Impact of extraction method on the lipids of Himalayan marmot oil with ultrahigh-performance liquid chromatography Q-Exactive Orbitrap mass spectrometry analysis.

RATIONALE: Himalayan marmot oil (SPO) has been used for pharmaceutical purposes for centuries, but its composition is still unclear. The bioactivity of SPO highly depends on the techniques used for its processing. This study focused on the comprehensive lipidomics of SPO, especially on the ones derived from dry rendering, wet rendering, cold pressing, and ultrasound-assisted solvent extraction. METHODS: We performed lipid profiling of SPO acquired by different extraction methods using ultrahigh-performance liquid chromatography Q-Exactive Orbitrap mass spectrometry, and 17 classes of lipids (2 BMPs, 12 LysoPCs, 9 LysoPEs, 41 PCs, 24 PEs, 23 Plasmenyl-PCs, 10 Plasmenyl-PEs, 10 MGs, 63 DGs, 187 TGs, 2 MGDGs, 3 Cer[NDS]s, 22 Cer[NS]s, 2 GlcCer[NS]s, 14 SMs, 14 CEs, and 6 AcylCarnitines) were characterized. RESULTS: Fifty-five lipids were differentially altered (VIP > 1.5, p < 0.05) between the extraction techniques, which can be used as potential biomarkers to differentiate SPO extracted by various methods. Additionally, the contents of oleic acid and arachidic acid were abundant in all samples that may suggest their medicinal values and are conducive to in-depth research. CONCLUSIONS: These findings reveal the alterations of lipid profile and free fatty acid composition in SPO obtained with different extraction methods, providing a theoretical foundation for investigating its important components as functional factors in medicines and cosmetics.

Taxonomic descriptions of Aeromicrobium duanguangcaii sp. nov., Aeromicrobium wangtongii sp. nov. and Aeromicrobium senzhongii sp. nov.

Six Gram-stain-positive, facultative anaerobic, nonmotile and rod-shaped strains, designated zg-Y50T, zg-Y1362, zg-Y1379T, zg-Y869, zg-629T and zg-Y636, were isolated from the intestinal contents of Marmota himalayana in Qinghai Province, PR China. Strains zg-Y50T, zg-Y1379T and zg-629T exhibited the highest 16S rRNA gene sequence similarities of 99.2, 98.9 and 98.8 % to Aeromicrobium choanae 9 H-4T, Aeromicrobium ginsengisoli JCM 14732T and Aeromicrobium flavum TYLN1T, respectively. Phylogenetic and phylogenomic analyses based on the 16S rRNA gene and genomic sequences, respectively, revealed that the six strains formed three distinct clades within the genus Aeromicrobium. The genome sizes of strains zg-Y50T, zg-Y1379T and zg-629T were 3.1-3.7 Mb, with DNA G+C contents of 69.6-70.4 mol%. Average nucleotide identity and digital DNA-DNA hybridization values between each novel strain and available members of the genus Aeromicrobium were all below species thresholds. All novel strains contained MK-9 (H4) as the major menaquinone and diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol as the polar lipids. The predominant fatty acid of the six isolates was C18 : 1 ω9c. The cell-wall peptidoglycan contained ʟʟ-diaminopimelic acid as the diagnostic diamino acid. Based on the results from this polyphasic taxonomic study, three novel species in the genus Aeromicrobium are proposed, namely, Aeromicrobium duanguangcaii sp. nov. (zg-Y50T=GDMCC 1.2981T=KCTC 49764T), Aeromicrobium wangtongii sp. nov. (zg-Y1379T=GDMCC 1.2982T=KCTC 49765T) and Aeromicrobium senzhongii sp. nov. (zg-629T=CGMCC 1.17414T=JCM 33888T).

Aborted infection of human sodium taurocholate cotransporting polypeptide (hNTCP) expressing woodchuck hepatocytes with hepatitis B virus (HBV).

Due to the limited host range of HBV, research progress has been hindered by the absence of a suitable animal model. The natural history of woodchuck hepatitis virus (WHV) infection in woodchuck closely mirrors that of HBV infection in human, making this species a promising candidate for establishing both in vivo and in vitro HBV infection models. Therefore, this animal may be a valuable species to evaluate HBV vaccines and anti-HBV drugs. A significant milestone in HBV and hepatitis D virus (HDV) infection is the discovery of sodium taurocholate cotransporting polypeptide (NTCP) as the functional receptor. In an effort to enhance susceptibility to HBV infection, we introduced hNTCP into the woodchuck hepatocytes by multiple approaches including transduction of vLentivirus-hNTCP in woodchuck hepatocytes, transfection of p-lentivirus-hNTCP-eGFP plasmids into these cells, as well as transduction of vAdenovirus-hNTCP-eGFP. Encouragingly, our findings demonstrated the successful introduction of hNTCP into woodchuck hepatocytes. However, it was observed that these hNTCP-expressing hepatocytes were only susceptible to HDV infection but not HBV. This suggests the presence of additional crucial factors mediating early-stage HBV infection that are subject to stringent species-specific restrictions.

