The oral bacterial microbiota facilitates the stratification for ulcerative colitis patients with oral ulcers.

BACKGROUND: Clinically, a large part of inflammatory bowel disease (IBD) patients is complicated by oral lesions. Although previous studies proved oral microbial dysbiosis in IBD patients, the bacterial community in the gastrointestinal (GI) tract of those IBD patients combined with oral ulcers has not been profiled yet. METHODS: In this study, we enrolled four groups of subjects, including healthy controls (CON), oral ulcer patients (OU), and ulcerative colitis patients with (UC_OU) and without (UC) oral ulcers. Bio-samples from three GI niches containing salivary, buccal, and fecal samples, were collected for 16S rRNA V3-V4 region sequencing. Bacterial abundance and related bio-functions were compared, and data showed that the fecal microbiota was more potent than salivary and buccal microbes in shaping the host immune system. ~ 22 UC and 10 UC_OU 5-aminosalicylate (5-ASA) routine treated patients were followed-up for six months; according to their treatment response (a decrease in the endoscopic Mayo score), they were further sub-grouped as responding and non-responding patients. RESULTS: We found those UC patients complicated with oral ulcers presented weaker treatment response, and three oral bacterial genera, i.e., Fusobacterium, Oribacterium, and Campylobacter, might be connected with treatment responding. Additionally, the salivary microbiome could be an indicator of treatment responding in 5-ASA routine treatment rather than buccal or fecal ones. CONCLUSIONS: The fecal microbiota had a strong effect on the host's immune indices, while the oral bacterial microbiota could help stratification for ulcerative colitis patients with oral ulcers. Additionally, the oral microbiota had the potential role in reflecting the treatment response of UC patients. Three oral bacteria genera (Fusobacterium, Oribacterium, and Campylobacter) might be involved in UC patients with oral ulcers lacking treatment responses, and monitoring oral microbiota may be meaningful in assessing the therapeutic response in UC patients.

Analysis for codon usage bias in membrane anchor of nonstructural protein 5A from BVDV.

The nonstructural protein 5A (NS5A) of the bovine viral diarrhea virus (BVDV) is a monotopic membrane protein. This protein can anchor to the cell membrane by an in-plane amphipathic ⍺-helix, which participates in the viral replication complex. In this study, the effects of synonymous codon usage pattern of NS5A and the overall transfer RNA (tRNA) abundance in cells on the formation of the in-plane membrane anchor of NS5A were analyzed, based on NS5A coding sequences of different BVDV genotypes. BVDV NS5A coding sequences represent the most potential for BVDV genotyping. Moreover, the nucleotide usage of BVDV NS5A dominates the genotype-specific pattern of synonymous codon usage. There is an obvious relationship between synonymous codon usage bias and the spatial conformation of the in-plane membrane anchor. Furthermore, the overall tRNA abundance profiling displays that codon positions with a high level of tRNA abundance are more than ones with a low level of tRNA abundance in the in-plane membrane anchor, implying that high translation speed probably acts on the spatial conformation of in-plane membrane anchor of BVDV NS5A. These results give a new opinion on the effect of codon usage bias in the formation of the in-plane membrane anchor of BVDV NS5A.

Local adrenomedullin gene delivery inhibits Leydig cell dysfunction by rescuing steroidogenic enzymes in vivo.

Adrenomedullin (ADM) has beneficial effects on Leydig cells under pathological conditions, including lipopolysaccharide (LPS)-induced orchitis. Our previous studies demonstrated that ADM exerts a restorative effect on steroidogenesis in LPS-treated primary rat Leydig cells by attenuating oxidative stress, inflammation and apoptosis. In this study, we aim to investigate whether ADM inhibits Leydig cell dysfunction by rescuing steroidogenic enzymes in vivo. Rats were administered with LPS and injected with Ad-ADM, an adeno-associated virus vector that expressed ADM. Then, rat testes were collected for 3ß-hydroxysteroid dehydrogenase (3ß-HSD) immunofluorescence staining. Steroidogenic enzymes or steroidogenic regulatory factors or protein, including steroidogenic factor-1 (SF-1), liver receptor homologue-1 (LRH1), Nur77, steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage enzyme (P450scc), 3ß-HSD, cytochrome P450 17α-hydroxylase/17, 20 lyase (CYP17) and 17ß-hydroxysteroid dehydrogenase (17ß-HSD), were detected via gene expression profiling and western blot analysis. Plasma testosterone concentrations were measured. Results showed that ADM may inhibit Leydig cell dysfunction by rescuing steroidogenic enzymes and steroidogenic regulatory factors in vivo. The reduction in the number of Leydig cells after LPS exposure was reversed by ADM. ADM rescued the gene or protein levels of SF-1, LRH1, Nur77, StAR, P450scc, 3ß-HSD, CYP17 and 17ß-HSD and plasma testosterone concentrations. To summarize ADM could rescue some important steroidogenic enzymes, steroidogenic regulatory factors and testosterone production in Leydig cells in vivo.

