Long-term administration of salvianolic acid A promotes endogenous neurogenesis in ischemic stroke rats through activating Wnt3a/GSK3ß/ß-catenin signaling pathway.

Stroke is the major cause of death and disability worldwide. Most stroke patients who survive in the acute phase of ischemia display various extents of neurological deficits. In order to improve the prognosis of ischemic stroke, promoting endogenous neurogenesis has attracted great attention. Salvianolic acid A (SAA) has shown neuroprotective effects against ischemic diseases. In the present study, we investigated the neurogenesis effects of SAA in ischemic stroke rats, and explored the underlying mechanisms. An autologous thrombus stroke model was established by electrocoagulation. The rats were administered SAA (10 mg/kg, ig) or a positive drug edaravone (5 mg/kg, iv) once a day for 14 days. We showed that SAA administration significantly decreased infarction volume and vascular embolism, and ameliorated pathological injury in the hippocampus and striatum as well as the neurological deficits as compared with the model rats. Furthermore, we found that SAA administration significantly promoted neural stem/progenitor cells (NSPCs) proliferation, migration and differentiation into neurons, enhanced axonal regeneration and diminished neuronal apoptosis around the ipsilateral subventricular zone (SVZ), resulting in restored neural density and reconstructed neural circuits in the ischemic striatum. Moreover, we revealed that SAA-induced neurogenesis was associated to activating Wnt3a/GSK3ß/ß-catenin signaling pathway and downstream target genes in the hippocampus and striatum. Edaravone exerted equivalent inhibition on neuronal apoptosis in the SVZ, as SAA, but edaravone-induced neurogenesis was weaker than that of SAA. Taken together, our results demonstrate that long-term administration of SAA improves neurological function through enhancing endogenous neurogenesis and inhibiting neuronal apoptosis in ischemic stroke rats via activating Wnt3a/GSK3ß/ß-catenin signaling pathway. SAA may be a potential therapeutic drug to promote neurogenesis after stroke.

Astragaloside IV reverses simvastatin-induced skeletal muscle injury by activating the AMPK-PGC-1α signalling pathway.

In this study, we investigated the effect of astragaloside IV on skeletal muscle energy metabolism disorder caused by statins and explored the possible mechanisms. High-fat diet-fed apolipoprotein E knockout (ApoE-/- ) mice performed aerobic exercise and were administered simvastatin, simvastatin + trimetazidine, or simvastatin + astragaloside IV by gavage. At the end of treatment, exercise performance was assessed by the hanging grid test, forelimb grip test, and running tolerance test. Moreover, plasma lipid and creatine kinase concentrations were measured. After sacrifice, the gastrocnemius muscle was used to assess muscle morphology, and energy metabolism was evaluated by determining the concentration of lactic acid and the storage capacity of adenosine triphosphate and glycogen. Mitochondrial function was assessed by measuring mitochondrial complex III and citrate synthase activity and membrane potential. In addition, oxidative stress was assessed by determining the level of hydrogen peroxide. Finally, using western blotting and reverse transcription polymerase chain reaction, we explored the mechanism of astragaloside IV in alleviating simvastatin-induced muscle injury. Our results demonstrated that astragaloside IV reversed simvastatin-induced muscle injury without affecting the lipid-lowering effect of simvastatin. Moreover, astragaloside IV promoted the phosphorylation of AMPK and activated PGC-1α, which upregulated the expression of NRF1 to enhance energy metabolism and inhibit skeletal muscle cell apoptosis.

Resolution Measurement from a Single Reconstructed Cryo-EM Density Map with Multiscale Spectral Analysis.

As a relatively new technology to solve the three-dimensional (3D) structure of a protein or protein complex, single-particle reconstruction (SPR) of cryogenic electron microscopy (cryo-EM) images shows much superiority and is in a rapidly developing stage. Resolution measurement in SPR, which evaluates the quality of a reconstructed 3D density map, plays a critical role in promoting methodology development of SPR and structural biology. Because there is no benchmark map in the generation of a new structure, how to realize the resolution estimation of a new map is still an open problem. Existing approaches try to generate a hypothetical benchmark map by reconstructing two 3D models from two halves of the original 2D images for cross-reference, which may result in a premature estimation with a half-data model. In this paper, we report a new self-reference-based resolution estimation protocol, called SRes, that requires only a single reconstructed 3D map. The core idea of SRes is to perform a multiscale spectral analysis (MSSA) on the map through multiple size-variable masks segmenting the map. The MSSA-derived multiscale spectral signal-to-noise ratios (mSSNRs) reveal that their corresponding estimated resolutions will show a cliff jump phenomenon, indicating a significant change in the SSNR properties. The critical point on the cliff borderline is demonstrated to be the right estimator for the resolution of the map.

