Systematic research on the pretreatment of peptides for quantitative proteomics using a C18 microcolumn

Reversed phase microcolumns have been widely used for peptide pretreatment to desalt and remove interferences before tandem LC–MS in proteomics studies. However, few studies have characterized the effects of experimental parameters as well as column characteristics on the composition of identified peptides. In this study, several parameters including the concentration of ACN in washing buffer, the microcolumn's purification effect, the peptide recovery rate, and the dynamic‐binding capacity were characterized in detail, based upon stable isotope labeling by amino acids in a cell culture quantitative approach. The results showed that peptide losses can be reduced with low ACN concentration in washing buffers resulting in a recovery rate of approximately 82%. Furthermore, the effects of ACN concentration and loading amount on the properties of identified peptides were also evaluated. We found that the dynamic‐binding capacity of the column was approximately 26 μg. With increased loading amounts, more hydrophilic peptides were replaced by hydrophobic peptides.     

Amino‐functionalized macroporous silica for efficient tryptic digestion in acidic solutions

Amino‐functionalized macroporous silica foam (NH₂‐MOSF) has been developed as a host reactor to realize highly efficient proteolysis in acidic solutions where normal tryptic reactions cannot occur. The digestion protocol consists simply of adding the functionalized NH₂‐MOSF into the protein and trypsin solutions without altering the bulk pH or preloading the enzymes on the materials. With this protocol, digestion of sample fractions from LC can be efficiently realized in the acidic solutions directly. Digestion of a protein fraction extracted from rat liver tissue after LC separation was performed to illustrate this principle, where 103 proteins were successfully identified at pH 3 after 1.5 h of tryptic digestion.     

一条新的柱层析纯化路径对HBsAg免疫原性的影响

摘要目的：建立一条新的毕赤酵母表达乙肝表面抗原（HepatitisBantigen,HBsAg）柱层析纯化方法,保持HBsAg结构完整性和提高免疫原性。方法：毕赤酵母发酵料液经过菌体破碎、聚乙二醇沉淀、疏水层析、超滤和凝胶分子筛精纯,收集HBsAg合格样品液适当稀释后加入铝佐荆吸附,制成乙肝疫苗半成品免疫BALB／c小鼠。结果：纯化产物经SDS-PAGE银染鉴定得单一条带,分子量在23kD左右,凝胶成像软件分析纯度超过95％;该纯化方法得到的HBsAg颗粒电镜观察得平均直径为22nm病毒样颗粒,结构较均一完整;自制疫苗免疫小鼠后,其血清抗体水平高于葛兰素史克生产的Engerix—B（安在时）,存在显著性差异（P〈0．05）。结论：通过该方法纯化的HBsAg结构完整性良好,疫苗免疫效果优于酵母表达的Engerix—B,纯化路径简单高效,易于放大用于工业化生产。     

类风湿性关节炎伴间质性肺病中肿瘤标志物对肺功能的影响分析

目的：探究类风湿性关节炎伴间质性肺病中肿瘤标志物对肺功能的影响。方法：选取2011年3月至2013年3月我院收治的类风湿性关节炎患者88例,根据其是否伴有ILD,分为RA组53例和RA-ILD组35例。检测两组肺功能指标用力肺活量（FVC）、1s用力呼气容积（FEV1）、最大通气量百分比（MVV）、一氧化碳弥散量（DLCO）,肿瘤标志物癌胚抗原CEA、癌抗原125（CA125）、癌抗原199（CA199）及抗环胍氨酸肽抗体（抗CCP抗体）的值,并利用统计学方法分析肿瘤标记物与肺功能指标、抗CCP抗体的相关性。结果：RA．ILD组FVC、FEV1、MVV、DLCO均比RA组低,CEA、CA125、CA199均比RA组高,结果比较差异显著具有统计学意义（P〈0．05）。但两组抗CCP抗体含量相比却无明显差异,不具有统计学意义（P〉0．05）。相关性分析显示,CEA与FVC、FEV1、MVV、DLCO呈负相关（P〈0．05）,而CA125、CA199却与肺功能指标无相关性,CEA、CA125、CA199与抗CCP抗体均无相关性（P〉0．05）。结论：RA-ILD的肺功能指标均有明显下降,而肿瘤标志物表达水平却明显增高,CEA对肺功能损害影响较大,却与关节损害的关系不大,这为临床对类风湿性关节炎伴间质性肺病早诊断、早治疗提供了依据。     

