Discovery of the First-in-Class Inhibitors of Hypoxia Up-Regulated Protein†1 (HYOU1) Suppressing Pathogenic Fibroblast Activation.

Fibroblasts are key regulators of inflammation, fibrosis, and cancer. Targeting their activation in these complex diseases has emerged as a novel strategy to restore tissue homeostasis. Here, we present a multidisciplinary lead discovery approach to identify and optimize small molecule inhibitors of pathogenic fibroblast activation. The study encompasses medicinal chemistry, molecular phenotyping assays, chemoproteomics, bulk RNA-sequencing analysis, target validation experiments, and chemical absorption, distribution, metabolism, excretion and toxicity (ADMET)/pharmacokinetic (PK)/in vivo evaluation. The parallel synthesis employed for the production of the new benzamide derivatives enabled us to a) pinpoint key structural elements of the scaffold that provide potent fibroblast-deactivating effects in cells, b) discriminate atoms or groups that favor or disfavor a desirable ADMET profile, and c) identify metabolic "hot spots". Furthermore, we report the discovery of the first-in-class inhibitor leads for hypoxia up-regulated protein 1 (HYOU1), a member of the heat shock protein 70 (HSP70) family often associated with cellular stress responses, particularly under hypoxic conditions. Targeting HYOU1 may therefore represent a potentially novel strategy to modulate fibroblast activation and treat chronic inflammatory and fibrotic disorders.

Combinatorial regulation by ERK1/2 and CK1δ protein kinases leads to HIF-1α association with microtubules and facilitates its symmetrical distribution during mitosis.

Hypoxia-inducible factor-1 (HIF-1) is the key transcriptional mediator of the cellular response to hypoxia and is also involved in cancer progression. Regulation of its oxygen-sensitive HIF-1α subunit involves post-translational modifications that control its stability, subcellular localization, and activity. We have previously reported that phosphorylation of the HIF-1α C-terminal domain by ERK1/2 promotes HIF-1α nuclear accumulation and stimulates HIF-1 activity while lack of this modification triggers HIF-1α nuclear export and its association with mitochondria. On the other hand, modification of the N-terminal domain of HIF-1α by CK1δ impairs HIF-1 activity by obstructing the formation of a HIF-1α/ARNT heterodimer. Investigation of these two antagonistic events by expressing double phospho-site mutants in HIF1A-/- cells under hypoxia revealed independent and additive phosphorylation effects that can create a gradient of HIF-1α subcellular localization and transcriptional activity. Furthermore, modification by CK1δ caused mitochondrial release of the non-nuclear HIF-1α form and binding to microtubules via its N-terminal domain. In agreement, endogenous HIF-1α could be shown to co-localize with mitotic spindle microtubules and interact with tubulin, both of which were inhibited by CK1δ silencing or inhibition. Moreover, CK1δ expression was necessary for equal partitioning of mother cell-produced HIF-1α to the daughter cell nuclei at the end of mitosis. Overall, our results suggest that phosphorylation by CK1δ stimulates the association of non-nuclear HIF-1α with microtubules, which may serve as a means to establish a symmetric distribution of HIF-1α during cell division under low oxygen conditions.

Proteome Analysis of the ROF-FKBP Mutants Reveals Functional Relations among Heat Stress Responses, Plant Development, and Protein Quality Control during Heat Acclimation in Arabidopsis thaliana.

