Single-Cell Sequencing Reveals MYOF-Enriched Monocyte/Macrophage Subcluster as a Favorable Prognostic Factor in Sepsis.

This research utilized single-cell RNA sequencing to map the immune cell landscape in sepsis, revealing 28 distinct cell clusters and categorizing them into nine major types. Delving into the monocyte/macrophage subclusters, 12 unique subclusters are identified and pathway enrichment analyses are conducted using KEGG and GO, discovering enriched pathways such as oxidative phosphorylation and antigen processing. Further GSVA and AUCell assessments show varied activation of interferon pathways, especially in subclusters 4 and 11. The clinical correlation analysis reveals genes significantly linked to survival outcomes. Additionally, cellular differentiation in these subclusters is explored. Building on these insights, the differential gene expression within these subclusters is specifically scrutinized, which reveal MYOF as a key gene with elevated expression levels in the survivor group. This finding is further supported by in-depth pathway enrichment analysis and the examination of cellular differentiation trajectories, where MYOF's role became evident in the context of immune response regulation and sepsis progression. Validating the role of the MYOF gene in sepsis, a dose-dependent response to LPS in THP-1 cells and C57 mice is observed. Finally, inter-cellular communications are analyzed, particularly focusing on the MYOF+Mono/Macro subcluster, which indicates a pivotal role in immune regulation and potential therapeutic targeting.

Circular olefin copolymers made de novo from ethylene and α-olefins.

Ethylene/α-olefin copolymers are produced in huge scale and widely used, but their after-use disposal has caused plastic pollution problems. Their chemical inertness made chemical re/upcycling difficult. Ideally, PE materials should be made de novo to have a circular closed-loop lifecycle. However, synthesis of circular ethylene/α-olefin copolymers, including high-volume, linear low-density PE as well as high-value olefin elastomers and block copolymers, presents a particular challenge due to difficulties in introducing branches while simultaneously installing chemical recyclability and directly using industrial ethylene and α-olefin feedstocks. Here we show that coupling of industrial coordination copolymerization of ethylene and α-olefins with a designed functionalized chain-transfer agent, followed by modular assembly of the resulting AB telechelic polyolefin building blocks by polycondensation, affords a series of ester-linked PE-based copolymers. These new materials not only retain thermomechanical properties of PE-based materials but also exhibit full chemical circularity via simple transesterification and markedly enhanced adhesion to polar surfaces.

The SMAC mimetic birinapant alleviates lipopolysaccharide-induced acute lung injury by inhibiting MAPK signaling.

The second mitochondria-derived activator of caspases (SMAC) mimetic birinapant attenuated liver injury by inhibited the degradation of tumor necrosis factor receptor-associated factor 3 (TRAF3) and activation of mitogen-activated protein kinase (MAPK) signaling pathway in liver macrophage, but its role in LPS induced acute lung injury (ALI) is not understood. The present study was to investigate the effects of birinapant on ALI and its possible mechanism. A dose of birinapant (30 mg/kg) or a vehicle was administered intravenously 24 hours before LPS (100 µg) stimulation in mice. The levels of TNF-α, IL-6 and IL-1ß in bronchoalveolar lavage fluid (BALF) were measured by ELISA. The infiltrated macrophages and expression of monocyte chemoattractant protein-1 (MCP-1) was determined by immunohistochemistry staining in the lung tissues. The JNK and p38 MAPK activation, protein expression and K48-linked polyubiquitination of TRAF3 were determined in alveolar macrophage cell line (MH-S cells) after 1µg/ml LPS stimulation. The results showed that the birinapant down-regulated the levels of TNF-α, IL-6 and IL-1ß in the BALF. In addition, birinapant markedly inhibited macrophages infiltration and MCP-1 protein expression in lung tissues. At last, birinapant suppressed the MAPKsignaling pathway and K48-linked ubiquitinated degradation of TRAF3 in MH-S cells after LPS administration. In conclusion, the results proved that birinapant protected against LPS-induced ALI through inhibiting MAPK activation and K48-linked ubiquitination of TRAF3 in alveolar macrophages.

Histone lactylation regulates cancer progression by reshaping the tumor microenvironment.