HDAC6 is critical for ketamine-induced impairment of dendritic and spine growth in GABAergic projection neurons.

Ketamine is widely used in infants and children for anesthesia; both anesthetic and sub-anesthetic doses of ketamine have been reported to preferentially inhibit the GABAergic neurons. Medium spiny neurons (MSNs), the GABAergic projection neurons in the striatum, are vulnerable to anesthetic exposure in the newborn brain. Growth of dendrites requires a deacetylase to remove acetyl from tubulin in the growth cone to destabilize the tubulin. Histone deacetylase 6 (HDAC6) affects microtubule dynamics, which are involved in neurite elongation. In this study we used a human induced pluripotent stem cells (iPSCs)-derived striatal GABA neuron system to investigate the effects of ketamine on HDAC6 and the morphological development of MSNs. We showed that exposure to ketamine (1-500 µM) decreased dendritic growth, dendrite branches, and dendritic spine density in MSNs in a time- and concentration-dependent manner. We revealed that ketamine treatment concentration-dependently inhibited the expression of HDAC6 or aberrantly translocated HDAC6 into the nucleus. Ketamine inhibition on HDAC6 resulted in α-tubulin hyperacetylation, consequently increasing the stability of microtubules and delaying the dendritic growth of MSNs. Finally, we showed that the effects of a single-dose exposure on MSNs were reversible and lasted for at least 10 days. This study reveals a novel role of HDAC6 as a regulator for ketamine-induced deficits in the morphological development of MSNs and provides an innovative method for prevention and treatment with respect to ketamine clinical applications.

Viral adaption of staphylococcal phage: A genome-based analysis of the selective preference based on codon usage Bias.

Given the high therapeutic value of the staphylococcal phage, the genome co-evolution of the phage and the host has gained great attention. Though the genome-wide AT richness in staphylococcal phages has been well-studied with nucleotide usage bias, here we proved that host factor, lifestyle and taxonomy are also important factors in understanding the phage nucleotide usages bias using information entropy formula. Such correlation is especially prominent when it comes to the synonymous codon usages of staphylococcal phages, despite the overall scattered codon usage pattern represented by principal component analysis. This strong relationship is explained by nucleotide skew which testified that the usage biases of nucleotide at different codon positions are acting on synonymous codons. Therefore, our study reveals a hidden relationship of genome evolution with host limitation and phagic phenotype, providing new insight into phage genome evolution at genetic level.

SP1-mediated upregulation of lncRNA LMCD1-AS1 functions a ceRNA for miR-106b-5p to facilitate osteosarcoma progression.

Growing studies have indicated the involvements of long noncoding RNAs (lncRNAs) in the initiation and progression of various tumors. We aimed to investigated the role of lncRNA LMCD1 antisense RNA 1 (LMCD1-AS1) in osteosarcoma development. We found that LMCD1-AS1 and SP1 were highly expressed in osteosarcoma tissues and cell lines. High levels of LMCD1-AS1 were correlated with positively metastasis and poor clinical prognosis. Moreover, we showed that SP1 can bind to the promoter region of LMCD1-AS1, resulting in its overexpression in osteosarcoma. Functionally, silencing of LMCD1-AS1 suppressed the proliferation, migration, invasion and EMT progress of osteosarcoma cells. Mechanistic studies revealed that LMCD1-AS1 was a sponge of miR-106b-5p activity. LMCD1-AS1 modulated survival of osteosarcoma via targeting miR-106b-5p. Overall, we firstly indicated that LMCD1-AS1 overexpression contributes to osteosarcoma development and poor clinical outcome, suggesting that LMCD1-AS1 may be a novel diagnostic and prognostic biomarker for osteosarcoma and a target for osteosarcoma therapy.