In the present study, a differential screening following heat stress acclimation was performed in Arabidopsis thaliana WT and ROF-FKBP mutated plants using mass spectrometry, and the results were used to understand and analyze the effect of the ROF PPIases during thermotolerance acquisition in plants. Our data highlight the central role of these two PPIases in heat stress and point to their direct or indirect effect on other proteins participating in cellular functions such as protein folding and quality control, cell division, photosynthesis, and other metabolic and signaling processes. Specifically, the heat stress response, protein folding, and protein ER processing pathways are enhanced following a 37 °C acclimation period independent of the mutation state. However, at 37 °C, and in the double-mutated rof1-/2- plants, a higher accumulation of proteins belonging to the above pathways is observed compared with all other conditions (WT, single mutants, control, and heat-acclimated plants). Furthermore, the proteasomal pathway, involving the common member of both the protasomal and the lysosomal degradation pathway, CDC48, is over-represented in the extracts of both the untreated and heat-stressed rof1-/2- mutants compared with the other extracts. In contrast, in the single rof1- mutation, the heat acclimation pathway is suppressed at 37 °C when compared to the WT. Protein accumulation related to the heat stress and the protein quality control pathways points to a differential but also synergistic role of the two proteins. Protein complexes of other biochemical and developmental mechanisms, such as the light-harvesting complex of the photosynthetic pathway and the phosphoinositide binding proteins involved in membrane-trafficking events during cell plate formation and cytokinesis (patellin 1, 2, and 4), are negatively regulated in the rof1-/2- mutant. Our results suggest that ROF1 and ROF2 FKBPs regulate stress response, and developmental and metabolic pathways via a complex feedback mechanism involving partners that ensure protein quality control and plant survival during heat stress.

Detection of Protein Tyrosine Phosphatase Interacting Partners by Mass Spectrometry.

Unraveling interacting partners of protein tyrosine (Tyr) phosphatases is considered a key aspect in resolving the regulation of signaling cascades either in a pathological or in developmental context. Mass spectrometry (MS)-based protein identification has emerged as the major approach in this arena, complemented by the development of novel biochemical methodologies for sample preparation. In this chapter, we highlight two methods that, combined with mass spectrometry, may help the investigator create an interactome map for the phosphatase of interest within a specific biological context.

Ferroptosis-protective membrane domains in quiescence.

Quiescence is a common cellular state, required for stem cell maintenance and microorganismal survival under stress conditions or starvation. However, the mechanisms promoting quiescence maintenance remain poorly known. Plasma membrane components segregate into distinct microdomains, yet the role of this compartmentalization in quiescence remains unexplored. Here, we show that flavodoxin-like proteins (FLPs), ubiquinone reductases of the yeast eisosome membrane compartment, protect quiescent cells from lipid peroxidation and ferroptosis. Eisosomes and FLPs expand specifically in respiratory-active quiescent cells, and mutants lacking either show accelerated aging and defective quiescence maintenance and accumulate peroxidized phospholipids with monounsaturated or polyunsaturated fatty acids (PUFAs). FLPs are essential for the extramitochondrial regeneration of the lipophilic antioxidant ubiquinol. FLPs, alongside the Gpx1/2/3 glutathione peroxidases, prevent iron-driven, PUFA-dependent ferroptotic cell death. Our work describes ferroptosis-protective mechanisms in yeast and introduces plasma membrane compartmentalization as an important factor in the long-term survival of quiescent cells.

A Peripheral Blood Signature of Increased Th1 and Myeloid Cells Combined with Serum Inflammatory Mediators Is Associated with Response to Abatacept in Rheumatoid Arthritis Patients.

Abatacept (CTLA4-Ig)-a monoclonal antibody which restricts T cell activation-is an effective treatment for rheumatoid arthritis (RA). Nevertheless, only 50% of RA patients attain clinical responses, while predictors of response are rather limited. Herein, we aimed to investigate for early biomarkers of response to abatacept, based on a detailed immunological profiling of peripheral blood (PB) cells and serum proteins. We applied flow cytometry and proteomics analysis on PB immune cells and serum respectively, of RA patients starting abatacept as the first biologic agent. After 6 months of treatment, 34.5% of patients attained response. At baseline, Th1 and FoxP3+ T cell populations were positively correlated with tender joint counts (p-value = 0.047 and p-value = 0.022, respectively). Upon treatment, CTLA4-Ig effectively reduced the percentages of Th1 and Th17 only in responders (p-value = 0.0277 and p-value = 0.0042, respectively). Notably, baseline levels of Th1 and myeloid cell populations were significantly increased in PB of responders compared to non-responders (p-value = 0.009 and p-value = 0.03, respectively). Proteomics analysis revealed that several inflammatory mediators were present in serum of responders before therapy initiation and strikingly 10 amongst 303 serum proteins were associated with clinical responses. Finally, a composite index based on selected baseline cellular and proteomics' analysis could predict response to abatacept with a high sensitivity (90%) and specificity (88.24%).

