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The study reported in this paper tested the hypothesis that the developmental and reproductive health of mosquitofish (Gambusia affinis) exposed to pulp and paper effluent in the Dengcun River would differ from that of mosquitofish living in a reference site. We also studied whether morphological characteristics such as the anal fin and hemal spines of mosquitofish could serve as indicators for evaluating the androgenic effect and mosquitofish population security in the Dengcun River. Male and female mosquitofish were captured at three sites contaminated by pulp and paper effluent in the Dengcun River in Sihui, South China, and at a nearby uncontaminated reference site. Samples were collected from the sampling sites on the same day in August 2009. We compared the populations by total length, wet body and liver mass, gonad mass, and population composition. We also compared the populations according to number of anal fin segments, oocyte and embryo count, anal fin and hemal spine morphology among females, and by sperm count and viability among males, and observed the gonadal and liver histology of both males and females. Female mosquitofish exposed to pulp and paper effluent in the Dengcun River were generally smaller in length and mass, had a greater number of anal fin segments and more embryos, but had significantly fewer oocytes in comparison with those living at the reference site. The higher number of anal fin ray 3 segments and the increased ray 4:6 length ratio observed among fish taken from the Dengcun River sites indicated that they might be subject to the androgenic effect. Furthermore, the significantly different hemal spine morphology of the effluent-affected females also indicated the pulp and paper mills effluents in Dengcun River might contain androgenic substance(s). Male mosquitofish at the sites exposed to effluent had a higher number of anal fin segments and greater testis mass in comparison with those living at the reference site. No evidence of intersex was found in either males or females, although histopathological tests on females revealed histologic abnormalities in the liver and gonads. It can be concluded that pulp and paper effluent contamination in the Dengcun River has affected a number of developmental parameters and reproductive characteristics in mosquitofish, with possible adverse effects on reproduction in this population.

The common bully (Gobiomorphus cotidianus), a smallbodied New Zealand native fish species, was used to monitor population impacts of multiple effluents in the Tarawera River, New Zealand. In an initial survey, the absence of reproductive development at the expected spawning time for common bully was observed in a population downstream of effluent discharges. Subsequently, we examined the hypotheses that the observed changes were due to effluent exposure, migratory patterns, or genetic differences between populations. Liver detoxification enzyme activity and stable isotopes provided evidence against upstream migration of sexually mature bully. The observed presence of developed gonads in the downstream population during winter season resulted in the rejection of the hypothesis that reproductive failure was due to effluent exposure, and it was concluded that there were substantial differences in reproductive timing. Genetic analyses of two upstream, one downstream, and one population from a nearby coastal river indicated the upstream (reference) and downstream (effluent exposed) bully in the river formed genetically distinct populations. The identification of a nearby river population with similar reproductive timing and high genetic similarity to the effluent-exposed population suggests that the observed differences in the genetics of the downstream population were not caused by effluent exposure. The genetic analysis did highlight the lack of donwstream dispersion and gene flow in the river which could possibly be related to anthropogenic stress.

Rodent liver tumor formation can be promoted by certain barbiturates and this may involve their ability to inhibit hepatocyte gap junctional intercellular communication (GJIC). In order to address the mechanisms and specificity of action of barbiturates on hepatocyte gap junctions, we have compared the effects of liver tumor-promoting barbiturates (phenobarbital, sodium barbital and amobarbital: PB, SB and AB, respectively) and a non-liver tumor-promoting barbiturate (barbituric acid: BA) on primary cultured rat hepatocyte GJIC and connexin32 (Cx32) expression after short (1-24 h) and long (2-14 days) treatment. GJIC was evaluated by fluorescent dye microinjection (dye-coupling); Cx32 expression was monitored by Northern blot, Western blot and immunohistochemistry. Both parameters were maintained at high levels over 14 days by coculture of the cells with WB-F344 rat liver epithelial cells in the presence of dexamethasone. Treatment with PB (2 mM) for 1 h sharply reduced dye-coupling from approximately 90-30%, but the cells fully recovered by 24 h. No inhibition was seen with the other barbiturates over this 1-day treatment period. Longer treatments (2-14 days) with the promoters PB, SB and AB, however, gradually reduced hepatocyte dye-coupling to approximately 30-50%. The non-promoter, BA, did not affect hepatocyte GJIC. These decreases in hepatocyte dye-coupling occurred without changes in Cx32 or gap junction expression. Dye-coupling of WB-F344 cells and expression of their predominant gap junction protein, connexin43 (Cx43), were also not affected. Thus, the inhibition of GJIC was specific to liver tumor promoting barbiturates in hepatocytes, was time-dependent and was not due to altered Cx32 expression.

A rapid filter paper disk assay for cyclic AMP based on the activation of a cyclic AMP dependent protein kinase from rabbit skeletal muscle is described. This procedure permits determination of as little as 0.2 picomoles of cyclic AMP. In addition this procedure employs ATP-γ-33P instead of ATP- γ-32P.

