diagnostic tool developed based on gender, age, alpha-fetoprotein, alpha-fetoprotein L3, and des-gamma-carboxy prothrombin, originally designed as a diagnostic tool for HCC in high-risk patients. Methods: We analyzed 212 patients with and without cirrhosis. The population study was divided into patients with liver cirrhosis without evidence of HCC at the time of serum sample collection for GALAD score determination and patients with liver cirrhosis and a confirmed diagnosis of HCC at the time of serum sample collection for GALAD score determination. Patients were followed up until death or liver transplantation. The association between variables and HCC mortality risk was performed, and the results were presented as hazard ratio (HR). The receiver operating characteristic (ROC) curve was used to assess the performance of the GALAD HCC diagnosis. The survival probability was explored using the non-parametric test, and the equality of survival amongst categories was assessed with the log-rank test. Results: Biomarkers were higher in the HCC group compared to cirrhosis. Kaplan–Meier survival probability analysis for individual GALAD categories revealed that a high GALAD level was associated with decreased survival during follow-up, and the difference between the curves was statistically significant (p = 0.01). Conclusions: Our findings suggest that the GALAD score has promise as a prognostic tool, with implications for improving patient management and treatment strategies for HCC.

Abstract Background Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive primary liver tumor with increasing incidence worldwide, dismal prognosis, and few therapeutic options. Mounting evidence underlines the role of the Hippo pathway in this disease; however, the molecular mechanisms whereby the Hippo cascade contributes to cholangiocarcinogenesis remain poorly defined. Methods We established novel iCCA mouse models via hydrodynamic transfection of an activated form of transcriptional coactivator with PDZ-binding motif (TAZ), a Hippo pathway downstream effector, either alone or combined with the myristoylated AKT (myr-AKT) protooncogene, in the mouse liver. Hematoxylin and eosin staining, immunohistochemistry, electron microscopy, and quantitative real-time RT-PCR were applied to characterize the models. In addition, in vitro cell line studies were conducted to address the growth-promoting roles of TAZ and its paralog YAP. Results Overexpression of TAZ in the mouse liver triggered iCCA development with very low incidence and long latency. In contrast, co-expression of TAZ and myr-AKT dramatically increased tumor frequency and accelerated cancer formation in mice, with 100% iCCA incidence and high tumor burden by 10 weeks post hydrodynamic injection. AKT/TAZ tumors faithfully recapitulated many of the histomolecular features of human iCCA. At the molecular level, the development of the cholangiocellular lesions depended on the binding of TAZ to TEAD transcription factors. In addition, inhibition of the Notch pathway did not hamper carcinogenesis but suppressed the cholangiocellular phenotype of AKT/TAZ tumors. Also, knockdown of YAP, the TAZ paralog, delayed cholangiocarcinogenesis in AKT/TAZ mice without affecting the tumor phenotype. Furthermore, human preinvasive and invasive iCCAs and mixed hepatocellular carcinoma/iCCA displayed widespread TAZ activation and downregulation of the mechanisms protecting TAZ from proteolysis. Conclusions Overall, the present data underscore the crucial role of TAZ in cholangiocarcinogenesis

IntroductionPaclitaxel (PTX) interferes with microtubule architecture by binding to β-tubulin, thereby blocking progression at the G2/M phase and inducing apoptosis. This study aimed to investigate molecular processes underlying PTX-mediated resistance in gastric cancer (GC) cells. MethodsPTX-mediated resistance involves many processes, and in this work some of the factors involved in the resistance mechanism were identified by comparing two GC lines with PTX induced resistance to their sensitive counterparts. ResultsThus, the key feature of PTX-resistant cells was the overexpression of pro-angiogenic factors such as VEGFA, VEGFC, and Ang2, known to support tumor cell growth. A second relevant change detected in PTX-resistant lines was the elevated level of TUBβIII, a tubulin isoform that opposes microtubule stabilization. A third identified factor contributing to PTX-resistance was P-glycoprotein (P-gp), a transporter responsible for chemotherapy efflux from the cells, highly expressed in PTX-resistant lines.Discussion These findings were in line with a greater sensitivity of resistant cells to treatment with both Ramucirumab and Elacridar. Ramucirumab significantly reduced the expression of angiogenic molecules and TUBβIII, while Elacridar restored the access of chemotherapy, recovering its anti-mitotic and pro-apoptotic effects. Finally, this study highlighted the role played by exosomes in spreading factors responsible for resistance in the tumor microenvironment.

