Comprehensive in silico CpG methylation analysis in hepatocellular carcinoma identifies tissue- and tumor-type specific marks disconnected from gene expression.

DNA methylation is crucial for chromatin structure, transcription regulation and genome stability, defining cellular identity. Aberrant hypermethylation of CpG-rich regions is common in cancer, influencing gene expression. However, the specific contributions of individual epigenetic modifications to tumorigenesis remain under investigation. In hepatocellular carcinoma (HCC), DNA methylation alterations are documented as in other tumor types. We aimed to identify hypermethylated CpGs in HCC, assess their specificity across other tumor types, and investigate their impact on gene expression. To this end, public methylomes from HCC, other liver diseases, and 27 tumor types as well as expression data from TCGA-LIHC and GTEx were analyzed. This study identified 39 CpG sites that were hypermethylated in HCC compared to control liver tissue, and were located within promoter, gene bodies, and intergenic CpG islands. Notably, these CpGs were predominantly unmethylated in healthy liver tissue and other normal tissues. Comparative analysis with 27 other tumors revealed both common and HCC-specific hypermethylated CpGs. Interestingly, the HCC-hypermethylated genes showed minimal expression in the different healthy tissues, with marginal changes in the level of expression in the corresponding tumors. These findings confirm previous evidence on the limited influence of DNA hypermethylation on gene expression regulation in cancer. It also highlights the existence of mechanisms that allow the selection of tissue-specific methylation marks in normally unexpressed genes during carcinogenesis. Overall, our study contributes to demonstrate the complexity of cancer epigenetics, emphasizing the need of better understanding the interplay between DNA methylation, gene expression dynamics, and tumorigenesis.

Non-mitogenic FGF19 mRNA-based therapy for the treatment of experimental metabolic dysfunction-associated steatotic liver disease (MASLD).

Metabolic dysfunction-associated steatohepatitis (MASH) represents a global health threat. MASH pathophysiology involves hepatic lipid accumulation and progression to severe conditions like cirrhosis and, eventually, hepatocellular carcinoma. Fibroblast growth factor (FGF)-19 has emerged as a key regulator of metabolism, offering potential therapeutic avenues for MASH and associated disorders. We evaluated the therapeutic potential of non-mitogenic (NM)-FGF19 mRNA formulated in liver-targeted lipid nanoparticles (NM-FGF19-mRNAs-LNPs) in C57BL/6NTac male mice with diet-induced obesity and MASH (DIO-MASH: 40% kcal fat, 20% kcal fructose, 2% cholesterol). After feeding this diet for 21 weeks, NM-FGF19-mRNAs-LNPs or control (C-mRNA-LNPs) were administered (0.5 mg/kg, i.v.) weekly for another six weeks, in which diet feeding continued. NM-FGF19-mRNAs-LNPs treatment in DIO-MASH mice resulted in reduced body weight, adipose tissue depots, and serum transaminases, along with improved insulin sensitivity. Histological analyses confirmed the reversal of MASH features, including steatosis reduction without worsening fibrosis. NM-FGF19-mRNAs-LNPs reduced total hepatic bile acids (BAs) and changed liver BA composition, markedly influencing cholesterol homeostasis and metabolic pathways as observed in transcriptomic analyses. Extrahepatic effects included the down-regulation of metabolic dysfunction-associated genes in adipose tissue. This study highlights the potential of NM-FGF19-mRNA-LNPs therapy for MASH, addressing both hepatic and systemic metabolic dysregulation. NM-FGF19-mRNA demonstrates efficacy in reducing liver steatosis, improving metabolic parameters, and modulating BA levels and composition. Given the central role played by BA in dietary fat absorption, this effect of NM-FGF19-mRNA may be mechanistically relevant. Our study underscores the high translational potential of mRNA-based therapies in addressing the multifaceted landscape of MASH and associated metabolic perturbations.

Impact of Cryopreserved Placental Allografts on Biochemical Recurrence in Prostate Cancer.

