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Our previous studies showed that ferroptosis plays an important role in the acute and subacute stages of spinal cord injury. High intracellular iron levels and low glutathione levels make oligodendrocytes vulnerable to cell death after central nervous system trauma. In this study, we established an oligodendrocyte (OLN-93 cell line) model of ferroptosis induced by RSL-3, an inhibitor of glutathione peroxidase 4 (GPX4). RSL-3 significantly increased intracellular concentrations of reactive oxygen species and malondialdehyde. RSL-3 also inhibited the main anti-ferroptosis pathway, i.e., SLC7A11/glutathione/glutathione peroxidase 4 (xCT/GSH/GPX4), and downregulated acyl-coenzyme A synthetase long chain family member 4. Furthermore, we evaluated the ability of several compounds to rescue oligodendrocytes from ferroptosis. Liproxstatin-1 was more potent than edaravone or deferoxamine. Liproxstatin-1 not only inhibited mitochondrial lipid peroxidation, but also restored the expression of GSH, GPX4 and ferroptosis suppressor protein 1. These findings suggest that GPX4 inhibition induces ferroptosis in oligodendrocytes, and that liproxstatin-1 is a potent inhibitor of ferroptosis. Therefore, liproxstatin-1 may be a promising drug for the treatment of central nervous system diseases.

Zebrafish are an effective vertebrate model to study the mechanisms underlying recovery after spinal cord injury. The subacute phase after spinal cord injury is critical to the recovery of neurological function, which involves tissue bridging and axon regeneration. In this study, we found that zebrafish spontaneously recovered 44% of their swimming ability within the subacute phase (2 weeks) after spinal cord injury. During this period, we identified 7762 differentially expressed genes in spinal cord tissue: 2950 were up-regulated and 4812 were down-regulated. These differentially expressed genes were primarily concentrated in the biological processes of the respiratory chain, axon regeneration, and cell-component morphogenesis. The genes were also mostly involved in the regulation of metabolic pathways, the cell cycle, and gene-regulation pathways. We verified the gene expression of two differentially expressed genes, clasp2 up-regulation and h1m down-regulation, in zebrafish spinal cord tissue in vitro. Pathway enrichment analysis revealed that up-regulated clasp2 functions similarly to microtubule-associated protein, which is responsible for axon extension regulated by microtubules. Down-regulated h1m controls endogenous stem cell differentiation after spinal cord injury. This study provides new candidate genes, clasp2 and h1m, as potential therapeutic intervention targets for spinal cord injury repair by neuroregeneration. All experimental procedures and protocols were approved by the Animal Ethics Committee of Tianjin Institute of Medical & Pharmaceutical Sciences (approval No. IMPS-EAEP-Q-2019-02) on September 24, 2019.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an important oncogenic virus previously shown to be neurotropic, but studies on neuronal cell infection and pathogenesis are still very limited. Here, we characterized the effects of KSHV infection on neuronal SH-SY5Y cells by the recombinant virus rKSHV.219, which expresses both green fluorescent protein (GFP) and red fluorescent protein (RFP) to reflect the latent and lytic phases of infection. We demonstrated that infected cells have a higher growth rate and that KSHV infection can be sustained. Interestingly, the infected cells can transition spontaneously back and forth between lytic and latent phases of infection, producing progeny viruses but without any adverse effects on cell growth. In addition, transcriptome analysis of viral and cellular genes in latent and lytic cells showed that unlike other infected cell lines, the latently infected cells expressed both latent and most, but not all, of the lytic genes required for infectious virion production. The viral genes uniquely expressed by the lytic cells were mainly involved in the early steps of virus binding. Some of the cellular genes that were deregulated in both latently and lytically infected cells are involved in cell adhesion, cell signal pathways, and tumorigenesis. The downregulated cellular CCDN1, PAX5, and NFASC and upregulated CTGF, BMP4, YAP1, LEF1, and HLA-DRB1 genes were found to be associated with cell adhesion molecules (CAMs), hippo signaling, and cancer. These deregulated genes may be involved in creating an environment that is unique in neuronal cells to sustain cell growth upon KSHV infection and not observed in other infected cell types. IMPORTANCE Our study has provided evidence that neuronal SH-SY5Y cells displayed unique cellular responses upon KSHV infection. Unlike other infected cells, this neuronal cell line displayed a higher growth rate upon infection and can spontaneously transition back and forth between latent and lytic phases of infection. Unlike other latently infected cells, a number of lytic genes were also expressed in the latent phase of infection in addition to the established latent viral genes. They may play a role in deregulating a number of host genes that are involved in cell signaling and tumorigenesis in order to sustain the infection and growth advantages for the cells. Our study has provided novel insights into KSHV infection of neuronal cells and a potential new model for further studies to explore the underlying mechanism in viral and host interactions for neuronal cells and the association of KSHV with neuronal diseases.

