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Prototype foamy virus (PFV) is a unique retrovirus that infects animals and humans and does not cause clinical symptoms. Long noncoding RNAs (lncRNAs) are believed to exert multiple regulatory functions during viral infections. Previously, we utilized RNA sequencing (RNA-seq) to characterize and identify the lncRNA lnc-RP5-1086D14.3.1-1:1 (lnc-RP5), which is markedly decreased in PFV-infected cells. However, little is known about the function of lnc-RP5 during PFV infection. In this study, we identified lnc-RP5 as a regulator of the PFV transcriptional transactivator (Tas). Lnc-RP5 enhanced the activity of the PFV internal promoter (IP). The expression of PFV Tas was found to be promoted by lnc-RP5. Moreover, miR-129-5p was found to be involved in the lnc-RP5-mediated promotion of PFV IP activity, while the Notch1 protein suppressed the activity of PFV IP and the expression of Tas. Our results demonstrate that lnc-RP5 promotes the expression of PFV Tas through the miR-129-5p/Notch1/PFV IP axis. This work provides evidence that host lncRNAs can manipulate PFV replication by employing miRNAs and proteins during an early viral infection. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12250-019-00168-3) contains supplementary material, which is available to authorized users.

Background Hypoxic-ischemic encephalopathy (HIE) is a serious birth complication with high incidence in both advanced and developing countries. Children surviving from HIE often have severe long-term sequela including cerebral palsy, epilepsy, and cognitive disabilities. The severity of HIE in infants is tightly associated with increased IL-1β expression and astrocyte activation which was regulated by transient receptor potential vanilloid 1 (TRPV1), a non-selective cation channel in the TRP family. Methods Neonatal hypoxic ischemia (HI) and oxygen-glucose deprivation (OGD) were used to simulate HIE in vivo and in vitro. Primarily cultured astrocytes were used for investigating the expression of glial fibrillary acidic protein (GFAP), IL-1β, Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), and activation of the nucleotide-binding, oligomerization domain (NOD)-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome by using Western blot, q-PCR, and immunofluorescence. Brain atrophy, infarct size, and neurobehavioral disorders were evaluated by Nissl staining, 2,3,5-triphenyltetrazolium chloride monohydrate (TTC) staining and neurobehavioral tests (geotaxis reflex, cliff aversion reaction, and grip test) individually. Results Astrocytes were overactivated after neonatal HI and OGD challenge. The number of activated astrocytes, the expression level of IL-1β, brain atrophy, and shrinking infarct size were all downregulated in TRPV1 KO mice. TRPV1 deficiency in astrocytes attenuated the expression of GFAP and IL-1β by reducing phosphorylation of JAK2 and STAT3. Meanwhile, IL-1β release was significantly reduced in TRPV1 deficiency astrocytes by inhibiting activation of NLRP3 inflammasome. Additionally, neonatal HI-induced neurobehavioral disorders were significantly improved in the TRPV1 KO mice. Conclusions TRPV1 promotes activation of astrocytes and release of astrocyte-derived IL-1β mainly via JAK2-STAT3 signaling and activation of the NLRP3 inflammasome. Our findings provide mechanistic insights into TRPV1-mediated brain damage and neurobehavioral disorders caused by neonatal HI and potentially identify astrocytic TRPV1 as a novel therapeutic target for treating HIE in the subacute stages (24 h). Electronic supplementary material The online version of this article (10.1186/s12974-019-1487-3) contains supplementary material, which is available to authorized users.

BRAFV600E mutation is found in ~50% of melanoma patients and BRAFV600E kinase activity inhibitor, vemurafenib, has achieved a remarkable clinical response rate. However, most patients treated with vemurafenib eventually develop resistance. Overcoming primary and secondary resistance to selective BRAF inhibitors remains one of the most critically compelling challenges for these patients. HDAC6 has been shown to confer resistance to chemotherapy in several types of cancer. Few studies focused on the role of HDAC6 in vemurafenib resistance. Here we showed that overexpression of HDAC6 confers resistance to vemurafenib in BRAF-mutant A375 cells. ACY-1215, a selective HDAC6 inhibitor, inhibits the proliferation and induces the apoptosis of A375 cells. Moreover, ACY-1215 sensitizes A375 cells to vemurafenib induced cell proliferation inhibition and apoptosis induction, which occur partly through induction of endoplasmic reticulum (ER) stress and inactivation of extracellular signal-regulated kinase (ERK). Taken together, our results suggest that the inhibition of HDAC6 may be a promising strategy for the treatment of melanoma and overcoming resistance to vemurafenib.

Aims: Chronic cocaine abuse decreases the inhibitory synaptic transmission via unknown mechanisms, while pharmacologically augmenting gamma-aminobutyric acid-ergic (GABAergic) transmission attenuates cocaine craving. Here, we propose that prolonged cocaine withdrawal downregulates GABAergic transmission and its important regulator gephyrin in medial prefrontal cortex (mPFC), in cocaine-conditioned place-preference (CPP) rats. Methods: CPP test, patch clamp, and Western blot analysis are engaged to test this proposal. Results: Two-week cocaine withdrawal further increased CPP score, as compared to the 24-hour withdrawn group. The amplitude of GABAergic inhibitory postsynaptic currents (IPSCs) was decreased in 2-week-withdrawn mPFC neurons from cocaine-CPP rats, compared to that of saline-CPP rats. Two-week withdrawal did not alter the amplitude of glutamatergic excitatory postsynaptic currents (EPSCs) in mPFC in cocaine-CPP rats. Two-week withdrawal increased the ratio of EPSCs/IPSCs (E/I) in the same mPFC neuron in cocaine-CPP rats. In addition, Western blots showed 2-week cocaine-withdrawn down-regulated gephyrin at postsynaptic density (PSD) sites of mPFC. Conclusion: We found decreased GABAergic IPSCs and downregulated gephyrin in PSD at mPFC in 2-week cocaine-withdrawn rats that showed increased CPP, suggesting that an increased E/I ratio and neuron excitability in mPFC may associate with a cocaine-seeking tendency. Strategies aimed at GABAergic synapses in mPFC may therapeutically benefit to cocaine addiction treatment.

