An unbiased ranking of murine dietary models based on their proximity to human metabolic dysfunction-associated steatotic liver disease (MASLD).

Metabolic dysfunction-associated steatotic liver disease (MASLD), previously known as non-alcoholic fatty liver disease, encompasses steatosis and metabolic dysfunction-associated steatohepatitis (MASH), leading to cirrhosis and hepatocellular carcinoma. Preclinical MASLD research is mainly performed in rodents; however, the model that best recapitulates human disease is yet to be defined. We conducted a wide-ranging retrospective review (metabolic phenotype, liver histopathology, transcriptome benchmarked against humans) of murine models (mostly male) and ranked them using an unbiased MASLD 'human proximity score' to define their metabolic relevance and ability to induce MASH-fibrosis. Here, we show that Western diets align closely with human MASH; high cholesterol content, extended study duration and/or genetic manipulation of disease-promoting pathways are required to intensify liver damage and accelerate significant (F2+) fibrosis development. Choline-deficient models rapidly induce MASH-fibrosis while showing relatively poor translatability. Our ranking of commonly used MASLD models, based on their proximity to human MASLD, helps with the selection of appropriate in vivo models to accelerate preclinical research.

Decoding the role of leptin and adiponectin in obesity-related gastrointestinal cancer.

The increasing prevalence of obesity brings forward its importance as a risk factor for cancer development, particularly in the gastrointestinal tract. Obesity may trigger cancer development through several mechanisms, where metabolic deregulation of adipokines can modulate multiple oncogenic molecular pathways. Leptin and adiponectin are the most well-studied adipokines, and their imbalance can trigger different tumorigenic responses. Both epidemiologic and experimental studies have associated leptin with increased cancer risk and cell responsiveness in carcinogenesis and tumor invasion. On the other hand, adiponectin is reported to elicit the opposite effect. In addition to circulating or tissue adipokine levels, adiponectin, and leptin receptors or genetic polymorphisms may also play a role in cancer development. Moreover, adiponectin and leptin modulation offer valuable therapeutic approaches. We will review the links underpinning obesity and cancer development and focus on discussing the pathophysiological roles of leptin and adiponectin.

miR-21-5p promotes NASH-related hepatocarcinogenesis.

BACKGROUND AND AIMS: The mechanisms governing the progression of non-alcoholic fatty liver disease (NAFLD) towards steatohepatitis (NASH) and hepatocellular carcinoma (HCC) remain elusive. Here, we evaluated the role of hsa-miRNA-21-5p in NASH-related hepatocarcinogenesis. METHODS: Hepatic hsa-miR-21-5p expression was evaluated in two cohorts of patients with biopsy-proven NAFLD (n = 199) or HCC (n = 366 HCC and n = 11 NAFLD-HCC). Serum/liver metabolomic profiles were correlated with hsa-miR-21-5p in NAFLD obese patients. Wild-type (WT) and Mir21 KO mice were fed a choline-deficient, amino acid-defined (CDAA) diet for 32 and 66 weeks to induce NASH and NASH-HCC, respectively. RESULTS: In obese individuals, hsa-miR-21-5p expression increased with NAFLD severity and associated with a hepatic lipotoxic profile. CDAA-fed WT mice displayed increased hepatic mmu-miR-21-5p levels and progressively developed NASH and fibrosis, with livers presenting macroscopically discernible pre-neoplastic nodules, hyperplastic foci and deregulated cancer-related pathways. Mir21 KO mice exhibited peroxisome-proliferator-activated receptor α (PPARα) activation, augmented mitochondrial activity, reduced liver injury and NAS below the threshold for NASH diagnosis, with the pro-inflammatory/fibrogenic milieu reversing to baseline levels. In parallel, Mir21 KO mice displayed reduced number of pre-neoplastic nodules, hepatocyte proliferation and activation of oncogenic signalling, being protected from NASH-associated carcinogenesis. The hsa-miRNA-21-5p/PPARα pathway was similarly deregulated in patients with HCC- or NASH-related HCC, correlating with HCC markers and worse prognosis. CONCLUSIONS: Hsa-miR-21-5p is a key inducer of whole-spectrum NAFLD progression, from simple steatosis to NASH and NASH-associated carcinogenesis. The inhibition of hsa-miR-21-5p, leading to a pro-metabolic profile, might constitute an appealing therapeutic approach to ameliorate NASH and prevent progression towards HCC.