Congenital hypothyroidism (CH) is the most common neonatal endocrine disorder with a genetic origin. The purpose of the present study was to analyze the mutation spectrum of CH patients in China. A targeted next-generation sequencing panel covering all exons of 29 CH-related causative genes was used in 43 Han Chinese patients with CH [11 dysgenesis and 32 glands in situ (GIS)]. The functional impact and pathogenicity of detected variants were analyzed using a comprehensive bioinformatics approach and co-segregation studies. A total of 47 rare non-polymorphic variants in 9 target genes associated with thyroid hormone synthesis (DUOX2, DUOXA2, TPO, TG, SLC26A4 and SLC5A5), thyroid stimulating hormone resistance (TSHR) and central hypothyroidism (PROP1 and TRHR) were identified in 31 patients (31/43, 72%). Of these variants, 8 were novel, including 3 in DUOX2, 2 in TPO, 3 in TSHR and 1 in SLC5A5. Variants were mostly affected by DUOX2, TG, TPO and TSHR. Approximately 44% of the patients (19/43) carried DUOX2 variants. The mutation detection rates in patients with GIS were higher compared with patients with dysgenesis [25/32 (78%) vs. 6/11 (54%)]. Oligogenic mutations were detected in 25.6% of the total cases and 35% of the mutated cases. Genetic basis was ascertained in 13 patients, reaching a diagnosis detection rate of 30%. In conclusion, genetic defects in dyshormonogenesis, mainly in DUOX2, were the main genetic cause of CH in the Chinese population. Oligogenicity is highly involved in CH pathogenesis and may thus be an important factor in common phenotypic variability observed in patients with CH.

Spinal cord injury (SCI) is a serious devastating condition and it has a high mortality rate and morbidity rate. The early pathological changes in the immediate phase of SCI may play a major part in the development of secondary injury. Alterations in the expression of many long noncoding RNAs (lncRNAs) have been shown to play fundamental roles in the diseases of the central nervous system. However, the roles of lncRNAs and messenger RNAs (mRNAs) in the immediate phase of SCI are not clear. We examined the expression of mRNAs and lncRNAs in a rat model at 2 h after SCI and identified the differentially expressed lncRNAs (DE lncRNAs) and differentially expressed mRNAs (DE mRNAs) using microarray analysis. 772 DE lncRNAs and 992 DE mRNAs were identified in spinal cord samples in the immediate phase following SCI compared with control samples. Moreover, Gene Ontology (GO) term annotation results showed that CXCR chemokine receptor binding, neutrophil apoptotic process, neutrophil migration, neutrophil extravasation, macrophage differentiation, monocyte chemotaxis and cellular response to interleukin-1 (IL-1) were the main significantly enriched GO terms. The results of Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the DEGs were enriched in toll−like receptor signaling pathway, p53 signaling pathway, MAPK signaling pathway and Jak–STAT signaling pathway. IL6, MBOAT4, FOS, TNF, JUN, STAT3, CSF2, MYC, CCL2 and FGF2 were the top 10 high-degree hub nodes and may be important targets in the immediate phase of SCI. The current study on provides novel insights into how lncRNAs and mRNAs regulate the pathogenesis of the immediate phase after SCI.

Despite the success of combined antiretroviral therapy in recent years, the prevalence of human immunodeficiency virus (HIV)-associated neurocognitive disorders in people living with HIV-1 is increasing, significantly reducing the health-related quality of their lives. Although neurons cannot be infected by HIV-1, shed viral proteins such as transactivator of transcription (Tat) can cause dendritic damage. However, the detailed molecular mechanism of Tat-induced neuronal impairment remains unknown. In this study, we first showed that recombinant Tat (1–72 aa) induced neurotoxicity in primary cultured mouse neurons. Second, exposure to Tat1–72 was shown to reduce the length and number of dendrites in cultured neurons. Third, Tat1–72 (0–6 h) modulates protein phosphatase 1 (PP1) expression and enhances its activity by decreasing the phosphorylation level of PP1 at Thr320. Finally, Tat1–72 (24 h) downregulates CREB activity and CREB-mediated gene (BDNF, c-fos, Egr-1) expression. Together, these findings suggest that Tat1–72 might impair cognitive function by regulating the activity of PP1 and the CREB/BDNF pathway.

Long non-coding RNAs (lncRNAs) are widely accepted as key players in various biological processes. However, the roles of lncRNA in peripheral nerve regeneration remain completely unknown. Thus, in this study, we performed microarray analysis to measure lncRNA expression in the distal segment of the sciatic nerve at 0, 3, 7 and 14 days following injury. We identified 5,354 lncRNAs that were differentially expressed: 3,788 lncRNAs were differentially expressed between days 0 and 3; 3,314 lncRNAs were differentially expressed between days 0 and 7; and 2,400 lncRNAs were differentially expressed between days 0 and 14. The results of RT-qPCR of two dysregulated lncRNAs were consistent with those of microarray analysis. Bioinformatics approaches, including lncRNA classification, gene ontology (GO) analysis and target prediction, were utilized to investigate the functions of these dysregulated lncRNAs in peripheral nerve damage. Importantly, we predicted that several lncRNA-mRNA pairs may participate in biological processes related to peripheral nerve injury. RT-qPCR was performed for the preliminary verification of three lncRNA-mRNA pairs. The overexpression of NONMMUG014387 promoted the proliferation of mouse Schwann cells. Thus, the findings of our study may enhance our knowledge of the role of lncRNAs in nerve injury.

Aims Many studies have demonstrated that circRNAs are closely associated with human diseases. Nonetheless, the potential mechanism by which circRNAs impacts spinal cord injury (SCI) is not fully understood. The aim of this study was to explore the regulatory roles of circRNAs in SCI. Main methods The sequencing data of circRNA, miRNA and mRNA were obtained from Gene Expression Omnibus (GEO) datasets. Candidates were identified to construct a circRNA-miRNA-mRNA network based on circRNA-miRNA interactions and miRNA-mRNA interactions. Protein-protein interactions (PPI) analysis was performed to determine hub genes, and a connectivity map (CMap) analysis was applied to determine potential therapeutic targets for SCI. Key findings A total of 1656 differentially expressed circRNAs (DEcircRNAs), 71 differentially expressed miRNAs (DEmiRNAs) and 2782 differentially expressed mRNAs (DEmRNAs) were identified. We integrated four overlapped circRNAs, six miRNAs and 101 target mRNAs into a circRNA–miRNA–mRNA network. We next identified two hub genes (DDIT4, EZR) based on the PPI network and identified five circRNA-miRNA-hub gene regulatory axes. In addition, we discovered three chemicals (tanespimycin, fulvestrant, carbamazepine) as potential treatment options for SCI. Significance Our study suggests a regulatory role for circRNAs in the pathogenesis and treatment of SCI from the view of a competitive endogenous RNA (ceRNA) network.