BACKGROUND: Human placental allografts are widely used to promote wound healing. Placental (or amniotic membrane/umbilical cord) allografts are placed along the neurovascular bundles during radical prostatectomy to improve continence and erectile function recovery. It is unknown whether placental allografts impact biochemical recurrence (BCR). METHODS: This was a single-surgeon retrospective study of 566 robotic radical prostatectomies performed from April 2015 to March 2021. The patients were divided into three groups: the negative control, Brand A, and Brand B. Brand A and Brand B were both cryopreserved amniotic membrane (CAM) allografts. A total of 324 cases were included for BCR Kaplan-Meier and risk-adjusted multivariate analyses (362 for continence analysis). In vitro analyses were performed to determine the effect of CAM allografts on prostate cancer (PCa) cell line growth. RESULTS: For propensity score-matched analysis (adjusting for pre-operative PSA, tumor stage, Gleason Grade, and margin status), (1) the allograft groups did not show differences in time to BCR vs. the negative control group (p = 0.7), and (2) combined allograft treatment groups showed better continence recovery vs. the negative controls (p = 0.01). In vitro, placental allografts reduced PCa cell line growth in co-culture assays. CONCLUSIONS: cryopreserved AM allografts (combined or individual brands) did not show a significant effect on BCR but improved continence recovery for PCa patients.

Identification of PRMT5 as a therapeutic target in cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CCA) is a very difficult-to-treat cancer. Chemotherapies are little effective and response to immune checkpoint inhibitors is limited. Therefore, new therapeutic strategies need to be identified. OBJECTIVE: We characterised the enzyme protein arginine-methyltransferase 5 (PRMT5) as a novel therapeutic target in CCA. DESIGN: We evaluated the expression of PRMT5, its functional partner MEP50 and methylthioadenosine phosphorylase (MTAP)-an enzyme that modulates the sensitivity of PRMT5 to pharmacological inhibitors-in human CCA tissues. PRMT5-targeting drugs, currently tested in clinical trials for other malignancies, were assessed in human CCA cell lines and organoids, as well as in two immunocompetent CCA mouse models. Transcriptomic, proteomic and functional analyses were performed to explore the underlying antitumoural mechanisms. RESULTS: PRMT5 and MEP50 proteins were correlatively overexpressed in most CCA tissues. MTAP was absent in 25% of intrahepatic CCA. PRMT5-targeting drugs markedly inhibited CCA cell proliferation, synergising with cisplatin and gemcitabine and hindered the growth of cholangiocarcinoma organoids. PRMT5 inhibition blunted the expression of oncogenic genes involved in chromatin remodelling and DNA repair, consistently inducing the formation of RNA loops and promoting DNA damage. Treatment with PRMT5-targeting drugs significantly restrained the growth of experimental CCA without adverse effects and concomitantly induced the recruitment of CD4 and CD8 T cells to shrinking tumourous lesions. CONCLUSION: PRMT5 and MEP50 are frequently upregulated in human CCA, and PRMT5-targeting drugs have significant antitumoural efficacy in clinically relevant CCA models. Our findings support the evaluation of PRMT5 inhibitors in clinical trials, including their combination with cytotoxic and immune therapies.

Genomic and transcriptomic landscape of HER2-low breast cancer.