Neisseria lisongii sp. nov. and Neisseria yangbaofengii sp. nov., isolated from the respiratory tracts of marmots.

Four Gram-stain-negative, oxidase-positive, non-motile, cocci-shaped bacteria strains (ZJ106T, ZJ104, ZJ785T and ZJ930) were isolated from marmot respiratory tracts. Phylogenetic analyses based on 16S rRNA genes, 53 ribosomal protein sequences and 441 core genes supported that all four strains belonged to the genus Neisseria with close relatives Neisseria weixii 10022T and Neisseria iguanae ATCC 51483T. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values were below the species-level thresholds (95-96 % for ANI, and 70 % for dDDH). The major fatty acids of all four strains were C16 : 1 ω7c /C16 : 1 ω6c, C16 : 0 and C18 : 1 ω9c. Major polar lipids were composed of diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. MK-8 was the major menaquinone. Based on Virulence Factor Database analysis, the four strains were found to contain NspA and PorB H-factor binding proteins that promote evasion of host immunity. Strains ZJ106T and ZJ104 contained structures similar to the capsule synthesis manipulator of Neisseria meningitidis. Based on phenotypic and phylogenetic evidence, we propose that strains ZJ106T and ZJ785T represent two novel species of the genus Neisseria, respectively, with the names Neisseria lisongii sp. nov. and Neisseria yangbaofengii sp. nov. The type strains are ZJ106T (=GDMCC 1.3111T=JCM 35323T) and ZJ785T (=GDMCC 1.1998T=KCTC 82336T).

Screening of Phyllanthus niruri Leaves Phytoconstituents for Antiviral and Antibacterial Activity by Molecular Docking Studies.

The genus Phyllanthus belongs to one of the largest plant families, the Phyllantaceae (L.). Phyllanthus niruri is an annual perennial herb that grows in tropical Asia, America, China, and the islands of the Indian Ocean. Numerous alkaloids, steroids, flavonoids, lignans, coumarins, polyphenols, and lipids are present in Phyllanthus. The effects of plants have been studied for a variety of purposes, including their antioxidant (Giribabu et al., Evid Based Complement Alternat Med, 2014), anti-inflammatory (Porto et al., Revista Brasileira de Pharmacognosy, 2013), antinociceptive (Sathisha et al., Indian Drugs, 2009), analgesic (Mostofa et al., BMC Complement Altern Med, 2017), antiulcer (Mali et al., Biomed Aging Pathol, 2011), antiarthritic (Obidike and Salawu, Planta Medica, 2010), antiplasmodial (Shilpa et al., Environ Dis, 2018), immunomodulatory (Manikkoth et al., Anticonvulsant activity of Phyllanthus amarus in experimental animal models), anticonvulsant (Wasnik et al., Int J Pharm Sci Rev Res, 2014), antidepressant (Venkateswaran et al., Effects of an extract from Phyllanthus niruri on hepatitis B and woodchuck hepatitis viruses: In vitro and in vivo studies (antiviral agent/Marmota monax/DNA polymerase/hepatitis B surface antigen/woodchuck hepatitis surface antigen). In Hepatitis B and The Prevention of Primary Cancer of The Liver: Selected Publications of Baruch S Blumberg, pp 535-539), antiviral (Venkateswaran et al., Effects of an extract from Phyllanthus niruri on hepatitis B and woodchuck hepatitis viruses: In vitro and in vivo studies (antiviral agent/Marmota monax/DNA polymerase/hepatitis B surface antigen/woodchuck hepatitis surface antigen). In Hepatitis B and The Prevention of Primary Cancer of The Liver: Selected Publications of Baruch S Blumberg, pp 535-539), antitumor (Sharma et al., Asian Pac J Cancer Prev, 2009), hyperlipidemia (Khanna et al., J Ethnopharmacol, 2002), and antifertility (Ezeonwu, Inquiries J, 2011). For additional docking investigations with distinct proteins, the leaf chemicals are assessed, that is, the crystal structure of serine protease hepsin in complex with inhibitor [PDB ID:5 CE1] for antiviral activity human topoisomerase II beta in complex with DNA and etoposide [PDB ID:3QX3] and crystal structure of E. coli GyraseB 24 kDa in complex with 4-(4-bromo-1H-pyrazol-1-yl)-6-[(ethylcarbamoyl)amino]-N-(pyridin-3-yl) pyridine-3-carboxamide [PDB ID: 6F86] for antibacterial activity and have been selected. To evaluate the in silico results and grading of virtual screening, or molecular docking, ritonavir antiviral activity and ampicillin for antibacterial activity were used as a benchmark.

