The in vitro replication phenotype of hepatitis B virus (HBV) splice variants Sp3 and Sp9 and their impact on wild-type HBV replication.

Prior to nuclear export, the hepatitis B virus (HBV) pregenomic RNA may be spliced by the host cell spliceosome to form shorter RNA sequences known as splice variants. Due to deletions in the open reading frames, splice variants may encode novel fusion proteins. Although not essential for HBV replication, the role of splice variants and their novel fusion proteins largely remains unknown. Some splice variants and their encoded novel fusion proteins have been shown to impair or promote wild-type HBV replication in vitro, and although splice variants Sp3 and Sp9 are two of the most common splice variants identified to date, their in vitro replication phenotype and their impact on wild-type HBV replication are unclear. Here, we utilize greater than genome-length Sp3 and Sp9 constructs to investigate their replication phenotype in vitro, and their impact on wild-type HBV replication. We show that Sp3 and Sp9 were incapable of autonomous replication, which was rescued by providing the polymerase and core proteins in trans. Furthermore, we showed that Sp3 had no impact on wild-type HBV replication, whereas Sp9 strongly reduced wild-type HBV replication in co-transfection experiments. Knocking out Sp9 novel precore-surface and core-surface fusion protein partially restored replication, suggesting that these proteins contributed to suppression of wild-type HBV replication, providing further insights into factors regulating HBV replication in vitro. IMPORTANCE: The role of hepatitis B virus (HBV) splice variants in HBV replication and pathogenesis currently remains largely unknown. However, HBV splice variants have been associated with the development of hepatocellular carcinoma, suggesting a role in HBV pathogenesis. Several in vitro co-transfection studies have shown that different splice variants have varying impacts on wild-type HBV replication, perhaps contributing to viral persistence. Furthermore, all splice variants are predicted to produce novel fusion proteins. Sp1 hepatitis B splice protein contributes to liver disease progression and apoptosis; however, the function of other HBV splice variant novel fusion proteins remains largely unknown. We show that Sp9 markedly impairs HBV replication in a cell culture co-transfection model, mediated by expression of Sp9 novel fusion proteins. In contrast, Sp3 had no effect on wild-type HBV replication. Together, these studies provide further insights into viral factors contributing to regulation of HBV replication.

Transcription and logic operations of magnetic skyrmions in bilayer cross structures.

Magnetic skyrmions are potential building blocks for future information storage and computing devices. Here, we computationally study the skyrmion dynamics in a cross structure made of two ferromagnetic nanotracks. We show that by controlling the skyrmion motion in the cross structure using spin currents, it is possible to realize the transcription of skyrmion at the intersection of the cross structure at certain conditions. Based on the transcription of skyrmion, we computationally demonstrate the AND, OR and NOT logical gates using the cross structures with modified geometries and appropriate magnetic parameters. Our results may provide guidelines to design future three-dimensional spintronics devices based on magnetic skyrmions.

CD8+ T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope display high naive precursor frequency and TCR promiscuity.

To better understand primary and recall T cell responses during coronavirus disease 2019 (COVID-19), it is important to examine unmanipulated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells. By using peptide-human leukocyte antigen (HLA) tetramers for direct ex vivo analysis, we characterized CD8+ T cells specific for SARS-CoV-2 epitopes in COVID-19 patients and unexposed individuals. Unlike CD8+ T cells directed toward subdominant epitopes (B7/N257, A2/S269, and A24/S1,208) CD8+ T cells specific for the immunodominant B7/N105 epitope were detected at high frequencies in pre-pandemic samples and at increased frequencies during acute COVID-19 and convalescence. SARS-CoV-2-specific CD8+ T cells in pre-pandemic samples from children, adults, and elderly individuals predominantly displayed a naive phenotype, indicating a lack of previous cross-reactive exposures. T cell receptor (TCR) analyses revealed diverse TCRαß repertoires and promiscuous αß-TCR pairing within B7/N105+CD8+ T cells. Our study demonstrates high naive precursor frequency and TCRαß diversity within immunodominant B7/N105-specific CD8+ T cells and provides insight into SARS-CoV-2-specific T cell origins and subsequent responses.