Epigenetic modifications including histone modifications are associated with seizure development and epileptogenesis; however, its underlying mechanism remains to be elucidated. Dipeptidyl peptidase 4 (DPP4) and IL6 are identified as febrile seizure (FS)-related genes using gene microarray analysis in hyperthermia prone (HP) rats. This purpose of the study focused on exploring whether epigenetic modifications marker histone H3 lysine 27 trimethylation (H3K27me3)-regulated DPP4 and IL6 expression further affected seizures development. Herein, we reported broad between-group differences in the global levels of H3K27me3 with increased seizure severity in vivo. Using chromatin immunoprecipitation (ChIP), we identified markedly decreased H3K27me3 enrichment at their promoters of DPP4 and IL6 in vivo. We further showed that hyperthermia significantly decreased protein levels of H3K27me3, increased mRNA levels of DPP4 and IL6 by decreasing H3K27me3 enrichment at their promoters of DPP4 and IL6 in vitro. Importantly, H3K27me3 loss via enhancer of zeste homolog 2 (EZH2) knockdown promoted expression of DPP4 and IL6 via the same mechanism in vitro. EZH2 knockdown also increased neuronal firing frequency in vitro and FS susceptibility in vivo companied with upregulation expression of DPP4 and IL6. Taken together, our study provided the first evidence that hyperthermia-induced decreased of H3K27me3 promoted seizure susceptibility via regulating the expression pattern of DPP4 and IL6. These findings suggested that the methylation level of H3K27me3 might be a key regulator of seizure susceptibility.

Coxsackievirus A16 (CVA16) is one of major pathogens of hand, foot and mouth disease (HFMD) in children. Long non-coding RNAs (IncRNAs) have been implicated in various biological processes, but they have not been associated with CVA16 infection. In this study, we comprehensively characterized the landscape of IncRNAs of normal and CVA16 infected rhabdomyosarcoma (RD) cells using RNA-Seq to investigate the functional relevance of IncRNAs. We showed that a total of 760 IncRNAs were upregulated and 1210 IncRNAs were downregulated. Out of these dysregulated IncRNAs, 43.64% were intergenic, 22.31% were sense, 15.89% were intronic, 8.67% were bidirectional, 5.59% were antisense, 3.85% were sRNA host IncRNAs and 0.05% were enhancer. Six dysregulated IncRNAs were validated by quantitative PCR assays and the secondary structures of these IncRNAs were projected. Moreover, we conducted a bioinformatics analysis of an IncRNAs (ENST00000602478) to elucidate the diversity of modification and functions of IncRNAs. In summary, the current study compared the dysregulated IncRNAs profile upon CVA16 challenge and illustrated the intricate relationship between coding and IncRNAs transcripts. These results may not only provide a complete picture of transcription in CVA16 infected cells but also provide novel molecular targets for treatments of HFMD. Electronic Supplementary Material Supplementary material is available for this article at 10.1007/s12250-015-3693-1 and is accessible for authorized users.

Artemisia annua produces artemisinin, an effective antimalarial drug. In recent decades, the later steps of artemisinin biosynthesis have been thoroughly investigated; however, little is known about the early steps of artemisinin biosynthesis. Comparative transcriptomics of glandular and filamentous trichomes and 13CO2 radioisotope study have shown that the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, rather than the mevalonate pathway, plays an important role in artemisinin biosynthesis. In this study, we have cloned three 1-deoxy-D-xylulose 5-phosphate synthase (DXS) genes from A. annua (AaDXS1, AaDXS2, and AaDXS3); the DXS enzyme catalyzes the first and rate-limiting enzyme of the MEP pathway. We analyzed the expression of these three genes in different tissues in response to multiple treatments. Phylogenetic analysis revealed that each of the three DXS genes belonged to a distinct clade. Subcellular localization analysis indicated that all three AaDXS proteins are targeted to chloroplasts, which is consistent with the presence of plastid transit peptides in their N-terminal regions. Expression analyses revealed that the expression pattern of AaDXS2 in specific tissues and in response to different treatments, including methyl jasmonate, light, and low temperature, was similar to that of artemisinin biosynthesis genes. To further investigate the tissue-specific expression pattern of AaDXS2, the promoter of AaDXS2 was cloned upstream of the β-glucuronidase gene and was introduced in arabidopsis. Histochemical staining assays demonstrated that AaDXS2 was mainly expressed in the trichomes of Arabidopsis leaves. Together, these results suggest that AaDXS2 might be the only member of the DXS family in A. annua that is involved in artemisinin biosynthesis.

Background Neonatal hypoxic-ischemic brain damage, characterized by tissue loss and neurologic dysfunction, is a leading cause of mortality and a devastating disease of the central nervous system. We have previously shown that vitexin has been attributed various medicinal properties and has been demonstrated to have neuroprotective roles in neonatal brain injury models. In the present study, we continued to reinforce and validate the basic understanding of vitexin (45 mg/kg) as a potential treatment for epilepsy and explored its possible underlying mechanisms. Methods P7 Sprague-Dawley (SD) rats that underwent right common carotid artery ligation and rat brain microvascular endothelial cells (RBMECs) were used for the assessment of Na+-K+-Cl− co-transporter1 (NKCC1) expression, BBB permeability, cytokine expression, and neutrophil infiltration by western blot, q-PCR, flow cytometry (FCM), and immunofluorescence respectively. Furthermore, brain electrical activity in freely moving rats was recorded by electroencephalography (EEG). Results Our data showed that NKCC1 expression was attenuated in vitexin-treated rats compared to the expression in the HI group in vivo. Oxygen glucose deprivation/reoxygenation (OGD) was performed on RBMECs to explore the role of NKCC1 and F-actin in cytoskeleton formation with confocal microscopy, N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide, and FCM. Concomitantly, treatment with vitexin effectively alleviated OGD-induced NKCC1 expression, which downregulated F-actin expression in RBMECs. In addition, vitexin significantly ameliorated BBB leakage and rescued the expression of tight junction-related protein ZO-1. Furthermore, inflammatory cytokine and neutrophil infiltration were concurrently and progressively downregulated with decreasing BBB permeability in rats. Vitexin also significantly suppressed brain electrical activity in neonatal rats. Conclusions Taken together, these results confirmed that vitexin effectively alleviates epilepsy susceptibility through inhibition of inflammation along with improved BBB integrity. Our study provides a strong rationale for the further development of vitexin as a promising therapeutic candidate treatment for epilepsy in the immature brain.

HIV is the causative pathogen of AIDS, which has generated worldwide concern. Long noncoding RNAs (lncRNAs) are a rising star in virus-host cross-talk pathways; they are differentially expressed during many viral infections and are involved in multiple biological processes. Currently, lncRNA growth arrest-specific transcript 5 (GAS5) is known to be downregulated during HIV-1 infection. However, the functions and mechanisms of GAS5 in HIV-1 infection remain largely unknown. In this report, it was found for the first time that GAS5 could inhibit HIV-1 replication. Interestingly, using bioinformatics analyses (with Genomica and starBase.v2.0), GAS5 was found to potentially interact with miR-873. It was further verified that GAS5 could suppress miR-873. Moreover, miR-873 could promote HIV-1 replication. Together, these results not only suggest that GAS5 may inhibit HIV-1 replication through interaction with miR-873 but the results may also provide novel biomarkers for antiviral drugs or potential targets for future therapeutics for HIV/AIDS.