As a major product of glycolysis and a vital signaling molecule, many studies have reported the key role of lactate in tumor progression and cell fate determination. Lactylation is a newly discovered post-translational modification induced by lactate. On the one hand, lactylation introduced a new era of lactate metabolism in the tumor microenvironment (TME), and on the other hand, it provided a key breakthrough point for elucidation of the interaction between tumor metabolic reprogramming and epigenetic modification. Studies have shown that the lactylation of tumor cells, tumor stem cells and tumor-infiltrating immune cells in TME can participate in the development of cancer through downstream transcriptional regulation, and is a potential and promising tumor treatment target. This review summarized the discovery and effects of lactylation, as well as recent research on histone lactylation regulating cancer progression through reshaping TME. We also focused on new strategies to enhance anti-tumor effects via targeting lactylation. Finally, we discussed the limitations of existing studies and proposed new perspectives for future research in order to further explore lactylation targets. It may provide a new way and direction to improve tumor prognosis.

Kupffer cells depletion alters cytokine expression and delays liver regeneration after Radio-frequency-assisted Liver Partition with Portal Vein Ligation.

Radio-frequency-assisted Liver Partition with Portal Vein Ligation (RALPP) induces comparable hypertrophy of the liver remnant compared to Associating Liver Partition and Portal vein ligation for Staged hepatectomy (ALPPS) in humans. However, whether it is significantly improved compared to ALPPS is unclear, and the underlying mechanisms of liver regeneration after RALPP need to further investigate. The present study was to develop an animal model mimicking RALPP and explore mechanisms of liver regeneration. The mice in RALPP group received liver radiofrequency ablation and 90% portal vein ligation (PVL), followed by resection of the targeted liver within two days after the first surgery. The mice in ALPPS group underwent 90% PVL combined with parenchyma transection. Controls received liver radiofrequency ablation (RAF group) or PVL (PVL group) or small left lateral lobe (LLL group) resection alone. Liver regeneration was assessed by liver weight and proliferation-associated molecules. The role of Kupffer cells (KCs) in liver regeneration was investigated after RALPP. The results showed that RALPP induced comparable liver regeneration compared to ALPPS, but with less liver injury and mortality in mice. RALPP led to over-expression of TNF-α and IL-6 in the circulating plasma compared with PVL. KCs infiltrating in liver tissues was a characteristic of mice in the RALPP group. KCs depletion markedly depressed cytokine expression and delayed liver regeneration after RALPP. These results suggested that RALPP in mice induced accelerated liver regeneration similar to ALPPS, but safer than ALPPS. KCs depletion altered cytokine expression and delayed liver regeneration after RALPP.

Development and validation of a simple and sensitive LC-MS/MS method for quantification of ampicillin and sulbactam in human plasma and its application to a clinical pharmacokinetic study.

Ampicillin-sulbactam is a broad-spectrum combination antibiotic used for a variety of clinical applications, including as a prophylactic agent to reduce the risk of surgical site infection. The pharmacokinetics of ampicillin-sulbactam after redosing during prolonged surgeries remains incompletely understood. In anticipation of further studying the intra-operative pharmacokinetics of this drug, we have developed a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of ampicillin and sulbactam. The plasma samples were prepared using a simple protein precipitation method. Gradient chromatographic elution was used to separate analytes, and MS/MS analysis was performed in negative ionization mode for both analytes via multiple reaction monitoring (MRM). All validation parameters were evaluated under a good laboratory practice (GLP) environment. For both ampicillin and sulbactam, the lower limit of quantitation (LLOQ) was established as 0.25 µg/mL. The calibration curve ranged from 0.25 to 200 µg/mL for ampicillin and 0.25-100 µg/mL for sulbactam. Inter- and intra-day precisions for both analytes were ≤11.5 % for quality controls and ≤17.4 % for LLOQ; accuracies ranged from -11.5 to 12.5% for 3 quality control levels and -18.1-18.7% for LLOQ. In addition to sensitivity, accuracy and precision, 13 other parameters were also validated for both analytes, and the results met the acceptance criteria. Our method was successfully applied to quantify ampicillin and sulbactam concentrations in patients undergoing surgery.