Role of LysM receptors in chitin-triggered plant innate immunity

Recent research findings clearly indicate that lysin motif (LysM)-containing cell surface receptors are involved in the recognition of specific oligosaccharide elicitors (chitin and peptidoglycan), which trigger an innate immunity response in plants. These receptors are either LysM-containing receptor-like kinases (LYKs) or LysM-containing receptor proteins (LYPs). In Arabidopsis, five LYKs (AtCERK1/AtLYK1 and AtLYK2–5) and three LYPs (AtLYP1–3) are likely expressed on the plasma membrane. In this review, we summarize recent research results on the role of these receptors in plant innate immunity, including the recent structural characterization of AtCERK1 and composition of the various receptor complexes in Arabidopsis.     

Role of recombinant plasmid pEGFP-N1-IGF-1 transfection in alleviating osteoporosis in ovariectomized rats

Decreased levels of serum insulin-like growth factor-1 (IGF-1) have been proven to cause osteoporosis. Gene transfer of IGF-1 offers an attractive technology to treat skeletal metabolic disorders including osteoporosis, but the viral vectors are limited by their high antigenicity and immune response. Our purpose was to investigate the expression of a non-invasive vector, recombinant plasmid enhanced green fluorescent protein-N1 (pEGFP-N1) that transferred IGF-1 gene into ovariectomized (OVX) rats in vivo and evaluate the effect of this therapy on osteoporosis. OVX or sham operations were performed in 60 female, 7-month-old unmated SD rats. 12 weeks after OVX operation, the vectors were transfected to the 10-month-old rats and experimental data were detected from 48 h to 7 week after transfection. Our results showed that remarkable expression of fluorescence and serum IGF-1 was observed in the rats transfected by recombinant plasmids, indicating that IGF-1 gene was successfully transferred to OVX rats by injecting the vector through hydrodynamic method via the tail vein. The bone metabolism index including serum alkaline phosphatase, the histomorphometric parameters of lumbar vertebra including trabecular area percentage, trabecular thickness, trabecular number and trabecular separation, and the bone mineral density (BMD) and biomechanical parameters of lumbar vertebra including BMD, maximum condensing force, crushing strength in OVX rats transfected by pEGFP-N1-IGF-1 were improved remarkably compared with OVX+pEGFP-N1 rats, indicating that the transfection of recombinant plasmid pEGFP-N1-IGF-1 played a significant role in alleviating osteoporosis in rats induced by OVX. This encouraged a potential approach of IGF-1 gene therapy to the treatment of osteoporosis.     

Saccharomyces cerevisiae DNA Ligase IV Supports Imprecise End Joining Independently of Its Catalytic Activity

DNA ligase IV (Dnl4 in budding yeast) is a specialized ligase used in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). Although point and truncation mutations arise in the human ligase IV syndrome, the roles of Dnl4 in DSB repair have mainly been examined using gene deletions. Here, Dnl4 catalytic point mutants were generated that were severely defective in auto-adenylation in vitro and NHEJ activity in vivo, despite being hyper-recruited to DSBs and supporting wild-type levels of Lif1 interaction and assembly of a Ku- and Lif1-containing complex at DSBs. Interestingly, residual levels of especially imprecise NHEJ were markedly higher in a deletion-based assay with Dnl4 catalytic mutants than with a gene deletion strain, suggesting a role of DSB-bound Dnl4 in supporting a mode of NHEJ catalyzed by a different ligase. Similarly, next generation sequencing of repair joints in a distinct single-DSB assay showed that dnl4-K466A mutation conferred a significantly different imprecise joining profile than wild-type Dnl4 and that such repair was rarely observed in the absence of Dnl4. Enrichment of DNA ligase I (Cdc9 in yeast) at DSBs was observed in wild-type as well as dnl4 point mutant strains, with both Dnl4 and Cdc9 disappearing from DSBs upon 5′ resection that was unimpeded by the presence of catalytically inactive Dnl4. These findings indicate that Dnl4 can promote mutagenic end joining independently of its catalytic activity, likely by a mechanism that involves Cdc9.     