Evaluation of Contrast-enhanced US LI-RADS version 2017: Application on 2020 Liver Nodules in Patients with Hepatitis B Infection.

Background Use of contrast material-enhanced (CE) US Liver Imaging Reporting and Data System (LI-RADS) version 2017 has not been validated in large populations where hepatitis B virus (HBV) is endemic. Purpose To evaluate the diagnostic performance of CE US LI-RADS version 2017 in a population with a high prevalence of HBV infection. Materials and Methods In this retrospective study, liver nodules in patients with HBV who were evaluated from January 2004 to December 2016 were categorized as CE US LR-1 to LR-5 through LR-M. A subgroup of LR-M nodules was reclassified as LR-5, and additional analysis was performed. The reference standard consisted of histologic evaluation or composite imaging and clinical follow-up findings. Diagnostic performance was assessed with sensitivity, specificity, positive predictive value (PPV), and negative predictive value. Results A total of 2020 nodules in 1826 patients (median age, 54 years ± 12 [standard deviation]; 1642 men) were included. Of the 1159 LR-5 lesions, 1141 were hepatocellular carcinoma (HCC); three, intrahepatic cholangiocarcinomas; six, other malignancies; six, atypical hyperplasia; and three, benign lesions. The PPV of LR-5 for HCC was 98% (95% confidence interval [CI]: 98%, 99%). In LR-M nodules, 153 showed arterial phase hyperenhancement, early washout, and absence of punched-out appearance within 5 minutes, and 142 of 153 (93%; 95% CI: 89%, 97%) were HCC. If these nodules were reclassified as LR-5, LR-M specificity and PPV as a predictor of non-HCC malignancy increased from 88% (95% CI: 87%, 89%) and 36% (95% CI: 31%, 41%) to 96% (95% CI: 95%, 97%) and 58% (95% CI: 51%, 65%), respectively (P < .001). Despite reclassification, LR-5 specificity and PPV remained high (94% [95% CI: 92%, 96%] and 98% [95% CI: 97%, 99%], respectively). Conclusion The contrast-enhanced US Liver Imaging Reporting and Data System version 2017 category LR-5 is effectively predictive of the presence of hepatocellular carcinoma. In patients with hepatitis B virus infection, performance may be further improved by reclassification of category LR-M nodules with arterial phase hyperenhancement, early washout, and no punched-out appearance to LR-5. Published under a CC BY 4.0 license. Online supplemental material is available for this article. See also the editorial by Sidhu in this issue.

Distinguishing intrahepatic cholangiocarcinoma from hepatocellular carcinoma in patients with and without risks: the evaluation of the LR-M criteria of contrast-enhanced ultrasound liver imaging reporting and data system version 2017.

PURPOSE: To assess the diagnostic performance of the LR-M criteria of Contrast-Enhanced Ultrasound Liver Imaging Reporting and Data System version 2017 in differentiating intrahepatic cholangiocarcinoma (ICC) from hepatocellular carcinoma (HCC) in patients with and without risk factors for HCC. METHODS: Fifty-four ICC in patients with risks and 55 ICC in patients without risks and matched control cases of HCC with and without risks (n = 59 and n = 55, respectively) were enrolled. The enhanced features of the lesions were retrospectively analyzed according to LR-M criteria. The diagnostic performances including the area under the receiver operating characteristic curve (AUC), sensitivity, and specificity of LR-M criteria were assessed. RESULT: Peripheral rim-like hyperenhancement, early washout (< 45 or 60s), and marked washout did not differ between ICCs with and without risks, while all of these features were more common in ICCs than in HCCs (p < 0.05) no matter if patients were with and without risk factors. Using the LR-M criteria to differentiate ICC from HCC, the AUC, sensitivity, specificity, and accuracy were 0.92, 97.25%, 87.72%, and 92.38%, respectively. If early washout onset was adjusted to < 45 s, the specificity was significantly increased to 95.61% (p = 0.004) without losing sensitivity (96.33%, p = 0.945). The rate of HCCs misdiagnosed as ICCs would decrease from 12.3 to 4.4%. CONCLUSION: Although the LR-M criteria showed high sensitivity in distinguishing ICCs from HCCs in patients with and without risks, the specificity would be significantly increased after adjustments to current criteria. KEY POINTS: • The LR-M criteria of CEUS-LI-RADS v2017 could be used for distinguishing ICC from HCC not only in patients with risk factors for HCC but also in those without risk factors. • The diagnostic performance of differentiating ICC from HCC by using the LR-M criteria showed high AUC (0.92), high sensitivity (97.25%), intermediate specificity (87.72%), and high accuracy (92.38%). • If the onset of early washout was adjusted to < 45 s, the specificity was significantly increased from 87.72 to 95.61% (p = 0.004) without losing sensitivity (p = 0.945), and the rate of HCCs misdiagnosed as ICCs would decrease from 12.3 to 4.4%.