Production and characterization of a fusion peptide derived from the rabies virus glycoprotein (RVG29).

Gene therapy targeting the brain holds great promise in curing nervous system degenerative diseases in clinical applications. With this in mind, in a previous study a 29 amino-acid peptide derived from the rabies virus glycoprotein (RVG29) with a nonamer stretch of arginine residues (RVG29-9R) at its carboxy-terminus was exploited as a ligand for brain-targeting gene delivery. Importantly, the report demonstrated that the RVG29-9R vector was able to cross the blood-brain barrier. RVG29-9R is currently synthesized by commercial companies with high associated costs. In this study, in order to reduce the costs of producing RVG29-9R, we have expressed and purified 6mg of a recombinant peptide (RVG29-9R-6His) from 0.4g of cultured Escherichia coli. We assessed the physiochemical properties of RVG29-9R-6His, its cytotoxicity, and the in vitro transfection efficiency in Neuro 2a cells (which express the acetylcholine receptor). Our results reveal that the RVG29-9R-6His peptide recognized Neuro 2a cells in a dose-dependent manner and it was also able to bind plasmid DNA and deliver it into the Neuro 2a cells effectively. Therefore, our study has demonstrated that the recombinant RVG29-9R-6His peptide retains the functions of RVG29-9R and so may provide an economically viable and alternative production method for the manufacture of RVG29-9R.

Biased gene expression reveals the contribution of subgenome to altitude adaptation in allopolyploid Isoetes sinensis.

Allopolyploids are believed to inherit the genetic characteristics of its progenitors and exhibit stronger adaptability and vigor. The allotetraploid Isoetes sinensis was formed by the natural hybridization and polyploidization of two diploid progenitors, Isoetes taiwanensis and Isoetes yunguiensis, and was believed to have the potential to adapt to plateau environments. To explore the expression pattern of homoeologous genes and their contributions to altitude adaptation, we transplanted natural allotetraploid I. sinensis (TnTnYnYn) along the altitude gradient for a long-term, and harvested them in summer and winter, respectively. One year after transplanting, it still lived well, even in the extreme environment of the Qinghai-Tibet Plateau. Then, we performed high-throughput RNA sequencing to measure their gene expression level. A total of 7801 homoeologous genes were expressed, among which 5786 were identified as shared expression in different altitudes and seasons. We further found that altitude variations could change the subgenome bias trend of I. sinensis, but season could not. Moreover, the functions of uniquely expressed genes indicated that temperature might be an important restrictive factor during the adaptation process. Through the analysis of DEGs and uniquely expressed genes, we found that Y subgenome provided more contributions to high altitude adaptation than T subgenome. These adaptive traits to high altitude may be inherited from its plateau progenitor I. yunguiensis. Through weighted gene co-expression network analysis, pentatricopeptide repeats gene family and glycerophospholipid metabolism pathway were considered to play important roles in high-altitude adaptation. Totally, this study will enrich our understanding of allopolyploid in environmental adaptation.

Canine parvovirus VP2 protein expressed in silkworm pupae self-assembles into virus-like particles with high immunogenicity.

The VP2 structural protein of parvovirus can produce virus-like particles (VLPs) by a self-assembly process in vitro, making VLPs attractive vaccine candidates. In this study, the VP2 protein of canine parvovirus (CPV) was expressed using a baculovirus expression system and assembled into parvovirus-like particles in insect cells and pupae. Electron micrographs of VLPs showed that they were very similar in size and morphology when compared to the wild-type parvovirus. The immunogenicity of the VLPs was investigated in mice and dogs. Mice immunized intramuscularly with purified VLPs, in the absence of an adjuvant, elicited CD4(+) and CD8(+) T cell responses and were able to elicit a neutralizing antibody response against CPV, while the oral administration of raw homogenates containing VLPs to the dogs resulted in a systemic immune response and long-lasting immunity. These results demonstrate that the CPV-VLPs stimulate both cellular and humoral immune responses, and so CPV-VLPs may be a promising candidate vaccine for the prevention of CPV-associated disease.