Activation of the Alternate Renin-Angiotensin System Correlates with the Clinical Status in Human Cirrhosis and Corrects Post Liver Transplantation.

INTRODUCTION: Recent animal studies have shown that the alternate renin-angiotensin system (RAS) consisting of angiotensin-converting enzyme 2 (ACE2), angiotensin-(1⁻7) (Ang-(1⁻7)) and the Mas receptor is upregulated in cirrhosis and contributes to splanchnic vasodilatation and portal hypertension. To determine the potential relevance of these findings to human liver disease, we evaluated its expression and relationship to the patients' clinical status in subjects with cirrhosis. METHODS: Blood sampling from peripheral and central vascular beds was performed intra-operatively for cirrhotic patients at the time of liver transplantation (LT) or trans-jugular intra-hepatic portosystemic shunt (TIPS) procedures to measure angiotensin II (Ang II) and Ang-(1⁻7) peptide levels and ACE and ACE2 enzyme activity. Relevant clinical and hemodynamic data were recorded pre-operatively for all subjects and peripheral blood sampling was repeated 3 months or later post-operatively. RESULTS: Ang-(1⁻-7) and ACE2 activity were up-regulated more than twofold in cirrhotic subjects both at the time of LT and TIPS and levels returned to comparable levels as control subjects post-transplantation. Ang-(1⁻7) levels correlated positively with the degree of liver disease severity, as measured by the model for an end-stage liver disease (MELD) and also with clinical parameters of pathological vasodilatation including cardiac output (CO). There were strong correlations found between the ACE2:ACE and the Ang-(1⁻7):Ang II ratio highlighting the inter-dependence of the alternate and classical arms of the RAS and thus their potential impact on vascular tone. CONCLUSIONS: In human cirrhosis, the alternate RAS is markedly upregulated and the activation of this system is associated strongly with features of the hyperdynamic circulation in advanced human cirrhosis.

The small molecule drug diminazene aceturate inhibits liver injury and biliary fibrosis in mice.

There is no established medical therapy to treat biliary fibrosis resulting from chronic inflammation in the biliary tree. We have recently shown that liver-specific over-expression of angiotensin converting enzyme 2 (ACE2) of the renin angiotensin system (RAS) ameliorated liver fibrosis in mice. Diminazene aceturate (DIZE), a small molecule drug approved by the US Food and Drug Administration, which is used to treat human trypanosomiasis, has been shown to have antifibrotic properties by enhancing ACE2 activity. In this study we sought to determine the therapeutic potential of DIZE in biliary fibrosis using bile duct ligated and multiple drug resistant gene-2 knockout mice. Additionally, human hepatic stellate (LX-2) and mouse Kupffer (KUP5) cell lines were used to delineate intracellular pathways. DIZE treatment, both in vivo and in vitro, markedly inhibited the activation of fibroblastic stellate cells which was associated with a reduced activation of Kupffer cells. Moreover, DIZE-inhibited NOX enzyme assembly and ROS generation, activation of profibrotic transcription factors including p38, Erk1/2 and Smad2/3 proteins and proinflammatory and profibrotic cytokine release. These changes led to a major reduction in biliary fibrosis in both models without affecting liver ACE2 activity. We conclude that DIZE has a potential to treat biliary fibrosis.

The Adeno-associated Virus - A Safe and Promising Vehicle for Liverspecific Gene Therapy of Inherited and Non-inherited Disorders.