Arthrobacter caoxuetaonis sp. nov., Arthrobacter zhangbolii sp. nov. and Arthrobacter gengyunqii sp. nov., isolated from Marmota himalayana faeces from Qinghai-Tibet Plateau.

Six aerobic or facultative anaerobic, motile, Gram-stain-positive, catalase-positive and oxidase-negative strains (zg-Y453T, zg-Y324, zg-Y462T, zg-Y411, zg-Y809T and zg-Y786) were isolated from different faecal samples of Marmota himalayana from the Qinghai-Tibet Plateau. Pale yellow, round, raised and moist colonies appeared 48 h after incubation at 28 °C on brain-heart infusion plates supplemented with 5 % defibrinated sheep blood. According to the 16S rRNA gene sequence alignment, two strain pairs (zg-Y453T/zg-Y324 and zg-Y462T/zg-Y411) shared the highest similarities to Arthrobacter luteolus (99.5 and 99.2 %), and the other one (zg-Y809T/zg-Y786) to Arthrobacter citreus (99.5 %). Results of phylogenetic analysis based on the 16S rRNA gene and genome sequences showed that these six strains represented three separate species within the genus Arthrobacter. The average nucleotide identity and digital DNA-DNA hybridization values between the three novel type strains (zg-Y453T/zg-Y462T/zg-Y809T) and other known species in this genus were all below respective thresholds (70.2-81.5/19.6-24.2 %, 70.6-81.8/19.8-25.0 %, and 70.4-88.2/19.9-35.3 %). Although phylogenetically related, there were obvious chemotaxonomic and phenotypic differences: strain pair zg-Y462T/zg-Y411 had anteiso-C15 : 0 as the only major fatty acid; the three novel species had different dominant quinones, MK-8(H2) in strains zg-Y462T/zg-Y809T (74.8/81.1 %) and MK-8(H2)/MK-9(H2) (43.1/53.0 %) in zg-Y453T; similarly, the ability to reduce nitrate in strains zg-Y453T and zg-Y462T could differentiate them from zg-Y809T. All strains had diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol, but differed slightly in the types of unidentified glycolipids, phospholipids and lipids. Based on the results of these polyphasic taxonomic analyses, three novel species within the genus Arthrobacter are proposed, namely Arthrobacter caoxuetaonis sp. nov. (type strain, zg-Y453T=GDMCC 1.2809T=JCM 35173T), Arthrobacter zhangbolii sp. nov. (type strain, zg-Y462T=GDMCC 1.2880T=JCM 35170T) and Arthrobacter gengyunqii sp. nov. (type strain, zg-Y809T=GDMCC 1.2808T=JCM 35168T).

Cellulomonas dongxiuzhuiae sp. nov., Cellulomonas wangleii sp. nov. and Cellulomonas fengjieae sp. nov., isolated from the intestinal contents of Marmota himalayana.