The present study aimed to explore the role of the PTEN/Akt/mTOR signaling pathway in the neurite outgrowth and apoptosis of cortical neurons. Cortical neurons were seeded on or adjacent to chondroitin sulfate proteoglycans. The length, number and crossing behavior of the neurites were calculated. Immunohistochemical staining and TUNEL data were analyzed. Neurites treated with PTEN inhibitor exhibited significant enhancements in elongation, initiation and crossing abilities when they encountered chondroitin sulfate proteoglycans in vitro. These effects disappeared when the PTEN/Akt/mTOR signaling pathway was blocked. Neurons exhibited significant enhancements in survival ability following PTEN inhibition. The present study demonstrated that PTEN inhibition can promote axonal elongation and initiation in cerebral cortical neurons, as well as the ability to cross the chondroitin sulfate proteoglycan border. In addition, PTEN inhibition is useful for protecting the neuron from apoptosis. The PTEN/Akt/mTOR signaling pathway is an important signaling pathway.

Introduction: The purpose of this study was to provide a comprehensive understanding of gene expression during Wallerian degeneration and axon regeneration after peripheral nerve injury. Methods: A microarray was used to detect gene expression in the distal nerve 0, 3, 7, and 14 days after sciatic nerve crush. Bioinformatic analysis was used to predict function of the differentially expressed mRNAs. Microarray results and the key pathways were validated by quantitative real-time polymerase chain reaction (qRT-PCR). Results: Differentially expressed mRNAs at different time-points (3, 7, and 14 days) after injury were identified and compared with a control group (0 day). Nine general trends of changes in gene expression were identified. Key signal pathways and 9 biological processes closely associated with nerve regeneration were identified and verified. Conclusions: Differentially expressed genes and biological processes and pathways associated with axonal regeneration may elucidate the molecular-biological mechanisms underlying peripheral nerve regeneration. Muscle Nerve 55: 373–383, 2017

Neural stem cells (NSCs) are characterized by their potential for self-renewal and ability to differentiate into neurons, astrocytes, and oligodendrocytes. They are of great value to scientific studies and clinical applications. Culturing NSCs in vitro is important for characterizing their properties under controlled environmental conditions that may be modified and monitored accurately. The present study explored a modified, detailed and efficient protocol for the isolation, culture and cryopreservation of rat embryonic NSCs. In particular, the viability, nestin expression, and self-renewal and multi-differentiation capabilities of NSCs cryopreserved for various periods of time (7 days, or 1, 6 or 12 months) were characterized and compared. Rat embryonic NSCs were successfully obtained and maintained their self-renewal and multipotent differentiation capabilities even following long-term cryopreservation (for up to 12 months).

Spinal cord injury (SCI) is a highly severe disease and it can lead to the destruction of the motor and sensory function resulting in temporary or permanent disability. Long noncoding RNAs (lncRNAs) are transcripts longer than 200 nt that play a critical role in central nervous system (CNS) injury. However, the exact roles of lncRNAs and messenger RNAs (mRNAs) in the early acute phase of SCI remain to be elucidated. We examined the expression of mRNAs and lncRNAs in a rat model at 2 days after SCI and identified the differentially expressed lncRNAs (DE lncRNAs) and differentially expressed mRNAs (DE mRNAs) using microarray analysis. Subsequently, a comprehensive bioinformatics analysis was also performed to clarify the interaction between DE mRNAs. A total of 3,193 DE lncRNAs and 4,308 DE mRNAs were identified between the injured group and control group. Classification, length distribution, and chromosomal distribution of the dysregulated lncRNAs were also performed. The gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were performed to identify the critical biological processes and pathways. A protein-protein interaction (PPI) network indicated that IL6, TOP2A, CDK1, POLE, CCNB1, TNF, CCNA2, CDC20, ITGAM, and MYC were the top 10 core genes. The subnetworks from the PPI network were identified to further elucidate the most significant functional modules of the DE mRNAs. These data may provide novel insights into the molecular mechanism of the early acute phase of SCI. The identification of lncRNAs and mRNAs may offer potential diagnostic and therapeutic targets for SCI.

Aim: To explore neurite growth/regeneration and spinal cord injury repair after PTEN silencing via lentivirus-mediated RNAi. Materials & methods: Cortical neurons were seeded on or adjacent to chondroitin sulfate proteoglycans. The length, number and crossing behavior of neurites were calculated. Lentivirus was locally injected into spinal cord contusion rats. The functional recovery and immunohistochemical staining were analyzed. Results: Neurites with PTEN silencing exhibited significant enhancements in elongation, initiation and crossing ability when they encountered chondroitin sulfate proteoglycans in vitro. In vivo PTEN silencing improved functional recovery significantly, and promoted axon and synapse formation, but not scar formation. Conclusions: PTEN silencing may be promising for spinal cord injury repair.