3'-tRF-CysGCA overexpression in HEK-293 cells alters the global expression profile and modulates cellular processes and pathways.

tRNA fragments (tRFs) are small non-coding RNAs generated through specific cleavage of tRNAs and involved in various biological processes. Among the different types of tRFs, the 3'-tRFs have attracted scientific interest due to their regulatory role in gene expression. In this study, we investigated the role of 3'-tRF-CysGCA, a tRF deriving from cleavage in the T-loop of tRNACysGCA, in the regulation of gene expression in HEK-293 cells. Previous studies have shown that 3'-tRF-CysGCA is incorporated into the RISC complex and interacts with Argonaute proteins, suggesting its involvement in the regulation of gene expression. However, the general role and effect of the deregulation of 3'-tRF-CysGCA levels in human cells have not been investigated so far. To fill this gap, we stably overexpressed 3'-tRF-CysGCA in HEK-293 cells and performed transcriptomic and proteomic analyses. Moreover, we validated the interaction of this tRF with putative targets, the levels of which were found to be affected by 3'-tRF-CysGCA overexpression. Lastly, we investigated the implication of 3'-tRF-CysGCA in various pathways using extensive bioinformatics analysis. Our results indicate that 3'-tRF-CysGCA overexpression led to changes in the global gene expression profile of HEK-293 cells and that multiple cellular pathways were affected by the deregulation of the levels of this tRF. Additionally, we demonstrated that 3'-tRF-CysGCA directly interacts with thymopoietin (TMPO) transcript variant 1 (also known as LAP2α), leading to modulation of its levels. In conclusion, our findings suggest that 3'-tRF-CysGCA plays a significant role in gene expression regulation and highlight the importance of this tRF in cellular processes.

Data-Independent Acquisition Mass Spectrometry Analysis of FFPE Rectal Cancer Samples Offers In-Depth Proteomics Characterization of the Response to Neoadjuvant Chemoradiotherapy.

Locally advanced rectal cancer (LARC) presents a challenge in identifying molecular markers linked to the response to neoadjuvant chemoradiotherapy (nCRT). This study aimed to utilize a sensitive proteomic method, data-independent mass spectrometry (DIA-MS), to extensively analyze the LARC proteome, seeking individuals with favorable initial responses suitable for a watch-and-wait approach. This research addresses the unmet need to understand the response to treatment, potentially guiding personalized strategies for LARC patients. Post-treatment assessment included MRI scans and proctoscopy. This research involved 97 LARC patients treated with intense chemoradiotherapy, comprising radiation and chemotherapy. Out of 97 LARC included in this study, we selected 20 samples with the most different responses to nCRT for proteome profiling (responders vs. non-responders). This proteomic approach shows extensive proteome coverage in LARC samples. The analysis identified a significant number of proteins compared to a prior study. A total of 915 proteins exhibited differential expression between the two groups, with certain signaling pathways associated with response mechanisms, while top candidates had good predictive potential. Proteins encoded by genes SMPDL3A, PCTP, LGMN, SYNJ2, NHLRC3, GLB1, and RAB43 showed high predictive potential of unfavorable treatment outcome, while RPA2, SARNP, PCBP2, SF3B2, HNRNPF, RBBP4, MAGOHB, DUT, ERG28, and BUB3 were good predictive biomarkers of favorable treatment outcome. The identified proteins and related biological processes provide promising insights that could enhance the management and care of LARC patients.

