In vitro antiviral activity of sulfated Auricularia auricula polysaccharides

Total Auricularia auricula polysaccharide (AAP(t)) was prepared by extracting and removing the proteins. Column chromatography was used to further graded it into AAP(1) and AAP(2). Three AAPs were modified by chlorosulfonic acid-pyridine method to obtain three sulfated AAPs (sAAPs), sAAP(t), sAAP(1) and sAAP(2), respectively. Three sAAPs and Newcastle disease virus (NDV) were added into cultivation system of chicken embryo fibroblast (CEF) in three manners, pre-, post- and simultaneous-adding polysaccharide with NDV respectively, taking three non-modified AAPs as control. Their anti-viral activities were compared by MTT method. The results showed that sAAPs and AAPs at a certain concentration could significantly inhibit the cellular infectivity of NDV in three manners. The effects of sAAPs were better than that of AAPs. It indicated that sulfated modification could enhance the antiviral activity of AAP. sAAP(1) and sAAP(t) possessed stronger activity and would be as the component of a new-type antiviral drug.     

Bcl-2 over-expression fails to prevent age-related loss of calretinin positive neurons in the mouse dentate gyrus

Background

Mutations in LRRK2 encoding leucine-rich repeat kinase 2 are thus far the most frequent genetic cause associated with autosomal dominant and idiopathic Parkinson's disease (PD). To examine whether LRRK2 is directly associated with neuropathology of PD and other related disorders, we analyzed LRRK2 in brains of patients affected by PD and dementia with Lewy bodies (DLB) using highly specific antibodies to LRRK2.

Results

We demonstrated that anti-LRRK2 antibodies strongly labelled brainstem and cortical Lewy bodies, the pathological hallmarks of PD and DLB, respectively. In addition, anti-LRRK2 also labelled brain vasculature, axons, and neuronal cell bodies. Interestingly, the immunocytochemical profile of LRRK2 varied with different antibodies depending upon specific antigenic sites along the LRRK2 protein. All anti-LRRK2 antibodies tested that were raised against various regions of LRRK2, were found to be immunoreactive to recombinant LRRK2 on Western blots. However, only the antibodies raised against the N-terminal and C-terminal regions of LRRK2, but not the regions containing folded protein domains, were positive in immunolabeling of Lewy bodies, suggesting a differential exposure of specific antigenic sites of LRRK2 on tissue sections.

Conclusion

We conclude that LRRK2 is a component of Lewy bodies in both PD and DLB, and therefore plays an important role in the Lewy body formation and disease pathogenesis. Information on the cellular localization of LRRK2 under normal and pathological conditions will deepen our understanding of its functions and molecular pathways relevant to the progression of PD and related disorders.     

Engineering imaging probes and molecular machines for nanomedicine

Nanomedicine is an emerging field that integrates nanotechnology, biomolecular engineering, life sciences and medicine; it is expected to produce major breakthroughs in medical diagnostics and therapeutics. Due to the size-compatibility of nano-scale structures and devices with proteins and nucleic acids, the design, synthesis and application of nanoprobes, nanocarriers and nanomachines provide unprecedented opportunities for achieving a better control of biological processes, and drastic improvements in disease detection, therapy, and prevention. Recent advances in nanomedicine include the development of functional nanoparticle based molecular imaging probes, nano-structured materials as drug/gene carriers for in vivo delivery, and engineered molecular machines for treating single-gene disorders. This review focuses on the development of molecular imaging probes and engineered nucleases for nanomedicine, including quantum dot bioconjugates, quantum dot-fluorescent protein FRET probes, molecular beacons, magnetic and gold nanoparticle based imaging contrast agents, and the design and validation of zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs) for gene targeting. The challenges in translating nanomedicine approaches to clinical applications are discussed.     

Genotype and haplotype analysis of the AZGP1 gene in cattle

Zinc-a2-glycoprotien (AZGP1) involved in lipid metabolism and associated with adipose tissue atrophy in cachexia. And it also related to sperm motility and in turn fertilization. To ascertain whether there were mutations in the bovine AZGP1 gene, this study investigated variation of the AZGP1 gene through PCR-SSCP and sequencing. Four missense mutations were identified in 649 cattle from six independent populations. Haplotype frequencies and linkage disequilibrium (LD) coefficients of these SNPs in three Chinese indigenous cattle breeds were analyzed. One LD block was found in three cattle breeds. The statistical analyses indicated that AC genotype of Z4 locus was associated with the high body weight, body length and chest girth in Jiaxian cattle breed (P?<?0.05). Our results provided evidence that polymorphisms in the AZGP1 gene were associated with growth traits, and may be used for marker-assisted selection and management in cattle breeding program.     

