Rongzhong Huang

Borna disease virus infection perturbs energy metabolites and amino acids in cultured human oligodendroglia cells.

Background Borna disease virus is a neurotropic, non-cytolytic virus that has been widely employed in neuroscientific research. Previous studies have revealed that metabolic perturbations are associated with Borna disease viral infection. However, the pathophysiological mechanism underlying its mode of action remains unclear. Methodology Human oligodendroglia cells infected with the human strain Borna disease virus Hu-H1 and non-infected matched control cells were cultured in vitro. At day 14 post-infection, a proton nuclear magnetic resonance-based metabonomic approach was used to differentiate the metabonomic profiles of 28 independent intracellular samples from Borna disease virus-infected cells (n = 14) and matched control cells (n = 14). Partial least squares discriminant analysis was performed to demonstrate that the whole metabonomic patterns enabled discrimination between the two groups, and further statistical testing was applied to determine which individual metabolites displayed significant differences between the two groups. Findings Metabonomic profiling revealed perturbations in 23 metabolites, 19 of which were deemed individually significant: nine energy metabolites (α-glucose, acetate, choline, creatine, formate, myo-inositol, nicotinamide adenine dinucleotide, pyruvate, succinate) and ten amino acids (aspartate, glutamate, glutamine, glycine, histidine, isoleucine, phenylalanine, threonine, tyrosine, valine). Partial least squares discriminant analysis demonstrated that the whole metabolic patterns enabled statistical discrimination between the two groups. Conclusion Borna disease viral infection perturbs the metabonomic profiles of several metabolites in human oligodendroglia cells cultured in vitro. The findings suggest that Borna disease virus manipulates the host cell’s metabolic network to support viral replication and proliferation.

Direct Blue 71 staining as a destaining‐free alternative loading control method for Western blotting

In Western blotting, a suitable loading control is indispensable for correcting errors in the total amount of loaded protein. Immunodetection of housekeeping proteins and total protein staining have traditionally been used as loading control methods. Direct Blue 71 (DB71) staining—a novel, sensitive, dye‐binding staining method compatible with immunodetection—may offer advantages over these traditional loading control methods. Three common neuroscientific samples (human plasma, human oligodendrocytes, and rat brain) were employed to assess DB71 staining as a loading control method for Western blotting. DB71, CBB, one traditional housekeeping protein, and one protein of interest were comparatively assessed for reliability and repeatability and linear dynamic range over 2.5–40 μg of protein loaded. DB71s effect on the reliability and repeatability and linear dynamic range of immunoreaction were also assessed. Across all three sample types, DB71 was either equivalent or superior to CBB and housekeeping protein‐based methods in terms of reliability and repeatability and linear dynamic range. Across all three sample types, DB71 staining did not impair the reliability and repeatability or linear dynamic range of immunoreaction. Our results demonstrate that the DB71 staining can be used as a destaining‐free alternative loading control method for Western blotting.

Systematic Review of All-Arthroscopic Versus Mini-Open Repair of Rotator Cuff Tears: A Meta-Analysis

The objective of this study was to compare outcomes in patients with rotator cuff tears undergoing all-arthroscopic versus mini-open rotator cuff repair. A systematic review and meta-analysis of outcomes of all-arthroscopic repair versus mini-open repair in patients with rotator cuff repair was conducted. Studies meeting the inclusion criteria were screened and included from systematic literature search for electronic databases including Medline, Embase, Cochrane CENTRAL, and CINAHL library was conducted from 1969 and 2015. A total of 18 comparative studies including 4 randomized clinical trials (RCTs) were included. Pooled results indicate that there was no difference in the functional outcomes, range of motion, visual analog scale (VAS) score, and short-form 36 (SF-36) subscales. However, Constant-Murley functional score was found to be significantly better in patients with mini-open repair. However, the results of the review should be interpreted with caution due to small size and small number of studies contributing to analysis in some of the outcomes. All-arthroscopic and mini-open repair surgical techniques for the management of rotator cuff repair are associated with similar outcomes and can be used interchangeably based on the patient and rotator tear characteristics.