Inactivation of Tumor Suppressor CYLD Inhibits Fibroblast Reprogramming to Pluripotency.

CYLD is a tumor suppressor gene coding for a deubiquitinating enzyme that has a critical regulatory function in a variety of signaling pathways and biological processes involved in cancer development and progression, many of which are also key modulators of somatic cell reprogramming. Nevertheless, the potential role of CYLD in this process has not been studied. With the dual aim of investigating the involvement of CYLD in reprogramming and developing a better understanding of the intricate regulatory system governing this process, we reprogrammed control (CYLDWT/WT) and CYLD DUB-deficient (CYLDΔ9/Δ9) mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPSCs) through ectopic overexpression of the Yamanaka factors (Oct3/4, Sox2, Klf4, c-myc). CYLD DUB deficiency led to significantly reduced reprogramming efficiency and slower early reprogramming kinetics. The introduction of WT CYLD to CYLDΔ9/Δ9 MEFs rescued the phenotype. Nevertheless, CYLD DUB-deficient cells were capable of establishing induced pluripotent colonies with full spontaneous differentiation potential of the three germ layers. Whole proteome analysis (Data are available via ProteomeXchange with identifier PXD044220) revealed that the mesenchymal-to-epithelial transition (MET) during the early reprogramming stages was disrupted in CYLDΔ9/Δ9 MEFs. Interestingly, differentially enriched pathways revealed that the primary processes affected by CYLD DUB deficiency were associated with the organization of the extracellular matrix and several metabolic pathways. Our findings not only establish for the first time CYLD's significance as a regulatory component of early reprogramming but also highlight its role as an extracellular matrix regulator, which has profound implications in cancer research.

The interactome of the UapA transporter reveals putative new players in anterograde membrane cargo trafficking.

Neosynthesized plasma membrane (PM) proteins co-translationally translocate to the ER, concentrate at regions called ER-exit sites (ERes) and pack into COPII secretory vesicles which are sorted to the early-Golgi through membrane fusion. Following Golgi maturation, membrane cargoes reach the late-Golgi, from where they exit in clathrin-coated vesicles destined to the PM, directly or through endosomes. Post-Golgi membrane cargo trafficking also involves the cytoskeleton and the exocyst. The Golgi-dependent secretory pathway is thought to be responsible for the trafficking of all major membrane proteins. However, our recent findings in Aspergillus nidulans showed that several plasma membrane cargoes, such as transporters and receptors, follow a sorting route that seems to bypass Golgi functioning. To gain insight on membrane trafficking and specifically Golgi-bypass, here we used proximity dependent biotinylation (PDB) coupled with data-independent acquisition mass spectrometry (DIA-MS) for identifying transient interactors of the UapA transporter. Our assays, which included proteomes of wild-type and mutant strains affecting ER-exit or endocytosis, identified both expected and novel interactions that might be physiologically relevant to UapA trafficking. Among those, we validated, using reverse genetics and fluorescence microscopy, that COPI coatomer is essential for ER-exit and anterograde trafficking of UapA and other membrane cargoes. We also showed that ArfAArf1 GTPase activating protein (GAP) Glo3 contributes to UapA trafficking at increased temperature. This is the first report addressing the identification of transient interactions during membrane cargo biogenesis using PDB and proteomics coupled with fungal genetics. Our work provides a basis for dissecting dynamic membrane cargo trafficking via PDB assays.

Transcriptomic and proteomic profiling reveals distinct pathogenic features of peripheral non-classical monocytes in systemic lupus erythematosus.

