TRIM44 Promotes Rabies Virus Replication by Autophagy-Dependent Mechanism.

Tripartite motif (TRIM) proteins are a multifunctional E3 ubiquitin ligase family that participates in various cellular processes. Recent studies have shown that TRIM proteins play important roles in regulating host-virus interactions through specific pathways, but their involvement in response to rabies virus (RABV) infection remains poorly understood. Here, we identified that several TRIM proteins are upregulated in mouse neuroblastoma cells (NA) after infection with the rabies virus using RNA-seq sequencing. Among them, TRIM44 was found to regulate RABV replication. This is supported by the observations that downregulation of TRIM44 inhibits RABV replication, while overexpression of TRIM44 promotes RABV replication. Mechanistically, TRIM44-induced RABV replication is brought about by activating autophagy, as inhibition of autophagy with 3-MA attenuates TRIM44-induced RABV replication. Additionally, we found that inhibition of autophagy with rapamycin reverses the TRIM44-knockdown-induced decrease in LC3B expression and autophagosome formation as well as RABV replication. The results suggest that TRIM44 promotes RABV replication by an autophagy-dependent mechanism. Our work identifies TRIM44 as a key host factor for RABV replication, and targeting TRIM44 expression may represent an effective therapeutic strategy.

Evolution and distribution of rabies viruses from a panorama view.

Rabies kills more than 59,000 people annually, mainly in developing countries. Previous studies on the evolution and distribution of rabies viruses (RABVs) were scattered. Here, we explore the evolution and distribution of this deadly virus from a novel panorama view. Multiple bioinformatic software tools were employed to analyze the phylogenetic diversity, evolution, spatiotemporal, and distribution of RABVs. The analyses were based on 1,202 qualified full-length genomes of RABVs and numerous literatures. Of the 10 distinct phylogenetic clades of RABV that we identified, more frequent intra- and inter-clade recombination occurs in the sequences of Asian-SEA, Arctic, and Cosmopolitan clades isolated from China, while according to existing sequence information, RABV might originate from bats (posterior probability, PP = 0.75, PP = 0.60 inferred from N and L genes, separately) in North America (PP = 0.57, PP = 0.62 inferred from N and L genes, separately). Due to the difference in evolutionary rate of N (2.22 × 10-4 subs/site/year, 95% HPD 1.99-2.47 × 10-4 subs/site/year) and L genes (1.67 × 10-4 subs/site/year, 95% HPD 1.59-1.74 × 10-4 subs/site/year), the root age was 1,406.6 (95% HPD 1,291.2-1,518.2) and 1,122.7 (95% HPD 1,052.4-1,193.9) inferred from N and L genes, separately. Among other findings, Mephitidae plays an important role in the interspecific transmission and communication of RABV, which we found tends to spread to populations genetically proximate to the host. We also identified amino acids under positive selection in different genes of different clades as well as single nucleotide variation sites important for different lineages. IMPORTANCE Rabies virus is widely distributed all over the world, and wild animals are its largest potential reservoir. Our study offers a panorama view about evolution and distribution of rabies viruses and emphasizes the need to monitor the transmission dynamics of animal rabies.

TRIM25 Suppresses Rabies Virus Fixed HEP-Flury Strain Production by Activating RIG-1-Mediated Type I Interferons.

Rabies remains a great threat to public health worldwide. So far, the mechanism of rabies virus (RABV) infection is not fully understood, and there is no effective treatment for rabies. Identifying more host restriction factors of RABV will spur the development of novel therapeutic interventions against rabies. Accumulating studies suggest that tripartite motif-containing (TRIM) proteins have great effects on virus replication. TRIMs control the antiviral responses through either direct interaction with viral proteins or indirect regulation of innate immune signaling molecules in the host. The role of TRIM25 in rabies virus (RABV) infection is poorly understood. Using next-generation sequencing, we found that TRIM25 is upregulated during HEP-Flury infection. Knockdown of TRIM25 enhances HEP-Flury production, while overexpression of TRIM25 suppresses HEP-Flury replication. Knockdown of interferon α and interferon ß weakens the anti-RABV response induced by TRIM25 overexpression, and potentiates RABV production. Furthermore, we found that TRIM25 regulates type-I interferon response by targeting retinoic acid-inducible gene I (RIG-I) during HEP-Flury infection. Knockdown of RIG-I weakens the anti-HEP-Flury response induced by TRIM25 overexpression, indicating that TRIM25 regulates RABV production via the RIG-I-IFN axis. In addition, we observed that TRIM25 does not directly interact with HEP-Flury structural proteins, suggesting that TRIM25 regulates HEP-Flury production indirectly. Taken together, our work identifies TRIM25 as a new host factor involved in HEP-Flury infection, which may be a potential target for the development of antiviral drugs against RABV.