Reversine suppresses osteosarcoma cell growth through targeting BMP-Smad1/5/8-mediated angiogenesis.

Reversine, or 2-(4-morpholinoanilino)-6cyclohexylaminopurine, is a 2,6-disubstituted purine derivative. This small molecule exhibits tumor-suppressive activities through different molecular mechanisms. In this study, in vitro and in vivo angiogenic models were used to elucidate the effect of Reversine on angiogenesis in the tumor suppression. Firstly, we grafted osteosarcoma-derived MNNG/HOS cell aggregates onto chick embryonic chorioallantoic membrane (CAM) to examine the vascularization of these grafts following Reversine treatment. Following culture, it was determined that Reversine inhibited MNNG/HOS grafts growth, and decreased the density of blood vessels in the chick CAM. We then used CAM and chick embryonic yolk-sac membrane (YSM) to investigate the effects of Reversine on angiogenesis. The results revealed Reversine inhibited the proliferation of endothelial cells, where cells were mainly arrested at G1/S phase of the cell cycle. Scratch-wound assay with HUVECs revealed that Reversine suppressed cell migration in vitro. Furthermore, endothelial cells tube formation assay and chick aortic arch sprouting assay demonstrated Reversine inhibited the sprouting, migration of endothelial cells. Lastly, qPCR and western blot analyses showed BMP-associated Smad1/5/8 signaling expressions were up-regulated by Reversine treatment. Our results showed that Reversine could suppress tumor growth by inhibiting angiogenesis through BMP signaling, and suggests a potential use of Reversine as an anti-tumor therapy.

The on-off action of Forkhead protein O3a in endotoxin tolerance of Kupffer cells depends on the PI3K/AKT pathway.

BACKGROUND: The endotoxin tolerance (ET) of Kupffer cells (KCs) is an important protective mechanism for limiting endotoxin shock. As a key anti-inflammatory molecule, the roles and mechanism of Forkhead protein O3a (Foxo3a) in ET of KCs are not yet well understood. METHODS: ET and nonendotoxin tolerance (NET) KCs models were established in vitro and in vivo. The levels of cytokines were detected by enzyme-linked immunosorbent assay (ELISA). The protein expression and phosphorylation levels were detected by western blotting (WB). Changes in the localization of nuclear factor kappa B (NF-κB) and Foxo3a in KCs were detected by immunofluorescence assays. KCs apoptosis and survival rates were detected by flow cytometry and an automatic cell counter, respectively. RESULTS: The activity of NF-κB and the levels of p-Foxo3a and tumor necrosis factor (TNF-α) in the ET group were significantly lower than those in the NET group, while the levels of Foxo3a and interleukin 10 (IL-10) in the ET group were significantly higher than those in the NET group. Overexpression of Foxo3a or the use of a phosphatidylinositol-3-hydroxykinase (PI3K) inhibitor suppressed the activation of NF-κB by decreasing the levels of p-Foxo3a by inhibiting the activity of PI3K/AKT, which improved the tolerance of KCs and mice to endotoxin. In contrast, silencing Foxo3a or the use of a PI3K agonist reduced the tolerance of KCs and mice to endotoxin. The PI3K agonist counteracted the inhibitory effects of Foxo3a overexpression on NF-κB, impairing the tolerance of KCs to endotoxin. CONCLUSIONS: The on-off action of Foxo3a in the ET of KCs depends on the PI3K/AKT pathway.

Cholesterol impairs hepatocyte lysosomal function causing M1 polarization of macrophages via exosomal miR-122-5p.