Peripheral blood monocytes propagate inflammation in systemic lupus erythematosus (SLE). Three major populations of monocytes have been recognized namely classical (CM), intermediate (IM) and non-classical monocytes (NCM). Herein, we performed a comprehensive transcriptomic, proteomic and functional characterization of the three peripheral monocytic subsets from active SLE patients and healthy individuals. Our data demonstrate extensive molecular disruptions in circulating SLE NCM, characterized by enhanced inflammatory features such as deregulated DNA repair, cell cycle and heightened IFN signaling combined with differentiation and developmental cues. Enhanced DNA damage, elevated expression of p53, G0 arrest of cell cycle and increased autophagy stress the differentiation potential of NCM in SLE. This immunogenic profile is associated with an activated macrophage phenotype of NCM exhibiting M1 characteristics in the circulation, fueling the inflammatory response. Together, these findings identify circulating SLE NCM as a pathogenic cell type in the disease that could represent an additional therapeutic target.

IQGAP1 mediates the communication between the nucleus and the mitochondria via NDUFS4 alternative splicing.

Constant communication between mitochondria and nucleus ensures cellular homeostasis and adaptation to mitochondrial stress. Anterograde regulatory pathways involving a large number of nuclear-encoded proteins control mitochondrial biogenesis and functions. Such functions are deregulated in cancer cells, resulting in proliferative advantages, aggressive disease and therapeutic resistance. Transcriptional networks controlling the nuclear-encoded mitochondrial genes are known, however alternative splicing (AS) regulation has not been implicated in this communication. Here, we show that IQGAP1, a scaffold protein regulating AS of distinct gene subsets in gastric cancer cells, participates in AS regulation that strongly affects mitochondrial respiration. Combined proteomic and RNA-seq analyses of IQGAP1KO and parental cells show that IQGAP1KO alters an AS event of the mitochondrial respiratory chain complex I (CI) subunit NDUFS4 and downregulates a subset of CI subunits. In IQGAP1KO cells, CI intermediates accumulate, resembling assembly deficiencies observed in patients with Leigh syndrome bearing NDUFS4 mutations. Mitochondrial CI activity is significantly lower in KO compared to parental cells, while exogenous expression of IQGAP1 reverses mitochondrial defects of IQGAP1KO cells. Our work sheds light to a novel facet of IQGAP1 in mitochondrial quality control that involves fine-tuning of CI activity through AS regulation in gastric cancer cells relying highly on mitochondrial respiration.

Endogenous Alpha-Synuclein is Essential for the Transfer of Pathology by Exosome-Enriched Extracellular Vesicles, Following Inoculation with Preformed Fibrils in vivo.

The main pathological hallmark of Parkinson's disease (PD) and related synucleinopathies is the presence of intracellular proteinaceous aggregates, enriched in the presynaptic protein alpha-Synuclein (α-Syn). α-Syn association with exosomes has been previously documented both as a physiological process of secretion and as a pathological process of disease transmission, however, critical information about the mechanisms governing this interplay is still lacking. To address this, we utilized the α-Syn preformed fibril (PFF) mouse model of PD, as a source of brain-derived exosome-enriched extracellular vesicles (ExE-EVs) and assessed their pathogenic capacity following intrastriatal injections in host wild type (WT) mouse brain. We further investigated the impact of the fibrillar α-Syn on the exosomal cargo independent of the endogenous α-Syn, by isolating ExE-EVs from PFF-injected α-Syn knockout mice. Although PFF inoculation does not alter the morphology, size distribution, and quantity of brain-derived ExE-EVs, it triggers changes in the exosomal proteome related to synaptic and mitochondrial function, as well as metabolic processes. Importantly, we showed that the presence of the endogenous α-Syn is essential for the ExE-EVs to acquire a pathogenic capacity, allowing them to mediate disease transmission by inducing phosphorylated-α-Syn pathology. Notably, misfolded α-Syn containing ExE-EVs when injected in WT mice were able to induce astrogliosis and synaptic alterations in the host brain, at very early stages of α-Syn pathology, preceding the formation of the insoluble α-Syn accumulations. Collectively, our data suggest that exosomal cargo defines their ability to spread α-Syn pathology.

