Transcriptomic changes behind Sparus aurata hepatic response to different aquaculture challenges: An RNA-seq study and multiomics integration.

Gilthead seabream (Sparus aurata) is an important species in Mediterranean aquaculture. Rapid intensification of its production and sub-optimal husbandry practices can cause stress, impairing overall fish performance and raising issues related to sustainability, animal welfare, and food safety. The advent of next-generation sequencing technologies has greatly revolutionized the study of fish stress biology, allowing a deeper understanding of the molecular stress responses. Here, we characterized for the first time, using RNA-seq, the different hepatic transcriptome responses of gilthead seabream to common aquaculture challenges, namely overcrowding, net handling, and hypoxia, further integrating them with the liver proteome and metabolome responses. After reference-guided transcriptome assembly, annotation, and differential gene expression analysis, 7, 343, and 654 genes were differentially expressed (adjusted p-value < 0.01, log2|fold-change| >1) in the fish from the overcrowding, net handling, and hypoxia challenged groups, respectively. Gene set enrichment analysis (FDR < 0.05) suggested a scenario of challenge-specific responses, that is, net handling induced ribosomal assembly stress, whereas hypoxia induced DNA replication stress in gilthead seabream hepatocytes, consistent with proteomics and metabolomics' results. However, both responses converged upon the downregulation of insulin growth factor signalling and induction of endoplasmic reticulum stress. These results demonstrate the high phenotypic plasticity of this species and its differential responses to distinct challenging environments at the transcriptomic level. Furthermore, it provides significant resources for characterizing and identifying potentially novel genes that are important for gilthead seabream resilience and aquaculture production efficiency with regard to fish welfare.

MCPIP1 Inhibits Hepatic Stellate Cell Activation in Autocrine and Paracrine Manners, Preventing Liver Fibrosis.

BACKGROUND & AIMS: Hepatic fibrosis is characterized by enhanced deposition of extracellular matrix (ECM), which results from the wound healing response to chronic, repeated injury of any etiology. Upon injury, hepatic stellate cells (HSCs) activate and secrete ECM proteins, forming scar tissue, which leads to liver dysfunction. Monocyte-chemoattractant protein-induced protein 1 (MCPIP1) possesses anti-inflammatory activity, and its overexpression reduces liver injury in septic mice. In addition, mice with liver-specific deletion of Zc3h12a develop features of primary biliary cholangitis. In this study, we investigated the role of MCPIP1 in liver fibrosis and HSC activation. METHODS: We analyzed MCPIP1 levels in patients' fibrotic livers and hepatic cells isolated from fibrotic murine livers. In vitro experiments were conducted on primary HSCs, cholangiocytes, hepatocytes, and LX-2 cells with MCPIP1 overexpression or silencing. RESULTS: MCPIP1 levels are induced in patients' fibrotic livers compared with their nonfibrotic counterparts. Murine models of fibrosis revealed that its level is increased in HSCs and hepatocytes. Moreover, hepatocytes with Mcpip1 deletion trigger HSC activation via the release of connective tissue growth factor. Overexpression of MCPIP1 in LX-2 cells inhibits their activation through the regulation of TGFB1 expression, and this phenotype is reversed upon MCPIP1 silencing. CONCLUSIONS: We demonstrated that MCPIP1 is induced in human fibrotic livers and regulates the activation of HSCs in both autocrine and paracrine manners. Our results indicate that MCPIP1 could have a potential role in the development of liver fibrosis.

Osteopontin characterizes bile duct-associated macrophages and correlates with liver fibrosis severity in primary sclerosing cholangitis.

BACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) is an immune-mediated cholestatic liver disease for which pharmacological treatment options are currently unavailable. PSC is strongly associated with colitis and a disruption of the gut-liver axis, and macrophages are involved in the pathogenesis of PSC. However, how gut-liver interactions and specific macrophage populations contribute to PSC is incompletely understood. APPROACH AND RESULTS: We investigated the impact of cholestasis and colitis on the hepatic and colonic microenvironment, and performed an in-depth characterization of hepatic macrophage dynamics and function in models of concomitant cholangitis and colitis. Cholestasis-induced fibrosis was characterized by depletion of resident KCs, and enrichment of monocytes and monocyte-derived macrophages (MoMFs) in the liver. These MoMFs highly express triggering-receptor-expressed-on-myeloid-cells-2 ( Trem2 ) and osteopontin ( Spp1 ), markers assigned to hepatic bile duct-associated macrophages, and were enriched around the portal triad, which was confirmed in human PSC. Colitis induced monocyte/macrophage infiltration in the gut and liver, and enhanced cholestasis-induced MoMF- Trem2 and Spp1 upregulation, yet did not exacerbate liver fibrosis. Bone marrow chimeras showed that knockout of Spp1 in infiltrated MoMFs exacerbates inflammation in vivo and in vitro , while monoclonal antibody-mediated neutralization of SPP1 conferred protection in experimental PSC. In human PSC patients, serum osteopontin levels are elevated compared to control, and significantly increased in advanced stage PSC and might serve as a prognostic biomarker for liver transplant-free survival. CONCLUSIONS: Our data shed light on gut-liver axis perturbations and macrophage dynamics and function in PSC and highlight SPP1/OPN as a prognostic marker and future therapeutic target in PSC.

Fibroblast-Derived Lysyl Oxidase Increases Oxidative Phosphorylation and Stemness in Cholangiocarcinoma.

BACKGROUND & AIMS: Metabolic and transcriptional programs respond to extracellular matrix-derived cues in complex environments, such as the tumor microenvironment. Here, we demonstrate how lysyl oxidase (LOX), a known factor in collagen crosslinking, contributes to the development and progression of cholangiocarcinoma (CCA). METHODS: Transcriptomes of 209 human CCA tumors, 143 surrounding tissues, and single-cell data from 30 patients were analyzed. The recombinant protein and a small molecule inhibitor of the LOX activity were used on primary patient-derived CCA cultures to establish the role of LOX in migration, proliferation, colony formation, metabolic fitness, and the LOX interactome. The oncogenic role of LOX was further investigated by RNAscope and in vivo using the AKT/NICD genetically engineered murine CCA model. RESULTS: We traced LOX expression to hepatic stellate cells and specifically hepatic stellate cell-derived inflammatory cancer-associated fibroblasts and found that cancer-associated fibroblast-driven LOX increases oxidative phosphorylation and metabolic fitness of CCA, and regulates mitochondrial function through transcription factor A, mitochondrial. Inhibiting LOX activity in vivo impedes CCA development and progression. Our work highlights that LOX alters tumor microenvironment-directed transcriptional reprogramming of CCA cells by facilitating the expression of the oxidative phosphorylation pathway and by increasing stemness and mobility. CONCLUSIONS: Increased LOX is driven by stromal inflammatory cancer-associated fibroblasts and correlates with diminished survival of patients with CCA. Modulating the LOX activity can serve as a novel tumor microenvironment-directed therapeutic strategy in bile duct pathologies.

Targeting the Heterogeneous Tumour-Associated Macrophages in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a prevalent and aggressive cancer that comprises a complex tumour microenvironment (TME). Tumour-associated macrophages (TAMs) are one of the most abundant immune cells present in the TME, and play a key role both in the development and in the progression of HCC. Thus, TAM-based immunotherapy has been presented as a promising strategy to complement the currently available therapies for HCC treatment. Among the novel approaches focusing on TAMs, reprogramming their functional state has emerged as a promising option for targeting TAMs as an immunotherapy in combination with the currently available treatment options. Nevertheless, a further understanding of the immunobiology of TAMs is still required. This review synthesizes current insights into the heterogeneous nature of TAMs in HCC and describes the mechanisms behind their pro-tumoural polarization focusing the attention on their interaction with HCC cells. Furthermore, this review underscores the potential involvement of TAMs' reprogramming in HCC therapy and highlights the urgency of advancing our understanding of these cells within the dynamic landscape of HCC.