Nonalcoholic steatohepatitis (NASH) is a major threat to health worldwide. Lipotoxicity and macrophage-mediated inflammation play key roles in the pathogenesis of NASH. In this study, we found that individuals with higher serum LDL-C levels have a higher prevalence of nonalcoholic fatty liver disease (NAFLD) and elevated levels of glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase and alkaline phosphatase. A logistic regression analysis revealed that serum LDL-C level is an independent risk factor for the prevalence and prognosis of NAFLD. In vitro, we used ox-LDL and MßCD-cholesterol to treat Huh7 cells and found that cholesterol loading reduced lysosomal quantity and impaired lysosomal acidification, reducing the number of multivesicular bodies (MVBs) colocalizing with lysosomes. The bafilomycin A1 inhibition of lysosomal function also inhibited lysosomal MVBs degradation, promoting the release of exosomes from the Huh7 cells. Next, we found that cholesterol loading promoted exosome release from the Huh7 cells. The exosomes from the cholesterol-loaded cells increased the ratio of the THP-1 cells positive for the M1 marker (iNOS-1) without affecting the ratio of the cells positive for the M2 marker (CD206). Moreover, an elevated level of miR-122-5p was observed in exosomes derived from the Huh7 cells loaded with cholesterol. While the miR-122-5p mimics promoted THP-1 M1 polarization, downregulating miR-122-5p in the Huh7 cells inhibited the exosome-induced activation of macrophages and macrophage-related inflammation. These findings suggest that cholesterol plays an important role in the development and progression of NASH. Cholesterol-induced lysosomal dysfunction increases exosome release from hepatocytes, resulting in M1 polarization and macrophage-induced inflammation in a miR-122-5p-dependent manner.

Shenfu Injection Attenuates Bile Duct Injury in Rats with Acute Obstructive Cholangitis.

Background: We investigated the effect of Shenfu injection (SFI) in Wistar rats with acute obstructive cholangitis (AOC) and considered the possible molecular mechanisms of the effects. Methods: The 96 rats were divided randomly into three groups. In one group, the common bile duct was subjected to ligation (BDL), and 0.2 mL of saline was injected into the proximal bile ducts. To create AOC, again, the common bile duct was ligated, and 0.2 mL of lipopolysaccharide (LPS)) (2 mg/mL) was injected into the proximal ducts. In the Shenfu injection (SFI) group, the material (10 mg/kg) was injected into the tail vein 2 hours before induction of AOC. The hepatic histopathologic changes were observed under a light microscope. The endotoxin, tumor necrosis factor-α (TNF-α), alanine transaminase (ALT), and total bilirubin (TB) concentrations in the serum were measured at different time points (0, 4, 8, and 16 hours) after ligation. The expression of nuclear transcription factor-κB (NF-κB) and CD14 in Kupffer cells also was analyzed at different times by Western blotting. Results: The TNF-α, ALT, and TB concentrations in the serum and the expression of CD14 and NF-κB in Kupffer cells were significantly higher in the SFI group than in the BDL group, but all were significantly lower than in the AOC group. Compared with the AOC group, the edema of cholangiocytes was alleviated in the SFI group, and the infiltration of inflammatory cells around cholangiocytes was reduced. Conclusion: Shenfu injection significantly alleviated bile duct injury. The potential mechanism may be associated with inhibition of CD14 expression and prevention of NF-κB activation in Kupffer cells.

USP25 promotes endotoxin tolerance via suppressing K48-linked ubiquitination and degradation of TRAF3 in Kupffer cells.

The inhibition of tumor necrosis factor receptor-associated factor 3 (TRAF3) degradation induces endotoxin tolerance (ET) in macrophages. However, the mechanisms leading to TRAF3 inhibition by ET are largely unknown. Here, we found that ubiquitin-specific peptidase 25 (USP25), a deubiquitinating enzyme (DUB), interacted with TRAF3 and stabilized ET in Kupffer cells (KCs). Lentiviral knockdown of USP25 activated K48-linked ubiquitination of TRAF3 and the cytoplasmic translocation of the Myd88-associated multiprotein complex in tolerized KCs. This outcome led to a subsequent activation of Myd88-dependent c-Jun N-terminal kinase (JNK) and p38-mediated downregulation of inflammatory cytokines. The overexpression of TRAF3 attenuated the proinflammatory effects of USP25 knockdown in tolerized KCs. Thus, our findings reveal a novel mechanism of endotoxin-mediated TRAF3 degradation in KCs.

Cell survival controlled by lens-derived Sema3A-Nrp1 is vital on caffeine-suppressed corneal innervation during chick organogenesis.