Transcriptome and proteome analysis reveals the anti-cancer properties of Hypnea musciformis marine macroalga extract in liver and intestinal cancer cells.

BACKGROUND: Marine seaweeds are considered as a rich source of health-promoting compounds by the food and pharmaceutical industry. Hypnea musciformis is a marine red macroalga (seaweed) that is widely distributed throughout the world, including the Mediterranean Sea. It is known to contain various bioactive compounds, including sulfated polysaccharides, flavonoids, and phlorotannins. Recent studies have investigated the potential anticancer effects of extracts from H. musciformis demonstrating their cytotoxic effects on various cancer cell lines. The anticancer effects of these extracts are thought to be due to the presence of bioactive compounds, particularly sulfated polysaccharides, which have been shown to have anticancer and immunomodulatory effects. However, further studies are needed to fully understand the molecular mechanisms that underlie their anticancer effects and to determine their potential as therapeutic agents for cancer treatment. METHODS: H. musciformis was collected from the Aegean Sea (Greece) and used for extract preparation. Transcriptome and proteome analysis was performed in liver and colon cancer human cell lines following treatment with H. musciformis seaweed extracts to characterize its anticancer effect in detail at the molecular level and to link transcriptome and proteome responses to the observed phenotypes in cancer cells. RESULTS: We have identified that treatment with the seaweed extract triggers a p53-mediated response at the transcriptional and protein level in liver cancer cells, in contrast to colon cancer cells in which the effects are more associated with metabolic changes. Furthermore, we show that in treated HepG2 liver cancer cells, p53 interacts with the chromatin of several target genes and facilitates their upregulation possibly through the recruitment of the p300 co-activator. CONCLUSIONS: Overall, the available evidence suggests that extracts from H. musciformis have the potential to serve as a source of anticancer agents in liver cancer cells mainly through activation of a p53-mediated anti-tumor response that is linked to inhibition of cellular proliferation and induction of cell death.

Differential intracellular trafficking of extracellular vesicles in microglia and astrocytes.

Extracellular vesicles (EVs) have emerged as key players in cell-to-cell communication in both physiological and pathological processes in the Central Nervous System. Thus far, the intracellular pathways involved in uptake and trafficking of EVs within different cell types of the brain are poorly understood. In our study, the endocytic processes and subcellular sorting of EVs were investigated in primary glial cells, particularly linked with the EV-associated α-synuclein (α-syn) transmission. Mouse microglia and astrocytic primary cultures were incubated with DiI-stained mouse brain-derived EVs. The internalization and trafficking pathways were analyzed in cells treated with pharmacological reagents that block the major endocytic pathways. Brain-derived EVs were internalized by both glial cell types; however, uptake was more efficient in microglia than in astrocytes. Colocalization of EVs with early and late endocytic markers (Rab5, Lamp1) indicated that EVs are sorted to endo-lysosomes for subsequent processing. Blocking actin-dependent phagocytosis and/or macropinocytosis with Cytochalasin D or EIPA inhibited EV entry into glial cells, whereas treatment with inhibitors that strip cholesterol off the plasma membrane, induced uptake, however differentially altered endosomal sorting. EV-associated fibrillar α-Syn was efficiently internalized and detected in Rab5- and Lamp1-positive compartments within microglia. Our study strongly suggests that EVs enter glial cells through phagocytosis and/or macropinocytosis and are sorted to endo-lysosomes for subsequent processing. Further, brain-derived EVs serve as scavengers and mediate cell-to-glia transfer of pathological α-Syn which is also targeted to the endolysosomal pathway, suggesting a beneficial role in microglia-mediated clearance of toxic protein aggregates, present in numerous neurodegenerative diseases.

Distinct modulation of cellular immunopeptidome by the allosteric regulatory site of ER aminopeptidase 1.