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.

Polycystic liver diseases: from molecular basis to development of effective treatments.

Polycystic liver diseases (PLDs) comprise a heterogeneous group of congenital genetic disorders that mainly affect bile duct epithelial cells, known as cholangiocytes. Patients with PLD usually present bile duct dilatation and/or progressive develop intrahepatic, fluid-filled biliary cysts (more than 10), which is the main cause of morbidity.

A new window into fish welfare: A proteomic discovery study of stress biomarkers in the skin mucus of gilthead seabream (Sparus aurata).

Fish skin mucus is a dynamic external mucosal layer that acts as the first line of defense in the innate immune system. Skin mucus' exudation and composition change severely under stress, making it a valuable biofluid to search for minimally invasive stress markers. This study focused on the skin mucus proteome response to repetitive handling, overcrowding, and hypoxia, using Sparus aurata, an important species in the Mediterranean aquaculture, as a model. Biomarker discovery analysis was performed using label-free shotgun proteomics coupled with bioinformatics to unveil the most predictive proteins for the stressed phenotype. A mean of 2166 proteins were identified at a < 0.2% false discovery rate, from which the differentially abundant proteins (DAPs) were mainly involved in the immune system and protein metabolism. A sparse partial least squares regression analysis revealed a high correlation between DAPs and plasma physiological stress indicators. Feature selection, performed by recursive feature elimination followed by logistic regression analysis of the selected proteins, disclosed 28 candidate biomarkers with values of area under the curve >0.75. These minimally invasive biomarkers could be used in forthcoming species-specific stress management protocols to improve fish welfare and promote farmed fish safety, positive societal outcomes, and business sustainability. SIGNIFICANCE: The fish skin mucus holds a great promise into fish welfare, as a valuable source of minimally invasive biomarkers for stress assessment. In this shotgun proteomics discovery study, we have identified 28 candidate biomarkers by combining a comprehensive functional analysis of the stress regulated proteome with predictive modeling, supported by a significant correlation (p < 0.01) with physiological stress indicators (cortisol, lactate and glucose). The candidate biomarkers showed a good predictive value in the testing set (AUC > 0.75), paving the way for the next step in their validation by targeted proteomics. An early and timely assessment of fish stressful events, by using minimally invasive biomarkers, as those that can be found in the fish skin mucus, can contribute to promote fish health/welfare in the aquaculture sector and its sustainability. The adoption of preventive and surveillance measures based on proteomics approaches can therefore help to avoid unnecessary adverse outcomes with a negative impact on this primordial food sector.