Background Spinal cord injury (SCI) is a disease associated with high disability and mortality rates. The transitional phase from subacute phase to intermediate phase may play a major role in the process of secondary injury. Changes in protein expression levels have been shown to play key roles in many central nervous system (CNS) diseases. Nevertheless, the roles of proteins in the transitional phase of SCI are not clear. Methods We examined protein expression in a rat model 2 weeks after SCI and identified differentially expressed proteins (DEPs) using isobaric tagging for relative and absolute protein quantification (iTRAQ). Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEPs were performed. Furthermore, we constructed a protein-protein interaction (PPI) network, and the top 10 high-degree core nodes were identified. Meanwhile, we validated protein level changes of five high-degree core regulated proteins using Western blots. Results A total of 162 DEPs were identified between the injury group and the control, of which 101 (62.35%) were up-regulated and 61 (37.65%) were down-regulated in the transitional phase of SCI. Key molecular function, cellular components, biological process terms and pathways were identified and may be important mechanisms in the transitional phase of SCI. Alb, Calm1, Vim, Apoe, Syp, P4hb, Cd68, Eef1a2, Rab3a and Lgals3 were the top 10 high-degree core nodes. Western blot analysis performed on five of these proteins showed the same trend as iTRAQ results. Conclusion The current study may provide novel insights into how proteins regulate the pathogenesis of the transitional phase after SCI.

Despite the notable success of combination antiretroviral therapy, how to eradicate latent HIV-1 from reservoirs poses a challenge. The Tat protein plays an indispensable role in HIV reactivation and histone demethylase LSD1 promotes Tat-mediated long terminal repeats (LTR) activation. However, the role of LSD1 in remodeling chromatin and the role of its component BHC80 in activation of latent HIV-1 in T cells are unknown. Our findings indicate that LSD1 could decrease the level of histone H3 lysine 4 trimethylation (H3K4me3) at the HIV-1 promoter by recruiting histone lysine demethylase 5A (KDM5A) and preventing histone methyltransferase Set1A and WD-40 repeat protein 5 (WDR5) from binding to LTR. Moreover, BHC80 is necessary for LSD1-triggered LTR activation and assists LSD1 in activating LTR by binding to nucleotides 305-631 of LTR. In activated J-Lat-A2 cells, BHC80 expression was elevated and its isoform BHC80-6 promoted the association of BHC80 with LSD1. These results suggest that the LSD1-BHC80 complex enhances HIV-1 transcription by a decrease of H3K4me3 level at the viral promoter. Therefore, it might be used as a new drug target to reactivate latent HIV-1.

Multipotent neural stem cells (NSCs) are currently under investigation as a candidate treatment for central nervous system (CNS) injury because of their potential to compensate for neuronal damage and to reconstruct disrupted neuronal connections. To maximize the regenerative effect of the derived neurons and to minimize the side effects of the derived astrocytes, it is necessary to regulate the fate determination of NSCs to produce more neurons and fewer astrocytes. Both valproic acid (VPA) and all-trans-retinoic acid (ATRA), two clinically established drugs, induce neuronal differentiation and facilitate neurite outgrowth at the expense of astrocytic differentiation in NSCs. However, the time-dependent activities and the long-term treatment effects of these drugs have not been explored in NSCs. More importantly, the efficacies of VPA and ATRA in neuronal promotion and astrocytic suppression remain unclear. In this study, we compare the time-dependent characteristics of VPA and ATRA in NSC differentiation and neurite outgrowth in vitro and, for the first time, demonstrate the improved efficacy of their combined application in neuronal induction and astrocytic suppression. These significant effects are closely coupled to the altered expression of a neurogenic transcription factor, a Wnt signaling component, a cell cycle regulator and a neural growth factor, indicating an underlying cross-talk between the mechanisms of action of ATRA and VPA. These findings indicate that a novel strategy combining these two therapeutic drugs may improve the restorative effect of NSC transplantation by altering the expression of their interconnected targets for fate determination.

Background/Aims: Low back pain has become one of the most common musculoskeletal diseases in the world. Studies have shown that intervertebral disc degeneration (IDD) is an important factor leading to low back pain, but the mechanisms underlying IDD remain largely unknown. Research over the past decade has suggested critical roles for microRNAs (miRNAs) in natural growth and disease progression. However, it remains poorly understood whether circular RNAs participate in IDD. Methods: Clinical IDD samples were collected from 20 patients who underwent discectomy. Weighted gene co-expression network analysis was used to identify the co-expression miRNA network modules (highly co-expressed clusters of miRNAs) that were associated with IDD grade. Results: miR-3150a-3p was the most significantly up-regulated miRNA in module “Blue.” Notably, aggrecan (ACAN) was identified as a direct target gene of miR-3150a-3p and ACAN expression was regulated by miR-3150a-3p. Overexpression of miR-3150a-3p decreased ACAN expression in nucleus pulposus cells, whereas inhibition of miR-3150a-3p increased ACAN expression. In addition, ACAN expression was negatively correlated with IDD grade. Conclusion: Our study suggests that the reduction of ACAN expression induced by the upregulation of miR-3150a-3p might participate in the development of IDD.