// Xiong Chen 1, * , Yingying Zhang 1, * , Yingying Shi 1 , Haiwei Lian 4 , Huilin Tu 1 , Song Han 1 , Jun Yin 1 , Biwen Peng 1, 2 , Beiyan Zhou 5 , Xiaohua He 1, 2 , Wanhong Liu 1, 3 1 School of Basic Medical Sciences, Wuhan University, Wuhan 430071, China 2 Hubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan 430071, China 3 Hubei Province Key Laboratory of Allergy and Immunology, Wuhan 430071, China 4 Department of Neurosurgery, Wuhan University Renmin Hospital, Wuhan 430060, China 5 Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A & M University, Texas 77843, USA * These authors contributed equally to this work Correspondence to: Xiaohua He, e-mail: hexiaohua@whu.edu.cn Wanhong Liu, e-mail: liuwanhong@whu.edu.cn Keywords: miR-129, E2F7, Notch-1, autophagy, glioma Received: July 07, 2015 Accepted: January 01, 2016 Published: January 25, 2016 ABSTRACT Abnormalities of autophagy have been implicated in an increasing number of human cancers, including glioma. To date, there is a wealth of evidence indicating that microRNAs (miRNAs) contribute significantly to autophagy in a variety of cancers. Previous studies have suggested that miR-129 functioned as an important inhibitor of the cell cycle and could promote the apoptosis of many cancer cell lines in vitro . Here, we reported that miR-129 acted as a potent inducer of autophagy. Forced expression of miR-129 could induce autophagic flux by targetedly suppressing Notch-1 in glioma cells. The autophagy induced by miR-129 could restrain the activity of mammalian target of rapamycin (mTOR) and upregulate Beclin-1. Moreover, we demonstrated that E2F transcription factor 7 (E2F7) could also trigger autophagic flux by upregulating Beclin-1 and mediating miR-129-induced autophagy. Additionally, knockdown of Notch-1 could upregulate the expression of E2F7, whereas downregulation of E2F7 alleviated shNotch-1-induced autophagic flux. In particular, knockdown of endogenous Beclin-1 could effectively reduce autophagic flux stimulated by miR-129 and E2F7. Interestingly, upon attenuation of miR-129- or E2F7-triggered autophagic flux rescued cell viability suppressed by them. More importantly, intratumoral injection of pHAGE-miR-129 lentivirus in a nude mouse xenograft model significantly restrained tumor growth and triggered autophagy. In conclusion, these findings identify a new function for miR-129 as a potent inducer of autophagy through a novel Notch-1/E2F7/Beclin-1 axis in glioma.

Previous studies have demonstrated that the early suppression of HIF-1α after hypoxia–ischemia (HI) injury provides neuroprotection. Vitexin (5, 7, 4-trihydroxyflavone-8-glucoside), an HIF-1α inhibitor, is a c-glycosylated flavone that has been identified in medicinal plants. Therefore, we hypothesized that treatment with vitexin would protect against HI brain injury. Newborn rat pups were subjected to unilateral carotid artery ligation followed by 2.5 h of hypoxia (8% O 2 at 37 °C). Vitexin (30, 45 or 60 mg/kg) was administered intraperitoneally at 5 min or 3 h after HI. Vitexin, administered 5 min after HI, was neuroprotective as seen by decreased infarct volume evaluated at 48 h post-HI. This neuroprotection was removed when vitexin was administered 3 h after HI. Neuronal cell death, blood–brain barrier (BBB) integrity, brain edema, HIF-1α and VEGF protein levels were evaluated using a combination of Nissl staining, IgG staining, brain water content, immunohistochemistry and Western blot at 24 and 48 h after HI. The long-term effects of vitexin were evaluated by brain atrophy measurement, Nissl staining and neurobehavioral tests. Vitexin (45 mg/kg) ameliorated brain edema, BBB disruption and neuronal cell death; Upregulation of HIF-1α by dimethyloxalylglycine (DMOG) increased the BBB permeability and brain edema compared to HI alone. Vitexin attenuated the increase in HIF-1α and VEGF. Vitexin also had long-term effects of protecting against the loss of ipsilateral brain and improveing neurobehavioral outcomes. In conclusion, our data indicate early HIF-1α inhibition with vitexin provides both acute and long-term neuroprotection in the developing brain after neonatal HI injury.

Enterovirus 71 (EV71) is a neurotropic virus that causes hand, foot and mouth disease (HFMD), occasionally leading to death. As a member of the RAS association domain family (RASSFs), RASSF4 plays important roles in cell death, tumor development and signal transduction. However, little is known about the relationship between RASSF4 and EV71. Our study reveals for the first time that RASSF4 promotes EV71 replication and then accelerates AKT phosphorylation inhibition in EV71-infected 293T cells, suggesting that RASSF4 may be a potential new target for designing therapeutic measures to prevent and control EV71 infection.

The first Enterovirus 71 (EV71) strain isolated in 1969 was classified as genotype A. It is interesting that the genotype A disappeared nearly 40 years until its re-emergence in mainland China in 2008-2010. Few studies on genetic characterization of the re-emerged genotype A viruses have been reported. In this study, a series of analyses were performed on molecular epidemiology and genome recombination of genotype A viruses in China. Phylogenetic analysis indicated that except for 17 reported genotype A strains and 3 orphan strains (C0, C3 and B5), almost all EV71 strains in mainland China were belonging to subgenotype C4 during 1987-2011. The subgenotype C4 was further divided into 3 clades C4a1, C4a2, and C4b. The genotype A viruses co-circulated with the predominant clade C4a2 and the re-emerged clade C4b both in eastern and central China in 2008-2009. Moreover, comprehensive recombination analysis showed that the genotype A viruses were "triple-recombinant" by combination of intratypic and intertypic recombination. Intertypic recombination between the oldest C4b strain (SHZH98) and Coxsackievirus A5 (CVA5) and intratypic recombination between the SHZH98 and C1 strains both with one junction in 5'-UTR were observed for some specific C4a2 strains and the re-emerged C4b strain, respectively. And intratypic recombination between the re-emerged C4b strain and the specific C4a2 strains with one junction in 5'-UTR was observed for the Chinese genotype A viruses. Taken together, these results provided potential explanations for the genesis of Chinese genotype A viruses which were significant for preventing and controlling outbreaks.