BACKGROUND: Novel agents have expanded the traditional HER2 definitions to include HER2-Low (HER2L) Breast Cancer (BC). We sought to evaluate the distinct molecular characteristics of HER2L BC to understand potential clinical/biologic factors driving resistance and clinical outcomes. METHODS: Retrospective analysis was performed on 13,613 BC samples, tested at Caris Life Sciences via NextGen DNA/RNA Sequencing. BC subtypes were defined by IHC/ISH. CODEai database was used to access clinical outcomes from insurance claims data. RESULTS: Overall, mutational landscape was similar between HER2L and classical subsets of HR+and HRneg cohorts. TP53 mutations were significantly higher in HRneg/HER2L group vs. HR+/HER2L tumors (p<0.001). A higher mutation rate of PIK3CA was observed in HRneg/HER2L tumors compared to TNBC subtype (p=0.016). PD-L1 positivity was elevated in HRneg/HER2L tumors compared to HR+/HER2L tumors, all p<0.01. Patients with HR+/HER2L tumors treated with CDK4/6 inhibitors had similar OS compared to pts with HR+/HER2-0 (HR=0.89, p=0.012). 27.2% of HR+/HER2L pts had activating PIK3CA mutations. Among HR+PIK3CA mutated tumors, HER2L pts treated with alpelisib showed no difference in OS vs. HER2-0 alpelisib-treated pts (HR=1.23, p=0.517). 13.9% of HER2L TNBC pts were PD-L1+. Interestingly, pts with PD-L1+ HER2L/HRneg (TNBC) treated with immune checkpoint inhibitors (ICI) showed improved OS than HER2-0 TNBC (HR=0.61, p=0.046). CONCLUSION: Our findings expand the understanding of the molecular profile of the HER2L subgroup and comparison to the classically defined breast cancer subgroups. Genomic risk assessments after progression on novel therapeutics can be assessed to better define implications for mechanisms of resistance.

Caspases compromise SLU7 and UPF1 stability and NMD activity during hepatocarcinogenesis.

Background & Aims: The homeostasis of the cellular transcriptome depends on transcription and splicing mechanisms. Moreover, the fidelity of gene expression, essential to preserve cellular identity and function is secured by different quality control mechanisms including nonsense-mediated RNA decay (NMD). In this context, alternative splicing is coupled to NMD, and several alterations in these mechanisms leading to the accumulation of aberrant gene isoforms are known to be involved in human disease including cancer. Methods: RNA sequencing, western blotting, qPCR and co-immunoprecipitation were performed in multiple silenced culture cell lines (replicates n ≥4), primary hepatocytes and samples of animal models (Jo2, APAP, Mdr2 -/- mice, n ≥3). Results: Here we show that in animal models of liver injury and in human HCC (TCGA, non-tumoral = 50 vs. HCC = 374), the process of NMD is inhibited. Moreover, we demonstrate that the splicing factor SLU7 interacts with and preserves the levels of the NMD effector UPF1, and that SLU7 is required for correct NMD. Our previous findings demonstrated that SLU7 expression is reduced in the diseased liver, contributing to hepatocellular dedifferentiation and genome instability during disease progression. Here we build on this by providing evidence that caspases activated during liver damage are responsible for the cleavage and degradation of SLU7. Conclusions: Here we identify the downregulation of UPF1 and the inhibition of NMD as a new molecular pathway contributing to the malignant reshaping of the liver transcriptome. Moreover, and importantly, we uncover caspase activation as the mechanism responsible for the downregulation of SLU7 expression during liver disease progression, which is a new link between apoptosis and hepatocarcinogenesis. Impact and implications: The mechanisms involved in reshaping the hepatocellular transcriptome and thereby driving the progressive loss of cell identity and function in liver disease are not completely understood. In this context, we provide evidence on the impairment of a key mRNA surveillance mechanism known as nonsense-mediated mRNA decay (NMD). Mechanistically, we uncover a novel role for the splicing factor SLU7 in the regulation of NMD, including its ability to interact and preserve the levels of the key NMD factor UPF1. Moreover, we demonstrate that the activation of caspases during liver damage mediates SLU7 and UPF1 protein degradation and NMD inhibition. Our findings identify potential new markers of liver disease progression, and SLU7 as a novel therapeutic target to prevent the functional decay of the chronically injured organ.

Cell-free and extracellular vesicle microRNAs with clinical utility for solid tumors.