Gli autori hanno studiato il comportamento delle proteine e delle glicoproteine solubili del fegato rigenerante di ratto mediante l'elettroforesi su carta. La colorazione con Amidoschwarz mostra che il comportamento elettroforetico delle proteine di fegato rigenerante e praticamente sovrapponibile a quello del fegato normale. La colorazione secondoKoiw eGronwall dimostra che le glicoproteine diminuiscono nei primi giorni dopo l'atto operatorio, tornando pero alla normalita circa 30 giorni dopo l'epatectomia.

Viene studiato il comportamento elettroforetico su carta delle proteine cellulari di fegato normale ed in degenerazione vacuolare e grassa. Nei ferogrammi di fegati in degenerazione vacuolare si ha la comparsa di una banda di tipo albuminico, che non compare nei ferogrammi normali. I ferogrammi di fegati in degenerazione grassa si rivelano piu semplici di quelli normali per la mancanza di una banda di tipo globulinico. Questi risultati vengono brevemente discussi.

Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent chronic liver disease, and patient susceptibility to its onset and progression is influenced by several factors. In this study, we investigated whether altered hepatic DNA methylation in liver tissue correlates with the degree of severity of NAFLD-like liver injury induced by a high-fat and high-sucrose (HF/HS) diet in Collaborative Cross (CC) mice. Using genome-wide targeted bisulphite DNA methylation next-generation sequencing, we found that mice with different non-alcoholic fatty liver (NAFL) phenotypes could be distinguished by changes in hepatic DNA methylation profiles. Specifically, NAFL-prone male CC042 mice exhibited more prominent DNA methylation changes compared with male CC011 mice and female CC011 and CC042 mice that developed only a mild NAFL phenotype. Moreover, these mouse strains demonstrated different patterns of DNA methylation. While the HF/HS diet induced both DNA hypomethylation and DNA hypermethylation changes in all the mouse strains, the NAFL-prone male CC042 mice demonstrated a global predominance of DNA hypermethylation, whereas a more pronounced DNA hypomethylation pattern developed in the mild-NAFL phenotypic mice. In a targeted analysis of selected genes that contain differentially methylated regions (DMRs), we identified NAFL phenotype-associated differences in DNA methylation and gene expression of the Apoa4, Gls2, and Apom genes in severe NAFL-prone mice but not in mice with mild NAFL phenotypes. These changes in the expression of Apoa4 and Gls2 coincided with similar findings in a human in vitro cell model of diet-induced steatosis and in patients with NAFL. These results suggest that changes in the expression and DNA methylation status of these three genes may serve as a set of predictive markers for the development of NAFLD.

As a common cause of liver injury, hepatic ischemia/reperfusion injury (HIRI) happens in various clinical conditions including trauma, hepatectomy and liver transplantation. Since transcription factor Yin Yang 1 (YY1) was reported to be downregulated after ischemia/reperfusion (I/R) injury, we focused on YY1 to explore its function in HIRI by functional assays like Cell Counting Kit-8 (CCK-8) assays and flow cytometry assays. The RT-qPCR assay revealed that YY1 was downregulated in hepatocytes after I/R injury. The function assays disclosed that YY1 facilitated cell viability and proliferation, but hindered cell apoptosis in hepatocytes after I/R injury. Through mechanism assays including luciferase reporter assay, RIP and RNA pulldown assay, miR-186-5p was found to bind with YY1 and promote hepatocyte apoptosis by targeting YY1. Subsequently, we verified that small nucleolar RNA host gene 1 (SNHG1) could sponge miR-186-5p to upregulate YY1. Importantly, we figured out that YY1 had a positive regulation on SNHG1. Along the way, YY1 was identified as the upstream transcription factor for SNHG1. In conclusion, our study unveiled a novel competing endogenous RNA (ceRNA) pattern of SNHG1/miR-186-5p/YY1 positive feedback loop in hepatic I/R injury, which might provide new insight into prevention of HIRI during liver transplantation or hepatic surgery.

Autoimmune hepatitis (AIH) is an immune-mediated chronic inflammatory liver disease, and its pathogenesis is not fully understood. Our previous study discovered that receptor interacting protein kinase 3 (RIP3) is correlated with serum transaminase levels in AIH patients. However, its role and underlying mechanism in AIH are poorly understood. Here, we detected the increased expression and activation of RIP3 in livers of patients and animal models with AIH. The inhibition of RIP3 kinase by GSK872 prevented concanavalin A (ConA)-induced immune-mediated hepatitis (IMH) by reduced hepatic proinflammatory cytokines and immune cells including Th17 cells and macrophages. Further experiments revealed that RIP3 inhibition resulted in an increase in CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs) with immunoregulatory properties in the liver, spleen, and peripheral blood. Moreover, the depletion of Gr-1+ MDSCs abrogated the protective effect and immune suppression function of GSK872 in ConA-induced IMH. Altogether, our data demonstrate that RIP3 blockade prevents ConA-induced IMH through promoting MDSCs infiltration. Inhibition of RIP3 kinase may be a novel therapeutic avenue for AIH treatment.