Six Gram-stain-positive, aerobic or facultative anaerobic, catalase-positive, urease- and oxidase-negative, rod-shaped bacteria (zg-ZUI157T/zg-ZUI40, zg-ZUI222T/zg-ZUI199 and zg-ZUI188T/ zg-ZUI168) were characterized by a polyphasic approach. Optimal growth of the six strains was observed at pH 7.0 and 28 °C. Phylogenetic analyses based on the 16S rRNA gene and 247 core genes revealed that they belong to genus Cellulomonas. The three type strains have low digital DNA-DNA hybridization (19.3-30.1%) and average nucleotide identity values (78.0-85.5%) with all available genomes in the genus Cellulomonas, and a DNA G+C content range of 73.0-74.6 mol%. The major fatty acids detected in strain pairs zg-ZUI157T/zg-ZUI40 and zg-ZUI 222T/zg-ZUI199 were C16:0, anteiso-C15:0 and anteiso A-C15:1, and C16:0, anteiso-C15:0, anteiso A-C15:1 and anteiso-C17:0 in strain pair zg-ZUI188T/zg-ZUI168. Diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol mannosides were the major polar lipids detected in the three novel species. MK-9(H4) was the predominant quinone detected in strains zg-ZUI222T (87.4 %) and zg-ZUI188T (91.4 %), and MK-9(H4) (49.1 %) and MK-8 (43.4 %) in strain zg-ZUI157T. The cell-wall sugars detected in the three novel species mainly contained rhamnose. The cell-wall peptidoglycan type of the three novel species was A4ß, with an inferred l-Orn-d-Asp interpeptide bridge for strains zg-ZUI157T and zg-ZUI222T, and l-Orn-d-Glu for strain zg-ZUI188T. Based on the results of the phenotypic, phylogenetic, genomic hybridization, average nucleotide identity and chemotaxonomic analyses, the six strains should be classified as belonging to three novel Cellulomonas species, for which the names Cellulomonas dongxiuzhuiae sp. nov. (zg-ZUI157T=GDMCC 1.2559T=KCTC 49678T), Cellulomonas wangleii sp. nov. (zg-ZUI222T=GDMCC 1.2501T=KCTC 49675T) and Cellulomonas fengjieae sp. nov. (zg-ZUI188T=GDMCC 1.2563T=KCTC 49674T) are proposed.

Recent Drug Development in the Woodchuck Model of Chronic Hepatitis B.

Infection with hepatitis B virus (HBV) is responsible for the increasing global hepatitis burden, with an estimated 296 million people being carriers and living with the risk of developing chronic liver disease and cancer. While the current treatment options for chronic hepatitis B (CHB), including oral nucleos(t)ide analogs and systemic interferon-alpha, are deemed suboptimal, the path to finding an ultimate cure for this viral disease is rather challenging. The lack of suitable laboratory animal models that support HBV infection and associated liver disease progression is one of the major hurdles in antiviral drug development. For more than four decades, experimental infection of the Eastern woodchuck with woodchuck hepatitis virus has been applied for studying the immunopathogenesis of HBV and developing new antiviral therapeutics against CHB. There are several advantages to this animal model that are beneficial for performing both basic and translational HBV research. Previous review articles have focused on the value of this animal model in regard to HBV replication, pathogenesis, and immune response. In this article, we review studies of drug development and preclinical evaluation of direct-acting antivirals, immunomodulators, therapeutic vaccines, and inhibitors of viral entry, gene expression, and antigen release in the woodchuck model of CHB since 2014 until today and discuss their significance for clinical trials in patients.

A novel cardiovirus species identified in feces of wild Himalayan marmots.

Recently a growing number of novel cardioviruses have been frequently discovered, which boosts interest in the search for the genetic diversity of cardioviruses. However, wild-marmot cardioviruses have been rarely reported. Here, a novel cardiovirus (tentatively named HHMCDV) was identified in fecal samples from wild Himalayan marmots in Qinghai Tibetan Plateau, China, by viral metagenomics analysis. 3 out of 99 fecal samples from Himalayan marmots were positive for HHMCDV, with the viral loads ranging from 2.7 × 105 to 1.3 × 107 gene copies/g. The complete genomic sequence of HHMCDV was 8108 nucleotides in length, with the typical cardiovirus genome organization and motifs. Coincidentally, while the data was analyzing, one marmot cardiovirus HT7 partial sequence was available in the Genbank, showing 95.1%, 95.6% and 96.0% amino acid (aa) identity in P1, P2 and P3, respectively. However, sequence analysis revealed that HHMCDV and HT7 are more closely related to species Cardiovirus F strain with 65.7%, 61.9-65.6%, 58.9-59.7%, 71.1-71.7%, 69.1-69.4% and 71.4-72.2% aa identity in polyprotein, P1, P2, P3, 2C and 3CD proteins, respectively. Phylogenetic analysis of P1, P2, P3 and 3CD aa sequences indicated that HHMCDV and HT7 clustered tightly and formed a distinct cluster in the Cardiovirus genus. Based on these data, we propose that HHMCDV and HT7 should be two different members of a potential novel species within the genus Cardiovirus. Further studies are needed to investigate the epidemiology and potential pathogenicity of the virus in Himalayan marmots.

Host cell-dependent late entry step as determinant of hepatitis B virus infection.