Combinatorial small interfering RNA duplexes (siRNAs) have the potential to be a gene therapy against HIV-1, and some studies have reported that transient combinatorial siRNA expression represses HIV replication, but the effects of long-term siRNA expression on HIV replication have not been studied in detail. In this study, HIV-1 replication under the influence of stable combinatorial siRNA expression from a single RNA transcript was analyzed. First, a series of cassettes encoding short hairpin RNA (shRNA)/long hairpin RNA (lhRNA)/double long hairpins (dlhRNA) was constructed and subjected to an analysis of inhibitory efficacy. Next, an optimized dlhRNA encoding cassette was selected and inserted into lentiviral delivery vector FG12. Transient dlhRNA expression reduced replication of HIV-1 in TZM-bl cells and CD4+ T cells successfully. HIV-1 susceptible TZM-bl cells were transducted with the dlhRNA expressing lentiviral vector and sorted by fluorescence-activated cell sorting to obtain stable dlhRNA expressing cells. The generation of four anti-HIV siRNAs in these dlhRNA expressing cells was verified by stem–loop RT-PCR assay. dlhRNA expression did not activate a non-specific interferon response. The dlhRNA expressing cells were also challenged with HIV-1 NL4-3, which revealed that stable expression of combinatorial siRNAs repressed HIV-1 replication for 8 days, after which HIV-1 overcame the inhibitory effect of siRNA expression by expressing mutant versions of RNAi targets. The results of this evaluation of the long-term inhibitory effects of combinatorial siRNAs against HIV-1 provide a reference for researchers who utilize combinatorial RNA interference against HIV-1 or other error-prone viruses.

Deferoxamine, a clinically safe drug used for treating iron overload, also repairs spinal cord injury although the mechanism for this action remains unknown. Here, we determined whether deferoxamine was therapeutic in a rat model of spinal cord injury and explored potential mechanisms for this effect. Spinal cord injury was induced by impacting the spinal cord at the thoracic T10 vertebra level. One group of injured rats received deferoxamine, a second injured group received saline, and a third group was sham operated. Both 2 days and 2 weeks after spinal cord injury, total iron ion levels and protein expression levels of the proinflammatory cytokines tumor necrosis factor-α and interleukin-1β and the pro-apoptotic protein caspase-3 in the spinal cords of the injured deferoxamine-treated rats were significantly lower than those in the injured saline-treated group. The percentage of the area positive for glial fibrillary acidic protein immunoreactivity and the number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells were also significantly decreased both 2 days and 2 weeks post injury, while the number of NeuN-positive cells and the percentage of the area positive for the oligodendrocyte marker CNPase were increased in the injured deferoxamine-treated rats. At 14–56 days post injury, hind limb motor function in the deferoxamine-treated rats was superior to that in the saline-treated rats. These results suggest that deferoxamine decreases total iron ion, tumor necrosis factor-α, interleukin-1β, and caspase-3 expression levels after spinal cord injury and inhibits apoptosis and glial scar formation to promote motor function recovery.

In conclusion,we first found that HIV infection upregulates the ATL1 mRNA.Up-regulation of ATL1 contributes to HIV-1 replication and down-regulation of ATL1 in host cells inhibited the viral replication.The further research found that ATL1 promoted the level of Env expression on the cell surface and then promoted the cell-to-cell fusion.Truncated mutation experiments revealed that the GTPase domain of ATL1 is the key domain of promoting Env-regulated cell-cell fusion.Finally, through the kinetic experiment,we found that ATL1 was able to promote long-term replication of HIV-1. We believe that the characterization of ATL1 contributes to a better understanding of HIV-host interactions and can identify potential novel antiviral targets.

Background/Aims: Neural stem cells (NSCs) reside in a hypoxic environment, and hypoxia plays an important role in their development and differentiation. This study aimed to explore the underlying mechanisms by which hypoxia affects NSC behavior. Methods: In the current study, we downloaded the gene expression dataset GSE68572 and identified the differentially expressed genes (DEGs) by analyzing high-throughput gene expression in hypoxic and normoxic NSCs. Subsequently, we analyzed these data using a combined bioinformatics approach and predicted the microRNAs (miRNAs) targeting the key gene using miRNA databases. Quantitative real-time PCR (qRT-PCR) was used to validate the expression of the top five DEGs. Results: In total, 1347 genes were identified as DEGs. We identified the predominant gene ontology categories and Kyoto Encyclopedia of Genes and Genomes pathways that were significantly over-represented in the hypoxic NSCs. A protein–protein interaction network he identification of miRNAs and their putative targets may offer new diagnostic and therapeutic strategies for liver cancer the top 10 core genes. Vascular endothelial growth factor A (VEGFA) had the highest degree and may be the key gene concerning NSC behavior under hypoxia. Further validation of the top five DEGs by qRT-PCR demonstrated that four DEGs were significantly higher and one DEG was significantly lower in the hypoxic group than in the control group. Seven miRNAs were predicted and proved to target VEGFA. Conclusion: This preliminary study can prompt the understanding of the molecular mechanisms by which hypoxia has an impact on NSC behavior and can help to optimize stem cell therapies for central nervous system injuries and diseases.

Laminectomy is a widely accepted treatment for lumbar disorders. Epidural Fibrosis (EF) is a common post-laminectomy or post-discectomy complication, which is thought to cause recurrent pain. RNA interference (RNAi) is a process by which double-stranded RNA triggers the destruction of mRNAs sharing the same sequence. Previously, extra-cellular signal-regulated kinase (ERK) 2 plays crucial roles in suppressing the collagen expression. To investigate the effects of lentiviral ERK2 siRNA on the prevention of post-laminectomy EF formation in a rat model, a controlled double-blinded study was conducted in 75 healthy adult Wistar rats that underwent laminectomy. They were divided randomly into 3 groups according to the treatment method: (1) control group; (2) ERK scrRNA group; (3) ERK siRNA group. All rats were euthanized humanely 4 weeks post-laminectomy. The hydroxyproline content, Rydell score, vimentin cells density, fibroblasts density, inflammatory cells density and inflammatory factors expressions were performed. The hydroxyproline content, Rydell score, vimentin cells density, fibroblasts density, inflammatory cells density and inflammatory factors expressions all suggested better results in ERK siRNA group than other two groups. None of the rats expired and no obvious adverse effects were observed. Local delivery of a lentiviral siRNA targeting ERK2 can prevent epidural scar adhesion in post-laminectomy rat via inhibiting collagen expression and inflammation.