Human but Not Laboratory Borna Disease Virus Inhibits Proliferation and Induces Apoptosis in Human Oligodendrocytes In Vitro

Borna disease virus (BDV) is a neurotropic virus that produces neuropsychiatric dysfunction in a wide range of warm-blooded species. Several studies have associated BDV with human psychiatric illness, but the findings remain controversial. Although oligodendrocytes are a major glial component of brain white matter and play a pivotal role in neuronal cell function, BDVs effects on human oligodendrocytes have not been clarified. Here, the effects of two BDV strains, Hu-H1 (isolated from a bipolar patient) and Strain V (a laboratory strain), on the proliferation and apoptosis of human oligodendrocytes were investigated. Three experimental cell lines were constructed: Hu-H1-infected oligodendroglioma (Hu-H1) cells, Strain V-infected oligodendroglioma (Strain V) cells, and non-infected oligodendroglioma (control) cells. BDV infection was assayed by BDV nucleoprotein (p40) immunofluorescence, cell proliferation was assayed by Cell Counting Kit-8 (CCK8), and cell cycle phases and apoptosis were assayed by flow cytometry. Expressions of the apoptosis-related proteins Bax and Bcl-2 were measured by Western blotting. p40 expression was confirmed in Hu-H1 and Strain V on and after day three post-infection. Strain V cells showed significantly greater cellular proliferation than Hu-H1 cells on and after day three post-infection. In Hu-H1 cells, Bax and Bcl-2 expression were significantly increased and decreased, respectively, on and after day three post-infection. In contrast, in Strain V cells, Bax and Bcl-2 expression were significantly decreased and increased, respectively, on and after day three post-infection. In conclusion, Hu-H1 inhibits cellular proliferation and promotes apoptosis in human oligodendrocytes via Bax upregulation and Bcl-2 downregulation. In contrast, Strain V promotes cellular proliferation and inhibits apoptosis in human oligodendrocytes via Bax downregulation and Bcl-2 upregulation. The effects of the Hu-H1 strain (isolated from a bipolar patient) are opposite from those of Strain V (a laboratory strain), thereby providing a proof of authenticity for both.

Real-Time qPCR Identifies Suitable Reference Genes for Borna Disease Virus-Infected Rat Cortical Neurons

Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is the most commonly-used technique to identify gene expression profiles. The selection of stably expressed reference genes is a prerequisite to properly evaluating gene expression. Here, the suitability of commonly-used reference genes in normalizing RT-qPCR assays of mRNA expression in cultured rat cortical neurons infected with Borna disease virus (BDV) was assessed. The expressions of eight commonly-used reference genes were comparatively analyzed in BDV-infected rat cortical neurons and non-infected control neurons mainly across 9 and 12 days post-infection. These reference genes were validated by RT-qPCR and separately ranked by four statistical algorithms: geNorm, NormFinder, BestKeeper and the comparative delta-Ct method. Then, the RankAggreg package was used to construct consensus rankings. ARBP was found to be the most stable internal control gene at Day 9, and ACTB at Day 12. As the assessment of the validity of the selected reference genes confirms the suitability of applying a combination of the two most stable references genes, combining the two most stable genes for normalization of RT-qPCR studies in BDV-infected rat cortical neurons is recommended at each time point. This study can contribute to improving BDV research by providing the means by which to obtain more reliable and accurate gene expression measurements.