Epilepsy is a common neurological disorder with a complex etiology. Our previous study demonstrated that dipeptidyl peptidase IV (DPP4) may be associated with the pathogenesis of epilepsy. However, whether the DPP4 inhibitor sitagliptin has an anticonvulsant effect and the underlying mechanism remain to be elucidated. In this study, we determined that sitagliptin remarkably attenuated the severity of seizures in a pentylenetetrazole (PTZ)-induced rat model. In addition, sitagliptin decreased epileptiform activity measured by electroencephalography (EEG) recordings and patch-clamp methods. Interestingly, sitagliptin pretreatment downregulated the RAGE-JAK2/STAT3 pathway and decreased the expression of CXCL4 and CXCR3. Moreover, CXCR3 knockdown decreased the expression of RAGE, JAK2 and STAT3 in cultured neurons, which suggests that CXCR3 is upstream of the RAGE-JAK2/STAT3 pathway. Altogether, our present data suggest that sitagliptin has an anticonvulsant effect, which might act via downregulation of the CXCL4/CXCR3 axis, followed by a decrease in RAGE and JAK2/STAT3 expression. Considering these effects, sitagliptin could be considered as a novel potential anticonvulsant drug.

Aim Multiple sclerosis (MS) is a neurological autoimmune disorder characterized by mistaken attacks of inflammatory cells against the central nervous system (CNS), resulting in demyelination and axonal damage. Kv1.3 channel blockers can inhibit T-cell activation and have been designed for MS therapy. However, little is known about the effects of Kv1.3 blockers on protecting myelin sheaths/axons in MS. This study aimed at investigating the neuroprotection efficacy of a selective Kv1.3 channel blocker ImKTx88 (ImK) in MS animal model. Methods Experimental autoimmune encephalomyelitis (EAE) rat model was established. The neuroprotective effect of ImK was assessed by immunohistochemistry and transmission electron microscopy (TEM). In addition, the antiinflammatory effect of ImK by suppressing T-cell activation was assessed by flow cytometry and ELISA in vitro. Results Our results demonstrated that ImK administration ameliorated EAE clinical severity. Moreover, ImK increased oligodendrocytes survival, preserved axons, and myelin integrity and reduced the infiltration of activated T cells into the CNS. This protective effect of the peptide may be related to its suppression of autoantigen-specific T-cell activation via calcium influx inhibition. Conclusion ImK prevents neurological damage by suppressing T-cell activation, suggesting the applicability of this peptide in MS therapy.

Febrile seizures (FS) are the most common seizure disorders in children aged 6 months to 5 years. Children suffering from complex FS have a high risk of developing subsequent temporal lobe epilepsy (TLE). Neuroinflammation is involved in the pathogenesis of FS although the mechanism remains unknown. Our previous study using the Whole Rat Genome Oligo Microarray determined that Dipeptidyl peptidase IV (DPP4) is potentially a related gene in FS rats. In this study, we demonstrated that DPP4 expression was significantly increased at both the protein and mRNA levels after hyperthermia induction. Sitagliptin, a specific enzyme inhibitor of DPP4, remarkably attenuated the severity of seizures in FS rats, and hyperthermia-induced astrocytosis was suppressed after DPP4 inhibition. Furthermore, sitagliptin significantly decreased the levels of the inflammatory cytokines IL-1β, TNF-α, and IL-6 but not IL-10. In addition, sitagliptin prevented NF-κB activation by decreasing phosphorylation of the p65 subunit. Taken together, our findings demonstrate that DPP4 functions as a critical regulator of neuroinflammation in hyperthermia-induced seizures and the DPP4 inhibitor may be a viable option for FS therapeutics.

The selection and validation of reference genes are essential for gene expression studies by real-time quantitative PCR. The genetic map of Artemisia annua L., a Chinese medicinal plant species producing anti-malarial artemisinin, has been reported. However, few reference genes of A. annua have been estimated for real-time quantitative PCR until now. In this study, ten putative housekeeping genes, including ACT, UBQ, TUB, 18S rRNA, EF1α, CYP, RPL13D, TUA, RPII and GAPDH, were chosen for identifying expression stability using geNorm and NormFinder software tools in 11 different sample pools, containing those from different plant organs and from plants treated with phytohormones and abiotic stresses. As expected, the variation in expression stability of the ten candidate reference genes tested in this study suggested there was no single reference gene that can be used for all experimental conditions in A. annua. The combination of RPII & EF1α was the most stably expressed reference genes for different organs. Under phytohormone treatment, the combination of EF1α & TUB was recommended as internal reference genes used for investigating target gene expression levels. In addition, the combination of ACT & EF1α was suitably chosen for normalization in temperature-shocked samples. In order to further verify the reliability of the experimental results, RPII & EF1α were used in combination as reference genes to examine the expression levels of ADS gene in different organs. Meanwhile, the expression levels of ADS, CYP71AV1 and DBR2 were tested by qPCR normalized with the combination of EF1α & TUB in MeJA treatment samples. Our study will benefit future research on the expression of genes related to artemisinin biosynthesis under different experimental conditions.

Methyl jasmonate (MeJA) is one of the most potent elicitors that can induce over accumulation of many natural products including artemisinin in plants. The 12 known genes (HMGR, DXS, DXR, HDS, HDR, FPS, ADS, CYP71AV1, DBR2, ALDH1, ORA and ERF1) of terpene metabolism in Artemisia annua were dynamically analyzed at the transcriptional levels in the treatment of MeJA from 0 to 48 h. HMGR (MVA pathway) showed higher expression level when the plants were treated with MeJA from 1 to 9 h and had the highest expression level at 3 h MeJA treatment. The expression levels of DXS and DXR (MEP pathway) reached the peak at 9 h. The last two genes of the MEP pathway, such as HDS and HDR, had the highest expression levels at 24 h. The expression of FPS increased significantly in the treatment of MeJA from 1 to 48 h, and the highest expression level appeared at 24 and 48 h after the MeJA treatment. Four genes in artemisinin-specific biosynthetic pathway including ADS, CYP71AV1, DBR2 and ALDH1 had higher expression levels in the treatment of MeJA from 1 to 48 h. The expression levels of two transcription factors such as ORA and ERF1 were also enhanced. The contents of artemisinin in the plants treated with MeJA for 24 and 48 h were respectively 0.971 and 0.973 mg/g DW, about 1.16-fold of the control (0.809 mg/g DW). Taken together, these results suggested that MeJA induced artemisinin biosynthesis by up-regulating the expression of the genes involved in artemisinin biosynthesis and the transcription factor.

