敲除trim47基因斑马鱼脑和脾的比较转录组学分析

用CRISPR/Cas9技术敲除斑马鱼(Danio Rerio)的trim47, 收集TRIM47–/–(trim47基因敲除)和WT(野生型)斑马鱼的大脑和脾脏进行RNA-seq分析, 以鉴定差异表达基因(DEGs)。总共确定了271个DEGs, 经过简要分析, 将这些DEGs注释为KEGG途径和GO富集分析, 与WT组相比, TRIM47-/-组的脑中DEGs集中在细胞黏附和谷氨酸能突触信号传导途径中。在脾脏中, 与野生型组相比, TRIM47-/-组的DEGs在补体和凝血级联信号通路中发生了变化。使用qRT-PCR验证了脑和脾中与补体途径相关的基因, 与转录组数据一致。这些结果表明, Trim47在脾脏和大脑中起着重要的生物学作用, 尤其是通过补体途径参与先天免疫功能。体内感染实验表明, trim47基因敲除可提高斑马鱼中鲤春病毒血症病毒(SVCV)的感染率。总之, 这些发现为TRIM成员在先天免疫中的功能提供了新线索。     

Visualizing the microtubule-associated protein tau in the nucleus

Although tau is mainly known as an axonal microtubule-associated protein,many studies indicate that it is not restricted to this subcellular compartment.Assessing tau’s subcellular distribution,however,is not trivial as is evident from transgenic mouse studies.When human tau is over-expressed,it can be immunohistochemically localized to axons and the somatodendritic domain,modeling what is found in neurodegenerative diseases such as Alzheimer’s disease.Yet,in wild-type mice,despite its abundance,tau is difficult to visualize even in the axon.It is even more challenging to detect this protein in the nucleus,where tau has been proposed to protect DNA from damage.To establish a framework for future studies into tau’s nuclear functions,we compared several methods to visualize endogenous nuclear tau in cell lines and mouse brain.While depending on the fixation and permeabilization protocol,we were able to detect nuclear tau in SH-SY5Y human neuroblastoma cells,we failed to do so in N2a murine neuroblastoma cells.As a second method we used subcellular fractionation of mouse tissue and found that in the nucleus tau is mainly present in a hypophosphorylated form.When either full-length or truncated human tau was expressed,both accumulated in the cytoplasm,but were also found in the nuclear fraction.Because subcellular fractionation methods have their limitations,we finally isolated nuclei to probe for nuclear tau and found that the nuclei were free of cytoplasmic contamination.Together our analysis identifies several protocols for detecting tau in the nucleus where it is found in a less phosphorylated form.     

Global signatures of protein binding on structured RNAs in Saccharomyces cerevisiae

Protein binding is essential to the transport,decay and regulation of almost all RNA molecules.However,the structural preference of protein binding on RNAs and their cellular functions and dynamics upon changing environmental conditions are poorly understood.Here,we integrated various high-throughput data and introduced a computational framework to describe the global interactions between RNA binding proteins(RBPs)and structured RNAs in yeast at single-nucleotide resolution.We found that on average,in terms of percent total lengths,~15%of mRNA untranslated regions(UTRs),~37%of canonical non-coding RNAs(ncRNAs)and~11%of long ncRNAs(lncRNAs)are bound by proteins.The RBP binding sites,in general,tend to occur at single-stranded loops,with evolutionarily conserved signatures,and often facilitate a specific RNA structure conformation in vivo.We found that four nucleotide modifications of tRNA are significantly associated with RBP binding.We also identified various structural motifs bound by RBPs in the UTRs of mRNAs,associated with localization,degradation and stress responses.Moreover,we identified>200 novel lncRNAs bound by RBPs,and about half of them contain conserved secondary structures.We present the first ensemble pattern of RBP binding sites in the structured non-coding regions of a eukaryotic genome,emphasizing their structural context and cellular functions.     

The herbal compound geniposide rescues formaldehyde-induced apoptosis in N2a neuroblastoma cells

The herbal medicine Tong Luo Jiu Nao(TLJN)contains geniposide(GP)and ginsenoside Rg1 at a molar ratio of 10:1.Rg1 is the major component of another herbal medicine,panax notoginseng saponin(PNS).TLJN has been shown to strengthen brain function in humans,and in animals it improves learning and memory.We have previously shown that TLJN reduces amyloidogenic processing in Alzheimer’s disease(AD)mouse models.Together this suggests TLJN may be a potential treatment for patients with dementia.Because chronic damage of the central nervous system by formaldehyde(FA)has been presented as a risk factor for age-associated cognitive dysfunction,in the present study we investigated the protective effect of both TLJN and GP in neuron-like cells exposed to FA.FA-exposed murine N2a neuroblastoma cells were incubated with TLJN,its main ingredient GP,as well as PNS,to measure cell viability and morphology,the rate of apoptosis and expression of genes encoding Akt,FOXO3,Bcl2 and p53.The CCK-8 assay,cytoskeletal staining and flow cytometry were used to test cell viability,morphology and apoptosis,respectively.Fluorescent quantitative real-time PCR(qRT-PCR)was used to monitor changes in gene expression,and HPLC to determine the rate of FA clearance.Treatment of N2a cells with 0.09 mmol L?1 FA for 24 h significantly reduced cell viability,changed cell morphology and promoted apoptosis.Both TLJN and GP conferred neuroprotection to FA-treated N2a cells,whereas PNS,which had to be used at lower concentrations because of its toxicity,did not.Our data demonstrate that TLJN can rescue neuronal damage caused by FA and that its main ingredient,GP,has a major role in this efficacy.This presents purified GP as a drug or lead compound for the treatment of AD.     