Assessment and enhancement of liver clearance are promising strategies for protection of liver from various liver diseases. Iron-tannic nanoparticles (FTs) were previously considered as imageable autophagic enhancers with biodegradation potential. Herein, we present a new approach for utilizing Iron-tannic nanoparticles (FTs) as a tool for imaging and increasing liver clearance. Pharmacokinetic profiling suggested that FTs were initially found in blood circulation and thereafter were distributed to the liver. By using MR imaging (T1 weighted), maximum MRI signal enhancement was found to occur after 30 minutes post-injection (i.v.) and gradually decreased afterward. Decreasing MRI signal may be due to FTs metabolism by the liver. By assessing imaging-derived pharmacokinetics, we can simply determine the rate constant of liver degradation of FTs. Potentially, we might use this parameter to monitor liver function, where its clearance is of concern. Once functional implication of FTs in liver clearance was investigated, FTs were found to induce hepatocyte autophagy along with activation of lysosomes. Consequently, the hepatocytes were capable of efficiently clearing cellular debris. From these results, it is clear that FTs should be considered as a molecular tool for quantitative MRI-derived liver function assessment, and for enhancing clearance function in liver parenchyma. Hopefully, our findings will pave the way to develop new strategies for non-invasive assessment and enhancement of liver clearance.

Perfluorooctanoic acid (PFOA) can accumulate in the livers of humans and animals via the food chain, resulting into liver injury, which is closely related to intestinal flora dysbiosis. Gastrodin has been reported to have hepatoprotective effect. However, whether gastrodin can alleviate PFOA-induced liver injury via modulating gut microbiota remains unclear. Herein, a PFOA-induced liver injury model was established by gavage of PFOA (5 mg/kg body weight) in 2% Tween 80 solution once daily for 6 weeks in mice, and then gastrodin in saline (20 mg/kg body weight) was used once daily for 8 weeks to treat liver damage. The biochemical indexes associated with liver function, oxidative stress, and inflammatory factors were examined. Hematoxylin-eosin staining was used to determine the liver histopathological changes. Besides, 16S rRNA sequencing was used to analyze the difference of gut microbiota between the model and treatment groups. The results showed that gastrodin significantly improved the oxidative stress caused by PFOA. Intestinal flora analysis showed that gastrodin treatment significantly increased the relative abundance of probiotics, such as Lactobacillus, Bifidobacterium, and Bacteroides, while the harmful bacteria, including Desulfovibrio were decreased. Gastrodin treatment also significantly increased the level of short-chain fatty acids (SCFAs), such as butyric acid and isobutyric acid. Spearman correlation analysis showed that the composition changes of gut microbiota and SCFAs increase were both beneficial to alleviate the liver injury caused by PFOA. To sum up, gastrodin can effectively alleviate PFOA-induced liver injury through regulating gut microbiota composition.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, and chemoprevention represents a feasible treatment to reduce the mortality of this carcinoma. Mulberry fruit polysaccharides (MFP) possess immunoregulatory and anti-inflammatory effects, which have been reported to alleviate liver damage evoked by CCl4 or alcohol in previous reports. However, its chemopreventive effect against liver carcinogenesis is insufficient. The present study was aimed to investigate the possible role of MFP as a pro-apoptosis, and anti-inflammatory agent to possess its chemoprevention property. Hepatocarcinogenesis was induced by diethylnitrosamine/phenobarbital (DEN/PB) for 14 weeks. The DEN/PB-administered rats were co-treated with different doses of MFP (50 or 100 mg/kg body weight) by oral gavage for 14 weeks. Basic hepatic function indexes (AST, ALT, ALP, GGT, total bilirubin, and albumin), and hepatic tumor biomarkers (AFP, CEA, and CA19.9), together with histological assessment were performed. Besides, the hepatic apoptosis markers (Bcl-2, Bax, caspase3, and caspase9), inflammation markers (IL-1β, TNF-α, and NF-κB), and mutT homologue gene 1 (MTH1) were examined. Oral gavage of MFP inhibited the elevations of hepatic function indexes and hepatic tumor biomarkers and alleviated pathological changes in hepatic tissue. In addition, the hepatic apoptosis markers, inflammation markers, and the mRNA level of MTH1 were abnormal in DEN/PB group, which were reversed by MFP treatment. In conclusion, MFP is an effective agent that provides chemoprevention against DEN/PB-evoked hepatocarcinogenesis via inhibition of inflammation and induction of apoptosis.

to explore the value of transcatheter arterial chemoembolization (TACE) combined with targeted nanoparticle delivery system for sorafenib (SFB) to treat hepatocellular carcinoma (HCC) with microvascular invasion. 42 HCC patients with microvascular invasion after liver cancer surgery were selected from our hospital from December 2020 and February 2021. Patients were divided into experimental group and control group based on their willingness. Patients in experimental group (18 cases) were treated with combination therapy of TACE and Ab-SFB-NP system; while patients in control group (24 cases) took TACE and non-nano drug delivery system. There was no obvious difference in liver function and blood test results between two groups of patients before treatment and one month after treatment (P > 0.05). Three months after treatment, differences of alanine aminotransferase (ALT) were statistically significant (P 0.05). The disease control rate (DCR) of patients in experimental group was higher slightly (P > 0.05). The incidence of adverse reactions of patients in experimental group was lower than the control group and the differences were statistically significant (P