Adeno-associated viruses serotype 2-mediated RNA interference efficiently inhibits rabies virus replication in vitro and in vivo.

To investigate the potential of adeno-associated viruses serotype 2 (AAV2)-mediated RNA interference (RNAi) as an antiviral agent against rabies, recombinant AAV2 vectors expressing siRNA targeting the nucleoprotein (N) gene of rabies virus (RABV) (rAAV-N796) were constructed and evaluated. When NA cells pretreated with rAAV-N796 were challenged with RABV, there was a 37.8 ± 3.4% to 55.1 ± 5.3% reduction in RABV virus titer. When cells pre-challenged with RABV were treated with rAAV-N796, there was a 4.4 ± 1.4 to 28.8 ± 3.2% reduction in RABV virus titer. Relative quantification of RABV transcripts using real-time PCR and Western blot revealed that the knockdown of RABV-N gene transcripts was based on the rAAV-N796 inoculation titer. When any NA cells were treated with rAAV-N796 before or after challenged with RABV, significant reduction in virus titer was observed in both administrations. Mice treated intracerebrally with rAAV-N796 exhibited 50 ± 5.3 and 62.5 ± 4.7% protection when challenged intracerebrally or intramuscally, respectively, with lethal RABV. When mice treated intramuscularly with rAAV-N796 were challenged intramuscularly with lethal RABV, they exhibited 37.5 ± 3.7% protection. When mice were intracerebrally and intramuscularly with rAAV-N796 24 hr after exposure to RABV infection, they exhibited 25 ± 4.1% protection The N gene mRNA levels in the brains of challenged mice with three different administrations were reduced (55, 68, 32 and 25%, respectively). These results indicated that AAV2 vector-mediated siRNA delivery in vitro in NA cells inhibited RABV multiplication, inhibited RABV multiplication in vivo in the mice brain and imparted partial protection against lethal rabies. So, it may have a potential to be used as an alternative antiviral approach against rabies.

Small interfering RNAs targeting the rabies virus nucleoprotein gene.

Rabies virus (RABV) infection continues to be a global threat to human and animal health, yet no curative therapy has been developed. RNA interference (RNAi) therapy, which silences expression of specific target genes, represents a promising approach for treating viral infections in mammalian hosts. We designed six small interfering (si)RNAs (N473, N580, N783, N796, N799 and N1227) that target the conserved region of the RABV challenge virus standard (CVS)-11 strain nucleoprotein (N) gene. Using a plasmid-based transient expression model, we demonstrated that N796, N580 and N799 were capable of significantly inhibiting viral replication in vitro and in vivo. These three siRNAs effectively suppressed RABV expression in infected baby hamster kidney-21 (BHK-21) cells, as evidenced by direct immunofluorescence assay, viral titer measurements, real-time PCR, and Western blotting. In addition, liposome-mediated siRNA expression plasmid delivery to RABV-infected mice significantly increased survival, compared to a non-liposome-mediated delivery method. Collectively, our results showed that the three siRNAs, N796, N580 and N799, targeting the N gene could potently inhibit RABV CVS-11 reproduction. These siRNAs have the potential to be developed into new and effective prophylactic anti-RABV drugs.

Isolation of ssDNA aptamers that inhibit rabies virus.

Aptamers, functional nucleic acids, capable of binding a variety of molecular targets with high affinity and specificity, have emerged as promising therapeutic agents. In this study, the cell surface-systematic evolution of ligands by exponential enrichment (Cell-SELEX) strategy was used to generate DNA aptamers which targeted to the intact rabies virus-infected live cells. Through 35 iterative rounds of selection, five high-affinity single-stranded DNA (ssDNA) aptamers were generated by cell-SELEX. Virus titer assay and real-time quantitative reverse transcription PCR (qRT-PCR) assay revealed that all five aptamers could inhibit replication of rabies virus (RABV) in cultured baby hamster kidney (BHK)-21 cells; and T14 and F34 aptamers were most effective. The qRT-PCR also showed a dose-dependent inhibitory effect in BHK-21 cells. Collectively, these data show the feasibility of generating functionally effective aptamers against rabies virus-infected cells by the Cell-SELEX iterative procedure. These aptamers may prove clinically useful as therapeutic molecules with specific antiviral potential against RABV infections.