Reproductive systems of termite colonies may involve the number of individuals in the reproductive caste and the copulatory selectivity of reproductive individuals (i.e., polyandry or polygamy), both of which directly impact the fertility and genetic diversity of the colony. Polygamy is widespread in the lower termites, whereas polyandry appears to be mostly absent in termites. In this paper, the differentiation of male and female neotenics were observed in orphaned experimental colonies of the subterranean termite Reticulitermes labralis. The artificial orphaned colonies began to produce neotenics a week after colony establishing, with more neotenics appearing in the same group over time. Finally, each experimental group reserved multi-neotenics that consisted of male and female neotenic individuals. Our results demonstrated that these neotenic individuals retained in the colony participated in reproduction. A genetic analysis at four microsatellite loci showed that in addition to the conspicuous morphologically male reproductives, there were inconspicuous males or workers that had copulated with the females in the orphaned colony. Multiple male and female reproductive individuals existed together in a single colony, and one female neotenic could mate with several male reproductives in a short time. Thus, multiple male and female reproductive systems and a polyandric mating system are present in R. labralis.

With laminectomy being widely accepted as the treatment for lumbar disorders, epidural fibrosis (EF) is a common complication for both the patients and the surgeons alike. Currently, EF is thought to cause recurrent postoperative pain after laminectomy or after discectomy.Angelica sinensisis a traditional Chinese medicine which has shown anti-inflammatory, antifibrotic, and antiproliferative properties. The object of this study was to investigate the effects ofAngelica sinensison the prevention of post-laminectomy EF formation in a rat model. A controlled double-blinded study was conducted in sixty healthy adult Wistar rats that underwent laminectomy at the L1-L2 levels. They were divided randomly into 3 groups according to the treatment method, with 20 in each group: (1)Angelica sinensistreatment group, (2) saline treatment group, and (3) sham group (laminectomy without treatment). All rats were euthanized humanely 4 weeks after laminectomy. The hydroxyproline content, Rydell score, vimentin cells density, fibroblasts density, inflammatory cells density, and inflammatory factors expressions all suggested better results inAngelica sinensisgroup than the other two groups. Topical application ofAngelica sinensiscould inhibit fibroblasts proliferation and TGF-β1 and IL-6 expressions and prevent epidural scar adhesion in postlaminectomy rat model.

Understanding the properties of viruses preferentially establishing infection during perinatal transmission of human immunodeficiency virus type 1 (HIV-1) is critical for the development of effective measures to prevent transmission. A previous study demonstrated that the newly transmitted viruses (in infants) of chronically infected mother–infant pairs (MIPs) were fitter in terms of growth, which was imparted by their envelope (Env) glycoprotein V1–V5 regions, than those in the corresponding chronically infected mothers. In order to investigate whether the higher fitness of transmitted viruses was conferred by their higher entry efficiency directed by the V1–V5 regions during perinatal transmission, the fusogenicity of Env containing V1–V5 regions derived from transmitted and non-tranmsmitted viruses of five chronically infected MIPs and two acutely infected MIPs was analysed using two different cell–cell fusion assays. The results showed that, in one chronically infected MIP, a higher fusion efficiency was induced by the infant Env V1–V5 compared with that of the corresponding mother. Moreover, the V4–V5 regions played an important role in discriminating the transmitted and non-transmitted viruses in this pair. However, neither a consistent pattern nor significant differences in fusogenicity mediated by the V1–V5 regions between maternal and infant variants was observed in the other MIPs. This study suggests that there is no consistent and significant correlation between viral fitness selection and entry efficiency directed by the V1–V5 regions during perinatal transmission. Other factors such as the route and timing of transmission may also be involved.

The ubiquitin-like modifier bISG15 is an antiviral protein found in fetal bovine lung (FBL) cells. Bovine Herpesvirus 1(BHV-1), which is a viral pathogen of cattle, can infect FBL cells and induce cytopathic effects. Real-time PCR assays showed that BHV-1's infection could repress the basal or inducible transcription of bISG15 in FBL cells. It demonstrates that this repression effect depends on BHV-1 viral infection and new protein synthesis. Our previous work showed that bIRF-3 was the key factor in the stimulation of bISG15 in FBL cells, so the effect of BHV-1 viral protein on bIRF-3 activating the promoter of bISG15 was confirmed. The luciferase assay showed the BHV-1 viral protein bICP0 inhibited the activation of bISG15 promoter stimulated by bIRF-3. Taken together, our work suggested that BHV-1 had some molecular mechanism to resist the cellular bISG15's antiviral functions.

Perinatal transmission of Human immunodeficiency virus (HIV), also called mother-to-child transmission (MTCT), accounts for 90% of infections in infants worldwide and occurs in 30%-45% of children born to untreated HIV-1 infected mothers. Among HIV-1 infected mothers, some viruses are transmitted from mothers to their infants while others are not. The relationship between virologic properties and the pathogenesis caused by HIV-1 remains unclear. Previous studies have demonstrated that one obvious source of selective pressure in the perinatal transmission of HIV-1 is maternal neutralizing antibodies. Recent studies have shown that viruses which are successfully transmitted to the child have growth advantages over those not transmitted, when those two viruses are grown together. Furthermore, the higher fitness is determined by the gp120 protein of the virus envelope. This suggests that the selective transmission of viruses with higher fitness occurred exclusively, regardless of transmission routes. There are many factors contributing to the selective transmission and HIV replicative fitness is an important one that should not be neglected. This review summarizes current knowledge of the role of HIV replicative fitness in HIV MTCT transmission and the determinants of viral fitness upon MTCT.