Purpose To build a physiologically based pharmacokinetic (PBPK) model of the clinical OATP1B1/OATP1B3/BCRP victim drug rosuvastatin for the investigation and prediction of its transporter-mediated drug-drug interactions (DDIs). Methods The Rosuvastatin model was developed using the open-source PBPK software PK-Sim®, following a middle-out approach. 42 clinical studies (dosing range 0.002–80.0 mg), providing rosuvastatin plasma, urine and feces data, positron emission tomography (PET) measurements of tissue concentrations and 7 different rosuvastatin DDI studies with rifampicin, gemfibrozil and probenecid as the perpetrator drugs, were included to build and qualify the model. Results The carefully developed and thoroughly evaluated model adequately describes the analyzed clinical data, including blood, liver, feces and urine measurements. The processes implemented to describe the rosuvastatin pharmacokinetics and DDIs are active uptake by OATP2B1, OATP1B1/OATP1B3 and OAT3, active efflux by BCRP and Pgp, metabolism by CYP2C9 and passive glomerular filtration. The available clinical rifampicin, gemfibrozil and probenecid DDI studies were modeled using in vitro inhibition constants without adjustments. The good prediction of DDIs was demonstrated by simulated rosuvastatin plasma profiles, DDI AUClast ratios (AUClast during DDI/AUClast without co-administration) and DDI Cmax ratios (Cmax during DDI/Cmax without co-administration), with all simulated DDI ratios within 1.6-fold of the observed values. Conclusions A whole-body PBPK model of rosuvastatin was built and qualified for the prediction of rosuvastatin pharmacokinetics and transporter-mediated DDIs. The model is freely available in the Open Systems Pharmacology model repository, to support future investigations of rosuvastatin pharmacokinetics, rosuvastatin therapy and DDI studies during model-informed drug discovery and development (MID3).

Aim: In the late stage of atherosclerosis, the endothelial barrier of plaque is destroyed. The rapid deposition of oxidized lipids in the circulation leads to migration of numerous smooth muscle cells and macrophages, as well as foaming necrosis. The plaque progresses rapidly, and vulnerable plaques can easily induce adverse cardiovascular events. Here, we take the principle of gene editing to transfer the liver to express the LOX-1 receptor which is more sensitive to Ox-LDL by using AAV8 containing a liver-specific promoter. In this way, we want to explore whether the progress of advanced atherosclerosis and the stability of advanced plaque can be improved when the liver continues to clear Ox-LDL from the circulation. Methods and Results: In order to explore the effect of the physiological and continuous elimination of Ox-LDL through the liver on advanced atherosclerosis, we chose ApoE-/- mice in high-fat diet for 20 weeks. After 16 weeks of high-fat diet, the baseline group was sacrificed and the specimens were collected. The virus group and the control group were injected with the same amount of virus dilution and normal saline through the tail vein, and continued to feed until 20 weeks of high-fat diet, and then sacrificed to collect specimens. The results showed that LOX-1 was ectopically and functionally expressed in the liver as an Ox-LDL receptor, reducing the content of it in circulation. Compared with the control group, the degree of plaque progression in the virus group was significantly reduced, similar to the baseline group, the plaque necrosis core decreased, and the collagen fiber content increased. In addition, there are more contractile smooth muscle cells in the plaques of the virus group instead of synthetic ones, and the content of macrophages was also reduced. These data suggested that the virus group mice have greatly increased advanced plaque stability compared with the control group mice. Conclusions: Due to the destruction of endothelial barrier in advanced plaques, rapid deposition of Ox-LDL can result in fast plaque progression, increased necrotic cores, and decreased stability. Our research shows that the use of AAV8 through gene editing allows the liver to express LOX-1 receptors that are more sensitive to Ox-LDL, so that it can continue to bind Ox-LDL in the circulation and exploit the liver's strong lipid metabolism ability to physiologically clear Ox-LDL, which can inhibit the rapid progress of advanced plaque and increase the stability of plaque.

Rationale: Hepatitis B x protein (HBx) is required to initiate and maintain the replication of hepatitis B virus (HBV). Protein arginine methyltransferases 5 (PRMT5) negatively regulates HBV transcription. WD repeat domain 77 protein (WDR77) greatly enhances the methyltransferase activity of PRMT5. However, the role of WDR77 in the modulation of cccDNA transcription and HBV replication is poorly understood. In this study, we investigated the mechanism by which HBx modulated HBV replication involving WDR77 in the liver. Methods: A human liver-chimeric mouse model was established. Immunohistochemistry (IHC) staining, Western blot analysis, Southern blot analysis, Northern blot analysis, immunofluorescence assays, ELISA, RT-qPCR, CoIP assays, and ChIP assays were performed in human liver-chimeric mouse model, primary human hepatocytes (PHHs), HepG2-NTCP, dHepaRG and HepG2 cell lines. Results: HBV infection and HBx expression remarkably reduced the protein levels of WDR77 in human liver-chimeric mice and HepG2-NTCP cells. WDR77 restricted cccDNA transcription and HBV replication in PHHs and HepG2-NTCP cells. Mechanically, WDR77 enhanced PRMT5-triggered symmetric dimethylation of arginine 3 on H4 (H4R3me2s) on the cccDNA minichromosome to control cccDNA transcription. HBx drove the cellular DDB1-containing E3 ubiquitin ligase to degrade WDR77 through recruiting WDR77, leading to the disability of methyltransferase activity of PRMT5. Thus, HBx promoted HBV replication by driving a positive feedback loop of HBx-DDB1/WDR77/PRMT5/H4R3me2s/cccDNA/HBV/HBx in the liver. Conclusions: HBx attenuates the WDR77-mediated HBV repression by driving DDB1-induced WDR77 degradation in the liver. Our finding provides new insights into the mechanism by which HBx enhances HBV replication in the liver.