Temporal expression of c-kit in spermatogenesis of two grasshopper species

Two species of grasshoppers, Calliptamus abbreviatus （Ikonn.） and Shirakiacris shirakii （I. Bol.）, were collected randomly in the Siping area of Jilin Province, China. By using immunohistochemical methods and statistical analysis, we observed and compared the temporal expression of c-kit protein in four representative stages of spermatogenesis of the two grasshoppers, namely： spermatogonia; primary spermatocyte; secondary spermatocyte; and mature sperm. Results showed that there was c-kit positive temporal expression at each stage of spermatogenesis, but there were different positive expression levels： （i） weak positive expression of c-kit protein appeared in spermatogonia and the positive granules were thinner; （ii） strong positive expression of c-kit protein existed in primary spermatocyte and positive granules became biggest among all developmental stages; （iii） c-kit positive expression stayed stronger in secondary spermatocyte while positive granules became thinner; （iv） there was a strong positive expression of c-kit and thinner positive granules in mature sperm, which distributed on head and tail; （v） the biggest c-kit positive granules had been found massing at the end of spermary; and （vi） significant differences of c-kit positive expression existed in spermatogenesis between two species of grasshoppers. The results indicated that c-kit protein may play a crucial role in spermatogenesis and even retain the physiological action of sperms and fertilization in grasshoppers.     

Characteristics of novel dental composites containing 2,2-bis[4-(2-methoxy-3-methacryloyloxy propoxy) phenyl] propane as a base resin

Many dental restorative dental composites still utilize 2,2-bis[4-(2-hydroxy-3-methacryloyloxy propoxy) phenyl] propane (Bis-GMA) as base resin. The high viscosity of Bis-GMA necessitates dilution with dimethacrylate ethers of low viscosity such as triethylene glycol dimethacrylate (TEGDMA). However, increased amounts of the TEGDMA have adverse effects on properties such as water uptake and curing shrinkage. The viscosity of the base resin should be as low as possible to enable the preparation of dental composites with a minimum content of diluent. To overcome the disadvantage of Bis-GMA, i.e., its high viscosity caused by hydrogen bonding between hydroxyl groups, 2,2-bis[4-(2-methoxy-3-methacryloyloxy propoxy) phenyl propane (Bis-M-GMA) was prepared by substituting methoxy groups for hydroxyl groups in Bis-GMA. The viscosity of Bis-GMA was dramatically decreased from 574 (Pa.s) to 3.7 (Pa.s) by substitution of methoxy group. Consequently, the amount of TEGDMA included in the resin matrix could be minimized. Dental composites were prepared from Bis-M-GMA (or Bis-GMA) mixtures with TEGDMA filled with 75 wt % filler. Comparing the curing shrinkage of dental composite containing Bis-M-GMA with that prepared from Bis-GMA, the reduction in curing shrinkage was about 47%. Dental composites prepared from new resin matrixes also exhibited low water uptake and better properties in mechanical strength.     

Relationship of HLA-A, -Cw Polymorphisms with HIV/AIDS in Chinese Yi Ethnic Group of Sichuan Province1

The relationship of HLA-A, -Cw alleles on HIV infection and AIDS disease progression in the Chinese Yi ethnic group of Sichuan province were investigated. The genetic polymorphisms of HLA-A, -Cw alleles of 102 unrelated healthy Chinese Yi ethnic individuals, 68 HIV-1 infected and 21 HIV positive long-time survivors were typed by PCR-SSP assay. Statistic signifiance was determined by the χ^2 test with the SPSS software. No significant differences were observed between the HLA-A, -Cw alleles of the 68 HIV-1 infected and 102 non-infected Chinese Yi control individuals. Whereas the prevalence of A＊3601,Cw＊14（01-03）and Cw＊0304 was significantly higher in 21 long time survivors compared with 102 healthy controls with P values of 0.016, 0.016 and 0.000 by χ^2 or the Fisher exact test respectively. The result implies that A＊3601,Cw＊14（01-03） and Cw＊0304 may be associated with slow AIDS disease progression in the Chinese Yi ethnic group, further studies on this association may yield insight on the pathogenesis of HIV-1 infection.     

异种细胞融合诱导熟前染色体凝聚(PCC)

在某种细胞促融剂介导下,同种或异种细胞能相互融合形成单个的新细胞。观察分析这种融合细胞的结构和机能的变化,在细胞生物学、细胞遗传学和细胞免疫学等研究中,已逐渐得到重视和应用。我们在开展同种和异种细胞融合选择杂种的同时,参考有关文献,进行了细胞融合诱导熟前染色体凝聚(premature     

Phylogenetic relationship of 16 Oedipodidae species （Insecta： Orthoptera） based on the 16S rRNA gene sequences

The sequences of the mitochondrial 16S rRNA gene of 16 Oedipodidae species were amplified and sequenced. All sequences were aligned and analyzed and the phylogenetic relationships were inferred. The properties of 16S gene in Oedipodidae showed typical patterns of many insects such as a high A＋T content and variable distance-dependent transition/transversion ratios. The 0.2 weight for sites of loops may be advisable for phylogeny reconstruction using the maximum parsimony method. The phylogenetic analysis results do not support the current subfamily classification systems of Oedipodidae. Bryodemellinae and Bryodeminae are closely related and should be merged as one subfamily. The status of Oedipodinae and Locustinae is also problematic.