Pharmacologic inhibition of acetyl-CoA carboxylase (ACC) enzymes, ACC1 and ACC2, offers an attractive therapeutic strategy for nonalcoholic fatty liver disease (NAFLD) through simultaneous inhibition of fatty acid synthesis and stimulation of fatty acid oxidation. However, the effects of ACC inhibition on hepatic mitochondrial oxidation, anaplerosis, and ketogenesis in vivo are unknown. Here, we evaluated the effect of a liver-directed allosteric inhibitor of ACC1 and ACC2 (Compound 1) on these parameters, as well as glucose and lipid metabolism, in control and diet-induced rodent models of NAFLD. Oral administration of Compound 1 preferentially inhibited ACC enzymatic activity in the liver, reduced hepatic malonyl-CoA levels, and enhanced hepatic ketogenesis by 50%. Furthermore, administration for 6 days to high-fructose-fed rats resulted in a 20% reduction in hepatic de novo lipogenesis. Importantly, long-term treatment (21 days) significantly reduced high-fat sucrose diet-induced hepatic steatosis, protein kinase C epsilon activation, and hepatic insulin resistance. ACCi treatment was associated with a significant increase in plasma triglycerides (approximately 30% to 130%, depending on the length of fasting). ACCi-mediated hypertriglyceridemia could be attributed to approximately a 15% increase in hepatic very low-density lipoprotein production and approximately a 20% reduction in triglyceride clearance by lipoprotein lipase (P ≤ 0.05). At the molecular level, these changes were associated with increases in liver X receptor/sterol response element-binding protein-1 and decreases in peroxisome proliferator-activated receptor-α target activation and could be reversed with fenofibrate co-treatment in a high-fat diet mouse model. Conclusion: Collectively, these studies warrant further investigation into the therapeutic utility of liver-directed ACC inhibition for the treatment of NAFLD and hepatic insulin resistance.

Vertical sleeve gastrectomy (VSG) is one of the most commonly performed clinical bariatric surgeries used for the remission of obesity and diabetes. However, the precise molecular mechanism by which VSG exerts its beneficial effects remains elusive. Here we report that the membrane-bound G protein-coupled bile acid receptor, GPBAR-1 (also known as TGR5), is required to mediate the effects of anti-obesity, anti-hyperglycemia, and improvements of fatty liver of VSG in mice. In the absence of TGR5, the beneficial metabolic effects of VSG in mice are lost. Moreover, we found that expression of TGR5 was significantly increased after VSG, and VSG alters both BA levels and composition in mice, resulting in enhancement of TGR5 signaling in the ileum and brown adipose tissues, concomitant with improved glucose control and increased energy expenditure. Conclusion Our study elucidates a novel underlying mechanism by which VSG achieves its postoperative therapeutic effects through enhanced TGR5 signaling.

Dual activity of ACC inhibitors provides anti-fibrotic benefit Hepatocytes Hepatic stellate cells ↓ Lipotoxicity ACC inhibitor ↓ Lipogenesis ↑ Fatty acid oxidation ↓ Glycolysis, mitochondrial respiration ↓ Collagen production ↓ α-SMA expression ↓ HSC activation Highlights ACC inhibition blocks TGF-b-induced hepatic stellate cell activation. ACC inhibition blocks tension-mediated activation of primary rat and human hepatic stellate cells. Inhibiting de novo lipogenesis targets hepatic stellate cells' reliance on increased glycolysis and oxidative phosphorylation. ACC inhibition in vivo significantly reduces fibrosis in 4 models of non-alcoholic steatohepatitis.