Liquid biopsy-based protein biomarkers for risk prediction, early diagnosis, and prognostication of cholangiocarcinoma.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA), heterogeneous biliary tumours with dismal prognosis, lacks accurate early diagnostic methods especially important for individuals at high-risk (i.e. those with primary sclerosing cholangitis [PSC]). Here, we searched for protein biomarkers in serum extracellular vesicles (EVs). METHODS: EVs from patients with isolated PSC (n = 45), concomitant PSC-CCA (n = 44), PSC who developed CCA during follow-up (PSC to CCA; n = 25), CCAs from non-PSC aetiology (n = 56), and hepatocellular carcinoma (n = 34) and healthy individuals (n = 56) were characterised by mass spectrometry. Diagnostic biomarkers for PSC-CCA, non-PSC CCA, or CCAs regardless of aetiology (Pan-CCAs) were defined and validated by ELISA. Their expression was evaluated in CCA tumours at a single-cell level. Prognostic EV biomarkers for CCA were investigated. RESULTS: High-throughput proteomics of EVs identified diagnostic biomarkers for PSC-CCA, non-PSC CCA, or Pan-CCA, and for the differential diagnosis of intrahepatic CCA and hepatocellular carcinoma, which were cross-validated by ELISA using total serum. Machine learning-based algorithms disclosed CRP/FIBRINOGEN/FRIL for the diagnosis of PSC-CCA (local disease [LD]) vs. isolated PSC (AUC = 0.947; odds ratio [OR] =36.9) and, combined with carbohydrate antigen 19-9, overpowers carbohydrate antigen 19-9 alone. CRP/PIGR/VWF allowed the diagnosis of LD non-PSC CCAs vs. healthy individuals (AUC = 0.992; OR = 387.5). It is noteworthy that CRP/FRIL accurately diagnosed LD Pan-CCA (AUC = 0.941; OR = 89.4). Levels of CRP/FIBRINOGEN/FRIL/PIGR showed predictive capacity for CCA development in PSC before clinical evidence of malignancy. Multi-organ transcriptomic analysis revealed that serum EV biomarkers were mostly expressed in hepatobiliary tissues, and single-cell RNA sequencing and immunofluorescence analysis of CCA tumours showed their presence mainly in malignant cholangiocytes. Multivariable analysis unveiled EV prognostic biomarkers, with COMP/GNAI2/CFAI and ACTN1/MYCT1/PF4V associated negatively and positively with patients' survival, respectively. CONCLUSIONS: Serum EVs contain protein biomarkers for the prediction, early diagnosis, and prognostication of CCA that are detectable using total serum, representing a tumour cell-derived liquid biopsy tool for personalised medicine. IMPACT AND IMPLICATIONS: The accuracy of current imaging tests and circulating tumour biomarkers for cholangiocarcinoma (CCA) diagnosis is far from satisfactory. Most CCAs are considered sporadic, although up to 20% of patients with primary sclerosing cholangitis (PSC) develop CCA during their lifetime, constituting a major cause of PSC-related death. This international study has proposed protein-based and aetiology-related logistic models with predictive, diagnostic, or prognostic capacities by combining two to four circulating protein biomarkers, moving a step forward into personalised medicine. These novel liquid biopsy tools may allow the (i) easy and non-invasive diagnosis of sporadic CCAs, (ii) identification of patients with PSC with higher risk for CCA development, (iii) establishment of cost-effective surveillance programmes for the early detection of CCA in high-risk populations (e.g. PSC), and (iv) prognostic stratification of patients with CCA, which, altogether, may increase the number of cases eligible for potentially curative options or to receive more successful treatments, decreasing CCA-related mortality.

A3907, a systemic ASBT inhibitor, improves cholestasis in mice by multiorgan activity and shows translational relevance to humans.

BACKGROUND AND AIMS: Cholestasis is characterized by intrahepatic accumulation of bile constituents, including bile acids (BAs), which promote liver damage. The apical sodium-dependent BA transporter (ASBT) plays an important role in BA reabsorption and signaling in ileum, bile ducts, and kidneys. Our aim was to investigate the pharmacokinetics and pharmacological activity of A3907, an oral and systemically available ASBT inhibitor in experimental mouse models of cholestasis. In addition, the tolerability, pharmacokinetics, and pharmacodynamics of A3907 were examined in healthy humans. APPROACH AND RESULTS: A3907 was a potent and selective ASBT inhibitor in vitro. In rodents, orally administered A3907 distributed to the ASBT-expressing organs, that is, ileum, liver, and kidneys, and dose dependently increased fecal BA excretion. A3907 improved biochemical, histological, and molecular markers of liver and bile duct injury in Mdr2-/- mice and also had direct protective effects on rat cholangiocytes exposed to cytotoxic BA concentrations in vitro . In bile duct ligated mice, A3907 increased urinary BA elimination, reduced serum BA levels, and prevented body weight loss, while improving markers of liver injury. A3907 was well tolerated and demonstrated target engagement in healthy volunteers. Plasma exposure of A3907 in humans was within the range of systemic concentrations that achieved therapeutic efficacy in mouse. CONCLUSIONS: The systemic ASBT inhibitor A3907 improved experimental cholestatic disease by targeting ASBT function at the intestinal, liver, and kidney levels, resulting in marked clearance of circulating BAs and liver protection. A3907 is well tolerated in humans, supporting further clinical development for the treatment of cholestatic liver diseases.