Distinct and Atypical Intrinsic and Extrinsic Cell Death Pathways between Photoreceptor Cell Types upon Specific Ablation of Ranbp2 in Cone Photoreceptors

Non-autonomous cell-death is a cardinal feature of the disintegration of neural networks in neurodegenerative diseases, but the molecular bases of this process are poorly understood. The neural retina comprises a mosaic of rod and cone photoreceptors. Cone and rod photoreceptors degenerate upon rod-specific expression of heterogeneous mutations in functionally distinct genes, whereas cone-specific mutations are thought to cause only cone demise. Here we show that conditional ablation in cone photoreceptors of Ran-binding protein-2 (Ranbp2), a cell context-dependent pleiotropic protein linked to neuroprotection, familial necrotic encephalopathies, acute transverse myelitis and tumor-suppression, promotes early electrophysiological deficits, subcellular erosive destruction and non-apoptotic death of cones, whereas rod photoreceptors undergo cone-dependent non-autonomous apoptosis. Cone-specific Ranbp2 ablation causes the temporal activation of a cone-intrinsic molecular cascade highlighted by the early activation of metalloproteinase 11/stromelysin-3 and up-regulation of Crx and CoREST, followed by the down-modulation of cone-specific phototransduction genes, transient up-regulation of regulatory/survival genes and activation of caspase-7 without apoptosis. Conversely, PARP1⁺-apoptotic rods develop upon sequential activation of caspase-9 and caspase-3 and loss of membrane permeability. Rod photoreceptor demise ceases upon cone degeneration. These findings reveal novel roles of Ranbp2 in the modulation of intrinsic and extrinsic cell death mechanisms and pathways. They also unveil a novel spatiotemporal paradigm of progression of neurodegeneration upon cell-specific genetic damage whereby a cone to rod non-autonomous death pathway with intrinsically distinct cell-type death manifestations is triggered by cell-specific loss of Ranbp2. Finally, this study casts new light onto cell-death mechanisms that may be shared by human dystrophies with distinct retinal spatial signatures as well as with other etiologically distinct neurodegenerative disorders.     

The Wilms Tumor Gene,Wt1, Is Critical for Mouse Spermatogenesis via Regulation of Sertoli Cell Polarity and Is Associated with Non-Obstructive Azoospermia in Humans

Azoospermia is one of the major reproductive disorders which cause male infertility in humans; however, the etiology of this disease is largely unknown. In the present study, six missense mutations of WT1 gene were detected in 529 human patients with non-obstructive azoospermia (NOA), indicating a strong association between WT1 mutation and NOA. The Wilms tumor gene, Wt1, is specifically expressed in Sertoli cells (SCs) which support spermatogenesis. To examine the functions of this gene in spermatogenesis, Wt1 was deleted in adult testis using Wt1^flox and Cre-ER^TM mice strains. We found that inactivation of Wt1 resulted in massive germ cell death and only SCs were present in most of the seminiferous tubules which was very similar to NOA in humans. In investigating the potential mechanism for this, histological studies revealed that the blood–testis barrier (BTB) was disrupted in Wt1 deficient testes. In vitro studies demonstrated that Wt1 was essential for cell polarity maintenance in SCs. Further studies found that the expression of cell polarity associated genes (Par6b and E-cadherin) and Wnt signaling genes (Wnt4, Wnt11) were downregulated in Wt1 deficient SCs, and that the expression of Par6b and E-cadherin was regulated by Wnt4. Our findings suggest that Wt1 is important in spermatogenesis by regulating the polarity of SCs via Wnt signaling pathway and that WT1 mutation is one of the genetic causes of NOA in humans.