Polyoxazoline-Based Nanovaccine Synergizes with Tumor-Associated Macrophage Targeting and Anti-PD-1 Immunotherapy against Solid Tumors.

Immune checkpoint blockade reaches remarkable clinical responses. However, even in the most favorable cases, half of these patients do not benefit from these therapies in the long term. It is hypothesized that the activation of host immunity by co-delivering peptide antigens, adjuvants, and regulators of the transforming growth factor (TGF)-ß expression using a polyoxazoline (POx)-poly(lactic-co-glycolic) acid (PLGA) nanovaccine, while modulating the tumor-associated macrophages (TAM) function within the tumor microenvironment (TME) and blocking the anti-programmed cell death protein 1 (PD-1) can constitute an alternative approach for cancer immunotherapy. POx-Mannose (Man) nanovaccines generate antigen-specific T-cell responses that control tumor growth to a higher extent than poly(ethylene glycol) (PEG)-Man nanovaccines. This anti-tumor effect induced by the POx-Man nanovaccines is mediated by a CD8+ -T cell-dependent mechanism, in contrast to the PEG-Man nanovaccines. POx-Man nanovaccine combines with pexidartinib, a modulator of the TAM function, restricts the MC38 tumor growth, and synergizes with PD-1 blockade, controlling MC38 and CT26 tumor growth and survival. This data is further validated in the highly aggressive and poorly immunogenic B16F10 melanoma mouse model. Therefore, the synergistic anti-tumor effect induced by the combination of nanovaccines with the inhibition of both TAM- and PD-1-inducing immunosuppression, holds great potential for improving immunotherapy outcomes in solid cancer patients.

Modulation of rectal cancer stemness, patient outcome and therapy response by adipokines

Response to chemoradiotherapy (CRT) in patients with locally advanced rectal cancer is highly variable. Identification of CRT non-responders and definite accurate biomarkers of response are unmet needs. In turn, adipokines might impact on colorectal cancer development. We hypothesized that imbalance in leptin and adiponectin modulates stemness potential CRT response in rectal cancer. Pre-CRT serum and tissue samples were collected from a cohort of locally advanced rectal cancer patients (n = 33), submitted to long-course CRT and proctectomy. Adiponectin and leptin were measured by ELISA in serum. In tumour biopsies, mRNA expression of stemness-related genes was evaluated by qRT-PCR and transcription factor STAT3 by immunoblotting. Correlations with clinical data and accuracy of potential CRT response biomarkers were evaluated. Carcinoembryonic antigen (CEA) but not leptin or adiponectin distinguished CRT responders from non-responders (p < 0.05). However, higher leptin and lower adiponectin serum levels were associated with positive extramesorectal nodes and extramural vascular invasion. mRNA expression of stemness factors was inversely correlated with adiponectin but positively correlated with leptin. STAT3 phosphorylation presented similar results. CEA levels together with STAT3 activation and OCT4/KLF4 expression accurately identified rectal cancer patients, CRT non-responders (AUROC 0.80; p < 0.05). Adipokines might impact rectal cancer stemness and patient prognosis. The leptin/STAT3 signalling axis provides the rational for a potential biomarker panel that identifies rectal cancer patients who will not benefit from CRT treatment. (AU)

Monocarbonyl Analogs of Curcumin with Potential to Treat Colorectal Cancer.

Curcumin has a plethora of biological properties, making this compound potentially effective in the treatment of several diseases, including cancer. However, curcumin clinical use is compromised by its poor pharmacokinetics, being crucial to find novel analogs with better pharmacokinetic and pharmacological properties. Here, we aimed to evaluate the stability, bioavailability and pharmacokinetic profiles of monocarbonyl analogs of curcumin. A small library of monocarbonyl analogs of curcumin 1a-q was synthesized. Lipophilicity and stability in physiological conditions were both assessed by HPLC-UV, while two different methods assessed the electrophilic character of each compound monitored by NMR and by UV-spectroscopy. The potential therapeutic effect of the analogs 1a-q was evaluated in human colon carcinoma cells and toxicity in immortalized hepatocytes. Our results showed that the curcumin analog 1e is a promising agent against colorectal cancer, with improved stability and efficacy/safety profile.