TRIM21 Promotes Rabies Virus Production by Degrading IRF7 through Ubiquitination.

Rabies, a highly fatal zoonotic disease, is a significant global public health threat. Currently, the pathogenic mechanism of rabies has not been fully elucidated, and no effective treatment for rabies is available. Increasing evidence shows that the tripartite-motif protein (TRIM) family of proteins participates in the host's regulation of viral replication. Studies have demonstrated the upregulated expression of tripartite-motif protein 21 (TRIM21) in the brain tissue of mice infected with the rabies virus. Related studies have shown that TRIM21 knockdown inhibits RABV replication, while overexpression of TRIM21 exerted the opposite effect. Knockdown of interferon-alpha and interferon-beta modulates the inhibition of RABV replication caused by TRIM21 knockdown and promotes the replication of the virus. Furthermore, our previous study revealed that TRIM21 regulates the secretion of type I interferon during RABV infection by targeting interferon regulatory factor 7 (IRF7). IRF7 knockdown reduced the inhibition of RABV replication caused by the knockdown of TRIM21 and promoted viral replication. TRIM21 regulates RABV replication via the IRF7-IFN axis. Our study identified TRIM21 as a novel host factor required by RABV for replication. Thus, TRIM21 is a potential target for rabies treatment or management.

Supercritical fluid coating of flavonoids on excipients enhances drug release and antioxidant activity.

Supercritical anti-solvent fluidized bed (SAS-FB) technology can be applied to reduce particle size, prevent particle aggregation, and improve the dissolution and bioavailability of poorly soluble drugs. In this work, drug-loaded microparticles of three similar structures, the flavonoids luteolin (LUT), naringenin (NGR), and dihydromyricetin (DMY) were prepared using SAS-FB technology, to explore its effect on the coating of flavonoid particles. Operating temperature, pressure, carrier, solvent, and concentration of drug solution were investigated for their effects on the yield and dissolution of flavonoid particles. The results showed that temperature, pressure, carrier, and drug solution concentration have a large effect on yield. Within the study range, low supercritical CO2 density at higher temperature and lower pressure, a larger surface area carrier, and moderate drug solution concentration led to a higher yield. The effect of the solvent on the yield of flavonoids is a result of multiple factors. Scanning electron microscopy (SEM) images showed that the drug-loaded particles prepared from different carriers and solvents have different precipitations pattern on the carrier surface, and their particle sizes were smaller than unprocessed particles and those prepared by the SAS process. Fluorescence microscopy (FM) results showed that the flavonoids were uniformly coated on the carrier. X-ray powder diffraction (XRPD) results showed that the crystalline morphology of SAS-FB particles remained unchanged after the SAS-FB process, although the diffraction peak intensity decreased. The cumulative dissolution of SAS-FB particles was more than four times faster in the first 5 min than that of the unprocessed flavonoids. The antioxidant activity of SAS-FB processed LUT, NGR and DMY was 1.89-3.78 times, 4.92-10.68 times and 0.99-2.57 times higher than that of the untreated flavonoids, respectively. The approach provides a reference for the application of SAS-FB technology in flavonoids.

Characterization of excipients to improve pharmaceutical properties of sirolimus in the supercritical anti-solvent fluidized process.

Enhanced drug release and bioavailability of poorly soluble active pharmaceutical ingredient (API) can be achieved via a fluidized bed coating integrated with supercritical anti-solvent (SAS-FB) - a process of precipitating drug particles onto carrier granules. However, in the absence of excipients, SAS-FB often results in crystalline of the API on the surface of carriers, limiting the improvement of pharmaceutical properties. Co-processing with excipients is considered an effective approach to improve drug release in the SAS-FB process. Our study used sirolimus, an immune suppressive agent, as the model API to characterize excipients for their effect on pharmaceutical properties in the SAS-FB process. We show that co-precipitation of excipients and sirolumus impacts on carrier specific surface area and drug yield. Among the tested excipients, formulation containing polyvinylpyrrolidone K30 achieved the highest drug yield. Importantly, compared with Rapamune® tablet, our optimized formulation displayed a superior in vivo oral bioavailability by 3.05-fold in Sprague-Dawley rats and 3.99-fold in beagle dogs. A series of characterization of the processed API was performed to understand the mechanism by which excipients contributed to drug dissolution properties. Our study provides a useful guidance for the use of excipients in the SAS-FB technology to improve pharmaceutical properties of sirolimus and other poorly soluble drugs.