Host-derived mannose glycans trigger a pathogenic γδ T cell/IL-17a axis in autoimmunity.

Autoimmune diseases are life-threatening disorders that cause increasing disability over time. Systemic lupus erythematosus (SLE) and other autoimmune diseases arise when immune stimuli override mechanisms of self-tolerance. Accumulating evidence has demonstrated that protein glycosylation is substantially altered in autoimmune disease development, but the mechanisms by which glycans trigger these autoreactive immune responses are still largely unclear. In this study, we found that presence of microbial-associated mannose structures at the surface of the kidney triggers the recognition of DC-SIGN-expressing γδ T cells, inducing a pathogenic interleukin-17a (IL-17a)-mediated autoimmune response. Mice lacking Mgat5, which have a higher abundance of mannose structures in the kidney, displayed increased γδ T cell infiltration into the kidney that was associated with spontaneous development of lupus in older mice. N-acetylglucosamine supplementation, which promoted biosynthesis of tolerogenic branched N-glycans in the kidney, was found to inhibit γδ T cell infiltration and control disease development. Together, this work reveals a mannose-γδ T cell-IL-17a axis in SLE immunopathogenesis and highlights glycometabolic reprogramming as a therapeutic strategy for autoimmune disease treatment.

Criteria for preclinical models of cholangiocarcinoma: scientific and medical relevance.

Cholangiocarcinoma (CCA) is a rare malignancy that develops at any point along the biliary tree. CCA has a poor prognosis, its clinical management remains challenging, and effective treatments are lacking. Therefore, preclinical research is of pivotal importance and necessary to acquire a deeper understanding of CCA and improve therapeutic outcomes. Preclinical research involves developing and managing complementary experimental models, from in vitro assays using primary cells or cell lines cultured in 2D or 3D to in vivo models with engrafted material, chemically induced CCA or genetically engineered models. All are valuable tools with well-defined advantages and limitations. The choice of a preclinical model is guided by the question(s) to be addressed; ideally, results should be recapitulated in independent approaches. In this Consensus Statement, a task force of 45 experts in CCA molecular and cellular biology and clinicians, including pathologists, from ten countries provides recommendations on the minimal criteria for preclinical models to provide a uniform approach. These recommendations are based on two rounds of questionnaires completed by 35 (first round) and 45 (second round) experts to reach a consensus with 13 statements. An agreement was defined when at least 90% of the participants voting anonymously agreed with a statement. The ultimate goal was to transfer basic laboratory research to the clinics through increased disease understanding and to develop clinical biomarkers and innovative therapies for patients with CCA.

Aberrant fucosylation sustains the NOTCH and EGFR/NF-κB pathways and has a prognostic value in human intrahepatic cholangiocarcinoma.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (iCCA) is a lethal malignancy, with increasing incidence worldwide and limited therapeutic options. Aberrant protein glycosylation is a hallmark of cancer. Here, we thoroughly investigated the possible involvement of fucosylation in cholangiocarcinogenesis. APPROACH AND RESULTS: We discovered that the levels of global fucosylation and members of the fucosylation pathway are ubiquitously upregulated in human iCCA tissues compared to nontumorous surrounding livers and normal biliary cells. In addition, total fucosylation levels correlate with poor patients' prognosis. Furthermore, fucosylation inhibition following 6-alkynylfucose (6AF) administration triggered a dose-dependent decrease in the proliferation and migration of iCCA cell lines. Notably, adding fucose to the cell medium annulled these effects. At the molecular level, 6AF administration or small interfering RNA-mediated silencing of GDP-L-fucose synthetase (FX) and the GDP-fucose transmembrane transporter (SLC35C1), both pivotal players of cellular fucosylation, decreased NOTCH activity, NOTCH1/Jagged1 interaction, NOTCH receptors, and related target genes in iCCA cell lines. In the same cells, EGFR, nuclear factor kappa-light-chain-enhancer of activated B cells p65, and Bcl-xL protein levels diminished, whereas IκBα (a critical cellular NF-κB inhibitor) increased after FX/SLC35C1 knockdown or 6AF administration. In the chick chorioallantoic membrane assay, 6AF treatment profoundly suppresses the growth of iCCA cells. CONCLUSIONS: Elevated global fucosylation characterizes human iCCA, contributing to cell growth and migration through the upregulation of the NOTCH and EGFR/NF-κB pathways. Thus, aberrant fucosylation is a novel pathogenetic player and a potential therapeutic target for human iCCA.