Gamma-amino-butyric acid (GABA)-containing interneurons are crucial to both development and function of the brain. Down-regulation of GABAergic inhibition may result in the generation of epileptiform activity. Loss, axonal sprouting, and dysfunction of interneurons are regarded as mechanisms involved in epileptogenesis. Recent evidence suggests that network connectivity and the properties of interneurons are responsible for excitatory-inhibitory neuronal circuits. The balance between excitation and inhibition in CA1 neuronal circuitry is considerably altered during epileptic changes. This review discusses interneuron diversity, the causes of interneuron dysfunction in epilepsy, and the possibility of using GABAergic neuronal progenitors for the treatment of epilepsy.

Artemisinic aldehyde Δ11 (13) reductase (DBR2) is the checkpoint enzyme catalyzing artemisinic aldehyde to form dihydroartemisinic aldehyde directly involved in artemisinin biosynthetic pathway. In the present study, DBR2 was employed to engineer the biosynthetic pathway of artemisinin in transgenic plants of Artemisia annua L. Seven independent transgenic plants of A. annua with DBR2 overexpression driven by the cauliflower mosaic virus 35S promoter were obtained by Agrobacterium-mediated genetic transformation and confirmed by genomic PCR. The results of real-time qPCR analysis showed that the expression levels of DBR2 gene in all the seven transgenic lines were significantly higher than in nontransgenic control. The high-performance liquid chromatography analysis of artemisinin and its relative metabolites demonstrated that the contents of artemisinin and its direct precursor dihydroartemisinic acid were remarkably increased in the transgenic plants of A. annua with DBR2 overexpression. Interestingly, it was also found that the contents of arteannuin B and its direct precursor artemisinic acid in the branch pathway competing against artemisinin biosynthesis were also improved in DBR2-overexpressed A. annua plants. The transgenic results in the present study indicated that DBR2 is a useful structural gene in engineering the artemisinin biosynthetic pathway to develop genetically modified A. annua with the higher yield of artemisinin.

Quantitative PCR (qPCR) is a powerful tool for measuring gene expression levels. Accurate and reproducible results are dependent on the correct choice of reference genes for data normalization. Atropa belladonna is a commercial plant species from which pharmaceutical tropane alkaloids are extracted. In this study, eight candidate reference genes, namely 18S ribosomal RNA (18S), actin (ACT), cyclophilin (CYC), elongation factor 1α (EF-1α), β-fructosidase (FRU), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), and beta-tubulin (TUB), were selected and their expression stabilities studied to determine their suitability for normalizing gene expression in A. belladonna. The expression stabilities of these genes were analyzed in the root, stem, and leaf under cold, heat, NaCl, UV-B, methyl jasmonate, salicylic acid, and abscisic acid treatments using geNorm, NormFinder, and BestKeeper. The statistical algorithms indicated that PGK was a reliable gene for normalizing gene expression under most of the experimental conditions. The pairwise value analysis showed that two genes were sufficient for proper expression normalization, except when analyzing gene expression in heat-treated roots. However, the choice of the second reference gene depended on specific conditions. Finally, the relative expression level of the PMT gene of A. belladonna was detected to validate the selection of PGK a reliable reference gene. In summary, our results should guide the selection of appropriate reference genes for gene expression studies in A. belladonna under different organs and abiotic stress conditions.

Enterovirus 71 (EV71) is a neurotropic virus that causes various clinical manifestations in young children, ranging from asymptomatic to fatal. Different pathotypes of EV71 notably differ in virulence. Several virulence determinants of EV71 have been predicted. However, these reported virulence determinants could not be used to identify the EV71 strains of subgenotype C4, which mainly circulate in China. In this study, VP1 sequences of 37 EV71 strains from severe cases (SC-EV71) and 192 EV71 strains from mild cases (MC-EV71) in mainland China were analyzed to determine the potential virulence determinants in the capsid protein VP1 of EV71. Although most SC-EV71 strains belonged to subgenotype C4a, no specific genetic lineages in C4a were correlated with EV71 virulence. Interestingly, amino acid substitutions at nine positions (H22Q, P27S, N31S/D, E98K, E145G/Q, D164E, T240A/S, V249I, and A289T) were detected by aligning the VP1 sequences of the SC-EV71 and MC-EV71 strains. Moreover, both the constituent ratios of the conservative or mutated residues in the MC-EV71 and SC-EV71 strains and the changes in the VP1 3D structure resulting from these mutations confirmed that the conservative residues (22H, 249V, and 289A) and the mutated residues (27S, 31S/D, 98K, 145G/Q, 164E, and 240A/S) might be potential virulence determinants in VP1 of EV71. Furthermore, these results led to the hypothesis that VP1 acts as a sandwich switch for viral particle stabilization and cellular receptors attachment, and specific mutations in this protein can convert mild cases into severe cases. These findings highlight new opportunities for diagnostic and therapeutic interventions.

microRNA (miRNA) dysregulation is associated with various types of human cancer by regulating cancer cell survival, proliferation and invasion. Aberrant expression of microRNA-503 (miR-503) has been reported in several cancer profiles. However, potential linkage of miR-503 levels and the underlying regulatory mechanisms in human glioblastoma multiforme (GBM) remain unclear. In the present study, we showed for the first time that the expression of miR-503 was significantly reduced in GBM tissues and cell lines (U251 and U87MG) relative to normal brain tissues. Furthermore, our results demonstrated that overexpression of miR-503 in GBM cell lines not only suppressed cell proliferation through inducing G0/G1 cell cycle arrest and apoptosis, but also inhibited cancer cell migration and tumor invasion. In addition, we identified insulin-like growth factor-1 (IGF-1R) receptor mRNA is a bona fide target of miR-503 by computational analysis followed by luciferase reporter assays. Of note, upregulation of miR-503 in GBM cells suppressed endogenous IGF-1R protein expression. Further mechanistic analysis revealed that forced expression of miR-503 inhibited AKT activation, suggesting the tumor suppressive effect of miR-503 in GBM cells is partially mediated by phosphatidylinositol 3-kinase/AKT signaling. Taken together, the results of the present study demonstrated that miR-503 is a tumor suppressor for GBM and a favorable factor against glioma progression through targeting IGF-1R, thus providing a new evidence-supported prognostic marker for GBM diagnosis.