INTRODUCTION: The first human adeno-associated virus (AAV) was originally discovered in 1960s as a contaminant of adenovirus stock preparation and thus it had not been of medical interest. Throughout the last three decades AAV has gained popularity to be used in gene therapy, mainly due to its replicative defectiveness and lack of pathogenicity in human. In addition, its ability to mediate stable and long-term expression in both non-dividing and dividing cells with specific tissue tropism makes AAV one of the most promising candidates for therapeutic gene transfer to treat many inherited as well as non-inherited disorders. Moreover, the use of AAV is not only restricted to overexpression of recombinant transgene, but also to over-express short hairpin RNA and microRNA to knockdown the expression of genes in targeted tissues. DISCUSSION AND CONCLUSION: This review is organized into four parts. In the first part of the review, we discuss about the discovery and history of AAV, followed by detailed AAV biology such as virus genome, virus structure and its life cycle. In the second part of the review, the discussion is centred on the molecular mechanisms of AAV and tissue transduction, including receptor recognition and cell binding, endosomal entry, virus uncoating, nuclear entry and genome replication. Advantages and limitations of using AAV as a safe vehicle for gene delivery is also discussed. In the third part of the review, we discuss about the most commonly used AAV serotypes and variants isolated from human and non-human primates, focusing on their diverse tissue tropisms, transduction efficiency, immunological profiles and their applications in animal studies. Final part of the review focuses on the recent progress of in-vivo gene transfer using AAV for inherited and non-inherited diseases in both preclinical and clinical settings with a special emphasis on potential clinical applications of AAV in the field of liver disease.

ACE2 Therapy Using Adeno-associated Viral Vector Inhibits Liver Fibrosis in Mice.

Angiotensin converting enzyme 2 (ACE2) which breaks down profibrotic peptide angiotensin II to antifibrotic peptide angiotensin-(1-7) is a potential therapeutic target in liver fibrosis. We therefore investigated the long-term therapeutic effect of recombinant ACE2 using a liver-specific adeno-associated viral genome 2 serotype 8 vector (rAAV2/8-ACE2) with a liver-specific promoter in three murine models of chronic liver disease, including carbon tetrachloride-induced toxic injury, bile duct ligation-induced cholestatic injury, and methionine- and choline-deficient diet-induced steatotic injury. A single injection of rAAV2/8-ACE2 was administered after liver disease has established. Hepatic fibrosis, gene and protein expression, and the mechanisms that rAAV2/8-ACE2 therapy associated reduction in liver fibrosis were analyzed. Compared with control group, rAAV2/8-ACE2 therapy produced rapid and sustained upregulation of hepatic ACE2, resulting in a profound reduction in fibrosis and profibrotic markers in all diseased models. These changes were accompanied by reduction in hepatic angiotensin II levels with concomitant increases in hepatic angiotensin-(1-7) levels, resulting in significant reductions of NADPH oxidase assembly, oxidative stress and ERK1/2 and p38 phosphorylation. Moreover, rAAV2/8-ACE2 therapy normalized increased intrahepatic vascular tone in fibrotic livers. We conclude that rAAV2/8-ACE2 is an effective liver-targeted, long-term therapy for liver fibrosis and its complications without producing unwanted systemic effects.

The role of autonomic and baroreceptor reflex control in blood pressure dipping and nondipping in rats.

AIMS: This study hypothesized that the sleep-wake cycle is the major determinant factor affecting blood pressure (BP) dipping in rats and that the sympathovagal imbalance during quiet sleep is associated with the degree of BP fall. METHODS: Polysomnographic recording was performed by telemetry on freely moving Wistar-Kyoto rats over 24  h. Active waking and quiet sleep stages were scored using electroencephalogram and electromyogram. BP dipping was assessed as the percentage decline in SBP from dark active waking to light quiet sleep. About 38% of the rats were classified as dippers (>10% dip) and 62% as nondippers (<10% dip). RESULTS: Among the dipper rats, as compared to dark active waking, the R-R interval and high-frequency power of heart rate variability (a cardiac vagal index) were increased, whereas low-frequency power of blood pressure variability [(BLF), a vascular sympathetic index)] was decreased in light quiet sleep. The sleep-wake cycle rather than the light-dark cycle played the major role in determining BP dipping. The light-dark change in R-R interval, BLF during dark active waking, and baroreflex sensitivity indices during quiet sleep were significantly lower among nondipper rats than among dipper rats. Correlation analysis revealed that BLF during dark active waking as well as high-frequency power of heart rate variability and baroreflex sensitivity indices during light quiet sleep were correlated with the BP dipping percentage. CONCLUSION: This study confirms that the sleep-wake cycle is more important than the light-dark cycle in determining BP dipping. Moreover, lower baroreflex control and parasympathetic activity during quiet sleep as well as lower sympathetic activity during active waking are associated with reduced BP dipping.