ER aminopeptidase 1 (ERAP1) is an ER-resident aminopeptidase that excises N-terminal residues of peptides that then bind onto Major Histocompatibility Complex I molecules (MHC-I) and indirectly modulates adaptive immune responses. ERAP1 contains an allosteric regulatory site that accommodates the C-terminus of at least some peptide substrates, raising questions about its exact influence on antigen presentation and the potential of allosteric inhibition for cancer immunotherapy. We used an inhibitor that targets this regulatory site to study its effect on the immunopeptidome of a human cancer cell line. The immunopeptidomes of allosterically inhibited and ERAP1 KO cells contain high-affinity peptides with sequence motifs consistent with the cellular HLA class I haplotypes but are strikingly different in peptide composition. Compared to KO cells, allosteric inhibition did not affect the length distribution of peptides and skewed the peptide repertoire both in terms of sequence motifs and HLA allele utilization, indicating significant mechanistic differences between the two ways of disrupting ERAP1 function. These findings suggest that the regulatory site of ERAP1 plays distinct roles in antigenic peptide selection, which should be taken into consideration when designing therapeutic interventions targeting the cancer immunopeptidome.

Integrated omics analysis for characterization of the contribution of high fructose corn syrup to non-alcoholic fatty liver disease in obesity.

BACKGROUND: High-Fructose Corn Syrup (HFCS), a sweetener rich in glucose and fructose, is nowadays widely used in beverages and processed foods; its consumption has been correlated to the emergence and progression of Non-Alcoholic Fatty Liver Disease (NAFLD). Nevertheless, the molecular mechanisms by which HFCS impacts hepatic metabolism remain scarce, especially in the context of obesity. Besides, the majority of current studies focuses either on the detrimental role of fructose in hepatic steatosis or compare separately the additive impact of fructose versus glucose in high fat diet-induced NAFLD. AIM: By engaging combined omics approaches, we sought to characterize the role of HFCS in obesity-associated NAFLD and reveal molecular processes, which mediate the exaggeration of steatosis under these conditions. METHODS: Herein, C57BL/6 mice were fed a normal-fat-diet (ND), a high-fat-diet (HFD) or a HFD supplemented with HFCS (HFD-HFCS) and upon examination of their metabolic and NAFLD phenotype, proteomic, lipidomic and metabolomic analyses were conducted to identify HFCS-related molecular alterations of the hepatic metabolic landscape in obesity. RESULTS: Although HFD and HFD-HFCS mice displayed comparable obesity, HFD-HFCS mice showed aggravation of hepatic steatosis, as analysis of the lipid droplet area in liver sections revealed (12,15 % of total section area in HFD vs 22,35 % in HFD-HFCS), increased NAFLD activity score (3,29 in HFD vs 4,86 in HFD-HFCS) and deteriorated hepatic insulin resistance, as compared to the HFD mice. Besides, the hepatic proteome of HFD-HFCS mice was characterized by a marked upregulation of 5 core proteins implicated in de novo lipogenesis (DNL), while an increased phosphatidyl-cholines(PC)/phosphatidyl-ethanolamines(PE) ratio (2.01 in HFD vs 3.04 in HFD-HFCS) was observed in the livers of HFD-HFCS versus HFD mice. Integrated analysis of the omics datasets indicated that Tricarboxylic Acid (TCA) cycle overactivation is likely contributing towards the intensification of steatosis during HFD-HFCS-induced NAFLD. CONCLUSION: Our results imply that HFCS significantly contributes to steatosis aggravation during obesity-related NAFLD, likely deriving from DNL upregulation, accompanied by TCA cycle overactivation and deteriorated hepatic insulin resistance.

A novel isolation method for spontaneously released extracellular vesicles from brain tissue and its implications for stress-driven brain pathology.