[Research Progress of the Roles of Ubiquitination/Deubiquitination in Androgen Receptor Abnormalities and Prostate Cancer].

Ubiquitin is a small molecule protein consisting of 76 amino acids,widely found in eukaryotic cells. The process by which ubiquitin binding to a specific protein is called ubiquitination. Deubiquitination is the reversed process of ubiquitination. Ubiquitination stimulates downstream signal,including complex assembly,protein conformation and activity changes,proteolysis,autophagy,guilt,chromatin remodeling,and DNA repair. More than 80% of eukaryotic protein degradation is mediated by the ubiquitination system,and ubiquitin-dependent proteolysis is an extremely complex process involving many biomolecular processes. By regulating protein homeostasis,ubiquitination can also regulate a variety of biological processes including cell cycle,cell proliferation,and apoptosis,which are closely related to tumorigenesis and progression. Many abnormalities of androgen receptor (AR) including AR gene amplification,mutation,shear mutation,and AR activity enhancement are closely related to prostate cancer progression. In particular,prostate cancer progression is regulated by the ubiquitination/deubiquitination processes. This article summarizes the recent research advances in the roles of ubiquitination/deubiquitination in AR abnormalities and prostate cancer.

Characterization of Equine Infectious Anemia Virus Long Terminal Repeat Quasispecies In Vitro and In Vivo.

The equine infectious anemia virus (EIAV) attenuated vaccine was developed by long-term passaging of a field-isolated virulent strain in cross-species hosts, followed by successive cultivation in cells in vitro To explore the molecular mechanism underlying the evolution of the EIAV attenuated vaccine, a systematic study focusing on long-terminal-repeat (LTR) variation in numerous virus strains ranging from virulent EIAV to attenuated EIAV was performed over time both in vitro and in vivo Two hypervariable regions were identified within the U3 region in the enhancer region (EHR) and the negative regulatory element (NRE) and within the R region in the transcription start site (TSS) and the Tat-activating region (TAR). Among these sites, variation in the U3 region resulted in the formation of additional transcription factor binding sites; this variation of the in vitro-adapted strains was consistent with the loss of pathogenicity. Notably, the same LTR variation pattern was observed both in vitro and in vivo Generally, the LTR variation in both the attenuated virus and the virulent strain fluctuated over time in vivo Interestingly, the attenuated-virus-specific LTR variation was also detected in horses infected with the virulent strain, supporting the hypothesis that the evolution of an attenuated virus might have involved branching from EIAV quasispecies. This hypothesis was verified by phylogenetic analysis. The present systematic study examining the molecular evolution of attenuated EIAV from EIAV quasispecies may provide an informative model reflecting the evolution of similar lentiviruses.IMPORTANCE The attenuated EIAV vaccine was the first lentiviral vaccine used to successfully control for equine infectious anemia in China. This vaccine provides an important reference for studying the relationship between EIAV gene variation and changes in biological characteristics. Importantly, the vaccine provides a model for the investigation of lentiviral quasispecies evolution. This study followed the "natural" development of the attenuated EIAV vaccine by use of a systematic analysis of LTR evolution in vitro and in vivo The results revealed that the increase in LTR variation with passaging was accompanied by a decrease in virulence, which indicated that LTR variability might parallel the attenuation of virulence. Interestingly, the attenuated-virus-specific LTR variation was also detected in virulent-strain-infected horses, a finding consistent with those of previous investigations of gp90 and S2 evolution. Therefore, we present a hypothesis that the evolution of the attenuated virus may involve branching from EIAV quasispecies present in vivo.