Coxsackievirus A16 (CA16) infection, which is responsible for hand, foot and mouth disease (HFMD), has become a common health problem in Asia due to the prevalence of the virus. Thus, it is important to understand the pathogenesis of CA16 infection. Viruses that induce endoplasmic reticulum (ER) stress are confronted with the unfolded protein response (UPR), which may lead to apoptotic cell death and influence viral replication. In this study, we found that CA16 infection could induce apoptosis and ER stress in RD cells. Interestingly, apoptosis via the activation of caspase-3, -8 and -9 in the extrinsic or intrinsic apoptotic pathways in RD cells was inhibited by 4-phenyl butyric acid (4PBA), a chemical chaperone that reduces ER stress. These results suggest that CA16 infection leads to ER stress, which in turn results in prolonged ER stress-induced apoptosis. This study provides a new basis for understanding CA16 infection and host responses.

The transient receptor potential vanilloid 1(TRPV1) channels are members of the transient receptor potential (TRP) superfamily. Members of this family are expressed in primary sensory neurons and are best known for their role in nociception and sensory transmission. Multiple painful stimuli can activate these channels. In this review, we discussed the mechanisms of different types of venoms that target TRPV1, such as scorpion venom, botulinum neurotoxin, spider toxin, ciguatera fish poisoning (CFP) and neurotoxic shellfish poisoning (NSP). Some of these toxins activate TRPV1; however, some do not. Regardless of TRPV1 inhibition or activation, they occur through different pathways. For example, BoNT/A decreases TRPV1 expression levels by blocking TRPV1 trafficking to the plasma membrane, although the exact mechanism is still under debate. Vanillotoxins from tarantula (Psalmopoeus cambridgei) are proposed to activate TRPV1 via interaction with a region of TRPV1 that is homologous to voltage-dependent ion channels. Here, we offer a description of the present state of knowledge for this complex subject.

Additional file 2. Primers used for the construction of various plasmids and qPCR.Â

Additional file 1: Tables S1. ImKTx88 reduces clinical signs in EAE rats.

Additional file 1. Additional tables and figures.

Background Human enterovirus 71 (EV71) causes severe hand, foot and mouse disease, accompanied by neurological complications. During the interaction between EV71 and the host, the virus subverts host cell machinery for its own replication. However, the roles of microRNAs (miRNAs) in this process remain obscure. Results In this study, we found that the miRNA hsa-let-7c-5p was significantly upregulated in EV71-infected rhabdomyosarcoma cells. The overexpression of hsa-let-7c-5p promoted replication of the virus, and the hsa-let-7c-5p inhibitor suppressed viral replication. Furthermore, hsa-let-7c-5p targeted mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) and inhibited its expression. Interestingly, downregulation of MAP4K4 expression led to an increase in EV71 replication. In addition, MAP4K4 knockdown or transfection with the hsa-let-7c-5p mimic led to activation of the c-Jun NH2-terminal kinase (JNK) signaling pathway, whereas the hsa-let-7c-5p inhibitor inhibited activation of this pathway. Moreover, EV71 infection promoted JNK pathway activation to facilitate viral replication. Conclusions Our data suggested that hsa-let-7c-5p facilitated EV71 replication by inhibiting MAP4K4 expression, which might be related to subversion of the JNK pathway by the virus. These results may shed light on a novel mechanism underlying the defense of EV71 against cellular responses. In addition, these findings may facilitate the development of new antiviral strategies for use in future therapies. Electronic supplementary material The online version of this article (doi:10.1186/s13578-017-0135-9) contains supplementary material, which is available to authorized users.

Human foamy virus (HFV) is a complex and unique retrovirus with the longest genomes among retroviruses that are used as vectors for gene therapy. Long non-coding RNAs (lncRNAs) are regarded as key regulators that are involved in diverse biological processes during viral infection. However, the role of lncRNAs in HFV infection remains unknown. In this study, we utilized next-generation sequencing to first characterize lncRNAs in 293T cells after HFV infection, evaluating length distribution, exon number distribution, volcano picture, and lncRNA class distribution. We identified 11,336 lncRNAs (4,729 upregulated lncRNAs and 6,588 downregulated lncRNAs) and 61,367 mRNAs (30,133 upregulated mRNAs and 31,220 downregulated mRNAs), which were differentially expressed in the HFV-infected 293T cells. Subsequently, six differentially expressed lncRNAs characterized using RNA-seq were confirmed by quantitative real-time polymerase chain reaction assays. Interestingly, Gene Ontology (GO)/Gene Ontology Tree Machine (GOTM) and Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analyses indicated that positive regulation of interleukin 8 (IL8) production and cytokine-cytokine receptor interaction might be involved in the functional enrichment of lncRNAs. Moreover, cis-acting and trans-acting regulatory networks show that NR_028036 may target the fas gene in a cis-acting manner and that ENST00000354838 may target the IL18 gene in a trans-acting manner. Overall, these results not only provide novel insights into the relationship between HFV and lncRNAs in the host response to infection but also have implications for the future wider application of HFV as a vector.

Rice ragged stunt disease caused leads to severe loss of rice yield. Recently, rice ragged stunt virus (RRSV) were found to be increasingly common in rice-growing regions of China and Vietnam. RRSV may cause problem by interacting with microRNAs (miRNAs) of host cells and the mechanism is not clear yet. In this study we identified 11 miRNAs in response to RRSV infection and predicted their possible targets to viral RNA segments (S1-S10) through the bioinformatics analysis. Interestingly, we found that Osa-miR-168b might bind to both the CDS region and 3'UTR of S5 and S8 and target eEF-1A to inhibit the activity of host cells to facilitate RRSV replication. These results suggest that miRNAs may be a potential target for developing rice against RRSV infection.

Histone deacetylases are considered to be among the most promising targets in drug development for cancer therapy. Histone deacetylase 6 (HDAC6) is a unique cytoplasmic enzyme that regulates many biological processes involved in tumorigenesis through its deacetylase and ubiquitin-binding activities. Here, we report that HDAC6 is overexpressed in glioblastoma tissues and cell lines. Overexpression of HDAC6 promotes the proliferation and spheroid formation of glioblastoma cells. HDAC6 overexpression confers resistance to temozolomide (TMZ) mediated cell proliferation inhibition and apoptosis induction. Conversely, knockdown of HDAC6 inhibits cell proliferation, impairs spheroid formation and sensitizes glioblastoma cells to TMZ. The inhibition of HDAC6 deacetylase activity by selective inhibitors inhibits the proliferation of glioblastoma cells and induces apoptosis. HDAC6 selective inhibitors can sensitize glioblastoma cells to TMZ. Moreover, we showed that HDAC6 mediated EGFR stabilization might partly account for its oncogenic role in glioblastoma. TMZ resistant glioblastoma cells showed higher expression of HDAC6 and more activation of EGFR. HDAC6 inhibitors decrease EGFR protein levels and impair the activation of the EGFR pathway. Taken together, our results suggest that the inhibition of HDAC6 may be a promising strategy for the treatment of glioblastoma.