As cutting-edge technologies applied for the study of body fluid molecular biomarkers are continuously evolving, clinical applications of these biomarkers improve. Diverse forms of circulating molecular biomarkers have been described, including cell-free DNA (cfDNA), circulating tumor cells (CTCs), and cell-free microRNAs (cfmiRs), although unresolved issues remain in their applicability, specificity, sensitivity, and reproducibility. Translational studies demonstrating the clinical utility and importance of cfmiRs in multiple cancers have significantly increased. This review aims to summarize the last 5 years of translational cancer research in the field of cfmiRs and their potential clinical applications to diagnosis, prognosis, and monitoring disease recurrence or treatment responses with a focus on solid tumors. PubMed was utilized for the literature search, following rigorous exclusion criteria for studies based on tumor types, patient sample size, and clinical applications. A total of 136 studies on cfmiRs in different solid tumors were identified and divided based on tumor types, organ sites, number of cfmiRs found, methodology, and types of biofluids analyzed. This comprehensive review emphasizes clinical applications of cfmiRs and summarizes underserved areas where more research and validations are needed.

Pilot study of plasma miRNA signature panel for differentiating single vs multiglandular parathyroid disease.

CONTEXT: The ability to differentiate sporadic primary hyperparathyroidism (sPHPT) caused by a single parathyroid adenoma (PTA) from multiglandular disease (MGD) pre-operatively, as well as definitely diagnose sPHPT in difficult patients, would enhance surgical decision making. OBJECTIVE: Identify miRNA (miR) signatures for MGD, single- and double-PTA, as well as cell-free miRNA (cfmiR) in plasma samples from patients with single-PTAs to use as biomarkers. DESIGN/SETTING/PATIENTS: 47 patients with sPHPT (single-PTA n=32, double-PTA n=12, MGD n=9). Pre-operative plasma samples from 16 single-PTA and 29 normal healthy donors (NHD). INTERVENTION: All specimens were processed and analyzed for 2,083 miRs using HTG EdgeSeq miR whole transcriptome assay and normalized using DESeq2 to identify differentially expressed (DE) miRs. MiR classifiers were identified using Random Forest. MAIN OUTCOME MEASURES: ROC curves and AUC. RESULTS: MiR signatures distinguished normal parathyroid from MGD and PTA as well as MGD from PTA in tissue samples. Common miRs were found in the single-PTA and double-PTAs. Data integration identified a 27-miR signature in single-PTA tissue samples compared to the rest of the tissue samples. In plasma samples analysis, significant cfmiRs were DE in single-PTA patients compared to NHD. Of those, only 9 miRNAs/cfmiRs were found DE in both tissue and plasma samples from patients diagnosed with a single-PTA (AUC=76%). CONCLUSIONS: Twenty-seven miRs were consistently found DE in single-PTA tissue and plasma samples. Data integration showed a 9-cfmiR signature with potential clinical utility to pre-operatively diagnose sPHPT caused by a single-PTA, which could decrease more invasive parathyroid explorations.

The Search for Risk, Diagnostic, and Prognostic Biomarkers of Cholangiocarcinoma and Their Biological and Clinicopathologic Significance.

Cholangiocarcinomas (CCAs) are a heterogeneous group of malignant tumors that originate from the biliary tract. They are usually diagnosed in advanced stages, leading to a dismal prognosis for affected patients. As CCA often arises as a sporadic cancer in individuals lacking specific risk factors or with heterogeneous backgrounds, and there are no defined high-risk groups, the implementation of effective surveillance programs for CCA is problematic. The identification and validation of new biomarkers useful for risk stratification, diagnosis, prognosis, and prediction of treatment response remains an unmet need for patients with CCA, even though numerous studies have been conducted lately to try to discover and validate CCA biomarkers. In this review, we overview the available information about the different types of biomarkers that have been investigated in recent years using minimally invasive biospecimens (blood, serum/plasma, bile, and urine) and their potential usefulness in diagnosis, prognosis, and risk stratification. It is widely accepted that early detection of CCA will impact patients' outcomes, by improving survival rates, quality of life, and the possibility of less invasive and/or curative treatments; however, challenges to its translation and clinical application for patients with CCA need to be resolved.

Application of Graph Models to the Identification of Transcriptomic Oncometabolic Pathways in Human Hepatocellular Carcinoma.