Hepatitis B virus (HBV) has a highly restricted host range and cell tropism. Other than the human sodium taurocholate cotransporting polypeptide (huNTCP), the HBV entry receptor, host determinants of HBV susceptibility are poorly understood. Woodchucks are naturally infected with woodchuck hepatitis virus (WHV), closely related to HBV, but not with HBV. Here, we investigated the capabilities of woodchuck hepatic and human non-hepatic cell lines to support HBV infection. DNA transfection assays indicated that all cells tested supported both HBV and WHV replication steps post entry, including the viral covalently closed circular DNA (cccDNA) formation, which is essential for establishing and sustaining infection. Ectopic expression of huNTCP rendered one, but not the other, woodchuck hepatic cell line and the non-hepatic human cell line competent to support productive HBV entry, defined here by cccDNA formation during de novo infection. All huNTCP-expressing cell lines tested became susceptible to infection with hepatitis D virus (HDV) that shares the same entry receptor and initial steps of entry with HBV, suggesting that a late entry/trafficking step(s) of HBV infection was defective in one of the two woodchuck cell lines. In addition, the non-susceptible woodchuck hepatic cell line became susceptible to HBV after fusion with human hepatic cells, suggesting the lack of a host cell-dependent factor(s) in these cells. Comparative transcriptomic analysis of the two woodchuck cell lines revealed widespread differences in gene expression in multiple biological processes that may contribute to HBV infection. In conclusion, other than huNTCP, neither human- nor hepatocyte-specific factors are essential for productive HBV entry. Furthermore, a late trafficking step(s) during HBV infection, following the shared entry steps with HDV and before cccDNA formation, is subject to host cell regulation and thus, a host determinant of HBV infection.

Cone-Beam Computed Tomography-Based Spatial Prediction of Drug Dose After Transarterial Chemoembolization Using Radiopaque Drug-Eluting Beads in Woodchuck Hepatocellular Carcinoma.

OBJECTIVES: The aims of this study were to develop a model to estimate drug dose delivered to tumors after transarterial chemoembolization (TACE) with radiopaque drug-eluting beads (DEBs) based on DEB density on cone-beam computed tomography (CT) and to evaluate drug penetration into tissue in a woodchuck hepatoma model. MATERIALS AND METHODS: Transarterial chemoembolization was performed in woodchucks with hepatocellular carcinoma (N = 5) using DEBs (70-150 µm, LC Bead LUMI) loaded with doxorubicin. Livers were resected 45 minutes after embolization, immediately frozen, and cut using liver-specific, 3D-printed sectioning molds. Doxorubicin levels in tumor specimens were measured by high-performance liquid chromatography and correlated with DEB iodine content that was measured using prototype cone-beam CT-based embolization treatment planning software. Doxorubicin penetration into tissue surrounding DEBs was assessed by fluorescence microscopy of tumor sections. Fluorescence intensity was converted into doxorubicin concentration using calibration standards. Intensity-thresholded color heatmaps were generated representing extravascular drug penetration. RESULTS: Consistent segmentation of DEBs on cone-beam CT was achieved using a semiautomated intensity thresholding method. A positive linear correlation (0.96) was found between DEB iodine content measured on cone-beam CT and the amount of doxorubicin measured in tumor specimens. Prediction of doxorubicin levels in tumor sections that were not included in model development was accurate, with a root-mean-square error of 0.08 mg of doxorubicin. Tumor penetration of eluted doxorubicin resulted in concentration gradients where drug content decreased with increasing distance from blood vessels containing DEBs. Drug penetration was greater for blood vessels containing DEB clusters compared with single DEB, with higher doxorubicin concentrations extending further away from the vessels. CONCLUSIONS: Estimation of drug dose delivered during transarterial chemoembolization in a woodchuck hepatocellular carcinoma model was possible using DEB radiopacity on cone-beam CT as a surrogate marker. Doxorubicin penetration was greatest adjacent to vessels containing DEB clusters compared with single DEB. Intraprocedural estimation of the spatial distribution of drug dose within the tumor could enable real-time adjustments to DEB delivery, to maximize treatment coverage or identify regions of tumor at risk for undertreatment.

Arthrobacter sunyaminii sp. nov. and Arthrobacter jiangjiafuii sp. nov., new members in the genus Arthrobacter.