治疗脊髓损伤之建立星形胶质细胞模型的研究进展

脊髓损伤(spinal cord injury,SCI)是一种极为复杂的破坏性疾病,一旦脊髓损伤发生,治疗棘手,对患者家庭、国家带来巨大的经济、社会负担。近年来,通过建立大鼠脊髓损伤细胞相关模型,对于脊髓损伤的病因病机治疗等方面有了进一步的认识,而星形胶质细胞模型的建立对脊髓损伤治疗有深远意义。研究发现,星形胶质细胞作为靶细胞通过血-脑脊液屏障直接或间接对脊髓损伤有双向调控作用。本文通过对近年来星形胶质细胞模型培养制备方案等研究进行总结,以期为建立一个客观化、定量化、可模拟化的星形胶质细胞模型提供指导对脊髓损伤的治疗提供新的思路。     

Non-specific phospholipase C4 hydrolyzes phosphosphingolipids and phosphoglycerolipids and promotes rapeseed growth and yield

Phosphorus is a major nutrient vital for plant growth and development, with a substantial amount of cellular phosphorus being used for the biosynthesis of membrane phospholipids. Here, we report that NON-SPECIFIC PHOSPHOLIPASE C4 (NPC4) in rapeseed (Brassica napus) releases phosphate from phospholipids to promote growth and seed yield, as plants with altered NPC4 levels showed significant changes in seed production under different phosphate conditions. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated knockout of BnaNPC4 led to elevated accumulation of phospholipids and decreased growth, whereas overexpression (OE) of BnaNPC4 resulted in lower phospholipid contents and increased plant growth and seed production. We demonstrate that BnaNPC4 hydrolyzes phosphosphingolipids and phosphoglycerolipids in vitro, and plants with altered BnaNPC4 function displayed changes in their sphingolipid and glycerolipid contents in roots, with a greater change in glycerolipids than sphingolipids in leaves, particularly under phosphate deficiency conditions. In addition, BnaNPC4-OE plants led to the upregulation of genes involved in lipid metabolism, phosphate release, and phosphate transport and an increase in free inorganic phosphate in leaves. These results indicate that BnaNPC4 hydrolyzes phosphosphingolipids and phosphoglycerolipids in rapeseed to enhance phosphate release from membrane phospholipids and promote growth and seed production.     

Histochemical examination of cathepsin K,MMP1 and MMP2 in compressed periodontal ligament during orthodontic tooth movement in periostin deficient mice

The purpose of this study was to investigate immunolocalization of collagenolytic enzymes including cathepsin K, matrix metalloproteinase (MMP) 1 and 2 in the compressed periodontal ligament (PDL) during orthodontic tooth movement using a periostin deficient (Pn-/-) mouse model. Twelve-week-old male mice homozygous for the disrupted periostin gene and their wild type (WT) littermates were used in these experiments. The tooth movement was performed according to Waldo’s method, in which elastic bands of 0.5 mm thickness were inserted between the first and second upper molars of mice under anesthesia. At 1 and 3 days after orthodontic force application, mice were fixed with transcardial perfusion of 4 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), and the first molars and peripheral alveolar bones were extracted for histochemical analyses. Compared with WT mice, immunolocalization of cathepsin K, MMP1 and MMP2 was significantly decreased at 1 and 3 days after orthodontic tooth movement in the compressed PDL of Pn-/- mice, although MMP1-reactivity and MMP2-reactivity decreased at different amounts. Very little cathepsin K-immunoreactivity was observed in the assessed regions of Pn-/- mice, both before and after orthodontic force application. Furthermore, Pn-/- mice showed a much wider residual PDL than WT mice. Taken together, we concluded that periostin plays an essential role in the function of collagenolytic enzymes like cathepsin K, MMP1 and MMP2 in the compressed PDL after orthodontic force application.     

Efficient generation of transgenic chickens using the spermatogonial stem cells in vivo and ex vivo transfection

The highly efficient novel methods to produce transgenic chickens were established by directly in-jecting the recombinant plasmid containing green fluorescent protein (GFP) gene into the cock's testis termed as testis-medianted gene transfer (TMGT), and transplanting transfected spermatogonial stem cells (TTSSCs). For the TMGT approach,four dosages of pEGFP-N1 DNA/cationic polymer complex were injected intratesticularly. The results showed: (1) 48 h after the injection,the percentages of testis cells expressing GFP were 4.0%, 8.7%, 10.2% and 13.6% in the 50, 100, 150 and 200 μg/mL group, re-spectively. The difference from the four dosage groups was significant (P<0.05). On day 25 after the injection, a dosage-dependent and time-dependent increase in the number of transgenic sperm was observed. The percentages of gene expression reached the summit and became stable from day 70 to 160, being 12.7%, 12.8%, 15.9% and 19.1%, respectively. The difference from the four dosage groups was also significant (P<0.05). (2) 70 d after the injection, strong green fluorescent could be observed in the seminiferous tubules by whole-mount in-situ hybridization. (3) 70 d after the injection, the semen was collected and used to artificially inseminate wild-type females. The blastoderms of F1 and F2 transgenic chicken expressed GFP were 56.2% (254/452) and 53.2% (275/517), respectively. The detec-tion of polymerase chain reaction (PCR) of F1 and F2 transgenic chicken blood genomic DNA showed that 56.5% (3/23) of F1 and 52.9% (9/17) of F2 were positive. Southern blot showed GFP DNA was in-serted in their genomic DNAs. (4) Frozen whole mount tissue sections of F1 and F2 transgenic chicken liver, heart, kidney and muscle showed that the rates of green fluorescent positive were between 50.0% and 66.7%. (5) With the TTSSCs method, SSCs ex vivo transfected with GFP were transplanted into recipient roosters whose endogenic SSCs had been resoluted. The donor SSCs settled and GFP ex-pression became readily detectable in the frozen whole mount tissue sections of recepient testes. Moreover, sperms carrying GFP could be produced normally. The results of artificially inseminating wild-type females with these sperms showed 12.5% (8/64) of offspring embryo expressed GFP and 11.1% (2/18) hatched chicks were tested transgenic. Our data therefore suggest TMGT and TTSSCs are the feasible methods for the generation of transgenic chickens.