Whole-tissue transcriptomic analyses have been helpful to characterize molecular subtypes of hepatocellular carcinoma (HCC). Metabolic subtypes of human HCC have been defined, yet whether these different metabolic classes are clinically relevant or derive in actionable cancer vulnerabilities is still an unanswered question. Publicly available gene sets or gene signatures have been used to infer functional changes through gene set enrichment methods. However, metabolism-related gene signatures are poorly co-expressed when applied to a biological context. Here, we apply a simple method to infer highly consistent signatures using graph-based statistics. Using the Cancer Genome Atlas Liver Hepatocellular cohort (LIHC), we describe the main metabolic clusters and their relationship with commonly used molecular classes, and with the presence of TP53 or CTNNB1 driver mutations. We find similar results in our validation cohort, the LIRI-JP cohort. We describe how previously described metabolic subtypes could not have therapeutic relevance due to their overall downregulation when compared to non-tumoral liver, and identify N-glycan, mevalonate and sphingolipid biosynthetic pathways as the hallmark of the oncogenic shift of the use of acetyl-coenzyme A in HCC metabolism. Finally, using DepMap data, we demonstrate metabolic vulnerabilities in HCC cell lines.

Sweet dreams could be made of this: carbohydrate-responsive element-binding protein (ChREBP) as a target for hepatocellular carcinoma therapy.

Rewiring of cellular metabolism is now fully recognized as a hallmark of cancer. Tumor cells reprogram metabolic pathways to meet the energetic and macromolecular demands to support unrestricted growth and survival under unfavorable conditions. It is becoming apparent that these adaptations underpin most of the traits that define a cancer cell's identity, including the ability to avoid immune surveillance, endure nutrient and oxygen restrictions, detach and migrate from their natural histological niche, and avert human-made aggressions (i.e., therapy). In a recent study, Benichou and collaborators identify carbohydrate-responsive element-binding protein (ChREBP), a master regulator of physiological glucose metabolism, as an oncogene in hepatocellular carcinoma (HCC) development. Upregulation of ChREBP expression results in a self-stimulatory loop interconnecting PI3K/AKT signaling and glucose metabolism to feed fatty acid and nucleotide synthesis supporting tumorigenesis. Importantly, pharmacological inhibition of ChREBP activity quells in vivo HCC tumor growth without causing systemic toxicity. This study identifies novel oncometabolic pathways and open up new avenues to improve the treatment of a deadly tumor.

Interruption of the intratumor CD8+ T cell:Treg crosstalk improves the efficacy of PD-1 immunotherapy.

PD-1 blockade unleashes potent antitumor activity in CD8+ T cells but can also promote immunosuppressive T regulatory (Treg) cells, which may worsen the response to immunotherapy. Tumor-Treg inhibition is a promising strategy to improve the efficacy of checkpoint blockade immunotherapy; however, our understanding of the mechanisms supporting tumor-Tregs during PD-1 immunotherapy is incomplete. Here, we show that PD-1 blockade increases tumor-Tregs in mouse models of melanoma and metastatic melanoma patients. Mechanistically, Treg accumulation is not caused by Treg-intrinsic inhibition of PD-1 signaling but depends on an indirect effect of activated CD8+ T cells. CD8+ T cells produce IL-2 and colocalize with Tregs in mouse and human melanomas. IL-2 upregulates the anti-apoptotic protein ICOS on tumor-Tregs, promoting their accumulation. Inhibition of ICOS signaling before PD-1 immunotherapy improves control over immunogenic melanoma. Thus, interrupting the intratumor CD8+ T cell:Treg crosstalk represents a strategy to enhance the therapeutic efficacy of PD-1 immunotherapy.

Ribosome-Associated Vesicles promote activity-dependent local translation.

Local protein synthesis in axons and dendrites underpins synaptic plasticity. However, the composition of the protein synthesis machinery in distal neuronal processes and the mechanisms for its activity-driven deployment to local translation sites remain unclear. Here, we employed cryo-electron tomography, volume electron microscopy, and live-cell imaging to identify Ribosome-Associated Vesicles (RAVs) as a dynamic platform for moving ribosomes to distal processes. Stimulation via chemically-induced long-term potentiation causes RAV accumulation in distal sites to drive local translation. We also demonstrate activity-driven changes in RAV generation and dynamics in vivo, identifying tubular ER shaping proteins in RAV biogenesis. Together, our work identifies a mechanism for ribosomal delivery to distal sites in neurons to promote activity-dependent local translation.