Inhibition of lysophosphatidic acid receptor 6 upregulated by the choline-deficient l-amino acid-defined diet prevents hepatocarcinogenesis in mice.

Hepatocellular carcinoma (HCC) is one of the most worrying tumors worldwide today, and its epidemiology is on the rise. Traditional pharmacological approaches have shown unfavorable results and exhibited many side effects. Hence, there is a need for new efficacious molecules with fewer side effects and improvements on traditional approaches. We previously showed that lysophosphatidic acid (LPA) supports hepatocarcinogenesis, and its effects are mainly mediated by LPA receptor 6 (LPAR6). We also reported that 9-xanthylacetic acid (XAA) acts as an antagonist of LPAR6 to inhibit the growth of HCC. Here, we report that LPAR6 is involved in the choline-deficient l-amino acid-defined (CDAA) diet-induced hepatocarcinogenesis in mice. Our data demonstrate that CDAA diet-induced metabolic imbalance stimulates LPAR6 expression in mice and that XAA counteracts diet-induced effects on hepatic lipid accumulation, fibrosis, inflammation, and HCC development. These conclusions are corroborated by results on LPAR6 gain and loss-of-function in HCC cells.

Human liver organoids: From generation to applications.

In the last decade, research into human hepatology has been revolutionized by the development of mini human livers in a dish. These liver organoids are formed by self-organizing stem cells and resemble their native counterparts in cellular content, multicellular architecture, and functional features. Liver organoids can be derived from the liver tissue or pluripotent stem cells generated from a skin biopsy, blood cells, or renal epithelial cells present in urine. With the development of liver organoids, a large part of previous hurdles in modeling the human liver is likely to be solved, enabling possibilities to better model liver disease, improve (personalized) drug testing, and advance bioengineering options. In this review, we address strategies to generate and use organoids in human liver disease modeling, followed by a discussion of their potential application in drug development and therapeutics, as well as their strengths and limitations.

Modulation of rectal cancer stemness, patient outcome and therapy response by adipokines.

Response to chemoradiotherapy (CRT) in patients with locally advanced rectal cancer is highly variable. Identification of CRT non-responders and definite accurate biomarkers of response are unmet needs. In turn, adipokines might impact on colorectal cancer development. We hypothesized that imbalance in leptin and adiponectin modulates stemness potential CRT response in rectal cancer. Pre-CRT serum and tissue samples were collected from a cohort of locally advanced rectal cancer patients (n = 33), submitted to long-course CRT and proctectomy. Adiponectin and leptin were measured by ELISA in serum. In tumour biopsies, mRNA expression of stemness-related genes was evaluated by qRT-PCR and transcription factor STAT3 by immunoblotting. Correlations with clinical data and accuracy of potential CRT response biomarkers were evaluated. Carcinoembryonic antigen (CEA) but not leptin or adiponectin distinguished CRT responders from non-responders (p < 0.05). However, higher leptin and lower adiponectin serum levels were associated with positive extramesorectal nodes and extramural vascular invasion. mRNA expression of stemness factors was inversely correlated with adiponectin but positively correlated with leptin. STAT3 phosphorylation presented similar results. CEA levels together with STAT3 activation and OCT4/KLF4 expression accurately identified rectal cancer patients, CRT non-responders (AUROC 0.80; p < 0.05). Adipokines might impact rectal cancer stemness and patient prognosis. The leptin/STAT3 signalling axis provides the rational for a potential biomarker panel that identifies rectal cancer patients who will not benefit from CRT treatment.

RIPK3 dampens mitochondrial bioenergetics and lipid droplet dynamics in metabolic liver disease.