Four novel bacterial strains (zg-ZUI122T/zg-ZUI10 and zg-ZUI227T/zg-ZUI100) were isolated from the intestinal contents of Marmota himalayana and characterized using a polyphasic approach. Cells were Gram-stain- and catalase-positive, urease- and oxidase-negative. Strains grew optimally at 28-30 °C, pH 7.0, with 0.5 % NaCl (w/v). A comparative analysis of 16S rRNA gene sequences revealed that strain pairs zg-ZUI122T/zg-ZUI10 and zg-ZUI227T/zg-ZUI100 belonged to the genus Arthrobacter and were most closely related to Arthrobacter citreus DSM 20133T, with similarities of 99.6 and 99.5 %, respectively. This was further confirmed by phylogenetic analyses based on the 16S rRNA gene and genome sequences. The digital DNA-DNA hybridization and average nucleotide identity values between the two new type strains (zg-ZUI122T and zg-ZUI227T) and other species in the genus Arthrobacter were 20.0-24.4/77.2-83.4% and 19.9-25.1/77.1-83.4%, all below the thresholds. The major cellular fatty acids detected in the two novel species included iso-C15 : 0 and anteiso-C15 : 0; the predominant polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol. MK-8(H2) (77.3%) was the predominant respiratory quinone detected in strain zg-ZUI122T, while MK-8(H2) (53.7%) and MK-9(H2) (46.3%) were detected in strain zg-ZUI227T. The shared cell-wall amino acids detected in the two novel species were alanine, glutamic acid and lysine; the shared whole cell wall sugars consisted of galactose, mannose and ribose. All these analyses concluded that these four strains represent two different novel species in the genus Arthrobacter, for which the names Arthrobacter sunyaminii sp. nov. (zg-ZUI122T = GDMCC 1.2502T = KCTC 49677T) and Arthrobacter jiangjiafuii sp. nov. (zg-ZUI227T = GDMCC 1.2500T = KCTC 49676T) are proposed.

Woodchuck Hepatic Anatomy and Vascular Alterations Due to Hepatocellular Carcinoma with Angiographic Atlas of the Abdomen and Pelvis.

PURPOSE: To characterize the hepatic and abdominal angiographic anatomy of woodchucks and vascular changes associated with hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Twenty-nine woodchucks (23 with viral-associated HCC, 6 without) underwent multiphasic computed tomography (CT). Fourteen woodchucks (8 with HCC) also underwent diagnostic angiography. Hepatic arterial diameters were measured on the CT scans. Woodchucks were divided into 3 groups: non-tumor-bearing, largest tumor supplied by the right hepatic artery (RHA), and largest tumor supplied by the left hepatic artery (LHA). Statistical analysis with a repeated measures model was performed to determine the effects of tumor location (right, left), vessel measured (RHA, LHA), and interaction between the 2 on vessel diameter. Lobar arteries supplying HCC were compared with those that did not. RESULTS: CT anatomy and normal and variant vascular anatomy were defined. In woodchucks with HCC, LHA and RHA supplying tumors had mean diameters of 2.0 mm ± 0.3 and 1.6 mm ± 0.3 versus 1.5 mm ± 0.3 and 1.1 mm ± 0.2 for non-tumor-supplying arteries (P = .0002 and P < .0001), respectively. Lobar arteries supplying tumors were similarly ectatic. The right lateral lobe artery had the most profound increase in the mean diameter when supplying tumors, measuring 1.7 mm ± 0.1 versus 1.0 mm ± 0.1 in the non-tumor-supplying artery (P < .0001). There were no differences in the diameters of the aorta and celiac, common, and proper hepatic arteries between tumor- and non-tumor-bearing woodchucks. An angiographic atlas of the abdominal vessels was generated. CONCLUSIONS: HCC tumoral vasculature in woodchucks was ectatic compared with normal vasculature. This phenomenon recapitulates human HCC and may facilitate investigation of transcatheter and drug delivery therapies in an HCC animal model.

Agonistic Activation of Cytosolic DNA Sensing Receptors in Woodchuck Hepatocyte Cultures and Liver for Inducing Antiviral Effects.