Background: Efficient and specific induction of cell death in liver cancer is urgently needed. In this study, we aimed to design an exosome-based platform to deliver ferroptosis inducer (Erastin, Er) and photosensitizer (Rose Bengal, RB) into tumor tissues with high specificity. Methods: Exosome donor cells (HEK293T) were transfected with control or CD47-overexpressing plasmid. Exosomes were isolated and loaded with Er and RB via sonication method. Hepa1-6 cell xenograft C57BL/6 model was injected with control and engineered exosomes via tail vein. In vivo distribution of the injected exosomes was analyzed via tracking the fluorescence labeled exosomes. Photodynamic therapy was conducted by 532 nm laser irradiation. The therapeutic effects on hepatocellular carcinoma and toxic side-effects were systemically analyzed. Results: CD47 was efficiently loaded on the exosomes from the donor cells when CD47 was forced expressed by transfection. CD47 surface functionalization (ExosCD47) made the exosomes effectively escape the phagocytosis of mononuclear phagocyte system (MPS), and thus increased the distribution in tumor tissues. Erastin and RB could be effectively encapsulated into exosomes after sonication, and the drug-loaded exosomes (Er/RB@ExosCD47) strongly induced ferroptosis both in vitro and in vivo in tumor cells after irradiation of 532 nm laser. Moreover, compared with the control exosomes (Er/RB@ExosCtrl), Er/RB@ExosCD47 displayed much lower toxicity in liver. Conclusion: The engineered exosomes composed of CD47, Erastin, and Rose Bengal, induce obvious ferroptosis in hepatocellular carcinoma (HCC) with minimized toxicity in liver and kidney. The proposed exosomes would provide a promising strategy to treat types of malignant tumors.

Histidine-rich calcium binding protein (HRC) is markedly overexpressed in hepatocellular carcinoma (HCC) and is significantly correlated with metastasis. Anoikis resistance and endoplasmic reticulum (ER) stress may have a critical effect on survival before metastasis. However, the potential functions of HRC in anoikis resistance in HCC remain unknown. Here, we uncovered the clinical value of HRC and its functional significance on anoikis in HCC. The positive expression of HRC was observably correlated with tumor size, tumor encapsulation, and tumor-node-metastasis (TNM) stage. The expression of HRC increased in HCC cells cultured in suspension. HRC enhanced the anoikis resistance of HCC, and promoted the HCC metastasis in vivo. Mechanistically, the anoikis resistance was probably dependent on endoplasmic reticulum stress. Modulating HRC level changed the ERS to affect anoikis resistance by acting protein kinase RNA-like ER kinase (PERK)-eIF2a-ATF4-CHOP signaling axis. In conclusion, we define HRC as a novel candidate oncogene involved in anoikis resistance and HCC metastasis, and provide a new potential therapeutic target for HCC.

Increasing proofs have declared that liver cancer stem cells (CSCs) are the main contributors to tumor initiation, metastasis, therapy resistance, and recurrence of hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying CSCs regulation remain largely unclear. Recently, PCNA-associated factor (PAF) was identified to play a key role in maintaining breast cancer cell stemness, but its role in liver cancer stem cells has not been declared yet. Herein, we found that both mRNA and protein expression levels of PAF were significantly higher in HCC tissues and cell lines than normal controls. CSC-enriched hepatoma spheres displayed an increase in PAF expression compared to monolayer-cultured cells. Both loss-of-function and gain-of-function experiments revealed that PAF enhanced sphere formation and the percentage of CD133(+) or EpCAM(+) cells in HCCLM3 and Huh7 cells. In the xenograft HCC tumor model, tumor initiation rates and tumor growth were suppressed by knockdown of PAF. Mechanistically, PAF can amplify the self-renewal of liver CSCs by activating β-catenin signaling. Taken together, our results demonstrate that PAF plays a crucial role in maintaining the hepatoma cell stemness by β-catenin signaling. Abbreviations: CSCs: cancer stem cells; HCC: hepatocellular carcinoma; PAF: pCNA-associated factor.