Type III Interferons in Viral Infection and Antiviral Immunity.

Interferons (IFNs) can serve as the first line of immune defense against viral infection. The identification of IFN-λs 1, 2, 3 & 4 (termed as type III IFNs) has revealed that the antiviral immune response to viruses contains more components than the type I IFNs that have been known for more than 50 years. IFN-λs are IFN-λ1 (IL-29), IFN-λ2 (IL-28a), IFN-λ3 (IL-28b) and IFN-λ4, which resembles IFN-λ3. IFN-λs have type I-IFN-like immune responses and biological activities, but our knowledge of these novel players in the antiviral response is not well established. In this review, we try to describe the current information on the expression and function of IFN-λs in the innate antiviral immune defense and IFN-λ2's role in regulating and shaping the adaptive immune response. We suggest that IFN-λs are key antiviral cytokines, directly performing an antiviral immune response at epithelial surfaces in the early stages of viral infection, and that these cytokines also skew the balance of Th1 and Th2 cells to Th1 phenotype. In addition, genetic polymorphisms in IFN-λ genes can impair antiviral immune responses in clinical treatment.

Efficacy of ultrasound-guided core needle biopsy in cervical lymphadenopathy: A retrospective study of 6,695 cases.

OBJECTIVES: To determine the diagnostic yield of ultrasound-guided core needle biopsy (US-CNB) in cervical lymphadenopathy and identify the factors influencing the diagnostic accuracy of US-CNB. METHODS: We retrospectively reviewed the records of 6,603 patients with cervical lymphadenopathy who underwent 6695 US-CNB procedures between 2004 and 2017. RESULTS: Adequate specimens were obtained in 92.19 % (6,172/6,695) of cases. Most lymph nodes (67.65 %) were malignant (metastatic carcinoma 4,131; lymphoma 398). The overall accuracy of US-CNB for differentiating benign from malignant lesions was 91.70 % (6,139/6,695). Among biopsies in which adequate material was obtained, the sensitivity, specificity and accuracy of US-CNB were 99.70 %, 100 % and 99.46 %, respectively. The success or failure of US-CNB for the diagnosis of lymphadenopathy was significantly correlated with node size, nature (malignant vs. benign), and location as well as penetration depth, but not with needle size (p = 0.665), number of core tissues obtained (p = 0.324), or history of malignancy (p = 0.060). There were no major procedure-related complications. CONCLUSIONS: US-CNB is a safe and effective method of diagnosing cervical lymphadenopathy, and our findings may help optimise the sampling procedure by maximising its diagnostic accuracy and preserving its minimally invasive nature. KEY POINTS: • US-CNB is useful for the diagnosis of cervical lymphadenopathy. • US-CNB is safe to perform on lymph nodes located near vital structures. • Larger, malignant, level IV lymph nodes yield sufficient tissue samples more easily.

The integration of a macrophage-adapted live vaccine strain of equine infectious anaemia virus (EIAV) in the horse genome.

Integration is an important feature of retroviruses and retrovirus-based therapeutic transfection vectors. The non-primate lentivirus equine infectious anaemia virus (EIAV) primarily targets macrophages/monocytes in vivo. Investigation of the integration features of EIAVDLV121 strains, which are adapted to donkey monocyte-derived macrophages (MDMs), is of great interest. In this study, we analysed the integration features of EIAVDLV121 in equine MDMs during in vitro infection. Our previously published integration sites (IS) for EIAVFDDV13 in fetal equine dermal (FED) cells were also analysed in parallel as references. Sequencing of the host genomic regions flanking the viral IS showed that reference sequence (RefSeq) genes were preferentially targeted for integration by EIAVDLV121. Introns, AT-rich regions, long interspersed nuclear elements (LINEs) and DNA transposons were also predominantly biased toward viral insertion, which is consistent with EIAVFDDV13 integration into the horse genome in FED cells. In addition, the most significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, specifically gag junctions for EIAVDLV121 and tight junctions for EIAVFDDV13, are regulators of metabolic function, which is consistent with the common bioprocesses, specifically cell cycle and chromosome/DNA organization, identified by gene ontology (GO) analysis. Our results demonstrate that EIAV integration occurs in regions that harbour structural and topological features of local chromatin in both macrophages and fibroblasts. Our data on EIAV will facilitate further understanding of lentivirus infection and the development of safer and more effective gene therapy vectors.