Immune modulation for the treatment of chronic hepatitis B (CHB) has gained more traction in recent years, with an increasing number of compounds designed for targeting different host pattern recognition receptors (PRRs). These agonistic molecules activate the receptor signaling pathway and trigger an innate immune response that will eventually shape the adaptive immunity for control of chronic infection with hepatitis B virus (HBV). While definitive recognition of HBV nucleic acids by PRRs during viral infection still needs to be elucidated, several viral RNA sensing receptors, including toll-like receptors 7/8/9 and retinoic acid inducible gene-I-like receptors, are explored preclinically and clinically as possible anti-HBV targets. The antiviral potential of viral DNA sensing receptors is less investigated. In the present study, treatment of primary woodchuck hepatocytes generated from animals with CHB with HSV-60 or poly(dA:dT) agonists resulted in increased expression of interferon-gamma inducible protein 16 (IFI16) or Z-DNA-binding protein 1 (ZBP1/DAI) and absent in melanoma 2 (AIM2) receptors and their respective adaptor molecules and effector cytokines. Cytosolic DNA sensing receptor pathway activation correlated with a decline in woodchuck hepatitis virus (WHV) replication and secretion in these cells. Combination treatment with HSV-60 and poly(dA:dT) achieved a superior antiviral effect over monotreatment with either agonist that was associated with an increased expression of effector cytokines. The antiviral effect, however, could not be enhanced further by providing additional type-I interferons (IFNs) exogenously, indicating a saturated level of effector cytokines produced by these receptors following agonism. In WHV-uninfected woodchucks, a single poly(dA:dT) dose administered via liver-targeted delivery was well-tolerated and induced the intrahepatic expression of ZBP1/DAI and AIM2 receptors and their effector cytokines, IFN-ß and interleukins 1ß and 18. Receptor agonism also resulted in increased IFN-γ secretion of peripheral blood cells. Altogether, the effect on WHV replication and secretion following in vitro activation of IFI16, ZBP1/DAI, and AIM2 receptor pathways suggested an antiviral benefit of targeting more than one cytosolic DNA receptor. In addition, the in vivo activation of ZBP1/DAI and AIM2 receptor pathways in liver indicated the feasibility of the agonist delivery approach for future evaluation of therapeutic efficacy against HBV in woodchucks with CHB.

Combination Treatment with the Vimentin-Targeting Antibody hzVSF and Tenofovir Suppresses Woodchuck Hepatitis Virus Infection in Woodchucks.

Current treatment options for patients infected with hepatitis B virus (HBV) are suboptimal, because the approved drugs rarely induce cure due to the persistence of the viral DNA genome in the nucleus of infected hepatocytes, and are associated with either severe side effects (pegylated interferon-alpha) or require life-long administration (nucleos(t)ide analogs). We report here the evaluation of the safety and therapeutic efficacy of a novel, humanized antibody (hzVSF) in the woodchuck model of HBV infection. hzVSF has been shown to act as a viral entry inhibitor, most likely by suppressing vimentin-mediated endocytosis of virions. Targeting the increased vimentin expression on liver cells by hzVSF after infection with HBV or woodchuck hepatitis virus (WHV) was demonstrated initially. Thereafter, hzVSF safety was assessed in eight woodchucks naïve for WHV infection. Antiviral efficacy of hzVSF was evaluated subsequently in 24 chronic WHV carrier woodchucks by monotreatment with three ascending doses and in combination with tenofovir alafenamide fumarate (TAF). Consistent with the proposed blocking of WHV reinfection, intravenous hzVSF administration for 12 weeks resulted in a modest but transient reduction of viral replication and associated liver inflammation. In combination with oral TAF dosing, the antiviral effect of hzVSF was enhanced and sustained in half of the woodchucks with an antibody response to viral proteins. Thus, hzVSF safely but modestly alters chronic WHV infection in woodchucks; however, as a combination partner to TAF, its antiviral efficacy is markedly increased. The results of this preclinical study support future evaluation of this novel anti-HBV drug in patients.

Molecular Mechanisms and Animal Models of HBV-Related Hepatocellular Carcinoma: With Emphasis on Metastatic Tumor Antigen 1.

Hepatocellular carcinoma (HCC) is an important cause of cancer death worldwide, and hepatitis B virus (HBV) infection is a major etiology, particularly in the Asia-Pacific region. Lack of sensitive biomarkers for early diagnosis of HCC and lack of effective therapeutics for patients with advanced HCC are the main reasons for high HCC mortality; these clinical needs are linked to the molecular heterogeneity of hepatocarcinogenesis. Animal models are the basis of preclinical and translational research in HBV-related HCC (HBV-HCC). Recent advances in methodology have allowed the development of several animal models to address various aspects of chronic liver disease, including HCC, which HBV causes in humans. Currently, multiple HBV-HCC animal models, including conventional, hydrodynamics-transfection-based, viral vector-mediated transgenic, and xenograft mice models, as well as the hepadnavirus-infected tree shrew and woodchuck models, are available. This review provides an overview of molecular mechanisms and animal models of HBV-HCC. Additionally, the metastatic tumor antigen 1 (MTA1), a cancer-promoting molecule, was introduced as an example to address the importance of a suitable animal model for studying HBV-related hepatocarcinogenesis.

A Lytic Yersina pestis Bacteriophage Obtained From the Bone Marrow of Marmota himalayana in a Plague-Focus Area in China.