In this study, we investigated the effect of caffeine overexposure on corneal innervation in the early chicken embryo. Caffeine administration restricted corneal innervation by affecting trigeminal nerve development. Immunohistochemistry for phospho-Histone3 (pHIS3) and C-caspase3 revealed that cell survival was repressed by caffeine administration. Whole-mount in situ hybridization against semaphorin 3A (Sema3A) and neuropilin-1 (Nrp1) showed that both caffeine and 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH, a free radical generator) administration upregulates the expression of both Sema3A and Nrp1. Next, we demonstrated that lens ablation in the developing chicken embryos significantly affected NF-labeled periocular nerve fascicles and innervation to the central eye region. Subsequently, we used a neuroblastoma cell line to investigate in vitro whether or not Sema3A-Nrp1 signaling exerts a key role on the caffeine-suppressed neuron survival. Knocking-down Sema3A through transfection with Sema3A-siRNA dramatically decreased the responsiveness of cells to caffeine administration, as well as cell apoptosis. We suggest that Sema3A-Nrp1 signaling regulates Trp53 and Cdkn1a through Slit2-Robo1 and Ephb2. Taken together, we speculate here that caffeine-enhanced reactive oxygen species upregulates Sema3A-Nrp1 expression in the lens and periocular tissues, resulting in corneal cell apoptosis, accompanied by its chemorepellent role on the invasion of the developing cornea by trigeminal sensory fibers.

Cathepsin B regulates non-canonical NLRP3 inflammasome pathway by modulating activation of caspase-11 in Kupffer cells.

OBJECTIVES: The non-canonical inflammasome pathway was described which engages caspase-11 to mediate pyroptosis and the subsequent release of IL-1α, IL-1ß and IL-18 in TLR4-independent way. Cathepsin B is capable of activating caspase-11 under cell-free conditions which may regulate non-canonical NLRP3 inflammasome pathway. In this study, we aimed to further investigate cathepsin B as potential activators of proinflammatory caspases which may be released upon proinflammatory stimuli and regulate non-canonical NLRP3 inflammasome pathway by modulating the activity of caspase-11. METHODS: Pharmacological and gene-silencing approaches were used to evaluate the impact of cathepsin B on regulating non-canonical NLRP3 inflammasome pathway in wild-type and TLR4-/- Kupffer cells. A sepsis model was also created to investigate the effect of cathepsin B on survival. Meanwhile, cathepsin B activity and the expression level of caspase-4 were detected in human peripheral blood mononuclear cells (PBMC) which were separated from patients suffered from SIRS or sepsis and healthy volunteers. RESULTS: LPS stimulation caused cathepsin B activity and caspase-11 expression increase in TLR4-/- mice. Cathepsin B activity inhibition reduced the activation of caspase-11 and inflammasome and benefited survival in TLR4-/- mice. Upregulation of cathepsin B activity and caspase-4 activation was found in PBMC of patients with SIRS or sepsis. CONCLUSION: Our results suggest a critical role for cathepsin B as activators of proinflammatory caspases-11 and the regulatory effect in LPS-induced caspases-11-dependent necrosis.

SIRT1 upregulation protects against liver injury induced by a HFD through inhibiting CD36 and the NF­κB pathway in mouse kupffer cells.

Sirtuin 1 (SIRT1) is an NAD(+)­dependent deacetylase, and a critical regulator in various metabolic processes, such as non­alcoholic fatty liver disease (NAFLD). The present study aimed to investigate whether activating SIRT1 could modulate the CD36 and nuclear factor (NF)­κB pathways to protect against liver injury induced by a high­fat diet (HFD) in mice. A mouse NAFLD model was established by administration of a HFD for 8 weeks. During the last 4 weeks, SRT1720, a specific SIRT1 activator, was added daily to the HFD feed. The hepatic morphological structure was observed using hematoxylin and eosin staining, and the ultrastructures in the liver tissue were observed using transmission electron microscopy. Protein expression of SIRT1, CD36 and P65 in liver tissues was detected by immunohistochemistry. Kupffer cells (KCs) from the livers of the mouse models were isolated to determine the mRNA and protein expression of SIRT1, CD36 and P65. SIRT1 activation attenuated the HFD­induced liver injury and significantly reduced the body weight and the levels of alanine transaminase, aspartate aminotransferase, triglyceride, tumor necrosis factor­α and interleukin­6. We observed an increased expression of SIRT1 in the liver tissues from the HFD+SRT1720 group compared with the HFD group. Simultaneously, the expression of CD36 and P65 in the liver tissues was downregulated in the HFD+SRT1720 group. The mRNA and protein expression of SIRT1 was elevated in the HFD+SRT1720 group, whereas the mRNA and protein expression of CD36 and P65 in KCs was significantly decreased in the HFD+SRT1720 group. The present study demonstrated that SIRT1 activation attenuated HFD­induced liver steatosis and inflammation by inhibiting CD36 expression and the NF­κB signaling pathway.