Human borna disease virus infection impacts host proteome and histone lysine acetylation in human oligodendroglia cells

Abstract Background Borna disease virus (BDV) replicates in the nucleus and establishes persistent infections in mammalian hosts. A human BDV strain was used to address the first time, how BDV infection impacts the proteome and histone lysine acetylation (Kac) of human oligodendroglial (OL) cells, thus allowing a better understanding of infection-driven pathophysiology in vitro. Methods Proteome and histone lysine acetylation were profiled through stable isotope labeling for cell culture (SILAC)-based quantitative proteomics. The quantifiable proteome was annotated using bioinformatics. Histone acetylation changes were validated by biochemistry assays. Results Post BDV infection, 4383 quantifiable differential proteins were identified and functionally annotated to metabolism pathways, immune response, DNA replication, DNA repair, and transcriptional regulation. Sixteen of the thirty identified Kac sites in core histones presented altered acetylation levels post infection. Conclusions BDV infection using a human strain impacted the whole proteome and histone lysine acetylation in OL cells.

Proteomics reveal energy metabolism and mitogen-activated protein kinase signal transduction perturbation in human Borna disease virus Hu-H1-infected oligodendroglial cells.

Abstract Borna disease virus (BDV) is a neurotropic, non-cytolytic RNA virus which replicates in the cell nucleus targeting mainly hippocampal neurons, but also astroglial and oligodendroglial cells in the brain. BDV is associated with a large spectrum of neuropsychiatric pathologies in animals. Its relationship to human neuropsychiatric illness still remains controversial. We could recently demonstrate that human BDV strain Hu-H1 promoted apoptosis and inhibited cell proliferation in a human oligodendroglial cell line (OL cells) whereas laboratory BDV strain V acted contrariwise. Here, differential protein expression between BDV Hu-H1-infected OL cells and non-infected OL cells was assessed through a proteomics approach, using two-dimensional electrophoresis followed by matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry. A total of 63 differential host proteins were identified in BDV Hu-H1-infected OL cells compared to non-infected OL cells. We found that most changes referred to alterations related to the pentose phosphate pathway, glyoxylate and dicarboxylate metabolism, the tricarboxylic acid (TCA) cycle, and glycolysis /gluconeogenesis. By manual querying, two differential proteins were found to be associated with mitogen-activated protein kinase (MAPK) signal transduction. Five key signaling proteins of this pathway (i.e., p-Raf, p-MEK, p-ERK1/2, p-RSK, and p-MSK) were selected for Western blotting validation. p-ERK1/2 and p-RSK were found to be significantly up-regulated, and p-MSK was found to be significantly down-regulated in BDV Hu-H1-infected OL cells compared to non-infected OL cell. Although BDV Hu-H1 constitutively activated the ERK–RSK pathway, host cell proliferation and nuclear translocation of activated pERK in BDV Hu-H1-infected OL cells were impaired. These findings indicate that BDV Hu-H1 infection of human oligodendroglial cells significantly perturbs host energy metabolism, activates the downstream ERK–RSK complex of the Raf/MEK/ERK signaling cascade, and disturbs host cell proliferation possibly through impaired nuclear translocation of pERK, a finding which warrants further research.

Metabolomic profiling of three brain regions from a postnatal infected Borna disease virus Hu-H1 rat model

Neonatal rat infection with Borna disease virus (BDV), termed neonatal Borna disease, is an established model for investigating the BDV-associated pathogenesis of neurodevelopmental abnormalities. BDV produces a persistent noncytolytic infection in all culture cell systems assayed to date, while persistent infection in neonatal rats results in a progressive loss of hippocampal granule cells, cerebellar Purkinje cells, and cortical GABA-ergic neurons. Persistent infection also results in behavioral deficits including hyperactivity, cognitive impairment, and abnormal social behavior. However, the molecular mechanisms underlying the neuronal degeneration and behavioral abnormalities remain unclear. Using a metabolomic approach based on gas chromatography coupled with mass spectrometry in conjunction with statistical pattern recognition, the metabolic changes in response to BDV Hu-H1 infection were characterized in the rat hippocampus, cerebellum, and cortex. Metabonomic profiling revealed significant perturbations in nucleotide (e.g., adenosine, uracil, inosine, adenosine-5′-monophosphate, uridine-5′-monophosphate, d-ribose 5-phosphate, and sedoheptulose 7-phosphate), amino acid (e.g., lysine, glycine, phenylalanine, tyrosine, proline, serine, cysteine, aspartic acid, pyroglutamic acid, and γ-aminobutyric acid), lipid (e.g., cholesterol, myristic acid, stearic acid, palmitic acid, 1-monopalmitoylglycerol, and arachidonic acid), and energy (e.g., glucose, lactose, 3-phosphoglyceric acid, and pyruvic acid) metabolites. These metabolites participate in pathways crucial to viral proliferation and neurotransmitter homeostasis. This metabolomic profiling study provides insight into the pathogenic mechanisms of BDV and new directions with which to investigate the in vivo effects of persistent BDV infection.