Dietary glycotoxins exacerbate progression of experimental fatty liver disease.

BACKGROUND & AIMS: Advanced glycation end-products (AGEs) levels are high in western diets and contribute to tissue injury via activation of RAGE (receptor for AGEs) and generation of reactive oxygen species (ROS). Here, we determined if high dietary AGE intake worsens progression of non-alcoholic fatty liver disease (NAFLD). METHODS: Male Sprague Dawley rats were fed a methionine choline deficient (MCD) diet for 6 weeks before 6 weeks of a high AGE MCD diet through baking. They were compared with animals on MCD diet or a methionine choline replete (MCR) diet alone for 12 weeks. Hepatic ROS, triglycerides, biochemistry, picro-sirius morphometry, hepatic mRNA expression and immunohistochemistry were determined. Primary hepatic stellate cells (HSCs) from both MCR and MCD animals were exposed to AGEs. ROS, proliferation and mRNA expression were determined. RESULTS: The high AGE MCD diet increased hepatic AGE content and elevated triglycerides, NADPH dependent superoxide production, HNE adducts, steatosis, steatohepatitis (CD43, IL-6, TNF-α) and fibrosis (α-SMA, CTGF, COL1A, picrosirius) compared to MCD alone. In HSCs, AGEs significantly increased ROS production, bromodeoxyuridine proliferation and MCP-1, IL-6, α-SMA, and RAGE expression in HSCs from MCD but not MCR animals. These effects were abrogated by RAGE or NADPH oxidase blockade. CONCLUSIONS: In the MCD model of NAFLD, high dietary AGEs increases hepatic AGE content and exacerbates liver injury, inflammation, and liver fibrosis via oxidative stress and RAGE dependent profibrotic effects of AGEs on activated HSCs. This suggests that pharmacological and dietary strategies targeting the AGE/RAGE pathway could slow the progression of NAFLD.

Angiotensin-(1-7) reduces the perfusion pressure response to angiotensin II and methoxamine via an endothelial nitric oxide-mediated pathway in cirrhotic rat liver.

Recent studies have shown that, in cirrhosis, portal angiotensin-(1-7) [Ang-(1-7)] levels are increased and hepatic expression of angiotensin converting enzyme 2 (ACE2) and the Mas receptor are upregulated, but the effects of Ang-(1-7) on hepatic hemodynamics in cirrhosis have not been studied. This study investigated the effects of Ang-(1-7) on vasoconstrictor-induced perfusion pressure increases in cirrhotic rat livers. Ang II or the alpha 1 agonist methoxamine (MTX) were injected in the presence or absence of Ang-(1-7), and the perfusion pressure response was recorded. Denudation of vascular endothelial cells with sodium deoxycholate was used to investigate the contribution of endothelium to the effects of Ang-(1-7). Ang-(1-7) alone had no effect on perfusion pressure. However, it reduced the maximal vasoconstriction response and area under the pressure response curve to Ang II and MTX by >50% (P < 0.05). This effect of Ang-(1-7) was not blocked by Mas receptor inhibition with A779 or by Ang II type 1 and type 2 receptor and bradykinin B(2) receptor blockade and was not reproduced by the Mas receptor agonist AVE0991. D-Pro(7)-Ang-(1-7), a novel Ang-(1-7) receptor antagonist, completely abolished the vasodilatory effects of Ang-(1-7), as did inhibition of endothelial nitric oxide synthase (eNOS) with N(G)-nitro-L-arginine methyl-ester, guanylate cyclase blockade with ODQ and endothelium denudation. The functional inhibition by D-Pro(7)-Ang-(1-7) was accompanied by significant (P < 0.05) inhibition of eNOS phosphorylation. This study shows that Ang-(1-7) significantly inhibits intrahepatic vasoconstriction in response to key mediators of increased vascular and sinusoidal tone in cirrhosis via a receptor population present on the vascular endothelium that is sensitive to D-Pro(7)-Ang-(1-7) and causes activation of eNOS and guanylate cyclase-dependent NO signaling pathways.

