A small molecule targeting ALOX12-ACC1 ameliorates nonalcoholic steatohepatitis in mice and macaques.

Nonalcoholic steatohepatitis (NASH) is a progressive liver disease and has become a leading indication for liver transplantation in the United States. The development of effective therapies for NASH is a major unmet need. Here, we identified a small molecule, IMA-1, that can treat NASH by interrupting the arachidonate 12-lipoxygenase (ALOX12)­acetyl-CoA carboxylase 1 (ACC1) interaction. IMA-1 markedly blocked diet-induced NASH progression in both male mice and Cynomolgus macaque therapeutic models. The anti-NASH efficacy of IMA-1 was comparable to ACC inhibitor in both species. Protein docking simulations and following functional experiments suggested that the anti-NASH effects of IMA-1 were largely dependent on its direct binding to a pocket in ALOX12 proximal to its ACC1-interacting surface instead of inhibiting ALOX12 lipoxygenase activity. IMA-1 treatment did not elicit hyperlipidemia, a known side effect of direct inhibition of ACC enzymatic activity, in both mice and macaques. These findings provide proof of concept across multiple species for the use of small molecule­based therapies for NASH.

Redefining Cardiac Biomarkers in Predicting Mortality of Inpatients With COVID-19.

The prognostic power of circulating cardiac biomarkers, their utility, and pattern of release in coronavirus disease 2019 (COVID-19) patients have not been clearly defined. In this multicentered retrospective study, we enrolled 3219 patients with diagnosed COVID-19 admitted to 9 hospitals from December 31, 2019 to March 4, 2020, to estimate the associations and prognostic power of circulating cardiac injury markers with the poor outcomes of COVID-19. In the mixed-effects Cox model, after adjusting for age, sex, and comorbidities, the adjusted hazard ratio of 28-day mortality for hs-cTnI (high-sensitivity cardiac troponin I) was 7.12 ([95% CI, 4.60-11.03] P<0.001), (NT-pro)BNP (N-terminal pro-B-type natriuretic peptide or brain natriuretic peptide) was 5.11 ([95% CI, 3.50-7.47] P<0.001), CK (creatine phosphokinase)-MB was 4.86 ([95% CI, 3.33-7.09] P<0.001), MYO (myoglobin) was 4.50 ([95% CI, 3.18-6.36] P<0.001), and CK was 3.56 ([95% CI, 2.53-5.02] P<0.001). The cutoffs of those cardiac biomarkers for effective prognosis of 28-day mortality of COVID-19 were found to be much lower than for regular heart disease at about 19%-50% of the currently recommended thresholds. Patients with elevated cardiac injury markers above the newly established cutoffs were associated with significantly increased risk of COVID-19 death. In conclusion, cardiac biomarker elevations are significantly associated with 28-day death in patients with COVID-19. The prognostic cutoff values of these biomarkers might be much lower than the current reference standards. These findings can assist in better management of COVID-19 patients to improve outcomes. Importantly, the newly established cutoff levels of COVID-19-associated cardiac biomarkers may serve as useful criteria for the future prospective studies and clinical trials.

Grazing exclusion promotes grasses functional group dominance via increasing of bud banks in steppe community.

To understand the bud banks response to grazing exclusion, we conducted a demographic experiment in long-term grazing exclusion (20 year and 30 year) typical steppe. Results showed that grass functional group constituted the vast majority of the aboveground vegetation and belowground bud bank in all treatments. Long-term grazing exclusion significantly increased total aboveground biomass (2.5 and 2.6 times in 20y and 30y grazing exclusion grasslands, respectively), and decreased total stem density (31% and 37% in 20y and 30y grazing exclusion grasslands, respectively). Grazing exclusion for 20 and 30 years increased grass aboveground biomass respectively by 6.0 and 8.0 times, and decreased grass stem density by 38% and 33%. Grazing exclusion had different effects on belowground bud density of grass and forb functional group. Long-term grazing exclusion significantly increased plant buds and bud bank size (25% and 37% in 20y and 30y grazing exclusion grasslands, respectively), especially for grass functional group (49% and 95% in 20y and 30y grazing exclusion grasslands, respectively), but had no significant effects on forb bud density. Changes of aboveground community were significantly related to changes of belowground bud bank under both grazing and grazing exclusion grasslands. The bud bank density of grass functional group was significantly positive with total (R2 = 0.33, P < 0.05) and grass aboveground biomass (R2 = 0.36, P < 0.01), while negative related with total (R2 = -0.27, P < 0.05) and grass stem density (R2 = -0.22, P < 0.05). Grazed grasslands, 20y and 30y grazing exclusion grasslands all were not meristem limited and had large reserve bud banks, which would completely replace the aboveground stem population during the growing season. These findings indicate that grazing exclusion could not only improve a large bud bank for grassland restoration but also improve the dominance of grass functional group by increasing grass belowground bud banks in typical steppe community. We propose that the belowground bud bank might be a good approach to indicating potential succession direction of aboveground community.