Selection of a minor viral genotype during perinatal transmission of human Immunodeficiency virus type 1 (HIV-1) has been observed, but there is a lack of information on the correlation of the restrictive transmission with biological properties of the virus, such as replicative fitness. Recombinant viruses expressing the enhanced green fluorescent protein or the Discosoma sp. red fluorescent (DsRed2) protein carrying the V1 to V5 regions of env from seven mother-infant pairs (MIPs) infected by subtype C HIV-1 were constructed, and competition assays were carried out to compare the fitness between the transmitted and nontransmitted viruses. Flow cytometry was used to quantify the frequency of infected cells, and the replicative fitness was determined based on a calculation that takes into account replication of competing viruses in a single infection versus dual infections. Transmitted viruses from five MIPs with the mothers chronically infected showed a restrictive env genotype, and all the recombinant viruses carrying the infants' Env had higher replicative fitness than those carrying the Env from the mothers. This growth fitness is lineage specific and can be observed only within the same MIP. In contrast, in two MIPs where the mothers had undergone recent acute infection, the viral Env sequences were similar between the mothers and infants and showed no further restriction in quasispecies during perinatal transmission. The recombinant viruses carrying the Env from the infants' viruses also showed replication fitness similar to those carrying the mothers' Env proteins. Our results suggest that newly transmitted viruses from chronically infected mothers have been selected to have higher replicative fitness to favor transmission, and this advantage is conferred by the V1 to V5 region of Env of the transmitted viruses. This finding has important implications for vaccine design or development of strategies to prevent HIV-1 transmission.

The multifunctional trans-activator Tat is an essential regulatory protein for HIV-1 replication and is characterized by high sequence diversity. Numerous experimental studies have examined Tat in HIV-1 subtype B, but research on subtype C Tat is lacking, despite the high prevalence of infections caused by subtype C worldwide. We hypothesized that amino acid differences contribute to functional differences among Tat proteins. In the present study, we found that subtype B NL4-3 Tat and subtype C isolate HIV1084i Tat exhibited differences in stability by overexpressing the fusion protein Tat-Flag. In addition, 1084i Tat can activate LTR and NF-κB more efficiently than NL4-3 Tat. In analyses of the activities of the truncated forms of Tat, we found that the carboxyl-terminal region of Tat regulates its stability and transactivity. According to our results, we speculated that the differences in stability between B-Tat and C-Tat result in differences in transactivation ability. [Image: see text]

The multifunctional transactivator Tat protein is an essentially regulatory protein for HIV-1 replication and it plays a role in pathogenesis of HIV-1 infection. At present, numerous experimental studies about HIV-1 Tat focus on subtype B, very few has been under study of subtype C-Tat. In view of the amino acid variation of the clade-specific Tat proteins, we hypothesized that the amino acid difference contributed to differential function of Tat proteins. In the present study, we documented that subtype B NL4-3 Tat and subtype C isolate HIV1084i Tat from pediatric patient in Zambia exhibited distinct nuclear localization by over-expressing fusion protein Tat-EGFP. Interestingly, 1084i Tat showed uniform nuclear distribution, whereas NL4-3 Tat primarily localized in nucleolus. The 57th amino acid, highly conserved between B-Tat (arginine) and C-Tat (serine), is located in the basic domain of Tat, and played an important role in this subcellular localization. Meanwhile, we found that substitution of arginine to serine at the site 57 decreases Tat transactivation of the HIV-1 LTR promoter.

Little is known about assembly of non-primate foamy virus (FV) such as bovine foamy virus (BFV). To help determine the requirements for assembly of BFV, we constructed BFV-Gag expression plasmids containing all or part of the gag gene, with or without modification by addition of myristate (Myr). Each construct was transfected alone, and with pFenv, into Sf-9 insect cells. The results showed that only the entire Gag could transit through nucleus, which is required for BFV viral assembly in the cytoplasm. Unlike other retroviruses (but like primate foamy viruses), BFV requires the coexpression of the Env protein for viral particle budding. In the case of BFV, this occurs at the plasma membrane rather than the endoplasmic reticulum (ER), due to lack of a functional ER retrieval signal (ERRS). The results also showed that addition of a Myr-membrane targeting signal to the C-terminus of Gag could restore the budding from plasma membrane, implying that Myr-membrane targeting signal could substitute for Env protein in budding.

The costs of spinal cord injury and its complications are high in personal, social and financial terms. Complications include bladder cancer, for which the risk is 16-28 times higher than that of the general population, There is currently little consensus regarding the cause of this discrepancy. As microRNAs are stable biomarkers and potential therapeutic targets of cancer, the present study aimed to explore the underlying mechanisms of this phenomenon by examining changes in the microRNAome. Rats were used to produce models of spinal cord injury. Microarrays and bioinformatics were used to investigate the cancer-associated microRNAs that are upregulated in rat bladders following spinal cord injury. In order to validate the results, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blotting and immunohistochemistry were performed. The expression of miR-1949 was found to be deregulated and abundant in the rat bladder following spinal cord injury. Bioinformatics demonstrated that retinoblastoma 1, which is involved in tumorigenesis, is a target gene of miR-1949. qRT-PCR, western blotting and immunohistochemistry confirmed the results of the microarray analysis. In addition, it was shown that miR-1949 expression was not influenced by aging. Furthermore, the expression of miR-1949 was stable until the third month following spinal cord injury, after which it significantly increased. If this increase was prolonged, the expression of retinoblastoma 1 may decline to a carcinogenic level. The present study suggests a role for miR-1949 in the translational regulation of retinoblastoma 1 and in subsequent bladder tumorigenesis following spinal cord injury.