Summary Beta‐thalassaemia is an inherited blood disorder characterised by ineffective erythropoiesis and anaemia. Consequently, hepcidin expression is reduced resulting in increased iron absorption and primary iron overload. Hepcidin is under the negative control of transmembrane serine protease 6 (TMPRSS6) via cleavage of haemojuvelin (HJV), a co‐receptor for the bone morphogenetic protein (BMP)‐mothers against decapentaplegic homologue (SMAD) signalling pathway. Considering the central role of the TMPRSS6/HJV/hepcidin axis in iron homeostasis, the inhibition of TMPRSS6 expression represents a promising therapeutic strategy to increase hepcidin production and ameliorate anaemia and iron overload in β‐thalassaemia. In the present study, we investigated a small interfering RNA (siRNA) conjugate optimised for hepatic targeting of Tmprss6 (SLN124) in β‐thalassaemia mice (Hbbth3/+). Two subcutaneous injections of SLN124 (3 mg/kg) were sufficient to normalise hepcidin expression and reduce anaemia. We also observed a significant improvement in erythroid maturation, which was associated with a significant reduction in splenomegaly. Treatment with the iron chelator, deferiprone (DFP), did not impact any of the erythroid parameters. However, the combination of SLN124 with DFP was more effective in reducing hepatic iron overload than either treatment alone. Collectively, we show that the combination therapy can ameliorate several disease symptoms associated with chronic anaemia and iron overload, and therefore represents a promising pharmacological modality for the treatment of β‐thalassaemia and related disorders.

The liver plays a pivotal role in mammalian aging. However, the mechanisms underlying liver aging remain unclear. Cisd2 is a pro‐longevity gene in mice. Cisd2 mediates lifespan and healthspan via regulation of calcium homeostasis and mitochondrial functioning. Intriguingly, the protein level of Cisd2 is significantly decreased by about 50% in the livers of old male mice. This down‐regulation of Cisd2 may result in the aging liver exhibiting non‐alcoholic fatty liver disease (NAFLD) phenotype. Here, we use Cisd2 transgenic mice to investigate whether maintaining Cisd2 protein at a persistently high level is able to slow down liver aging. Our study identifies four major discoveries. Firstly, that Cisd2 expression attenuates age‐related dysregulation of lipid metabolism and other pathological abnormalities. Secondly, revealed by RNA sequencing analysis, the livers of old male mice undergo extensive transcriptomic alterations, and these are associated with steatosis, hepatitis, fibrosis, and xenobiotic detoxification. Intriguingly, a youthful transcriptomic profile, like that of young 3‐month‐old mice, was found in old Cisd2 transgenic male mice at 26 months old. Thirdly, Cisd2 suppresses the age‐associated dysregulation of various transcription regulators (Nrf2, IL‐6, and Hnf4a), which keeps the transcriptional network in a normal pattern. Finally, a high level of Cisd2 protein protects the liver from oxidative stress, and this is associated with a reduction in mitochondrial DNA deletions. These findings demonstrate that Cisd2 is a promising target for the development of therapeutic agents that, by bringing about an effective enhancement of Cisd2 expression, will slow down liver aging., The protein level of CDGSH iron‐sulfur domain‐containing protein 2 (Cisd2) is decreased by about 50% in the aged livers of old mice. RNA sequencing and histopathological analyses reveal that in the Cisd2 transgenic mice, a persistently high level of Cisd2 slows down liver aging via attenuating oxidative stress and mitochondrial DNA deletion, as well as preserving a youthful transcriptomic profile, thereby protecting the liver against age‐associated structural injury and functional decline.

Pig’s blood and liver are valuable edible slaughter by-products which are also the major ingredients of offal-derived foodstuffs. The aim of the study was an evaluation of the occurrence of hepatitis E virus (HEV) and porcine adenovirus (pAdV) as an index virus of faecal contamination in pig’s blood and liver for human consumption. In total, 246 samples of retail liver (n = 100) and pooled pig’s blood (n = 146) were analysed for the presence of HEV and pAdV. Blood samples were individually collected from 1432 pigs at slaughter age. Viral genomic material, including RNA of a sample process control virus was isolated from food samples using a QIAamp® Viral RNA Mini Kit. Virus-specific IAC-controlled real-time PCR methods were used for detection of target viruses. HEV RNA was found in 6 (2.4%; 95% CI: 0.9–5.2) out of 246 samples of tested foodstuffs. The virus was detected in pig’s blood (3.4%; 95% CI: 1.1–7.8) and liver (1.0%; 95% CI: 0.0–5.0) with no significant differences observed in the frequency of its occurrence between the two by-products (t = 1.33; p = 0.182 > 0.05); however PAdV was detected more frequently in pig’s blood than in liver (t = 4.65; p = 0.000 F = 0.81, p = 0.447 > 0.05 for HEV; F = 0.42, p = 0.655 > 0.05 for pAdV). Although HEV was detected in pig’s offal only sporadically, consumers cannot treat its occurrence with disregard as it demonstrates that HEV-contaminated pig tissues can enter the food chain.

Background Liver biopsy is a widely used, safe diagnostic tool utilised by clinicians for the histopathological assessment of the liver. Our study aims to report our experience in patients who underwent ultrasound-guided plugged percutaneous liver biopsy in a tertiary care hospital in India. Material and methods The Institutional Ethical Review Board approved this retrospective study, and informed consent was obtained from all the patients. A total of 830 liver biopsies were performed between January 2014 and December 2018, of which 782 were plugged percutaneous liver biopsies. The tract was plugged using Gelfoam slurry. Various observations related to the procedures were recorded. Results Seven hundred and eighty-two were plugged percutaneous liver biopsies, which were performed during the study period. Of the 782 patients, 163 were male, and 619 were female (20.8 % and 79.2 %, respectively), with a mean age of 49.6 ± 2 years (1 month to 86 years). A 100% technical success rate was seen. No immediate major complications were documented in any of the patients who underwent plugged biopsies. No significant complications were seen in any patient. Conclusions Percutaneous liver biopsy is an extensively performed diagnostic tool. We found that ultrasound-guided percutaneous plugged liver biopsy is an easy to perform procedure, which is associated with a lower risk of a bleeding complications.