Rabies Virus-Induced Autophagy Is Dependent on Viral Load in BV2 Cells.

An increasing number of studies are showing that autophagy plays a vital role in viral replication and escape. Rabies virus (RABV), a typical neurotropic virus, has been proven to induce autophagy in neurons. However, there are no reports indicating that RABV can cause autophagy in other cells of the central nervous system. Thus, we aimed to explore the relationship between autophagy and RABV infection in BV2 cells in this study. Results of viral growth curves showed that the titers of microglial BV2 cells infected with RABV peaked at 12 hours post-infection (hpi) and then decreased continuously over time. However, it was found that the viral genome RNA and structural proteins can express normally in BV2 cells. In addition, Western blotting indicated that RABV infection increased LC3-II and p62 expression in BV2 cells. LC3 punctate increased with RABV infection in BV2 cells after the transfection of fluorescent protein-tagged LC3 plasmids. Moreover, autophagy cargo protein further accumulated with RABV infection in Bafilomycin A1-treated cells. Subsequently, RABV infection inhibited the fusion of autophagosomes with lysosomes by using a tandem fluorescent marker. Furthermore, a higher multiplicity of infection induced stronger autophagy. Thus, RABV can induce autophagy in BV2 cells, and the autophagy is positively associated with the viral load.

Artesunate and Dihydroartemisinin Inhibit Rabies Virus Replication.

Rabies is caused by infection of rabies virus (RABV) and remains a serious threat to the global public health. Except for the requirement for cold chain and high cost of human rabies immune globulin, no small molecule drugs are currently available for clinical treatment of rabies. So, it is of great importance to identify novel compounds that can effectively inhibit RABV infection. Artesunate (ART) and dihydroartemisinin (DHA), two derivatives of artemisinin, are widely used for treatment of malaria in adults and children, showing high safety. In this study, we found that both ART and DHA were able to inhibit RABV replication in host cells at a low concentration (0.1 µmol/L). The antiviral effects of ART and DHA were independent of viral strains and cell lines. Pre-treatment with ART or DHA for 2 h in vitro did not affect the viral replication in host cells, implying that ART and DHA neither reduced the viability of RABV directly nor inhibited the binding and entrance of the virus to host cells. Further studies revealed that ART and DHA inhibited RABV genomic RNA synthesis and viral gene transcription. Treatment with ART or DHA (5 mg/kg) by intramuscular injection improved, to some extent, the survival rate of RABV-challenged mice. Combination treatment with derivatives of artemisinin and mannitol significantly improved the survival rate of RABV-challenged mice. The results suggest that ART and DHA have a great potential to be explored as new anti-rabies agents for treatment of rabies.

Optimization of the supercritical fluidized bed process for sirolimus coating and drug release.

Directly coating an active pharmaceutical ingredient (API) onto excipient granules has been a common approach to prepare solid dosage forms. The combination of supercritical anti-solvent (SAS) and fluidized bed (FB) coating technology (SAS-FB) has the advantages of preventing nanoparticles aggregation, oxidation and light exposure. However individual operating parameters and factors which contribute to the overall coating efficiency remain to be defined. Sirolimus is an immunosuppressive agent for preventing the rejection of organ transplants and this drug is sensitive to light exposure and high temperature. Our study used sirolimus as the model API to evaluate parameters including temperature, pressure, drug concentration, mass, material and diameter of carrier, CO2 flow rate and solvent in the SAS-FB process. By optimizing these parameters, we achieved a 3.5-fold enhancement of the coating efficiency over the standard condition. A series of characterizations of the sirolimus coated particles were performed from which scanning electron microscopy and Raman mapping confirmed that the sirolimus particles were uniformly coated on carriers as cuboid particles; X-ray powder diffraction showed that processed sirolimus is crystalline but has lower crystallinity than the API, and fourier transform infrared spectroscopy and differential scanning calorimeter confirmed that there is no chemical interaction between sirolimus and carriers after SAS-FB processing. Finally compared to sirolimus alone, sirolimus coated particles displayed a faster dissolution and higher bioavailability. Collectively, our optimized operation parameters for SAS-FB coating technique provide a useful guidance for achieving higher efficiency of drug coating and faster release rate of sirolimus pellets, which has the potential to apply to other APIs.

Artesunate enhances the immune response of rabies vaccine as an adjuvant.