BACKGROUND AND AIMS: Receptor-interacting protein kinase 3 (RIPK3) mediates NAFLD progression, but its metabolic function is unclear. Here, we aimed to investigate the role of RIPK3 in modulating mitochondria function, coupled with lipid droplet (LD) architecture in NAFLD. APPROACH AND RESULTS: Functional studies evaluating mitochondria and LD biology were performed in wild-type (WT) and Ripk3-/- mice fed a choline-deficient, amino acid-defined (CDAA) diet for 32 and 66 weeks and in CRISPR-Cas9 Ripk3 -null fat-loaded immortalized hepatocytes. The association between hepatic perilipin (PLIN) 1 and 5, RIPK3, and disease severity was also addressed in a cohort of patients with NAFLD and in PLIN1 -associated familial partial lipodystrophy. Ripk3 deficiency rescued impairment in mitochondrial biogenesis, bioenergetics, and function in CDAA diet-fed mice and fat-loaded hepatocytes. Ripk3 deficiency was accompanied by a strong upregulation of antioxidant systems, leading to diminished oxidative stress upon fat loading both in vivo and in vitro. Strikingly, Ripk3-/- hepatocytes displayed smaller size LD in higher numbers than WT cells after incubation with free fatty acids. Ripk3 deficiency upregulated adipocyte and hepatic levels of LD-associated proteins PLIN1 and PLIN5. PLIN1 upregulation controlled LD structure and diminished mitochondrial stress upon free fatty acid overload in Ripk3-/- hepatocytes and was associated with diminished human NAFLD severity. Conversely, a pathogenic PLIN1 frameshift variant was associated with NAFLD and fibrosis, as well as with increased hepatic RIPK3 levels in familial partial lipodystrophy. CONCLUSIONS: Ripk3 deficiency restores mitochondria bioenergetics and impacts LD dynamics. RIPK3 inhibition is promising in ameliorating NAFLD.

Towards personalized antibody cancer therapy: development of a microfluidic cell culture device for antibody selection.

Antibody therapy has been one of the most successful therapies for a wide range of diseases, including cancer. One way of expediting antibody therapy development is through phage display technology. Here, by screening thousands of randomly assembled peptide sequences, it is possible to identify potential therapeutic candidates. Conventional screening technologies do not accommodate perfusion through the system, as is the case of standard plate-based cultures. This leads to a poor translation of the experimental results obtained in vitro when moving to a more physiologically relevant setting, such as the case of preclinical animal models or clinical trials. Microfluidics is a technology that can improve screening efficacy by replicating more physiologically relevant conditions such as shear stress. In this work, a polydimethylsiloxane/polystyrene-based microfluidic system for a continuously perfused culture of cancer cells is reported. Human colorectal adenocarcinoma cells (HCT116) expressing CXCR4 were used as a cell target. Fluorescently labeled M13 phages anti-CXCR4 were used to study the efficiency of the microfluidic system as a tool to study the binding kinetics of the engineered bacteriophages. Using our microfluidic platform, we estimated a dissociation constant of 0.45 pM for the engineered phage. Additionally, a receptor internalization assay was developed using SDF-1α to verify phage specificity to the CXCR4 receptor. Upon receptor internalization there was a signal reduction, proving that the anti-CXCR4 fluorescently labelled M13 phages bound specifically to the CXCR4 receptor. The simplicity and ease of use of the microfluidic device design presented in this work can form the basis of a generic platform that facilitates the study and optimization of therapies based on interaction with biological entities such as mammalian cells.

The necroptosis-inducing pseudokinase mixed lineage kinase domain-like regulates the adipogenic differentiation of pre-adipocytes.

Receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like (MLKL) proteins are key regulators of necroptosis, a highly pro-inflammatory mode of cell death, which has been involved in various human diseases. Necroptotic-independent functions of RIPK3 and MLKL also exist, notably in the adipose tissue but remain poorly defined. Using knock-out (KO) cell models, we investigated the role of RIPK3 and MLKL in adipocyte differentiation. Mlkl-KO abolished white adipocyte differentiation via a strong expression of Wnt10b, a ligand of the Wnt/ß-catenin pathway, and a downregulation of genes involved in lipid metabolism. This effect was not recapitulated by the ablation of Ripk3. Conversely, Mlkl and Ripk3 deficiencies did not block beige adipocyte differentiation. These findings indicate that RIPK3 and MLKL have distinct roles in adipogenesis. The absence of MLKL blocks the differentiation of white, but not beige, adipocytes highlighting the therapeutic potential of MLKL inhibition in obesity.

Disclosing the antitumour potential of the marine bromoditerpene sphaerococcenol A on distinct cancer cellular models.