Update on new aspects of the renin-angiotensin system in liver disease: clinical implications and new therapeutic options.

The RAS (renin-angiotensin system) is now recognized as an important regulator of liver fibrosis and portal pressure. Liver injury stimulates the hepatic expression of components of the RAS, such as ACE (angiotensin-converting enzyme) and the AT(1) receptor [AngII (angiotensin II) type 1 receptor], which play an active role in promoting inflammation and deposition of extracellular matrix. In addition, the more recently recognized structural homologue of ACE, ACE2, is also up-regulated. ACE2 catalyses the conversion of AngII into Ang-(1-7) [angiotensin-(1-7)], and there is accumulating evidence that this 'alternative axis' of the RAS has anti-fibrotic, vasodilatory and anti-proliferative effects, thus counterbalancing the effects of AngII in the liver. The RAS is also emerging as an important contributor to the pathophysiology of portal hypertension in cirrhosis. Although the intrahepatic circulation in cirrhosis is hypercontractile in response to AngII, resulting in increased hepatic resistance, the splanchnic vasculature is hyporesponsive, promoting the development of the hyperdynamic circulation that characterizes portal hypertension. Both liver fibrosis and portal hypertension represent important therapeutic challenges for the clinician, and there is accumulating evidence that RAS blockade may be beneficial in these circumstances. The present review outlines new aspects of the RAS and explores its role in the pathogenesis and treatment of liver fibrosis and portal hypertension.

Studying permeability in a commonly used epithelial cell line: T84 intestinal epithelial cells.

The integrity, or barrier function, of the intestinal epithelium is of paramount importance in -maintaining good health. This is largely imparted by a single layer of epithelial cells linked by the transmembrane tight junction protein complex near their apical surface. Disruption of epithelial permeability via the tight junctions can contribute to disease progression. The cytokine IFNγ is involved in many inflammatory processes and has been shown to dramatically increase permeability via changes at the tight junction in experimental models. One of its key effectors is the transcription factor, -IRF-1. In our studies of the role of IRF-1 in barrier function using the human T84 intestinal epithelial cell monolayer model, we have found that induction of IRF-1 alone is insufficient to change permeability and that if IRF-1 is involved in mediating the permeability effects of IFNγ, then other factors must also be required.

Regulation of epithelial apical junctions and barrier function by Galpha13.

The epithelial tight junction forms a barrier to paracellular solute movement. In this study we show that the heterotrimeric G-protein Galpha13 regulates the epithelial tight junction barrier. We generated MDCKII kidney epithelial cell lines in which the expression of an active Galpha13 mutant (Galpha13Q226L) could be induced. We demonstrated that Galpha13Q226L expression increased paracellular permeability and caused the disruption and redistribution of proteins comprising the tight junction and the adherens junction away from sites of cell contact and the appearance of basal stress fibers. The effects on the junctional proteins and the actin cytoskeleton were abrogated by the Rho kinase inhibitor Y27632 but not by the Src kinase inhibitor PP2. The Galpha13 mediated increase in permeability was also Src kinase independent but was partly dependent on Rho kinase signalling. Our data establish a link between Galpha13, Rho kinase signaling and epithelial barrier function and not only demonstrate that Galpha13 regulates epithelial apical junction properties but that it does so via signaling pathways that are distinct from the closely related protein Galpha12.