Rabies is an ancient zoonosis that continues to be an important health problem worldwide. Vaccination with rabies vaccine is the most important strategy to prevent rabies. Adjuvants contribute to the immune response of viral vaccine. The aim of this study was to investigate whether artemisinin derivatives artesunate and dihydroartemisinin could enhance the immunogenicity of inactivated rabies virus in mice. Administration of artesunate or dihydroartemisinin by intramuscular injection at a dose of 5 mg/kg did not cause body weight loss and unusual symptoms in mice. Mice were immunized with inactivated CVS-11 or inactivated rHEP-dG together with either artesunate or dihydroartemisinin through intramuscular injection. Blood samples were collected to investigate the virus-neutralizing antibody (VNA) titers, and challenge assays were then conducted. The results showed that the rabies VNA titers in mice co-treated with artesunate rather than dihydroartemisinin were significantly higher than in the control animals treated with the phosphate buffered saline (PBS). In addition, mice co-treated with artesunate survived from lethal rabies virus challenge compared with those treated with PBS. In contrast, co-treatment with dihydroartemisinin did not improve the survival rate of the challenged mice. The findings indicate that artesunate could be used as a new candidate adjuvant for rabies vaccination.

Fushiming Capsule Attenuates Diabetic Rat Retina Damage via Antioxidation and Anti-Inflammation.

AIMS: Diabetic retinopathy (DR) remains one of the leading causes of acquired blindness. Fushiming capsule (FSM), a compound traditional Chinese medicine, is clinically used for DR treatment in China. The present study was to investigate the effect of FSM on retinal alterations, inflammatory response, and oxidative stress triggered by diabetes. MAIN METHODS: Diabetic rat model was induced by 6-week high-fat and high-sugar diet combined with 35 mg/kg streptozotocin (STZ). 30 days after successful establishment of diabetic rat model, full field electroretinography (ffERG) and optical coherence tomography (OCT) were performed to detect retinal pathological alterations. Then, FSM was administered to diabetic rats at different dosages for 42-day treatment and diabetic rats treated with Calcium dobesilate (CaD) capsule served as the positive group. Retinal function and structure were observed, and retinal vascular endothelial growth factor-α (VEGF-α), glial fibrillary acidic (GFAP), and vascular cell adhesion protein-1 (VCAM-1) expressions were measured both on mRNA and protein levels, and a series of blood metabolic indicators were also assessed. KEY FINDINGS: In DR rats, FSM (1.0 g/kg and 0.5 g/kg) treatment significantly restored retinal function (a higher amplitude of b-wave in dark-adaptation 3.0 and OPs2 wave) and prevented the decrease of retinal thickness including inner nuclear layer (INL), outer nuclear layer (ONL), and entire retina. Additionally, FSM dramatically decreased VEGF-α, GFAP, and VCAM-1 expressions in retinal tissues. Moreover, FSM notably improved serum antioxidative enzymes glutathione peroxidase, superoxide dismutase, and catalase activities, whereas it reduced serum advanced glycation end products, methane dicarboxylic aldehyde, nitric oxide, and total cholesterol and triglycerides levels. SIGNIFICANCE: FSM could ameliorate diabetic rat retina damage possibly via inhibiting inflammation and improving antioxidation.

Over-expressed LOC101927196 suppressed oxidative stress levels and neuron cell proliferation in a rat model of autism through disrupting the Wnt signaling pathway by targeting FZD3.

Accumulating evidence indicates that long non-coding RNAs (lncRNAs) play an important role in autism. Herein, we delineated the functions of LOC101927196 and its potential mitigation effect on a rat model of autism. We retrieved various bioinformatics databases and websites to screen differentially expressed lncRNAs associated with autism. Next, a rat model of autism was established with the neuron cells extracted for transfection of different plasmids. The regulatory effect of LOC101927196 on neuron cell proliferation, apoptosis as well as oxidative stress was also investigated. Firstly, microarray dataset GSE18123 revealed that LOC101927196 was poorly expressed in a rat model of autism. Poor development and growth and oxidative stress disorder were also observed in a rat model of autism. In addition, LOC101927196 targeting FZD3 played a vital role in a rat model of autism through the Wnt signaling pathway. Furthermore, we further demonstrated that over-expressed LOC101927196 blocked neuron cell proliferation and reduced oxidative stress levels, while promoting apoptosis by suppressing the activation of the Wnt signaling pathway. Our findings illustrate that up-regulated LOC101927196 attenuated oxidative stress disorder in a rat model of autism through suppressing the activation of Wnt signaling pathway by targeting FZD3.

Carbon pools in China's terrestrial ecosystems: New estimates based on an intensive field survey.