Nature has revealed to be a key source of innovative anticancer drugs. This study evaluated the antitumour potential of the marine bromoditerpene sphaerococcenol A on different cancer cellular models. Dose-response analyses (0.1-100 µM; 24 h) were accomplished in eight different tumour cell lines (A549, CACO-2, HCT-15, MCF-7, NCI-H226, PC-3, SH-SY5Y, SK-MEL-28). Deeper studies were conducted on MFC-7 cells, namely, determination of hydrogen peroxide (H2O2) levels and evaluation of apoptosis biomarkers (phosphatidylserine membrane translocation, mitochondrial dysfunction, Caspase-9 activity, and DNA changes). The ability of the compound to induce genotoxicity was verified in L929 fibroblasts. Sphaerococcenol A capacity to impact colorectal-cancer stem cells (CSCs) tumourspheres (HT29, HCT116, SW620) was evaluated by determining tumourspheres viability, number, and area, as well as the proteasome inhibitory activity. Sphaerococcenol A hepatoxicity was studied in AML12 hepatocytes. The compound exhibited cytotoxicity in all malignant cell lines (IC50 ranging from 4.5 to 16.6 µM). MCF-7 cells viability loss was accompanied by H2O2 generation, mitochondrial dysfunction, Caspase-9 activation and DNA nuclear morphology changes. Furthermore, the compound displayed the lowest IC50 on HT29-derived tumourspheres (0.70 µM), followed by HCT116 (1.77 µM) and SW620 (2.74 µM), impacting the HT29 tumoursphere formation by reducing their number and area. Finally, the compound displayed low cytotoxicity on AML12 hepatocytes without genotoxicity. Overall, sphaerococcenol A exhibits broad cytotoxic effects on different tumour cells, increasing H2O2 production and apoptosis. It also affects colorectal CSCs-enriched tumoursphere development. These data highlight the relevance to include sphaerococcenol A in further pharmacological studies aiming cancer treatments.

The role of RIPK3 in liver mitochondria bioenergetics and function.

BACKGROUND: Receptor-interacting protein kinase 3 (RIPK3) is a key player of regulated necrosis or necroptosis, an inflammatory form of cell death possibly governing outcomes in chronic liver diseases, such as nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. METHODS: This narrative review is based on literature search using PubMed. RESULTS: RIPK3 activation depends on post-transcriptional modifications, including phosphorylation, hence coordinating the assembly of macromolecular death complex named 'necrosome', which may also involve diverse mitochondrial components. Curiously, recent studies suggested a potential link between RIPK3 and mitochondrial bioenergetics. RIPK3 can modulate mitochondrial function and quality through the regulation of mitochondrial reactive oxygen species production, sequestration of metabolic enzymes and resident mitochondrial proteins, activity of mitochondrial respiratory chain complexes, mitochondrial biogenesis and fatty acid oxidation. CONCLUSIONS: Since mitochondrial dysfunction and RIPK3-mediated necroptosis are intimately involved in chronic liver disease pathogenesis, understanding the role of RIPK3 in mitochondrial bioenergetics and its potential translational application are of great interest.

Adiponectin, Leptin, and IGF-1 Are Useful Diagnostic and Stratification Biomarkers of NAFLD.

Background: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease where liver biopsy remains the gold standard for diagnosis. Here we aimed to evaluate the role of circulating adiponectin, leptin, and insulin-like growth factor 1 (IGF-1) levels as non-invasive NAFLD biomarkers and assess their correlation with the metabolome. Materials and Methods: Leptin, adiponectin, and IGF-1 serum levels were measured by ELISA in two independent cohorts of biopsy-proven obese NAFLD patients and healthy-liver controls (discovery: 38 NAFLD, 13 controls; validation: 194 NAFLD, 31 controls) and correlated with clinical data, histology, genetic parameters, and serum metabolomics. Results: In both cohorts, leptin increased in NAFLD vs. controls (discovery: AUROC 0.88; validation: AUROC 0.83; p < 0.0001). The leptin levels were similar between obese and non-obese healthy controls, suggesting that obesity is not a confounding factor. In the discovery cohort, adiponectin was lower in non-alcoholic steatohepatitis (NASH) vs. non-alcoholic fatty liver (AUROC 0.87; p < 0.0001). For the validation cohort, significance was attained for homozygous for PNPLA3 allele c.444C (AUROC 0.63; p < 0.05). Combining adiponectin with specific serum lipids improved the assay performance (AUROC 0.80; p < 0.0001). For the validation cohort, IGF-1 was lower with advanced fibrosis (AUROC 0.67, p < 0.05), but combination with international normalized ratio (INR) and ferritin increased the assay performance (AUROC 0.81; p < 0.01). Conclusion: Serum leptin discriminates NAFLD, and adiponectin combined with specific lipids stratifies NASH. IGF-1, INR, and ferritin distinguish advanced fibrosis.