Combined Hepatocellular Cholangiocarcinoma (Biphenotypic) Tumors: Potential Role of Contrast-Enhanced Ultrasound in Diagnosis.

OBJECTIVE: The objective of this study was to evaluate the diagnostic performance of contrast-enhanced ultrasound (CEUS) in differentiating combined hepatocellular cholangiocarcinomas (CHCs) from hepatocellular carcinomas (HCCs) and intrahepatic cholangiocarcinomas (ICCs). MATERIALS AND METHODS: Thirty-three patients with pathologically confirmed CHC and matched control subjects with pathologically confirmed HCC (n = 30) or ICC (n = 32) who underwent preoperative CEUS from January 2005 to December 2015 were enrolled in this study. The CEUS images of the hepatic lesions were subjectively analyzed in consensus by two radiologists. The diagnostic performances were evaluated by ROC analysis. RESULTS: In the arterial phase, hyperenhancement was more common in CHCs (76%) and HCCs (100%) than in ICCs (22%), whereas in the late phase marked washout was more common in CHCs (76%) and ICCs (100%) than in HCCs (10%). Using marked washout in the late phase to differentiate CHC from HCC, the area under the ROC curve (AUC) was 0.829, and the sensitivity, specificity, and accuracy were 78%, 90%, and 83%, respectively. Using hyperenhancement in the arterial phase followed by marked washout in the late phase to distinguish CHC from ICC, the AUC value was 0.663, and the sensitivity, specificity, and accuracy were 55%, 78%, and 66%. CONCLUSION: Although the imaging features of CHC, HCC, and ICC on CEUS may overlap, CEUS could be used in the differential diagnosis of CHC from HCC and ICC.

Genetic Evolution during the development of an attenuated EIAV vaccine.

BACKGROUND: The equine infectious anemia virus (EIAV) vaccine is the only attenuated lentiviral vaccine applied on a large scale that has been shown to be effective in controlling the prevalence of EIA in China. This vaccine was developed by successive passaging of a field-isolated virulent strain in different hosts and cultivated cells. To explore the molecular basis for the phenotype alteration of this vaccine strain, we systematically analyzed its genomic evolution during vaccine development. RESULTS: Sequence analysis revealed that the genetic distance between the wild-type strain and six representative strains isolated from key development stages gradually increased with the number of passages. Env gene, but not gag and pol, showed a clear evolutionary flow similar to that of the whole genomes of different generations during the attenuation. Stable mutations were identified in multiple regions of multiple genes along with virus passaging. The adaption of the virus to the growth environment of cultured cells with accumulated genomic and genetic variations was positively correlated with the reduction in pathogenicity and rise of immunogenicity. Statistical analyses revealed significant differences in the frequency of the most stable mutations between in vivo and ex vivo-adapted strains and between virulent and attenuated strains. CONCLUSIONS: These data indicate that EIAV evolution during vaccine development generated an accumulation of mutations under the selective drive force, which helps to better understand the molecular basis of lentivirus pathogenicity and immunogenicity.

Double-stranded-RNA-specific adenosine deaminase 1 (ADAR1) is proposed to contribute to the adaptation of equine infectious anemia virus from horses to donkeys.