After spinal cord injury (SCI), the rapidly upregulated chondroitin sulfate proteoglycans (CSPGs), the prominent chemical constituents and main repulsive factors of the glial scar, play an important role in the extremely limited ability to regenerate in adult mammals. Although many methods to overcome the inhibition have been tested, no successful method with clinical feasibility has been devised to date. It was recently discovered that receptor protein tyrosine phosphatase sigma (RPTPσ) is a functional receptor for CSPGs-mediated inhibition. In view of the potential clinical application of RNA interference (RNAi), here we investigated whether silencing RPTPσ via lentivirus-mediated RNA interference can promote axon regeneration and functional recovery after SCI. Neurites of primary rat cerebral cortical neurons with depleted RPTPσ exhibited a significant enhancement in elongation and crossing ability when they encountered CSPGs in vitro. A contusion model of spinal cord injury in Wistar rats (the New York University (NYU) impactor) was used for in vivo experiments. Local injection of lentivirus encoding RPTPσ shRNA at the lesion site promoted axon regeneration and synapse formation, but did not affect the scar formation. Meanwhile, in vivo functional recovery (motor and sensory) was also enhanced after RPTPσ depletion. Therefore, strategies directed at silencing RPTPσ by RNAi may prove to be a beneficial, efficient and valuable approach for the treatment of SCI.

Increasing evidence indicates that HIV-1 infection has an impact on cell autophagy, and a susceptible cell line is required for studying the relationship of HIV-1 with autophagy. However, there is limited information on the optimal cell line to evaluate the changes of autophagy affected by HIV infection. In this study cell line TZM-tfLC3B was constructed to express mCherry-EGFP tandem fluorescent tagged LC3B (tfLC3B) by stable transfection of tfLC3B as well as allowing X4/R5 tropic HIV-1 replication. The monitoring of autophagic flux in TZM-tfLC3B was achieved by observing fluorescent puncta. HIV-1 virus-like particles lacking replicative nucleic acid could induce autophagy in TZM-tfLC3B in an envelope glycoprotein dependent manner. These data suggest that TZM-tfLC3B will be a useful tool for studying the HIV-1-induced autophagy modulation of host cells.

Small molecular CCR5 inhibitors represent a new class of drugs for treating HIV-1 infection. The evaluation of the primary resistance mutations associated with entry inhibitors during HIV-1 perinatal transmission is required because they may have a profound impact on the clinical management in MTCT.To evaluate the primary resistance mutations to maraviroc and vicriviroc during perinatal transmission and analyze the sensitivity of Env derived from mother-infant pairs to maraviroc.Nine MIPs infected by subtype C HIV-1 were recruited to analyze the prevalence and transmission of primary resistance mutations to maraviroc and vicriviroc. Moreover, Env derived from six MIPs were employed to construct provirus clones and to analyze the sensitivity to maraviroc.Mutations A316T, conferring partial resistance to maraviroc, T307I and R315Q, both conferring partial resistance to vicriviroc are prevalent in mother and infant cohorts, indicating the transmission of primary resistance mutations during HIV-1 perinatal transmission. However, the mutations of acutely infected mothers seem to directly transmit to their corresponding infants, while some mutations at low frequency of chronically infected mothers would be lost during transmission. Moreover, provirus clones derived from acutely infected MIPs are less susceptible to maraviroc than those from chronically infected MIPs.Our study suggests that the transmission mode of primary resistance mutations and the sensitivity to maraviroc are dependent on infection status of MIPs either acutely or chronically infected. These results may indicate that higher dose of maraviroc could be needed for treatment of acutely infected MIPs compared to chronically infected MIPs.

The cellular protein tetherin tethers the HIV-1 viral particles on the cellular membrane to inhibit the replication of HIV-1. However, the HIV-1 accessory protein Vpu counteracts the antiviral function of tetherin. In this study, two retroviral vector plasmids were constructed. One inhibited the vpu gene expression; the other one over-expressed the tetherin. Both retroviral vector plasmids could be packaged in the packaging cell line PT67 to obtain the corresponding retroviruses. The retroviral vector plasmids’ functions of tetherin over-expression or vpu-RNAi were detected at the cell level. Retroviral vector plasmids were transfected to PT67 cells at different ratios from 0T3V to 3T0V, and then mixed retroviruses were harvested. The antiviral functions of mixed retroviruses were detected in HIV-1 infected TZM-bl cells. The results showed that packaged mixed retroviruses could repress the replication of HIV-1 in TZM-bl cells.

Bovine ISG15 (bISG15) is an interferon inducible ubiquitin-like protein that is responsible for the establishment of early pregnancy in ruminant, understanding the properties of bISG15 capable of being inducible in fetal bovine lung (FBL) cells upon infection of bovine immunodeficiency virus (BIV) is of significant importance. In this study, we investigated the expression of bISG15 in poly I:C treated FBL cells. The increased expression of bISG15 was observed, and the inhibition of BIV replication was also detected in FBL cells. Elimination of bISG15 expression by small interfering RNA reversed the bISG15 mediated inhibition of BIV replication. These findings demonstrate that bISG15 plays an important role in inhibition of the BIV replication in FBL cells. Furthermore, real-time PCR and western blot assay revealed that bISG15's expression can also be induced in BIV infected FBL cells. Taken together, bISG15 is an antiviral and inducible protein in BIV infected FBL cells.