T-cell selection in the thymus: New routes toward the identification of the self-peptide ligandome presented by thymic epithelial cells.

Within the thymus, thymic epithelial cells (TECs) provide a dedicated niche for the selection of functional T cells expressing a highly variable and self-tolerant T-cell receptor (TCR) repertoire. In this minireview, we start by summarizing recent studies that have improved our understanding on the composition of cortical TEC and medullary TEC microenvironments. Next, we focus on the molecular processes that control the function of TECs in T-cell selection. In particular, we discuss the role of cortical TECs in positive selection and the pathways employed by these cells to generate and present selecting self-peptides:MHC II complexes. Several studies have underscored the role of the ß5t-containing thymoproteasome in the production of unique MHC I-bound peptides critical for CD8 T-cell selection. Contrarily, the identity of the molecular determinants that regulate the generation of MHC II-bound self-peptides capable of positive selecting CD4 T cells is far more uncertain. We highlight recent advances that interconnect the autophagy-lysosomal pathway, the presentation of specific sets of self-peptide:MHC II complexes, and the diversification of CD4 TCR repertoire. Lastly, we discuss how these findings may open up new avenues for deciphering the identity of the MHC I and MHC II ligandome in the thymus.

Voice Disorders Detection Through Multiband Cepstral Features of Sustained Vowel.

This study aims to detect voice disorders related to vocal fold nodule, Reinke's edema and neurological pathologies through multiband cepstral features of the sustained vowel /a/. Detection is performed between pairs of study groups and multiband analysis is accomplished using the wavelet transform. For each pair of groups, a parameters selection is carried out. Time series of the selected parameters are used as input for four classifiers with leave-one-out cross validation. Classification accuracies of 100% are achieved for all pairs including the control group, surpassing the state-of-art methods based on cepstral features, while accuracies higher than 88.50% are obtained for the pathological pairs. The results indicated that the method may be adequate to assist in the diagnosis of the voice disorders addressed. The results must be updated in the future with a larger population to ensure generalization.

LAMP2 regulates autophagy in the thymic epithelium and thymic stroma-dependent CD4 T cell development.