Equine infectious anemia virus (EIAV) is a member of the genus Lentivirus of the family Retroviridae. Horses are the most susceptible equids to EIAV infection and are therefore the primary hosts of this virus. In contrast, infected donkeys do not develop clinically active equine infectious anemia (EIA). This phenomenon is similar to what has been observed with HIV-1, which fails to induce AIDS in non-human primates. Interestingly, Shen et al. developed a donkey-tropic pathogenic virus strain (EIAVDV117, DV117) by serially passaging a horse-tropic pathogenic strain, EIAVLN40 (LN40), in donkeys. LN40, which was generated by passaging a field isolate in horses, displayed enhanced virulence in horses but caused no clinical symptoms in donkeys. Infection with DV117 induced acute EIA in nearly 100 % of donkeys. Genomic analysis of DV117 revealed a significantly higher frequency of A-to-G substitutions when compared to LN40. Furthermore, detailed analysis of dinucleotide editing showed that A-to-G mutations had a preference for 5'TpA and 5'ApA. These results strongly implicated the activity of the adenosine deaminase, ADAR1, in this type of mutation. Further investigation demonstrated that overexpression of donkey ADAR1 increased A-to-G mutations within the genome of EIAV. Together with our previous finding that multiple mutations in multiple genes are generated in DV117 during its adaptation from horses to donkeys, the present study suggests that ADAR1-induced A-to-G mutations occur during virus adaption to related new hosts contributing to the alteration of EIAV host tropism.

Predictive Values of Red Blood Cell Distribution Width in Assessing Severity of Chronic Heart Failure.

BACKGROUND This retrospective study was performed to evaluate the value of baseline red blood cell distribution width (RDW) for predicting the severity of chronic heart failure (CHF) compared with N-terminal prohormone brain natriuretic peptide (NT-ProBNP) and other hematological and biochemical parameters. MATERIAL AND METHODS Hematological and biochemical parameters were obtained from 179 patients with New York Heart Association (NYHA) CHF class I (n=44), II (n=39), III (n=41), and IV (n=55). Receiver operator characteristic (ROC) curves were used for assessing predictive values. RESULTS RDW increased significantly in class III and IV compared with class I (14.3±2.3% and 14.3±1.7% vs. 12.9±0.8%, P<0.01). Areas under ROCs (AUCs) of RDW and NT-ProBNP for class IV HF were 0.817 and 0.840, respectively. RDW was markedly elevated in the mortality group compared with the survival group (13.7±1.7 vs. 15.8±1.8, P<0.01). The predictive value of RDW was lower than that of NT-ProBNP but was comparable to white blood cell (WBC), neutrophil (NEU), lymphocyte (L), and neutrophil/lymphocyte ratio (N/L) for mortality during hospitalization, with AUCs of 0.837, 0.939, 0.858, 0.891, 0.885, and 0.885, respectively. RDW and NT-proBNP showed low predictive values for repeated admission (≥3). RDW was an independent risk factor for mortality (OR=2.531, 95% CI: 1.371-4.671). CONCLUSIONS RDW increased significantly in class III and IV patients and in the mortality group. The predictive value of RDW is comparable to NT-proBNP for class IV and lower than that of NT-proBNP for mortality. Elevated RDW is an independent risk factor for mortality.

Proteomic alteration of equine monocyte-derived macrophages infected with equine infectious anemia virus.

Similar to the well-studied viruses human immunodeficiency virus (HIV)-1 and simian immunodeficiency virus (SIV), equine infectious anemia virus (EIAV) is another member of the Lentivirus genus in the family Retroviridae. Previous studies revealed that interactions between EIAV and the host resulted in viral evolution in pathogenicity and immunogenicity, as well as adaptation to the host. Proteomic analysis has been performed to examine changes in protein expression and/or modification in host cells infected with viruses and has revealed useful information for virus-host interactions. In this study, altered protein expression in equine monocyte-derived macrophages (eMDMs, the principle target cell of EIAV in vivo) infected with the EIAV pathogenic strain EIAV(DLV34) (DLV34) was examined using 2D-LC-MS/MS coupled with the iTRAQ labeling technique. The expression levels of 210 cellular proteins were identified to be significantly upregulated or downregulated by infection with DLV34. Alterations in protein expression were confirmed by examining the mRNA levels of eight selected proteins using quantitative real-time reverse-transcription PCR, and by verifying the levels of ten selected proteins using parallel reaction monitoring (PRM). Further analysis of GO and Kyoto Encyclopedia of Genes and Genomes (KEGG)-Pathway enrichment demonstrated that these differentially expressed proteins are primarily related to the biological processes of oxidative phosphorylation, protein folding, RNA splicing, and ubiquitylation. Our results can facilitate a better understanding of the host response to EIAV infection and the cellular processes required for EIAV replication and pathogenesis.

Mice transgenic for equine cyclin T1 and ELR1 are susceptible to equine infectious anemia virus infection.