Applying chlorogenic acid in an alginate scaffold of chondrocytes can improve the repair of damaged articular cartilage.

Damaged cartilage has very low regenerative potential which has led to the search for novel tissue-engineering approaches to help treat cartilage defects. While various approaches have been reported, there is no perfect treatment currently. In this study we evaluated the effects of a plant extract, chlorogenic acid (CGA), as part of chondrocyte transplantation on a model of knee joint injury in chicks. First, primary cultured chondrocytes used to evaluate the effects of CGA on chondrogenesis. Then using an articular cartilage injury model of chick knee we assessed the functional recovery after transplantation of the complexes containing chondrocytes and CGA in an alginate scaffold. Histological analysis, PCR, and western blot were further used to understand the underlying mechanisms. We showed that 60 µM CGA in alginate exhibited notable effects on stimulating chondrogenesis in vitro. Secondly, it was shown that the application of these complexes accelerated the recovery of injury-induced dysfunction by gait analysis when followed for 21 days. Histochemical analysis demonstrated that there was less abnormal vasculature formation, more chondrocyte proliferation and cartilage matrix synthesis in the presence of the complexes containing CGA. We discovered CGA treated transplantation up-regulated the expressions of Sox9 and Col2a1 which were responsible for the stimulation of chondrogenesis. Furthermore, the application of these complexes could suppress the abnormal angiogenesis and fibrosis at the injury site. Lastly, the elevated levels of inflammatory cytokines IL-1ß, TNF-α, p-p65, and MMPs expression were decreased in the presence of CGA. This may be caused through adjusting cellular redox homeostasis associated with Nrf2. This study suggests that combining chondrocytes and CGA on an alginate scaffold can improve the recovery of damaged articular cartilage.

Comparison and validation of the prognostic value of preoperative systemic immune cells in hepatocellular carcinoma after curative hepatectomy.

In this study, we aimed to compare and validate the prognostic abilities of preoperative systemic immune cells in hepatocellular carcinoma (HCC) after curative hepatectomy. We developed two nomograms to predict the postoperative recurrence-free survival (RFS) and overall survival (OS) after comparisons of the systemic immune cell prognostic scores. The two nomograms were constructed based on 305 patients who underwent curative hepatectomy for HCC. The predictive accuracy and discriminative ability of the nomograms were compared with six commonly used staging systems for HCC. The results were validated using bootstrap resampling and an internal validation cohort of 142 patients and an external validation cohort of 169 patients. Necroinflammatory activity in peritumoral liver tissues in the primary cohort was evaluated by hematoxylin and eosin (H&E) staining. Neutrophil, monocyte, and lymphocyte ratio (NMLR) had a higher area under the receiver operating characteristic curves (AUROC) value at both RFS (AUC = 0.603) and OS (AUC = 0.726) compared to that of other systemic immune cell prognostic scores. The independent predictors of RFS or OS, including α-fetoprotein (AFP), tumor size, tumor number, microvascular invasion, and NMLR, were incorporated into the two nomograms. In the primary cohort, the C-indexes of the RFS and OS nomograms were 0.705 and 0.797, respectively. The ROC analyses showed that the two nomograms had larger AUC values (0.664 for RFS and 0.821 for OS) than those of the American Joint Commission on Cancer seventh edition, Barcelona Clinic Liver Cancer, Cancer of the Liver Italian Program, Japan Integrated Staging Score, Okuda stage, and the Vauthey's system. These results were verified by internal and external validations. The nomogram-predicted probability of RFS was associated with peritumoral necroinflammatory activity scores (r = 0.304, P < 0.001). The proposed nomograms had accurate prognostic prediction in patients with HCC after curative hepatectomy.