BACKGROUND: Extracellular vesicles (EVs), including small EVs (sEVs) such as exosomes, exhibit great potential for the diagnosis and treatment of brain disorders, representing a valuable tool for precision medicine. The latter demands high-quality human biospecimens, especially in complex disorders in which pathological and specimen heterogeneity, as well as diverse individual clinical profile, often complicate the development of precision therapeutic schemes and patient-tailored treatments. Thus, the collection and characterization of physiologically relevant sEVs are of the utmost importance. However, standard brain EV isolation approaches rely on tissue dissociation, which can contaminate EV fractions with intracellular vesicles. METHODS: Based on multiscale analytical platforms such as cryo-EM, label-free proteomics, advanced flow cytometry, and ExoView analyses, we compared and characterized the EV fraction isolated with this novel method with a classical digestion-based EV isolation procedure. Moreover, EV biogenesis was pharmacologically manipulated with either GW4869 or picrotoxin to assess the validity of the spontaneous-release method, while the injection of labelled-EVs into the mouse brain further supported the integrity of the isolated vesicles. RESULTS: We hereby present an efficient purification method that captures a sEV-enriched population spontaneously released by mouse and human brain tissue. In addition, we tested the significance of the release method under conditions where biogenesis/secretion of sEVs was pharmacologically manipulated, as well as under animals' exposure to chronic stress, a clinically relevant precipitant of brain pathologies, such as depression and Alzheimer's disease. Our findings show that the released method monitors the drug-evoked inhibition or enhancement of sEVs secretion while chronic stress induces the secretion of brain exosomes accompanied by memory loss and mood deficits suggesting a potential role of sEVs in the brain response to stress and related stress-driven brain pathology. CONCLUSIONS: Overall, the spontaneous release method of sEV yield may contribute to the characterization and biomarker profile of physiologically relevant brain-derived sEVs in brain function and pathology. Video Abstract.

"Hit" to lead optimization and chemoinformatic studies for a new series of Autotaxin inhibitors.

Robust experimental evidence has highlighted the role of Autotaxin (ATX)/Lysophosphatidic acid (LPA) axis not only in the pathogenesis of chronic inflammatory conditions and especially in fibroproliferative diseases but also in several types of cancer. As a result, different series of substrate-, lipid-based and small-molecule ATX inhibitors have been identified thus far by both academia and pharma. The "crowning achievement" of these drug discovery campaigns was the development and entry of the first-in-class ATX inhibitor (ziritaxestat, GLPG-1690) in advanced clinical trials against idiopathic pulmonary fibrosis. Herein, the potency optimization efforts of a new series of Autotaxin inhibitors, namely 2-substituted-2,6-dihydro-4H-thieno[3,4-c]pyrazol-1-substituted amide, is described using a previously identified novel chemical scaffold as a "hit". The mode of inhibition of the most promising ATX inhibitors was investigated, while their cellular activity, aqueous solubility and cytotoxicity were evaluated. Our pharmacological results were corroborated by chemoinformatic tools (molecular docking and molecular dynamics simulations) deployed, to provide insight into the binding mechanism of the synthesized inhibitors to ATX.

A wide foodomics approach coupled with metagenomics elucidates the environmental signature of potatoes.

The term "terroir" has been widely employed to link differential geographic phenotypes with sensorial signatures of agricultural food products, influenced by agricultural practices, soil type, and climate. Nowadays, the geographical indications labeling has been developed to safeguard the quality of plant-derived food that is linked to a certain terroir and is generally considered as an indication of superior organoleptic properties. As the dynamics of agroecosystems are highly intricate, consisting of tangled networks of interactions between plants, microorganisms, and the surrounding environment, the recognition of the key molecular components of terroir fingerprinting remains a great challenge to protect both the origin and the safety of food commodities. Furthermore, the contribution of microbiome as a potential driver of the terroir signature has been underestimated. Herein, we present a first comprehensive view of the multi-omic landscape related to transcriptome, proteome, epigenome, and metagenome of the popular Protected Geographical Indication potatoes of Naxos.