Mass spectrometry imaging (MSI) provides insight into the molecular distribution of a broad range of compounds and, therefore, is frequently applied in the pharmaceutical industry. Pharmacokinetic and toxicological studies deploy MSI to localize potential drugs and their metabolites in biological tissues but currently require other analytical tools to quantify these pharmaceutical compounds in the same tissues. Quantitative mass spectrometry imaging (Q-MSI) is a field with challenges due to the high biological variability in samples combined with the limited sample cleanup and separation strategies available prior to MSI. In consequence, more selectivity in MSI instruments is required. This can be provided by multiple reaction monitoring (MRM) which uses specific precursor ion-product ion transitions. This targeted approach is in particular suitable for pharmaceutical compounds because their molecular identity is known prior to analysis. In this work, we compared different analytical platforms to assess the performance of MRM detection compared to other MS instruments/MS modes used in a Q-MSI workflow for two drug candidates (A and B). Limit of detection (LOD), linearity, and precision and accuracy of high and low quality control (QC) samples were compared between MS instruments/modes. MRM mode on a triple quadrupole mass spectrometer (QqQ) provided the best overall performance with the following results for compounds A and B: LOD 35.5 and 2.5 μg/g tissue, R2 0.97 and 0.98 linearity, relative standard deviation QC R2 0.86–0.98 and 0.86–0.98 linearity, relative standard deviation QC Graphical abstract

Intestinal microbiota regulates multiple host metabolic and immunological processes. Consequently, any difference in its qualitative and quantitative composition is susceptible to exert significant effects, in particular along the gut-liver axis. Indeed, recent findings suggest that such changes modulate the severity and the evolution of a wide spectrum of hepatobiliary disorders. However, the mechanisms linking intestinal microbiota and the pathogenesis of liver disease remain largely unknown. In this work, we investigated how a distinct composition of the intestinal microbiota, in comparison with germ-free conditions, may lead to different outcomes in an experimental model of acute cholestasis. Acute cholestasis was induced in germ-free (GF) and altered Schaedler’s flora (ASF) colonized mice by common bile duct ligation (BDL). Studies were performed 5 days after BDL and hepatic histology, gene expression, inflammation, lipids metabolism, and mitochondrial functioning were evaluated in normal and cholestatic mice. Differences in plasma concentration of bile acids (BA) were evaluated by UHPLC-HRMS. The absence of intestinal microbiota was associated with significant aggravation of hepatic bile infarcts after BDL. At baseline, we found the absence of gut microbiota induced altered expression of genes involved in the metabolism of fatty and amino acids. In contrast, acute cholestasis induced altered expression of genes associated with extracellular matrix, cell cycle, autophagy, activation of MAPK, inflammation, metabolism of lipids, and mitochondrial functioning pathways. Ductular reactions, cell proliferation, deposition of collagen 1 and autophagy were increased in the presence of microbiota after BDL whereas GF mice were more susceptible to hepatic inflammation as evidenced by increased gene expression levels of osteopontin, interleukin (IL)-1β and activation of the ERK/MAPK pathway as compared to ASF colonized mice. Additonally, we found that the presence of microbiota provided partial protection to the mitochondrial functioning and impairment in the fatty acid metabolism after BDL. The concentration of the majority of BA markedly increased after BDL in both groups without remarkable differences according to the hygiene status of the mice. In conclusion, acute cholestasis induced more severe liver injury in GF mice compared to mice with limited intestinal bacterial colonization. This protective effect was associated with different hepatic gene expression profiles mostly related to tissue repair, metabolic and immune functions. Our findings suggest that microbial-induced differences may impact the course of cholestasis and modulate liver injury, offering a background for novel therapies based on the modulation of the intestinal microbiota.

Flavin adenine dinucleotide (FAD) is engaged in several metabolic diseases. Its main role is being a cofactor essential for the activity of many flavoproteins, which play a crucial role in electron transport pathways in living systems. The aim of this study was to apply a pegylated flavins formulation named FAD-PEG diacide complex as theranostic pathway in cancer therapy. For this purpose, a mouse liver cancer model induced by Hepa1-6 cells was used to evaluate the therapeutic efficacy of FAD (named NP1) and FAD-PEG diacide complex (named NP2). The cytokines were applied to screen the serum inflammatory factors, to establish the blood cell content of different groups of nude mice. The highlights follows that FAD formulations (NP1; NP2) significantly suppressed the tumor growth and reduced the tumor index without effects on the body weight of mice. Furthermore, NP2 significantly reduced the serum levels of cytokines IL-6, TNF-α and IL-12 (P70). The reported results provide the proof-of-concept for the synthesis of a smart adjuvant for liver cancer therapy and support their further development in the field of nanomedicine.