The deubiquitinating enzyme cylindromatosis mitigates nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a common clinical condition that can lead to advanced liver diseases. Lack of effective pharmacotherapies for NASH is largely attributable to an incomplete understanding of its pathogenesis. The deubiquitinase cylindromatosis (CYLD) plays key roles in inflammation and cancer. Here we identified CYLD as a suppressor of NASH in mice and in monkeys. CYLD is progressively degraded upon interaction with the E3 ligase TRIM47 in proportion to NASH severity. We observed that overexpression of Cyld in hepatocytes concomitantly inhibits lipid accumulation, insulin resistance, inflammation and fibrosis in mice with NASH induced in an experimental setting. Mechanistically, CYLD interacts directly with the kinase TAK1 and removes its K63-linked polyubiquitin chain, which blocks downstream activation of the JNK-p38 cascades. Notably, reconstitution of hepatic CYLD expression effectively reverses disease progression in mice with dietary or genetically induced NASH and in high-fat diet-fed monkeys predisposed to metabolic syndrome. Collectively, our findings demonstrate that CYLD mitigates NASH severity and identify the CYLD-TAK1 axis as a promising therapeutic target for management of the disease.

The deubiquitinating enzyme TNFAIP3 mediates inactivation of hepatic ASK1 and ameliorates nonalcoholic steatohepatitis.

Activation of apoptosis signal-regulating kinase 1 (ASK1) in hepatocytes is a key process in the progression of nonalcoholic steatohepatitis (NASH) and a promising target for treatment of the condition. However, the mechanism underlying ASK1 activation is still unclear, and thus the endogenous regulators of this kinase remain open to be exploited as potential therapeutic targets. In screening for proteins that interact with ASK1 in the context of NASH, we identified the deubiquitinase tumor necrosis factor alpha-induced protein 3 (TNFAIP3) as a key endogenous suppressor of ASK1 activation, and we found that TNFAIP3 directly interacts with and deubiquitinates ASK1 in hepatocytes. Hepatocyte-specific ablation of Tnfaip3 exacerbated nonalcoholic fatty liver disease- and NASH-related phenotypes in mice, including glucose metabolism disorders, lipid accumulation and enhanced inflammation, in an ASK1-dependent manner. In contrast, transgenic or adeno-associated virus-mediated TNFAIP3 gene delivery in the liver in both mouse and nonhuman primate models of NASH substantially blocked the onset and progression of the disease. These results implicate TNFAIP3 as a functionally important endogenous suppressor of ASK1 hyperactivation in the pathogenesis of NASH and identify it as a potential new molecular target for NASH therapy.

Corrigendum: Targeting CASP8 and FADD-like apoptosis regulator ameliorates nonalcoholic steatohepatitis in mice and nonhuman primates.

This corrects the article DOI: 10.1038/nm.4290.

USP18 protects against hepatic steatosis and insulin resistance through its deubiquitinating activity.