BACKGROUND: As a member of the tumor necrosis factor receptor (TNFR) protein superfamily, equine lentivirus receptor 1 (ELR1) has been shown to be expressed in various equine cells that are permissive for equine infectious anemia virus (EIAV) replication. The EIAV Tat protein (eTat) activates transcription initiated at the viral long terminal repeat (LTR) promoter through a unique mechanism that requires the recruitment of the equine cyclin T1 (eCT1) cofactor into the viral TAR RNA target element. In vitro studies have demonstrated that mouse fibroblast cell lines (e.g., NIH 3T3 cells) that express the EIAV receptor ELR1 and eCT1 support the productive replication of EIAV. Therefore, we constructed transgenic eCT1- and ELR1-expressing mice to examine whether they support in vivo EIAV replication. FINDINGS: For the first time, we constructed mice transgenic for ELR1 and eCT1. Real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis confirmed that ELR1 and eCT1 were expressed in the transgenic mouse tissues, particularly in the intestines, spleen and lymph nodes. Consistent with the results of EIAV infection in NIH 3T3 cells expressing ELR1 and eCT1, mouse embryonic fibroblasts (MEFs) from the transgenic mice could support EIAV replication. More importantly, this virus could infect and replicate in mouse blood monocyte-derived macrophages (mMDMs). Macrophages are the principle target cell of EIAV in its natural hosts. Furthermore, after the transgenic mice were inoculated with EIAV, the virus could be detected not only in the plasma of the circulating blood but also in multiple organs, among which, the spleen and lymph nodes were the predominant sites of EIAV replication. Finally, we found that consistent with high viral replication levels, the relevant pathological changes occurred in the spleen and lymph nodes. CONCLUSIONS: Our results show that mice transgenic for ELR1 and eCT1 are susceptible to EIAV infection and replication. Further, EIAV infection can cause lesions on the spleen and lymph nodes, similar to those frequently observed in horses, the natural hosts. Therefore, ELR1 and eCT1 are essential in vivo for EIAV invasion and replication.

Equine viperin restricts equine infectious anemia virus replication by inhibiting the production and/or release of viral Gag, Env, and receptor via distortion of the endoplasmic reticulum.

UNLABELLED: Viperin is an endoplasmic reticulum (ER)-associated multifunctional protein that regulates virus replication and possesses broad antiviral activity. In many cases, viperin interferes with the trafficking and budding of viral structural proteins by distorting the membrane transportation system. The lentivirus equine infectious anemia virus (EIAV) has been studied extensively. In this study, we examined the restrictive effect of equine viperin (eViperin) on EIAV replication and investigated the possible molecular basis of this restriction to obtain insights into the effect of this cellular factor on retroviruses. We demonstrated that EIAV infection of primary equine monocyte-derived macrophages (eMDMs) upregulated the expression of eViperin. The overexpression of eViperin significantly inhibited the replication of EIAV in eMDMs, and knockdown of eViperin transcription enhanced the replication of EIAV in eMDMs by approximately 45.8%. Further experiments indicated that eViperin restricts EIAV at multiple steps of viral replication. The overexpression of eViperin inhibited EIAV Gag release. Both the α-helix domain and radical S-adenosylmethionine (SAM) domain were required for this activity. However, the essential motifs in SAM were different from those reported for the inhibition of HIV-1 Gag by human viperin. Furthermore, eViperin disrupted the synthesis of both EIAV Env and receptor, which consequently inhibited viral production and entry, respectively, and this disruption was dependent on the eViperin α-helix domain. Using immunofluorescence assays and electron microscopy, we demonstrated that the α-helix domain is responsible for the distortion of the endoplasmic reticulum (ER). Finally, EIAV did not exhibit counteracting eViperin at the protein level. IMPORTANCE: In previous studies, viperin was indicated as restricting virus replications primarily by the inhibition of virus budding. Here, we show that viperin may have multiple antiviral mechanisms, including the reduction of EIAV Gag budding and Env expression, and these activities are dependent on different viperin domains. We especially demonstrate that the overexpression of viperin inhibits EIAV entry by decreasing the level of virus receptor. Therefore, viperin restriction of viruses is determined largely by the dependence of virus on the cellular membrane transportation system.