RIPK3 acts as a lipid metabolism regulator contributing to inflammation and carcinogenesis in non-alcoholic fatty liver disease.

OBJECTIVE: Receptor-interacting protein kinase 3 (RIPK3) is a key player in necroptosis execution and an emerging metabolic regulator, whose contribution to non-alcoholic fatty liver disease (NAFLD) is controversial. We aimed to clarify the impact of RIPK3 signalling in the pathogenesis of human and experimental NAFLD. DESIGN: RIPK3 levels were evaluated in two large independent cohorts of patients with biopsy proven NAFLD diagnosis and correlated with clinical and biochemical parameters. Wild-type (WT) or Ripk3-deficient (Ripk3-/-) mice were fed a choline-deficient L-amino acid-defined diet (CDAA) or an isocaloric control diet for 32 and 66 weeks. RESULTS: RIPK3 increased in patients with non-alcoholic steatohepatitis (NASH) in both cohorts, correlating with hepatic inflammation and fibrosis. Accordingly, Ripk3 deficiency ameliorated CDAA-induced inflammation and fibrosis in mice at both 32 and 66 weeks. WT mice on the CDAA diet for 66 weeks developed preneoplastic nodules and displayed increased hepatocellular proliferation, which were reduced in Ripk3-/- mice. Furthermore, Ripk3 deficiency hampered tumourigenesis. Intriguingly, Ripk3-/- mice displayed increased body weight gain, while lipidomics showed that deletion of Ripk3 shifted hepatic lipid profiles. Peroxisome proliferator-activated receptor γ (PPARγ) was increased in Ripk3-/- mice and negatively correlated with hepatic RIPK3 in patients with NAFLD. Mechanistic studies established a functional link between RIPK3 and PPARγ in controlling fat deposition and fibrosis. CONCLUSION: Hepatic RIPK3 correlates with NAFLD severity in humans and mice, playing a key role in managing liver metabolism, damage, inflammation, fibrosis and carcinogenesis. Targeting RIPK3 and its intricate signalling arises as a novel promising approach to treat NASH and arrest disease progression.

Host miRNA-21 promotes liver dysfunction by targeting small intestinal Lactobacillus in mice.

New evidence shows that host-microbiota crosstalk can be modulated via endogenous miRNAs. We have previously reported that miR-21 ablation protects against liver injury in cholestasis. In this study, we investigated the role of miR-21 in modulating the gut microbiota during cholestasis and its effects in liver dysfunction. Mice lacking miR-21 had reduced liver damage and were protected against small intestinal injury as well as from gut microbiota dysbiosis when subjected to bile duct ligation surgery. The unique microbiota profile of miR-21KO mice was characterized by an increase in Lactobacillus, a key microbiome genus for gut homeostasis. Interestingly, in vitro incubation of synthetic miR-21 directly reduced Lactobacillus load. Moreover, supplementation with Lactobacillus reuteri revealed reduced liver fibrosis in acute bile duct-ligated mice, mimicking the protective effects in miR-21 knockout mice. D-lactate, a main product of Lactobacillus, regulates gut homeostasis that may link with reduced liver fibrosis. Altogether, our results demonstrate that miR-21 promotes liver dysfunction through direct modulation of the gut microbiota and highlight the potential therapeutic effects of Lactobacillus supplementation in gut and liver homeostasis.

Phenotypic high-throughput screening platform identifies novel chemotypes for necroptosis inhibition.