Within the thymus, thymic epithelial cells (TECs) provide dedicated thymic stroma microenvironments for T cell development. Because TEC functionality is sensitive to aging and cytoablative therapies, unraveling the molecular elements that coordinate their thymopoietic role has fundamental and clinical implications. Particularly, the selection of CD4 T cells depends on interactions between TCRs expressed on T cell precursors and self-peptides:MHC II complexes presented by cortical TECs (cTECs). Although the macroautophagy/autophagy-lysosomal protein degradation pathway is implicated in CD4 T cell selection, the molecular mechanism that controls the generation of selecting MHC II ligands remains elusive. LAMP2 (lysosomal-associated membrane protein 2) is a well-recognized mediator of autolysosome (AL) maturation. We showed that LAMP2 is highly expressed in cTECs. Notably, genetic inactivation of Lamp2 in thymic stromal cells specifically impaired the development of CD4 T cells that completed positive selection, without misdirecting MHC II-restricted cells into the CD8 lineage. Mechanistically, defects in autophagy in lamp2-deficient cTECs were linked to alterations in MHC II processing, which was associated with a marked reduction in CD4 TCR repertoire diversity selected within the lamp2-deficient thymic stroma. Together, our findings suggest that LAMP2 interconnects the autophagy-lysosomal axis and the processing of selecting self-peptides:MHC II complexes in cTECs, underling its implications for the generation of a broad CD4 TCR repertoire.Abbreviations: AIRE: autoimmune regulator (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy); AL: autolysosome; AP: autophagosome; Baf-A1: bafilomycin A1; B2M: beta-2 microglobulin; CTSL: cathepsin L; CD74/Ii: CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated); CFSE: carboxyfluorescein succinimidyl ester; CFU: colony-forming unit; CLIP: class II-associated invariant chain peptides; cTECs: cortical TECs dKO: double knockout; DN: double negative; DP: double positive; ENPEP/LY51: glutamyl aminopeptidase; FOXP3: forkhead box; P3 IFNG/IFNγ: interferon gamma; IKZF2/HELIOS: IKAROS family zinc finger 2; IL2RA/CD25: interleukin 2 receptor, alpha chain; KO: knockout; LAMP2: lysosomal-associated membrane protein 2; LIP: lymphopenia-induced proliferation; Lm: Listeria monocytogenes; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MHC: major histocompatibility complex; mTECs: medullary TECs; PRSS16/TSSP: protease, serine 16 (thymus); SELL/CD62L: selectin, lymphocyte; SP: single positive; TCR: T cell receptor; TCRB: T cell receptor beta chain; TECs: thymic epithelial cells; UEA-1: Ulex europaeus agglutinin-1; WT: wild-type.

Gilthead Seabream Liver Integrative Proteomics and Metabolomics Analysis Reveals Regulation by Different Prosurvival Pathways in the Metabolic Adaptation to Stress.

The study of the molecular mechanisms of stress appraisal on farmed fish is paramount to ensuring a sustainable aquaculture. Stress exposure can either culminate in the organism's adaptation or aggravate into a metabolic shutdown, characterized by irreversible cellular damage and deleterious effects on fish performance, welfare, and survival. Multiomics can improve our understanding of the complex stressed phenotype in fish and the molecular mediators that regulate the underlying processes of the molecular stress response. We profiled the stress proteome and metabolome of Sparus aurata responding to different challenges common to aquaculture production, characterizing the disturbed pathways in the fish liver, i.e., the central organ in mounting the stress response. Label-free shotgun proteomics and untargeted metabolomics analyses identified 1738 proteins and 120 metabolites, separately. Mass spectrometry data have been made fully accessible via ProteomeXchange, with the identifier PXD036392, and via MetaboLights, with the identifier MTBLS5940. Integrative multivariate statistical analysis, performed with data integration analysis for biomarker discovery using latent components (DIABLO), depicted the 10 most-relevant features. Functional analysis of these selected features revealed an intricate network of regulatory components, modulating different signaling pathways related to cellular stress, e.g., the mTORC1 pathway, the unfolded protein response, endocytosis, and autophagy to different extents according to the stress nature. These results shed light on the dynamics and extent of this species' metabolic reprogramming under chronic stress, supporting future studies on stress markers' discovery and fish welfare research.

Fish Processing and Digestion Affect Parvalbumins Detectability in Gilthead Seabream and European Seabass.