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis, impaired insulin sensitivity, and chronic low-grade inflammation. However, the pathogenic mechanism of NAFLD is poorly understood, which hinders the exploration of possible treatments. Here, we report that ubiquitin-specific protease 18 (USP18), a member of the deubiquitinating enzyme family, plays regulatory roles in NAFLD progression. Expression of USP18 was down-regulated in the livers of nonalcoholic steatohepatitis patients and high-fat diet (HFD)-induced or genetically obese mice. When challenged with HFD, hepatocyte-specific USP18 transgenic mice exhibited improved lipid metabolism and insulin sensitivity, whereas mice knocked out of USP18 expression showed adverse trends regarding hepatic steatosis and glucose metabolic disorders. Furthermore, the concomitant inflammatory response was suppressed in USP18-hepatocyte-specific transgenic mice and promoted in USP18-hepatocyte-specific knockout mice treated with HFD. Mechanistically, hepatocyte USP18 ameliorates hepatic steatosis by interacting with and deubiquitinating transforming growth factorß-activated kinase 1 (TAK1), which inhibits TAK1 activation and subsequently suppresses the downstream c-Jun N-terminal kinase and nuclear factor kappa B signaling pathways. This is further validated by alleviated steatotic phenotypes and highly activated insulin signaling in HFD-fed USP18-hepatocyte-specific knockout mice administered a TAK1 inhibitor. The therapeutic effect of USP18 on NAFLD relies on its deubiquitinating activity because HFD-fed mice injected with active-site mutant USP18 failed to inhibit hepatic steatosis. CONCLUSION: USP18 associates with and deubiquitinates TAK1 to protect against hepatic steatosis, insulin resistance, and the inflammatory response. (Hepatology 2017;66:1866-1884).

The novel intracellular protein CREG inhibits hepatic steatosis, obesity, and insulin resistance.

Cellular repressor of E1A-stimulated genes (CREG), a novel cellular glycoprotein, has been identified as a suppressor of various cardiovascular diseases because of its capacity to reduce hyperplasia, maintain vascular homeostasis, and promote endothelial restoration. However, the effects and mechanism of CREG in metabolic disorder and hepatic steatosis remain unknown. Here, we report that hepatocyte-specific CREG deletion dramatically exacerbates high-fat diet and leptin deficiency-induced (ob/ob) adverse effects such as obesity, hepatic steatosis, and metabolic disorders, whereas a beneficial effect is conferred by CREG overexpression. Additional experiments demonstrated that c-Jun N-terminal kinase 1 (JNK1) but not JNK2 is largely responsible for the protective effect of CREG on the aforementioned pathologies. Notably, JNK1 inhibition strongly prevents the adverse effects of CREG deletion on steatosis and related metabolic disorders. Mechanistically, CREG interacts directly with apoptosis signal-regulating kinase 1 (ASK1) and inhibits its phosphorylation, thereby blocking the downstream MKK4/7-JNK1 signaling pathway and leading to significantly alleviated obesity, insulin resistance, and hepatic steatosis. Importantly, dramatically reduced CREG expression and hyperactivated JNK1 signaling was observed in the livers of nonalcoholic fatty liver disease (NAFLD) patients, suggesting that CREG might be a promising therapeutic target for NAFLD and related metabolic diseases. CONCLUSION: The results of our study provides evidence that CREG is a robust suppressor of hepatic steatosis and metabolic disorders through its direct interaction with ASK1 and the resultant inactivation of ASK1-JNK1 signaling. This study offers insights into NAFLD pathogenesis and its complicated pathologies, such as obesity and insulin resistance, and paves the way for disease treatment through targeting CREG. (Hepatology 2017;66:834-854).

Targeting CASP8 and FADD-like apoptosis regulator ameliorates nonalcoholic steatohepatitis in mice and nonhuman primates.

Nonalcoholic steatohepatitis (NASH) is a progressive disease that is often accompanied by metabolic syndrome and poses a high risk of severe liver damage. However, no effective pharmacological treatment is currently available for NASH. Here we report that CASP8 and FADD-like apoptosis regulator (CFLAR) is a key suppressor of steatohepatitis and its metabolic disorders. We provide mechanistic evidence that CFLAR directly targets the kinase MAP3K5 (also known as ASK1) and interrupts its N-terminus-mediated dimerization, thereby blocking signaling involving ASK1 and the kinase MAPK8 (also known as JNK1). Furthermore, we identified a small peptide segment in CFLAR that effectively attenuates the progression of steatohepatitis and metabolic disorders in both mice and monkeys by disrupting the N-terminus-mediated dimerization of ASK1 when the peptide is expressed from an injected adenovirus-associated virus 8-based vector. Taken together, these findings establish CFLAR as a key suppressor of steatohepatitis and indicate that the development of CFLAR-peptide-mimicking drugs and the screening of small-molecular inhibitors that specifically block ASK1 dimerization are new and feasible approaches for NASH treatment.