Regulated necrosis or necroptosis, mediated by receptor-interacting kinase 1 (RIPK1), RIPK3 and pseudokinase mixed lineage kinase domain-like protein (MLKL), contributes to the pathogenesis of inflammatory, infectious and degenerative diseases. Recently identified necroptosis inhibitors display moderate specificity, suboptimal pharmacokinetics, off-target effects and toxicity, preventing these molecules from reaching the clinic. Here, we developed a cell-based high-throughput screening (HTS) cascade for the identification of small-molecule inhibitors of necroptosis. From the initial library of over 250,000 compounds, the primary screening phase identified 356 compounds that strongly inhibited TNF-α-induced necroptosis, but not apoptosis, in human and murine cell systems, with EC50 < 6.7 µM. From these, 251 compounds were tested for RIPK1 and/or RIPK3 kinase inhibitory activity; some were active and several have novel mechanisms of action. Based on specific chemical descriptors, 110 compounds proceeded into the secondary screening cascade, which then identified seven compounds with maximum ability to reduce MLKL activation, IC50 >100 µM, EC50 2.5-11.5 µM under long-term necroptosis execution in murine fibroblast L929 cells, and full protection from ATP depletion and membrane leakage in human and murine cells. As a proof of concept, compound SN-6109, with binding mode to RIPK1 similar to that of necrostatin-1, confirmed RIPK1 inhibitory activity and appropriate pharmacokinetic properties. SN-6109 was further tested in mice, showing efficacy against TNF-α-induced systemic inflammatory response syndrome. In conclusion, a phenotypic-driven HTS cascade promptly identified robust necroptosis inhibitors with in vivo activity, currently undergoing further medicinal chemistry optimization. Notably, the novel hits highlight the opportunity to identify new molecular mechanisms of action in necroptosis.

Antitumour Potential of Gigartina pistillata Carrageenans against Colorectal Cancer Stem Cell-Enriched Tumourspheres.

Gigartina pistillata is a red seaweed common in Figueira da Foz, Portugal. Here, the antitumour potential of G. pistillata carrageenan, with a known variable of the life cycle, the female gametophyte (FG) and tetrasporophyte (T) was evaluated against colorectal cancer stem cell (CSC) -enriched tumourspheres. FTIR-ATR analysis of G. pistillata carrageenan extracts indicated differences between life cycle phases, being FG a κ/ι hybrid carrageenan and T a ʎ/ξ hybrid. Both carrageenan extracts presented IC50 values inferior to 1 µg/mL in HT29-derived CSC-enriched tumourspheres, as well as reduced tumoursphere area. The two extracts were also effective at reducing cellular viability in SW620- and SW480-derived tumourspheres. These results indicate that carrageenans extracted from two G. pistillata life cycle phases have antitumour potential against colorectal cancer stem-like cells, specially the T carrageenan.

Diet-dependent gut microbiota impacts on adult neurogenesis through mitochondrial stress modulation.

The influence of dietary factors on brain health and mental function is becoming increasingly recognized. Similarly, mounting evidence supports a role for gut microbiota in modulating central nervous system function and behaviour. Still, the molecular mechanisms responsible for the impact of diet and associated microbiome in adult neurodegeneration are still largely unclear. In this study, we aimed to investigate whether and how changes in diet-associated microbiome and its metabolites impact on adult neurogenesis. Mice were fed a high-fat, choline-deficient diet, developing obesity and several features of the metabolic syndrome, including non-alcoholic steatohepatitis. Strikingly, our results showed, for the first time, that animals fed with this specific diet display premature increased neurogenesis, possibly exhausting the available neural stem cell pool for long-term neurogenesis processes. The high-fat, choline-deficient diet further induced neuroinflammation, oxidative stress, synaptic loss and cell death in different regions of the brain. Notably, this diet-favoured gut dysbiosis in the small intestine and cecum, up-regulating metabolic pathways of short-chain fatty acids, such as propionate and butyrate and significantly increasing propionate levels in the liver. By dissecting the effect of these two specific short-chain fatty acids in vitro, we were able to show that propionate and butyrate enhance mitochondrial biogenesis and promote early neurogenic differentiation of neural stem cells through reactive oxygen species- and extracellular signal-regulated kinases 1/2-dependent mechanism. More importantly, neurogenic niches of high-fat, choline-deficient-fed mice showed increased expression of mitochondrial biogenesis markers, and decreased mitochondrial reactive oxygen species scavengers, corroborating the involvement of this mitochondrial stress-dependent pathway in mediating changes of adult neurogenesis by diet. Altogether, our results highlight a mitochondria-dependent pathway as a novel mediator of the gut microbiota-brain axis upon dietary influences.