A lytic Yersinia pestis phage vB_YpP-YepMm (also named YepMm for briefly) was first isolated from the bone marrow of a Marmota himalayana who died of natural causes on the Qinghai-Tibet plateau in China. Based on its morphologic (isometric hexagonal head and short non-contractile conical tail) and genomic features, we classified it as belonging to the Podoviridae family. At the MOI of 10, YepMm reached maximum titers; and the one-step growth curve showed that the incubation period of the phage was about 10 min, the rise phase was about 80 min, and the lysis amount of the phage during the lysis period of 80 min was about 187 PFU/cell. The genome of the bacteriophage YepMm had nucleotide-sequence similarity of 99.99% to that of the Y. pestis bacteriophage Yep-phi characterized previously. Analyses of the biological characters showed that YepMm has a short latent period, strong lysis, and a broader lysis spectrum. It could infect Y. pestis, highly pathogenic bioserotype 1B/O:8 Y. enterocolitica, as well as serotype O:1b Y. pseudotuberculosis-the ancestor of Y. pestis. It could be further developed as an important biocontrol agent in pathogenic Yersinia spp. infection.

Electromagnetic Tracking and Optical Molecular Imaging Guidance for Liver Biopsy and Point-of-Care Tissue Assessment in Phantom and Woodchuck Hepatocellular Carcinoma.

PURPOSE: To evaluate an integrated liver biopsy platform that combined CT image fusion, electromagnetic (EM) tracking, and optical molecular imaging (OMI) of indocyanine green (ICG) to target hepatocellular carcinoma (HCC) lesions and a point-of-care (POC) OMI to assess biopsy cores, all based on tumor retention of ICG compared to normal liver, in phantom and animal model. MATERIAL: A custom CT image fusion and EM-tracked guidance platform was modified to integrate the measurement of ICG fluorescence intensity signals in targeted liver tissue with an OMI stylet or a POC OMI system. Accuracy was evaluated in phantom and a woodchuck with HCC, 1 day after administration of ICG. Fresh biopsy cores and paraffin-embedded formalin-fixed liver tissue blocks were evaluated with the OMI stylet or POC system to identify ICG fluorescence signal and ICG peak intensity. RESULTS: The mean distance between the initial guided needle delivery location and the peak ICG signal was 5.0 ± 4.7 mm in the phantom. There was complete agreement between the reviewers of the POC-acquired ICG images, cytology, and histopathology in differentiating HCC-positive from HCC-negative biopsy cores. The peak ICG fluorescence intensity signal in the ex vivo liver blocks was 39 ± 12 and 281 ± 150 for HCC negative and HCC positive, respectively. CONCLUSION: Biopsy guidance with fused CT imaging, EM tracking, and ICG tracking with an OMI stylet to detect HCC is feasible. Immediate assessment of ICG uptake in biopsy cores with the POC OMI system is feasible and correlates with the presence of HCC in the tissue.

Characterization and Application of Precore/Core-Related Antigens in Animal Models of Hepatitis B Virus Infection.

BACKGROUND AND AIMS: The hepatitis B core-related antigen (HBcrAg), a composite antigen of precore/core gene including classical hepatitis B core protein (HBc) and HBeAg and, additionally, the precore-related antigen PreC, retaining the N-terminal signal peptide, has emerged as a surrogate marker to monitor the intrahepatic HBV covalently closed circular DNA (cccDNA) and to define meaningful treatment endpoints. APPROACH AND RESULTS: Here, we found that the woodchuck hepatitis virus (WHV) precore/core gene products (i.e., WHV core-related antigen [WHcrAg]) include the WHV core protein and WHV e antigen (WHeAg) as well as the WHV PreC protein (WPreC) in infected woodchucks. Unlike in HBV infection, WHeAg and WPreC proteins were N-glycosylated, and no significant amounts of WHV empty virions were detected in WHV-infected woodchuck serum. WHeAg was the predominant form of WHcrAg, and a positive correlation was found between the serum WHeAg and intrahepatic cccDNA. Both WHeAg and WPreC antigens displayed heterogeneous proteolytic processing at their C-termini, resulting in multiple species. Analysis of the kinetics of each component of the precore/core-related antigen, along with serum viral DNA and surface antigens, in HBV-infected chimpanzees and WHV-infected woodchucks revealed multiple distinct phases of viral decline during natural resolution and in response to antiviral treatments. A positive correlation was found between HBc and intrahepatic cccDNA but not between HBeAg or HBcrAg and cccDNA in HBV-infected chimpanzees, suggesting that HBc can be a better marker for intrahepatic cccDNA. CONCLUSIONS: In conclusion, careful monitoring of each component of HBcrAg along with other classical markers will help understand intrahepatic viral activities to elucidate natural resolution mechanisms as well as guide antiviral development.