GS-9688 (selgantolimod) is an oral selective small molecule agonist of toll-like receptor 8 (TLR8) in clinical development for the treatment of chronic hepatitis B (CHB). In this study, we evaluated the antiviral efficacy of GS-9688 in woodchucks chronically infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to hepatitis B virus (HBV). WHV-infected woodchucks received eight weekly oral doses of vehicle, 1 mg/kg GS-9688 or 3 mg/kg GS-9688. Vehicle and 1 mg/kg GS-9688 had no antiviral effect, whereas 3 mg/kg GS-9688 induced a >5 log 10 reduction in serum viral load and reduced WHV surface antigen (WHsAg) levels to below the limit of detection in half of the treated woodchucks. In these animals, the antiviral response was maintained until the end of the study (>5 months after the end of treatment). GS-9688 treatment reduced intrahepatic WHV RNA and DNA levels by >95% in animals in which the antiviral response was sustained after treatment cessation, and these woodchucks also developed detectable anti-WHsAg antibodies. The antiviral efficacy of weekly oral dosing with 3 mg/kg GS-9688 was confirmed in a second woodchuck study. The antiviral response to GS-9688 did not correlate with systemic GS-9688 or cytokine levels but was associated with transient elevation of liver injury biomarkers and enhanced proliferative response of peripheral blood mononuclear cells (PBMC) to WHV peptides. Transcriptomic analysis of liver biopsies taken prior to treatment suggested that T follicular helper cells (T FH) and various other immune cell subsets may play a role in the antiviral response to GS-9688. Conclusion: Finite, short-duration treatment with a clinically relevant dose of GS-9688 is well tolerated and can induce a sustained antiviral response in WHV-infected woodchucks. The identification of a baseline intrahepatic transcriptional signature associated with response to GS-9688 treatment provides insights into the immune mechanisms that mediate this antiviral effect. Approximately 260 million individuals are chronically infected with hepatitis B virus (HBV), and over half a million people are estimated to die each year due to liver diseases associated with chronic hepatitis B (CHB), such as cirrhosis and hepatocellular carcinoma (HCC). Immunological control of CHB ("functional cure") is defined as sustained loss of HBV surface antigen (HBsAg) off treatment, with or without seroconversion to anti-HBs antibody. Several nucleos(t)ide analogs, as well as interferon-alpha (IFN-α), are approved for the treatment of CHB. These therapies reduce viremia and

HIV infection is controlled immunologically in a small subset of infected individuals without antiretroviral therapy (ART), though the mechanism of control is unclear. CD8 ϩ T cells are a critical component of HIV control in many immunological controllers. NK cells are also believed to have a role in controlling HIV infection, though their role is less well characterized. We used mass cytometry to simultaneously measure the levels of expression of 24 surface markers on peripheral NK cells from HIV-infected subjects with various degrees of HIV natural control; we then used machine learning to identify NK cell subpopulations that differentiate HIV controllers from noncontrollers. Using CITRUS (cluster identification, characterization, and regression), we identified 3 NK cell subpopulations that differentiated subjects with chronic HIV viremia (viremic noncontrollers [VNC]) from individuals with undetectable HIV viremia without ART (elite controllers [EC]). In a parallel approach, we identified 11 NK cell subpopulations that differentiated HIV-infected subject groups using k-means clustering after dimensionality reduction by t-neighbor stochastic neighbor embedding (tSNE) or linear discriminant analysis (LDA). Among these additional 11 subpopulations, the frequencies of 5 correlated with HIV DNA levels; importantly, significance was retained in 2 subpopulations in analyses that included only cohorts without detectable viremia. By comparing the surface marker expression patterns of all identified subpopulations, we revealed that the CD11b ϩ CD57 Ϫ CD161 ϩ Siglec-7 ϩ subpopulation of CD56 dim CD16 ϩ NK cells are more abundant in EC and HIV-negative controls than in VNC and that the frequency of these cells correlated with HIV DNA levels. We hypothesize that this population may have a role in immunological control of HIV infection. IMPORTANCE HIV infection results in the establishment of a stable reservoir of latently infected cells; ART is usually required to keep viral replication under control and disease progression at bay, though a small subset of HIV-infected subjects can control HIV infection without ART through immunological mechanisms. In this study, we sought to identify subpopulations of NK cells that may be involved in the natural immunological control of HIV infection. We used mass cytometry to measure surface marker expression on peripheral NK cells. Using two distinct semisupervised machine learning approaches, we identified a CD11b ϩ CD57 Ϫ CD161 ϩ Siglec-7 ϩ subpopulation of CD56 dim CD16 ϩ NK cells that differentiates HIV controllers from noncontrollers. These cells can be sorted out for future functional studies to assess their potential role in the immunological control of HIV infection.