KCNQ2 (KV7.2) plays a key role in the regulation of neuronal excitability. Although temperature is thought to modulate all ion channels by different magnitudes, temperature dependence of KCNQ channels has not been demonstrated. In the present work, we reported that temperature elevation caused an increased expression of KCNQ2. Notably, the increased protein was retained intracellular. Meanwhile, we found that elevated temperature had a significant effect on the voltage-dependence of KCNQ2 channel activation. These data suggested that temperature can increase the rates of activation of KCNQ2 channel and has a critical role in the trafficking of KCNQ2 subunits to the surface membrane.

Many studies have been performed on venomous peptides derived from animals. However, little of this research has focused on peptides from centipede venoms. Here, a venom gland cDNA library was suc-cessfully constructed for the centipede Scolopendra subspinipes mutilans. A new cDNA encoding the precursor of a venom peptide, named SsmTx, was cloned from the venomous gland cDNA library of the centipede S. subspinipes mutilans. The full-length SsmTx cDNA sequence is 465 nt, including a 249 nt ORF, a 45 nt 5′ UTR and a 171 nt 3′ UTR. There is a signal tail AATAAA 31 nt upstream of the poly (A) tail. The precursor nucleotide sequence of SsmTx encodes a signal peptide of 25 residues and a mature peptide of 57 residues, which is bridged by two pairs of disulfide bonds. SsmTx displays a unique cysteine motif that is completely different from that of other venomous animal toxins. This is the first reported cDNA sequence encoding a venom peptide from the centipede S. subspinipes mutilans.

Febrile seizures (FSs) are the most common convulsions in childhood. Studies have demonstrated a significant relationship between a history of prolonged FSs during early childhood and temporal sclerosis, which is responsible for intractable mesial temporal lobe epilepsy. It has been shown that interleukin-1β (IL-1β) is intrinsically involved in the febrile response in children and in the generation of FSs. We summarize the gene polymorphisms, changes of IL-1β levels and the putative role of IL-1β in the generation of FSs. IL-1β could play a role either in enhancing or in reducing neural excitability. If the enhancing and reducing effects are balanced, an FS does not occur. When the enhancing effect plays the leading role, an FS is generated. A mild imbalance can cause simple FSs while a severe imbalance can cause complex FSs and febrile status epilepticus. Therefore, anti-IL-1β therapy may help to treat FSs.

One of the most fascinating findings in retrovirology is the construction of viral vectors based on foamy viruses (FVs) for gene therapy. The envelope glycoprotein (Env), one of the structural proteins of FV, is an important antigen in the immunoassays, as it is highly specific. To compare the characteristics of all 15 available FV Envs, the phylogenesis, hydrophobicity, modifications, and conserved motifs were analyzed based on the Env sequences. Meanwhile, the secondary structures of transmembrane (TM) domains of FV Envs were predicted. The results of phylogenetic analyses based on Envs indicated that the foamy viruses from different hosts could form three groups. The hydrophobicity analysis revealed that FV Envs had two prominent hydrophobic regions, which was similar to other retroviruses. Though the glycosylation, ubiquitination, and the secondary structures of TM domains of FV Envs were in line with other retroviruses, the roles were distinctly different. Interestingly, the analyses of conserved motifs suggested that FV Envs possessed several specific functional motifs.

Background: Hereditary neuropathy with liability to pressure palsy (HNPP), mainly associated with the peripheral myelin protein 22 (PMP22) gene, is generally an autosomal-dominant inherited peripheral neuropathy. The present large family including four generations provides an exciting opportunity to gain important insights into HNPP in China. Patients and Methods: A large 43-member family with ten members suspected to be affected by HNPP was studied. Neurologic examinations, electrophysiological and neuropathological studies and molecular genetic testing were used for these kindred. Results: Clinically, the proband had limb hyposthenia and atrophy, and his mother showed declined tendon reflexes in the right lower limb. Electrophysiologically, sensory and motor nerve conduction velocities were generalized reduced. Sural nerve biopsy for the proband showed focal thickesning of the myelin sheaths. Furthermore, real-time quantitative PCR demonstrated that the PMP22 gene has a higher Ct value than reference gene in all suspected patients. Conclusions: These results indicated that the family is indeed a rare and large pedigree of HNPP caused by the deletion of PMP22 gene. Given that the suspected patient in the fourth generation is absent, this family is still worthy of further follow-up study.

Selective degeneration of nigrostriatal dopaminergic neurons in Parkinson's disease (PD) can be modeled by the administration of the neurotoxin 1-methyl-4-phenylpyridinium (MPP(+) ). Because abnormal mitochondrial dynamics are increasingly implicated in the pathogenesis of PD, in this study, we investigated the effect of MPP(+) on mitochondrial dynamics and assessed temporal and causal relationship with other toxic effects induced by MPP(+) in neuronal cells. In SH-SY5Y cells, MPP(+) causes a rapid increase in mitochondrial fragmentation followed by a second wave of increase in mitochondrial fragmentation, along with increased DLP1 expression and mitochondrial translocation. Genetic inactivation of DLP1 completely blocks MPP(+) -induced mitochondrial fragmentation. Notably, this approach partially rescues MPP(+) -induced decline in ATP levels and ATP/ADP ratio and increased [Ca(2+) ](i) and almost completely prevents increased reactive oxygen species production, loss of mitochondrial membrane potential, enhanced autophagy and cell death, suggesting that mitochondria fragmentation is an upstream event that mediates MPP(+) -induced toxicity. On the other hand, thiol antioxidant N-acetylcysteine or glutamate receptor antagonist D-AP5 also partially alleviates MPP(+) -induced mitochondrial fragmentation, suggesting a vicious spiral of events contributes to MPP(+) -induced toxicity. We further validated our findings in primary rat midbrain dopaminergic neurons that 0.5 μm MPP(+) induced mitochondrial fragmentation only in tyrosine hydroxylase (TH)-positive dopaminergic neurons in a similar pattern to that in SH-SY5Y cells but had no effects on these mitochondrial parameters in TH-negative neurons. Overall, these findings suggest that DLP1-dependent mitochondrial fragmentation plays a crucial role in mediating MPP(+) -induced mitochondria abnormalities and cellular dysfunction and may represent a novel therapeutic target for PD.