China's terrestrial ecosystems have functioned as important carbon sinks. However, previous estimates of carbon budgets have included large uncertainties owing to the limitations of sample size, multiple data sources, and inconsistent methodologies. In this study, we conducted an intensive field campaign involving 14,371 field plots to investigate all sectors of carbon stocks in China's forests, shrublands, grasslands, and croplands to better estimate the regional and national carbon pools and to explore the biogeographical patterns and potential drivers of these pools. The total carbon pool in these four ecosystems was 79.24 ± 2.42 Pg C, of which 82.9% was stored in soil (to a depth of 1 m), 16.5% in biomass, and 0.60% in litter. Forests, shrublands, grasslands, and croplands contained 30.83 ± 1.57 Pg C, 6.69 ± 0.32 Pg C, 25.40 ± 1.49 Pg C, and 16.32 ± 0.41 Pg C, respectively. When all terrestrial ecosystems are taken into account, the country's total carbon pool is 89.27 ± 1.05 Pg C. The carbon density of the forests, shrublands, and grasslands exhibited a strong correlation with climate: it decreased with increasing temperature but increased with increasing precipitation. Our analysis also suggests a significant sequestration potential of 1.9-3.4 Pg C in forest biomass in the next 10-20 years assuming no removals, mainly because of forest growth. Our results update the estimates of carbon pools in China's terrestrial ecosystems based on direct field measurements, and these estimates are essential to the validation and parameterization of carbon models in China and globally.

A network meta-analysis of short-term efficacy of different single-drug targeted therapies in the treatment of renal cell carcinoma.

The network meta-analysis was conducted to compare the short-term efficacy of different single-drug targeted therapies in the treatment of renal cell carcinoma (RCC). We initially searched databases for randomized controlled trials (RCTs) on different single-drug targeted therapies in treating RCC. The meta-analysis combined the direct and indirect evidence to calculate the pooled odds ratios (OR) and draw surface under the cumulative ranking curves (SUCRA). A total of 14 eligible RCTs were ultimately selected. The partial response (PR) of Cabozantinib in the treatment of RCC was better than Sunitinib (OR = 2.7, 95%CI = 1.0-7.8), Everolimus (OR = 8.1, 95%CI = 3.1-25.0), and Temsirolimus (OR = 4.8, 95%CI = 1.0-31.0); the overall response rate (ORR) of Cabozantinib was better than Sorafenib, Sunitinib, Everolimus, and Temsirolimus (OR = 5.5, 95%CI = 1.1-27.0; OR = 2.6, 95%CI = 1.1-6.6; OR = 8.3, 95%CI = 3.5-20.0; OR = 5.7, 95%CI = 1.3-28.0 respectively). In addition, as for complete response (CR), PR, stable disease (SD), progressive disease (PD), ORR, and disease control rate (DCR), Cabozantinib had the best short-term efficacy among nine single-drug targeted therapies in the treatment of RCC (CR: 50.3%; PR: 93.6%; SD: 75.1%; PD: 68.0%; ORR: 95.5%; DCR: 73.2%); while Everolimus had the worst short-term efficacy (CR: 33.6%; PR: 22.3%; SD: 78.0%; PD: 35.9%; ORR: 22.9%; DCR: 19.9%). Our network meta-analysis indicated that Cabozantinib might have better short-term efficacy than other regimens in the treatment of RCC, while Everolimus might have poor short-term efficacy.

[Effects of Montessori education on the intellectual development in children aged 2 to 4 years].

OBJECTIVE: To compare the effects of Montessori education and traditional education on the intellectual development in children aged 2 to 4 years. METHODS: Children aged between 2 to 3 years who were enrolled in a kindergarten in September 2006 were randomly assigned to the Montessori education and the traditional education groups. In addition to receiving the traditional education, the Montessori education group participated in the two-hour Montessori pedagogical activities every day. The intellectual development was evaluated by the Neuropsychological Development Examination Format for Children Aged 0~6 years published by Capital Pediatrics Research Institute at enrollment and one year after the trial. RESULTS: There were no significant differences in the intelligence growth level between the Montessori education and the traditional education groups at enrollment. After one year, the levels of fine movements, adaptation ability, language, and social behavior developments in the Montessori education group were significantly higher than those in the traditional education group (p<0.05 or 0.01). The intelligence increasing scores of the large motor ability, fine movements, language, social behavior and development quotient in the Montessori education group were also higher than those in the traditional education group (p<0.05 or 0.01). CONCLUSIONS: Montessori education can promote the development of large motor ability, fine movements, language, and social behavior in children.