Predictive and preventive significance of AMPK activation on hepatocarcinogenesis in patients with liver cirrhosis.

Metformin has been demonstrated to prevent hepatocellular carcinoma (HCC). Metformin acts mainly by phosphorylation of AMPK. However, the phosphorylation status of AMPK and its role in the prediction and prevention of HCC in cirrhotic patients remains unclear. The phosphorylation status of AMPK (Thr172) was determined by immunostaining in tissue microarrays of 426 cirrhotic liver tissues. Low expression of p-AMPK was observed in 94 (22.1%) cases. The median follow-up time was 87 months. HCC occurrence probability at 1/3/5/10 years after Hassab procedure was 3.1/9.6/13.8/30.6% in patients with p-AMPK low expression and 0/0.3/0.3/8% in patients with p-AMPK high expression, respectively. HCC occurrence risk was significantly higher in patients with p-AMPK low expression in univariable analysis (HR, 6.25; 95% CI: 3.36-11.60; P < 0.001) and multivariable analysis (HR, 6.0; 95% CI: 3.24-11.10; P < 0.001). An independent external cohort validated the significance of p-AMPK low expression. In addition, in vivo experiments demonstrated that AMPK activation status was negatively related to HCC occurrence and blocking autophagy by chloroquine counteracted the protective effect of AMPK phosphorylation. These results present novel insight into a critical predictive role of AMPK activation in hepatocarcinogenesis and AMPK activation seems to be a potential target for the prevention of hepatocellular carcinoma in patients with liver cirrhosis.

Determinants of tuberculosis trends in six Indigenous populations of the USA, Canada, and Greenland from 1960 to 2014: a population-based study.

BACKGROUND: Tuberculosis continues to disproportionately affect many Indigenous populations in the USA, Canada, and Greenland. We aimed to investigate whether population-based tuberculosis-specific interventions or changes in general health and socioeconomic indicators, or a combination of these factors, were associated with changes in tuberculosis incidence in these Indigenous populations. METHODS: For this population-based study we examined annual tuberculosis notification rates between 1960 and 2014 in six Indigenous populations of the USA, Canada, and Greenland (Inuit [Greenland], American Indian and Alaska Native [Alaska, USA], First Nations [Alberta, Canada], Cree of Eeyou Istchee [Quebec, Canada], Inuit of Nunavik [Quebec, Canada], and Inuit of Nunavut [Canada]), as well as the general population of Canada. We used mixed-model linear regression to estimate the association of these rates with population-wide interventions of bacillus Calmette-Guérin (BCG) vaccination of infants, radiographic screening, or testing and treatment for latent tuberculosis infection (LTBI), and with other health and socioeconomic indicators including life expectancy, infant mortality, diabetes, obesity, smoking, alcohol use, crowded housing, employment, education, and health expenditures. FINDINGS: Tuberculosis notification rates declined rapidly in all six Indigenous populations between 1960 and 1980, with continued decline in Indigenous populations in Alberta, Alaska, and Eeyou Istchee thereafter but recrudescence in Inuit populations of Nunavut, Nunavik, and Greenland. Annual percentage reductions in tuberculosis incidence were significantly associated with two tuberculosis control interventions, relative to no intervention, and after adjustment for infant mortality and smoking: BCG vaccination (-11%, 95% CI -6 to -17) and LTBI screening and treatment (-10%, -3 to -18). Adjusted associations were not significant for chest radiographic screening (-1%, 95% CI -7 to 5). Declining tuberculosis notification rates were significantly associated with increased life expectancy (-37·8 [95% CI -41·7 to -33·9] fewer cases per 100 000 for each 1-year increase) and decreased infant mortality (-9·0 [-9·5 to -8·6] fewer cases per 100 000 for each death averted per 1000 livebirths) in all six Indigenous populations, but no significant associations were observed for other health and socioeconomic indicators examined. INTERPRETATION: Population-based BCG vaccination of infants and LTBI screening and treatment were associated with significant decreases in tuberculosis notification rates in these Indigenous populations. These interventions should be reinforced in populations still affected by tuberculosis, while also addressing the persistent health and socioeconomic disparities. FUNDING: Public Health Department of the Cree Board of Health and Social Services of James Bay.