Long Noncoding RNA MIR4697HG Promotes Cell Growth and Metastasis in Human Ovarian Cancer.

Ovarian cancer is one of the three most common gynecological malignant tumors worldwide. The prognosis of patients suffering from this malignancy remains poor because of limited therapeutic strategies. Herein, we investigated the role of a long noncoding RNA named MIR4697 host gene (MIR4697HG) in the cell growth and metastasis of ovarian cancer. Results showed that the transcriptional level of MIR4697HG in cancerous tissues increased twofold compared with that in adjacent noncancerous tissues. MIR4697HG was differentially expressed in ovarian cancer cell lines, with the highest levels in OVCAR3 and SKOV3 cells. MIR4697HG knockdown by specific shRNA significantly inhibited cell proliferation and colony formation in both OVCAR3 and SKOC3 cells. Consistently, in a xenograft model of ovarian cancer, MIR4697HG depletion also significantly restricted tumor volumes and weights. Furthermore, MIR4697HG knockdown inhibited cell migration and invasion capacities. Invasion ability was inhibited by 58% in SKOV3 cells and 40% in OVCAR3 cells, and migration ability was inhibited by 73% in SKOV3 cells and 62% in OVCAR3 cells after MIR4697HG knockdown. MIR4697HG knockdown also caused a decrease in matrix metalloprotease-9, phosphorylated ERK, and phosphorylated AKT. These data suggested that MIR4697HG promoted ovarian cancer growth and metastasis. The aggressive role of MIR4697HG in ovarian cancer may be related to the ERK and AKT signaling pathways.

Computational identification of microRNAs and their targets in perennial Ryegrass (Lolium perenne).

MicroRNAs (miRNAs) are small non-coding RNA molecules of 22 nucleotides in length that have been characterized as regulators of messenger RNA (mRNA) regulating a number of developmental processes in plants and animals by silencing genes using multiple mechanisms. miRNAs have been extensively studied in various plant species; however, few information are available about miRNAs in perennial ryegrass, animal feed, and industrial raw materials. In this study, the 12 potential perennial ryegrass miRNAs were identified for the first time by computational approach. Using the newly identified miRNA sequences, the perennial ryegrass mRNA database was further used for BLAST search and detected 33 potential targets of miRNAs. Prediction of potential miRNA target genes revealed their functions involved in various important plant biological processes. Our result should be useful for further investigation into the biological functions of miRNAs in perennial ryegrass. The selected miRNAs representing four families were verified by RT-PCR experiment, indicating that the prediction method that we used to identify the miRNAs was effective.

Identification of microRNA genes and their mRNA targets in Festuca arundinacea.

MicroRNAs (miRNAs) have emerged as a novel class of endogenous, small, non-coding RNAs of 22 nucleotides (nts) in length, which plays important roles in post-transcriptional degradation of target mRNA or inhibition of protein synthesis through binding the specific sites of target mRNA. Growing evidences have shown that miRNAs play an important role in various biological processes, including growth and development, signal transduction, apoptosis, proliferation, stress responses, maintenance of genome stability, and so on. In our study, we used bioinformatic tools to predict miRNA and the corresponding target genes of Festuca arundinacea. We used known miRNAs of other plants from miRBase to search against expressed sequence tags (EST) databases and genome survey sequences (GSS) of F. arundinacea. A total of 8 potential miRNAs were predicted. Phylogenetic analysis of the predicted miRNAs revealed that miRNA398c of F. arundinacea species was evolutionary highly conserved with Populus trichocarpa. The 8 potential miRNAs corresponding to 20 target genes were found. Most of the miRNA target genes were predicted to encode transcription factors that regulate cell growth and development, signaling, metabolism, and other biology processes. By bioinformatics methods, we can effectively predict novel miRNAs and its target genes and add information to F. arundinacea miRNA database. Moreover, it shows a path for the prediction and analysis of miRNAs to those species whose genomes are not available through bioinformatics tools.