Hepatitis B virus (HBV) can integrate into the chromosomes of infected hepatocytes, creating potentially oncogenic lesions that can lead to hepatocellular carcinoma (HCC). However, our current understanding of integrated HBV DNA architecture, burden and transcriptional activity is incomplete due to technical limitations. A combination of genomics approaches was used to describe HBV integrations and corresponding transcriptional signatures in three HCC cell lines: huH-1, PLC/PRF/5 and Hep3B. To generate high coverage long-read sequencing data, a custom panel of HBV-targeting biotinylated oligonucleotide probes was designed. Targeted long-read DNA sequencing captured entire HBV integration events within individual reads, revealing that integrations may include deletions and inversions of viral sequences. Surprisingly, all three HCC cell lines contain integrations that are associated with host chromosomal translocations. In addition, targeted long-read RNA sequencing allowed for the assignment of transcriptional activity to specific integrations and resolved the contribution of overlapping HBV transcripts. HBV transcripts chimeric with host sequences were resolved in their entirety and often included >1000bp of host sequence. This study provides the first comprehensive description of HBV integrations and associated transcriptional activity in three commonly utilized HCC-derived cell lines. The application of novel methods sheds new light on the complexity of these integrations, including HBV bidirectional transcription, nested transcripts, silent integrations and host genomic rearrangements. The observation of multiple HBV-associated chromosomal translocations gives rise to the hypothesis that HBV may be a driver of genetic instability and provides a potential new mechanism for HCC development. Importance HCC-derived cell lines have served as practical models to study HBV biology for decades. These cell lines harbor multiple HBV integrations and express only HBV surface antigen (HBsAg). To date, an accurate description of the integration burden, architecture and transcriptional profile of these cell lines has been limited due to technical constraints. We have developed a targeted long-read sequencing assay which reveals the entire architecture of integrations in these cell lines. In addition, we identified five chromosomal translocations with integrated HBV DNA at the inter-chromosomal junctions. Incorporation of long-read RNA-Seq data indicated that many integrations and translocations were transcriptionally silent. The observation of multiple HBV-associated translocations has strong implications regarding the potential mechanisms for the development of HBV-associated HCC.

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Curcumin is an antioxidant and anti-inflammatory bioflavonoid that has been recently identified as an anti-amyloid agent as well. To make it more available in its potent form as a potential amyloid disaggregation agent, we employed high-density lipoproteins (HDL), which are lipidprotein complexes that transport plasma cholesterol, to transport curcumin. The objective of this study was to employ reconstituted HDL containing human apoE3 N-terminal (NT) domain, as a vehicle to transport curcumin. The NT domain serves as a ligand to mediate binding and uptake of lipoprotein complexes via the low-density lipoprotein receptor (LDLr) family of proteins located at the cell surface. Reconstituted HDL was prepared with phospholipids and recombinant apoE3-NT domain in the absence or presence of curcumin. Non-denaturing polyacrylamide gel electrophoresis indicated that the molecular mass and Stokes' diameter of HDL bearing curcumin were ∼670 kDa and ∼17 nm, respectively, while electron microscopy revealed the presence of discoidal particles. Fluorescence emission spectra of HDL bearing (the intrinsically fluorescent) curcumin indicated that the wavelength of maximal fluorescence emission (λmax) of curcumin was ∼495 nm, which is highly blue-shifted compared to λmax of curcumin in solvents of varying polarity (λmax ranging from 515- 575 nm) or in aqueous buffers. In addition, an enormous enhancement in fluorescence emission intensity was noted in curcumin-containing HDL compared to curcumin in aqueous buffers. Curcumin fluorescence emission was quenched to a significant extent by lipid-based quenchers but not by aqueous quenchers. These observations indicate that curcumin has partitioned efficiently into the hydrophobic milieu of the phospholipid bilayer of HDL. Functional assays indicated that the LDLr-binding ability of curcumin-containing HDL with apoE3-NT is similar to that of HDL without curcumin. Taken together, we report that apoE-containing HDL has tremendous potential as a ‘nanovehicle’ with a homing device to transport curcumin to target sites.