Strategies to enhance the response of liver cancer to pharmacological treatments.

In contrast to other types of cancers, there is no available efficient pharmacological treatment to improve the outcomes of patients suffering from major primary liver cancers, i.e., hepatocellular carcinoma and cholangiocarcinoma. This dismal situation is partly due to the existence in these tumors of many different and synergistic mechanisms of resistance, accounting for the lack of response of these patients, not only to classical chemotherapy but also to more modern pharmacological agents based on the inhibition of tyrosine kinase receptors (TKIs) and the stimulation of the immune response against the tumor using immune checkpoint inhibitors (ICIs). This review summarizes the efforts to develop strategies to overcome this severe limitation, including searching for novel drugs derived from synthetic, semisynthetic, or natural products with vectorial properties against therapeutic targets to increase drug uptake or reduce drug export from cancer cells. Besides, immunotherapy is a promising line of research that is already starting to be implemented in clinical practice. Although less successful than in other cancers, the foreseen future for this strategy in treating liver cancers is considerable. Similarly, the pharmacological inhibition of epigenetic targets is highly promising. Many novel "epidrugs," able to act on "writer," "reader," and "eraser" epigenetic players, are currently being evaluated in preclinical and clinical studies. Finally, gene therapy is a broad field of research in the fight against liver cancer chemoresistance, based on the impressive advances recently achieved in gene manipulation. In sum, although the present is still dismal, there is reason for hope in the non-too-distant future.

Exploring current and emerging therapies for porphyrias.

Porphyrias are rare, mostly inherited disorders resulting from altered activity of specific enzymes in the haem synthesis pathway that lead to accumulation of pathway intermediates. Photocutaneous symptoms occur when excess amounts of photoreactive porphyrins circulate in the blood to the skin, whereas increases in potentially neurotoxic porphyrin precursors are associated with neurovisceral symptoms. Current therapies are suboptimal and their mechanisms are not well established. As described here, emerging therapies address underlying disease mechanisms by introducing a gene, RNA or other specific molecule with the potential to cure or slow progression of the disease. Recent progress in nanotechnology and nanoscience, particularly regarding particle design and formulation, is expanding disease targets. More secure and efficient drug delivery systems have extended our toolbox for transferring specific molecules, especially into hepatocytes, and led to proof-of-concept studies in animal models. Repurposing existing drugs as molecular chaperones or haem synthesis inhibitors is also promising. This review summarizes key examples of these emerging therapeutic approaches and their application for hepatic and erythropoietic porphyrias.

The Genomic, Transcriptomic, and Immunologic Landscape of HRAS Mutations in Solid Tumors.

Tipifarnib is the only targeted therapy breakthrough for HRAS-mutant (HRASmt) recurrent or metastatic head and neck squamous cell carcinoma (HNSCC). The molecular profiles of HRASmt cancers are difficult to explore given the low frequency of HRASmt. This study aims to understand the molecular co-alterations, immune profiles, and clinical outcomes of 524 HRASmt solid tumors including urothelial carcinoma (UC), breast cancer (BC), non-small-cell lung cancer (NSCLC), melanoma, and HNSCC. HRASmt was most common in UC (3.0%), followed by HNSCC (2.82%), melanoma (1.05%), BC (0.45%), and NSCLC (0.44%). HRASmt was absent in Her2+ BC regardless of hormone receptor status. HRASmt was more frequently associated with squamous compared to non-squamous NSCLC (60% vs. 40% in HRASwt, p = 0.002). The tumor microenvironment (TME) of HRASmt demonstrated increased M1 macrophages in triple-negative BC (TNBC), HNSCC, squamous NSCLC, and UC; increased M2 macrophages in TNBC; and increased CD8+ T-cells in HNSCC (all p < 0.05). Finally, HRASmt was associated with shorter overall survival in HNSCC (HR: 1.564, CI: 1.16-2.11, p = 0.003) but not in the other cancer types examined. In conclusion, this study provides new insights into the unique molecular profiles of HRASmt tumors that may help to identify new targets and guide future clinical trial design.