‘Angiodiversity’ refers to the structural and functional heterogeneity of endothelial cells (EC) along the segments of the vascular tree and especially within the microvascular beds of different organs. Organotypically differentiated EC ranging from continuous, barrier-forming endothelium to discontinuous, fenestrated endothelium perform organ-specific functions such as the maintenance of the tightly sealed blood–brain barrier or the clearance of macromolecular waste products from the peripheral blood by liver EC-expressed scavenger receptors. The microvascular bed of the liver, composed of discontinuous, fenestrated liver sinusoidal endothelial cells (LSEC), is a prime example of organ-specific angiodiversity. Anatomy and development of LSEC have been extensively studied by electron microscopy as well as linage-tracing experiments. Recent advances in cell isolation and bulk transcriptomics or single-cell RNA sequencing techniques allowed the identification of distinct LSEC molecular programs and have led to the identification of LSEC subpopulations. LSEC execute homeostatic functions such as fine tuning the vascular tone, clearing noxious substances from the circulation, and modulating immunoregulatory mechanisms. In recent years, the identification and functional analysis of LSEC-derived angiocrine signals, which control liver homeostasis and disease pathogenesis in an instructive manner, marks a major change of paradigm in the understanding of liver function in health and disease. This review summarizes recent advances in the understanding of liver vascular angiodiversity and the functional consequences resulting thereof.

Recent reports indicated that mitophagy protects against alcohol-induced liver injury, which helps remove damaged mitochondria to reduce the accumulation of reactive oxygen species (ROS). AMP-activated protein kinase (AMPK) has been recently used in ALD (alcoholic liver disease) and mitochondrial dysfunction research. However, the inner mechanism, whether AMPK can regulate mitophagy in ALD, remains unknown. Here we found that AMPK can significantly reduce alcohol-induced liver injury and enhances hepatocytes��� mitophagy level. Next, we identified that AMPK rescued alcohol-induced low expression of UQCRC2 (ubiquinol-cytochrome c reductase core protein 2). Interestingly, UQCRC2 knockdown (KD) treatment causes impaired mitophagy, whereas UQCRC2 overexpression (OE) can significantly increase mitophagy to attenuate liver injury. Also, we identified that AMPK indirectly upregulates UQCRC2 protein level, and RNA-seq, chromatin immunoprecipitation (ChIP) assay, bioinformatics, and luciferase assays helped us understand that AMPK enhanced UQCRC2 gene transcription through activating NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2). Our results demonstrate that AMPK regulating UQCRC2 is a significant mitochondrial event in mitophagy. It identifies a new signaling axis, AMPK-NFE2L2-UQCRC2, in the regulation of mitophagy levels in the liver, suggesting a possible therapeutic strategy to treat ALD. Abbreviations: AAV: AENO-associated virus; ALD: alcoholic liver disease; AMPK: AMP-activated protein kinase; BUN: blood urea nitrogen; H&E: hematoxylin and eosin; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ChIP: chromatin immunoprecipitation assay; CO-IP: co-immunoprecipitation; COPD: chronic obstructive pulmonary disease; EM: electron microscope; GOT1/AST: glutamic-oxaloacetic transaminase 1; GPT/ALT: glutamic���pyruvic transaminase; IF: immunofluorescence; IHC: immunohistochemistry; KD: knockdown; MAP1LC3/LC3: microtubule associated protein 1 light chain protein 3; MTDR: MitoTracker Deep Red; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; mtDNA: mitochondrial DNA; MTRC: MitoTracker Red CMXRos; OCR: Oxygen consumption rate; OE: overexpress; PINK1: PTEN induced kinase 1; qRT-PCR: quantitative real-time PCR; ROS: reactive oxygen species; SD: standard deviation; SOD2: superoxide dismutase 2; UQCRC2: ubiquinol-cytochrome c reductase core protein 2; WB: western blot; ����: mitochondrial membrane potential

When confined in pairs, juvenile rainbow trout (Oncorhynchus mykiss) form dominance hierarchies in which subordinate fish exhibit characteristic physiological changes including reduced growth rates and chronically elevated plasma cortisol concentrations. We hypothesized that alterations in protein metabolism contribute to the reduced growth rate of socially stressed trout, and predicted that subordinate trout would exhibit reduced rates of protein synthesis coupled with increases in protein degradation. Protein metabolism was assessed in dominant and subordinate fish after 4 days of social interaction, and in fish that were separated after 4 days of interaction for a 4 days recovery period, to determine whether effects on protein metabolism recovered when social stress was alleviated. Protein metabolism was assessed in liver and white muscle by measuring the fractional rate of protein synthesis and markers of protein degradation. In the white muscle of subordinate fish, protein synthesis was inhibited and activities of the ubiquitin-proteasome pathway (UPP) and the autophagy lysosomal system (ALS) were elevated. By contrast, the liver of subordinate fish exhibited increased rates of protein synthesis and activation of the ALS. When allowed to recover from chronic social stress for 4 days, differences in protein metabolism observed in white muscle of subordinate fish during the interaction period disappeared. In liver, protein synthesis returned to baseline levels during recovery from social stress, but markers of protein degradation did not. Collectively, these data support the hypothesis that inhibition of muscle protein synthesis coupled with increases in muscle protein breakdown contribute to the reduced growth rates of subordinate rainbow trout. Supplementary Information The online version contains supplementary material available at 10.1007/s00360-021-01340-6.