Health care professionals at risk of infection with Borna disease virus - evidence from a large hospital in China (Chongqing)

BackgroundHuman Borna disease virus (BDV) infections have recently been reported in China. BDV causes cognitive and behavioural disturbances in animals. The impact on human mental disorders is subject to debate, but previous studies worldwide have found neuropsychiatric patients more frequently infected than healthy controls. A few isolates were recovered from severely depressed patients, but contagiousness of BDV strain remains unknown.MethodWe addressed the risk of infection in health care settings at the first affiliated hospital of Chongqing Medical University (CQMU), located in downtown Chongqing, a megacity in Southwest China. Between February 2012 and March 2013, we enrolled 1529 participants, of whom 534 were outpatients with major depressive disorder (MDD), 615 were hospital personnel, and 380 were healthy controls who underwent a health check. Infection was determined through BDV-specific circulating immune complexes (CIC), RNA, and selective antibodies (blood).ResultsOne-fifth of the hospital staff (21.8%) were found to be infected (CIC positive), with the highest prevalence among psychiatry and oncology personnel, which is twice as many as were detected in the healthy control group (11.1%), and exceeds the prevalence detected in MDD patients (18.2%).ConclusionBDV circulates unnoticed in hospital settings in China, putting medical staff at risk and warranting clarification of infection modes and introduction of prevention measures.

GC-MS-Based Metabonomic Profiling Displayed Differing Effects of Borna Disease Virus Natural Strain Hu-H1 and Laboratory Strain V Infection in Rat Cortical Neurons.

Borna disease virus (BDV) persists in the central nervous systems of a wide variety of vertebrates and causes behavioral disorders. Previous studies have revealed that metabolic perturbations are associated with BDV infection. However, the pathophysiological effects of different viral strains remain largely unknown. Rat cortical neurons infected with human strain BDV Hu-H1, laboratory BDV Strain V, and non-infected control (CON) cells were cultured in vitro. At day 12 post-infection, a gas chromatography coupled with mass spectrometry (GC–MS) metabonomic approach was used to differentiate the metabonomic profiles of 35 independent intracellular samples from Hu-H1-infected cells (n = 12), Strain V-infected cells (n = 12), and CON cells (n = 11). Partial least squares discriminant analysis (PLS-DA) was performed to demonstrate discrimination between the three groups. Further statistical testing determined which individual metabolites displayed significant differences between groups. PLS-DA demonstrated that the whole metabolic pattern enabled statistical discrimination between groups. We identified 31 differential metabolites in the Hu-H1 and CON groups (21 decreased and 10 increased in Hu-H1 relative to CON), 35 differential metabolites in the Strain V and CON groups (30 decreased and 5 increased in Strain V relative to CON), and 21 differential metabolites in the Hu-H1 and Strain V groups (8 decreased and 13 increased in Hu-H1 relative to Strain V). Comparative metabonomic profiling revealed divergent perturbations in key energy and amino acid metabolites between natural strain Hu-H1 and laboratory Strain V of BDV. The two BDV strains differentially alter metabolic pathways of rat cortical neurons in vitro. Their systematic classification provides a valuable template for improved BDV strain definition in future studies.