[Cathepsin B in hepatic Kupffer cells regulates activation of TLR4-independent inflammatory pathways in mice with lipopolysaccharide-induced sepsis].

OBJECTIVE: To investigate the role of cathepsin B in hepatic Kupffer cells (KCs) in activating Toll-like receptor 4(TLR- 4)-independent inflammatory pathways in mice with lipopolysaccharide (LPS)-induced sepsis. METHODS: Eighteen wild-type (WT) mice and 18 TLR4-knockout (TLR4-/-) mice were both divided into 3 groups for intraperitoneal injections of a lethal dose (54 mg/kg) of LPS, LPS and CA-074(a cathepsin B inhibitor), or normal saline, and the survival of the mice were observed. Another 36 WT mice and 36 TLR4-/-mice were also divided into 3 groups and subjected to intraperitoneal injections of normal saline, 20 mg/kg LPS, or LPS with CA-074 pretreatment.After the treatments, KCs were collected from the mice for assessing the protein level and activity of cathepsin B.The histopathological changes of the liver were observed with HE staining, and the serum levels of IL-1α, IL-1ß, TNF-α and IL-18 were detected. RESULTS: Compared with the WT mice,TLR4-/-mice receiving the lethal dose of LPS had significantly longer survival time (up to 84 h) after the injection,but were still unable to fully resist LPS challenge.CA-074 pretreatment prolonged the survival time of WT mice and TLR4-/-mice to 60 h and 132 h,respectively.In the mouse models of sepsis,20 mg/kg LPS induced significantly enhanced activity of cathepsin B without affecting its expression level in the KCs (P<0.05) and increased the serum levels of the inflammatory cytokines.CA-074 pretreatment of the mice obviously lessened the detrimental effects of LPS in TLR4-/-mice by significantly lowering cathepsin B activity in the KCs,alleviating hepatocyte apoptosis and reducing the serum levels of inflammatory cytokines. CONCLUSIONS: Cathepsin B plays an important role in activating TLR4-independent inflammatory pathways in mice with LPS-induced sepsis.

Overexpression of RIP140 suppresses the malignant potential of hepatocellular carcinoma by inhibiting NF­κB­mediated alternative polarization of macrophages.

Tumor-associated macrophages (TAMs) and their alternative activation contribute greatly to the development of hepatocellular carcinoma (HCC). Receptor-interacting protein 140 (RIP140) is widely expressed in macrophages and regulates macrophage-mediated energy metabolism, the inflammatory response and tumorigenesis. However, whether RIP140 is involved in the activation of TAMs has not been reported. In the present study, we determined the expression of RIP140 in macrophages after treatment with HCC-conditioned medium (HCM) for 24 h. We also analyzed the effects of RIP140 overexpression on macrophage polarization, invasion and apoptosis of HepG2 and Huh7 cells. Transwell and apoptosis assays were used to estimated cell invasion and apoptosis. In addition, we investigated the effects of RIP140 overexpression in macrophages on the growth of H22 cells by subcutaneous injection of H22 cells along with macrophages in BALB/c nude mice. Western blotting and qRT-PCR were used to detect protein and mRNA expression associated with the NF-κB/IL-6 axis in TAMs. Immunohistochemical and immunofluorescence staining were used to evaluate the protein expression of RIP140 or F4/80 in human HCC samples. The protein expression of RIP140 in peripheral blood mononuclear cells was detected by western blotting. Kaplan­Meier survival curve estimation of overall survival for patients with HCC was based on RIP140 or F4/80 expression in HCC samples. We found that HCM inhibited RIP140 expression and fostered the alternative activation of macrophages. RIP140 overexpression in TAMs significantly inhibited the alternative activation of macrophages by inhibiting the NF-κB/IL-6 axis in TAMs, and suppressed HCC cell growth both in vitro and in vivo. In addition, the protein expression of RIP140 in peripheral blood monocytes was significantly lower in patients with HCC than in healthy people, and this result was consistent with the expression of RIP140 in HCC samples. Furthermore, low RIP140 expression and high F4/80 expression were found to be closely correlated with shorter survival time of the patients with HCC.