Abstract Background Intrahepatic Cholangiocarcinoma (iCCA) is characterized by a strong stromal reaction playing a role in tumor progression. Thymus cell antigen 1 (THY1), also called Cluster of Differentiation 90 (CD90), is a key regulator of cell–cell and cell–matrix interaction. In iCCA, CD90 has been reported to be associated with a poor prognosis. In an iCCA PDX model, we recently found that CD90 was downregulated in mice treated with the Notch γ-secretase inhibitor Crenigacestat. The study aims to investigate the role of CD90 in relation to the NOTCH pathway. Methods THY1/CD90 gene and protein expression was evaluated in human iCCA tissues and xenograft models by qRT-PCR, immunohistochemistry, and immunofluorescence. Notch1 inhibition was achieved by siRNA. THY1/CD90 functions were investigated in xenograft models built with HuCCT1 and KKU-M213 cell lines, engineered to overexpress or knockdown THY1, respectively. Results CD90 co-localized with EPCAM, showing its epithelial origin. In vitro, NOTCH1 silencing triggered HES1 and THY1 down-regulation. RBPJ, a critical transcriptional regulator of NOTCH signaling, exhibited putative binding sites on the THY1 promoter and bound to the latter, implying CD90 as a downstream NOTCH pathway effector. In vivo, Crenigacestat suppressed iCCA growth and reduced CD90 expression in the PDX model. In the xenograft model, Crenigacestat inhibited tumor growth of HuCCT1 cells transfected to overexpress CD90 and KKU-M213 cells constitutively expressing high levels of CD90, while not affecting the growth of HuCCT1 control cells and KKU-M213 depleted of CD90. In an iCCA cohort, patients with higher expression levels of NOTCH1/HES1/THY1 displayed a significantly shorter survival. Conclusions iCCA patients with higher NOTCH1/HES1/THY1 expression have the worst prognosis, but they are more likely to benefit from Notch signaling inhibition. These findings represent the scientific rationale for testing NOTCH1 inhibitors in clinical trials, taking the first step toward precision medicine for iCCA.

Background: Extra-virgin olive oil (EVOO) is the main source of seasoning fat in the Mediterranean diet and it is one of the components with known protective factors on chronic-degenerative disease. We aimed to evaluate the effect of a medium-high level of oil consumption on mortality in a cohort with good adherence to the Mediterranean diet. Methods: A total of 2754 subjects who had completed the food questionnaire in the Multicenter Italian study on Cholelithiasis (MICOL) cohort were included in the study. EVOO consumption was categorized in four levels (40 g/die). We performed a flexible parametric survival model to assess mortality by EVOO consumption level adjusted for some covariates. We also performed the analysis on subjects with and without non-alcoholic fatty liver disease (NAFLD) to evaluate the effects of oil in this more fragile sub-cohort. Results: We found a statistically significant negative effect on mortality for the whole sample when EVOO consumption was used, both as a continuous variable and when categorized. The protective effect was stronger in the sub-cohort with NAFLD, especially for the highest levels of EVOO consumption (HR = 0.58 with p < 0.05). Conclusions: Our study has shown a protective effect of EVOO consumption towards all causes of mortality. Despite the higher caloric intake, the protective power is greater for a consumption >40 g/day in both the overall cohort and the sub-cohorts with and without NAFLD.

Obesity and metabolic dysfunction-associated steatotic liver disease (MASLD) are frequently associated conditions characterized by low-grade inflammation. Very low-calorie ketogenic diet (VLCKD) strategies are commonly used to simultaneously obtain weight loss and an improvement of liver steatosis. We evaluated the efficacy of 8 weeks’ VLCKD in decreasing the white blood cell (WBC) and platelet (PLT) counts, as well as liver steatosis and fibrosis, diagnosed using transient elastography (FibroScan). Metabolic and anthropometric parameters commonly associated with MASLD were also evaluated. This study included 87 participants; 58 women and 29 men aged between 18 and 64 years with overweight (18%) or obesity (82%), but not taking any medication. Anthropometric measurements, bioimpedance analysis, and biochemical assays were performed before and after the dietary intervention. BMI (kg/m2) (p-value < 0.001), waist circumference (cm) (p-value < 0.001), and fat mass (kg) (p-value < 0.001) were significantly decreased following VLCKD. After VLCKD, the FibroScan parameter CAP (db/m), which measures the accumulation of fatty liver, significantly decreased (p-value < 0.001), as did liver stiffness (kPA), the FibroScan parameter quantifying liver fibrosis (p-value < 0.05). Seemingly, WBC (p-value < 0.001) and PLT (p-value < 0.001) counts were lowered by VLCKD in the whole group; however, the decrease in WBC and platelet counts were significant only in patients with steatosis (CAP ≥ 215 dB/m). Fasting blood glucose (p-value < 0.001), insulin (p-value < 0.001), HbA1c (p-value < 0.001), triglycerides (p-value < 0.001), total cholesterol (p-value < 0.001), LDL-cholesterol (p-value < 0.001), HDL-cholesterol (p-value < 0.001); γGT (p-value < 0.001) blood levels and insulin resistance (as measured by HOMAIR) (p-value < 0.001); and systolic (p-value < 0.001), and diastolic (p-value < 0.001) blood pressure levels, were all significantly lower after VLCKD. In contrast, blood levels of vitamin D were higher following the diet (p-value < 0.001). We conclude that treating subjects with overweight and obesity with VLCKD is followed by a simultaneous reduction in WBCs and platelets, the expression of low-grade inflammation, and of liver steatosis and fibrosis. Therefore, we can hypothesize that VLCKD decreases general and liver low-grade inflammation, thus improving liver health.