Chronic pain is a major health concern that affects millions of people. There are no adequate long-term therapies for chronic pain sufferers, leading to significant cost for both society and the individual. The most commonly used therapy for chronic pain is the application of opioid analgesics and nonsteroidal anti-inflammatory drugs, but these drugs can lead to addiction and may cause side effects. Further studies of the mechanisms of chronic pain have opened the way for development of new treatment strategies, one of which is gene therapy. The key to gene therapy is selecting safe and highly efficient gene delivery systems that can deliver therapeutic genes to overexpress or suppress relevant targets in specific cell types. Here we review several promising viral vectors that could be applied in gene transfer for the treatment of chronic pain and further discuss the possible mechanisms of genes of interest that could be delivered with viral vectors for the treatment of chronic pain.

The Hepadnavirus core protein is a viral structural protein with an N-terminal self-assembling domain and a C-terminal protamine-like arginine-rich domain (ARD). The ARD contains four clusters of arginine residues involved in RNA binding, genome DNA synthesis, and nuclear localization. Characterization of the multi-functions of ARD has been impeded due to the insoluble nature of the core protein expressed in vitro. A GST (glutathione-S-transferase) and ARD fusion protein, GST–ARD, was expressed and purified in this study. Gel mobility shift assays using purified GST–ARD fusion proteins demonstrated that, similar to protamine, the ARD domain of the core protein bound to oligonucleotides without sequence preference. In vitro affinity chromatography binding assays showed further that the ARD bound to tested random plasmid DNA in a sequence-independent manner. The GST–ARD fusion protein-based approach can be employed further to study other biochemical properties of the core protein.

To determine whether actions on sodium channels contribute to ethanol's depressant effects on the autonomic nervous system, the acute effects of ethanol on Na + currents in primary cultured superior cervical ganglion were examined by whole-cell patch clamp recordings. Ethanol inhibited Na + currents concentration dependently, and decreased action potential firing. Ethanol (100 mM) did not affect activation curve, but resulted in a left shift of the inactivation curve and prolonged the recovery from inactivation.This finding indicates that the channels in the inactivated state are more susceptible to ethanol than those in the resting state. For the first time, this study demonstrates acute inhibitory effects of ethanol on sodium channel gating in sympathetic neurons.

BRCA1 is an important tumor suppressor gene associated with inherited breast and ovarian cancers. In this investigation, two novel BRCA1 splicing variants were cloned from breast cancer cell line ZR-75-30. These transcripts, named BRCA1-PI21-Delta2-21 and BRCA1-Delta2-14, lacked most exons of full length BRCA1 gene, but maintained the original reading frame. We also demonstrated the presence of BRCA1-PI21-Delta2-21 in several human cell lines. Expression of both variants fused with green fluorescent protein (GFP) showed that they targeted different subcellular compartments in the transfected cells. Viability of the cells expressing both fusion proteins decreased notably compared with the viability of cells expressing only GFP. Fluorescence activated cell sorting assay confirmed that the overexpression of two splicing variants resulted in cell apoptosis. Taken together, the different subcellular localization and cell effects of two BRCA1 splicing variants imply that they can have different biological functions in breast cancer cells. Elucidating the functions of BRCA1 splicing variants would help to understand the exact roles of the BRCA1 gene in tumor suppression.

In recent years, although several related genes had been cloned and characterized, the role of aldehyde dehydrogenase 1 (ALDH1), the newly cloned gene involved in artemisinin biosynthesis pathway, is still not clear. In this study, a 2,100-bp ALDH1 promoter region fused with GUS reporter gene was stably transferred into Arabidopsis thaliana. Histochemical staining showed the methyl jasmonate (MeJA) and wounding treatment induced the GUS gene expression specifically in the trichomes of transgenic A. thaliana, consistent with the results that the expression level of ALDH1 gene was increased in the A. annua under MeJA and wounding treatments. Two RAA motifs (AP2/ERF binding site) but no W box (WRKY binding site) motif were identified in the ALDH1 promoter by the analysis through PLACE and plantCARE. Through the dual luciferase reporter assay, we revealed that both AaORA and AaERF2, rather than AaWRKY1, could activate the expression of ALDH1 promoter. Our study shed light on the in-depth understanding of the role of ALDH1 in artemisinin biosynthesis.

Previous publications reported that the artemisinin level was increased in Artemisia annua following a night-frost period. However, the molecular mechanism was not clear. In this study, we found that exogenous jasmonate (JA) effectively enhanced the freezing tolerance of A. annua. The JA biosynthetic genes (LOX1, LOX2, allene oxide cyclase [AOC], and jasmonate resistant 1 [JAR1]) were induced by cold stress, leading to an increase in endogenous JA in cold-treated A. annua. Increased endogenous JA enhanced the expression of three JA-responsive transcription factors, ethylene response factor 1, ethylene response factor 2, and octadecanoid-responsive AP2/ERF, all of which were reported to transcriptionally activate the expression of artemisinin biosynthetic genes, such as amorpha-4,11-diene synthase (ADS), CYP71AV1, DBR2, and aldehyde dehydrogenase 1 (ALDH1). Furthermore, the expression levels of the four artemisinin biosynthetic genes were also significantly increased under cold stress. Consequently, the levels of artemisinin and related secondary metabolites, such as dihydroartemisinic acid, artemisinin B, and artemisinic acid, were increased in A. annua under cold stress. Our study points to a molecular mechanism in which the production of artemisinin is regulated by cold stress in A. annua.

Treatment with cisplatin, a chemotherapeutic agent commonly used in glioma patients, often results in chemoresistance. Increasing evidence has shown that microRNAs (miRNAs) are implicated in the drug resistance of gliomas. However, the function of miR‑873 in cisplatin resistance of gliomas remains unknown. In this study, we found that many miRNAs, including miR‑873, are differentially expressed in cisplatin-resistant glioma cells compared to wild-type glioma cells. Moreover, cisplatin reduced the expression of miR‑873 in a time-dependent manner. Overexpression of miR‑873 decreased the cell proliferation, migration and invasion while increased apoptosis of cisplatin-resistant glioma cells and sensitized the cells to cisplatin-induced cell growth arrest and apoptosis. Furthermore, miR‑873 was downregulated while Bcl-2 was upregulated in the tissues of twelve high-grade glioma patients compared to seven normal brain tissues, and the miR‑873 level was negatively correlated with the Bcl-2 protein level. A luciferase reporter assay further confirmed that Bcl-2 was a direct target of miR‑873, and miR‑873 decreased the level of the Bcl-2 protein in cisplatin-resistant glioma cells. Notably, re-expression of Bcl-2 attenuated the function of miR‑873 in cisplatin-resistant glioma cells and the sensitivity of the cells to cisplatin. Taken together, these data suggest that miR‑873 might be a potential marker for cisplatin resistance and a promising sensitizer in cisplatin treatment.