Consumption of aquatic food, including fish, accounts for 17% of animal protein intake. However, fish consumption might also result in several side-effects such as sneezing, swelling and anaphylaxis in sensitized consumers. Fish allergy is an immune reaction to allergenic proteins in the fish muscle, for instance parvalbumin (PV), considered the major fish allergen. In this study, we characterize PV in two economically important fish species for southern European aquaculture, namely gilthead seabream and European seabass, to understand its stability during in vitro digestion and fish processing. This information is crucial for future studies on the allergenicity of processed fish products. PVs were extracted from fish muscles, identified by mass spectrometry (MS), and detected by sandwich enzyme-linked immunosorbent assay (ELISA) after simulated digestion and various food processing treatments. Secondary structures were determined by circular dichroism (CD) after purification by anion exchange and gel filtration chromatography. In both species, PVs presented as α-helical and ß-sheet structures, at room temperature, were shown to unfold at boiling temperatures. In European seabass, PV detectability decreased during the simulated digestion and after 240 min (intestinal phase) no detection was observed, while steaming showed a decrease (p < 0.05) in PVs detectability in comparison to raw muscle samples, for both species. Additionally, freezing (−20 °C) for up to 12 months continued to reduce the detectability of PV in tested processing techniques. We concluded that PVs from both species are susceptible to digestion and processing techniques such as steaming and freezing. Our study obtained preliminary results for further research on the allergenic potential of PV after digestion and processing.

Validation of StepTest4all for Assessing Cardiovascular Capacity in Young Adults.

BACKGROUND: Cardiovascular capacity, expressed as maximal oxygen uptake (VO2max), is a strong predictor of health and fitness and is considered a key measure of physiological function in the healthy adult population. The purpose of this study was to validate a specific step test (StepTest4all) as an adequate procedure to estimate cardiovascular capacity in young adults. METHODS: The sample was composed of 56 participants, including 19 women (aged 21.05 ± 2.39 years, body mass = 57.50 ± 6.64 kg, height = 1.62 ± 0.05 m, body mass index = 22.00 ± 2.92 kg/m2) and 37 men (aged 22.05 ± 3.14 years, body mass = 72.50 ± 7.73 kg, height = 1.76 ± 0.07 m, body mass index = 23.34 ± 2.17 kg/m2). Participants were included in one of the following groups: (i) the group used to predict the VO2max, and (ii) the group used to validate the prediction model. All participants performed the StepTest4all protocol. The step height and the intensity of the effort was determined individually. Heart rate and oxygen uptake were measured continuously during rest, effort, and recovery phases. The validation process included the following three stages: (i) mean data comparison, (ii) simple linear regression, and (iii) Bland-Altman analysis. RESULTS: The linear regression retained, as significant predictors of the VO2max, sex (p < 0.001) and heart rate recovery for one minute (p = 0.003). The prediction equation revealed a high relationship between measurements (R2 = 63.0%, SEE = 5.58). The validation procedure revealed non-significant differences (p > 0.05) between the measured and estimated maximal oxygen uptake, high relationship (R2 = 63.3%), and high agreement with Bland-Altman plots. Thus, VO2max can be estimated with the formula: VO2max = 22 + 0.3 · (HRR1min) + 12 · (sex), where HRR1min is the magnitude of the HR decrease (bpm) in one minute immediately after the step was stopped, and sex: men = 1, women = 0. CONCLUSIONS: The StepTest4all is an adequate procedure to estimate cardiovascular capacity, expressed as VO2max, in young adults. In addition, it is possible to determine the qualitative level of cardiovascular capacity from the heart rate recovery for one minute, more specifically, poor: <20, moderate: 20 to 34, good: 35 to 49, and excellent: ≥50. This procedure has the benefit of being simple to apply and can be used by everyone, even at home, without specialist supervision.

Fish Allergenicity Modulation Using Tailored Enriched Diets-Where Are We?

Food allergy is an abnormal immune response to specific proteins in a certain food. The chronicity, prevalence, and the potential fatality of food allergy, make it a serious socio-economic problem. Fish is considered the third most allergenic food in the world, affecting part of the world population with a higher incidence in children and adolescents. The main allergen in fish, responsible for the large majority of fish-allergic reactions in sensitized patients, is a small and stable calcium-binding muscle protein named beta-parvalbumin. Targeting the expression or/and the 3D conformation of this protein by adding specific molecules to fish diets has been the innovative strategy of some researchers in the fields of fish allergies and nutrition. This has shown promising results, namely when the apo-form of ß-parvalbumin is induced, leading in the case of gilthead seabream to a 50% reduction of IgE-reactivity in fish allergic patients.