Refinement of paramagnetic bead-based digestion protocol for automatic sample preparation using an artificial neural network.

Despite technological advances in the proteomics field, sample preparation still represents the main bottleneck in mass spectrometry (MS) analysis. Bead-based protein aggregation techniques have recently emerged as an efficient, reproducible, and high-throughput alternative for protein extraction and digestion. Here, a refined paramagnetic bead-based digestion protocol is described for Opentrons® OT-2 platform (OT-2) as a versatile, reproducible, and affordable alternative for the automatic sample preparation for MS analysis. For this purpose, an artificial neural network (ANN) was applied to maximize the number of peptides without missed cleavages identified in HeLa extract by combining factors such as the quantity (µg) of trypsin/Lys-C and beads (MagReSyn® Amine), % (w/v) SDS, % (v/v) acetonitrile, and time of digestion (h). ANN model predicted the optimal conditions for the digestion of 50 µg of HeLa extract, pointing to the use of 2.5% (w/v) SDS and 300 µg of beads for sample preparation and long-term digestion (16h) with 0.15 µg Lys-C and 2.5 µg trypsin (≈1:17 ratio). Based on the results of the ANN model, the manual protocol was automated in OT-2. The performance of the automatic protocol was evaluated with different sample types, including human plasma, Arabidopsis thaliana leaves, Escherichia coli cells, and mouse tissue cortex, showing great reproducibility and low sample-to-sample variability in all cases. In addition, we tested the performance of this method in the preparation of a challenging biological fluid such as rat bile, a proximal fluid that is rich in bile salts, bilirubin, cholesterol, and fatty acids, among other MS interferents. Compared to other protocols described in the literature for the extraction and digestion of bile proteins, the method described here allowed identify 385 unique proteins, thus contributing to improving the coverage of the bile proteome.

New insights into the regulation of bile acids synthesis during the early stages of liver regeneration: A human and experimental study.

BACKGROUND AND AIMS: Liver regeneration is essential for the preservation of homeostasis and survival. Bile acids (BAs)-mediated signaling is necessary for liver regeneration, but BAs levels need to be carefully controlled to avoid hepatotoxicity. We studied the early response of the BAs-fibroblast growth factor 19 (FGF19) axis in healthy individuals undergoing hepatectomy for living donor liver transplant. We also evaluated BAs synthesis in mice upon partial hepatectomy (PH) and acute inflammation, focusing on the regulation of cytochrome-7A1 (CYP7A1), a key enzyme in BAs synthesis from cholesterol. METHODS: Serum was obtained from twelve human liver donors. Mice underwent 2/3-PH or sham-operation. Acute inflammation was induced with bacterial lipopolysaccharide (LPS) in mice fed control or antoxidant-supplemented diets. BAs and 7α-hydroxy-4-cholesten-3-one (C4) levels were measured by HPLC-MS/MS; serum FGF19 by ELISA. Gene expression and protein levels were analyzed by RT-qPCR and western-blot. RESULTS: Serum BAs levels increased after PH. In patients with more pronounced hypercholanemia, FGF19 concentrations transiently rose, while C4 levels (a readout of CYP7A1 activity) dropped 2 h post-resection in all cases. Serum BAs and C4 followed the same pattern in mice 1 h after PH, but C4 levels also dropped in sham-operated and LPS-treated animals, without marked changes in CYP7A1 protein levels. LPS-induced serum C4 decline was attenuated in mice fed an antioxidant-supplemented diet. CONCLUSIONS: In human liver regeneration FGF19 upregulation may constitute a protective response from BAs excess during liver regeneration. Our findings suggest the existence of post-translational mechanisms regulating CYP7A1 activity, and therefore BAs synthesis, independent from CYP7A1/Cyp7a1 gene transcription.