Immunochemical studies on blood groups: The internal structure and immunological properties of water-soluble human blood group A substance studied by Smith degradation,liberation, and fractionation of oligosaccharides and reaction with lectins

Carbohydrate structures in the interior of a blood group A active substance (MSS) were exposed by one and by two Smith degradations. Reactivities of the original glycoprotein and its Smith degraded products with 13 different lectins and with anti-I Ma were studied by quantitative precipitin assay. MSS and its first Smith degraded product completely precipitated Ricinus communis hemagglutinin with five times less of the first Smith degraded glycoprotein being required for 50% precipitation. The second Smith degraded material precipitated only 90% of the lectin. MSS did not precipitate peanut lectin, whereas its first and second Smith degraded products completely precipitated the lectin. The first Smith degraded glycoprotein also reacted well with Wistaria floribunda, Maclura pomifera, Bauhinia purpurea alba, and Geodia lectins indicating that its carbohydrate moiety could contain dGalNAc, dGalβ1 → 3dGalNAc, dGalβ1 → 4dGlcNAc, dGalβ1 → 3dGlcNAcβ1 → 3dGal and/or dGalβ1 → 4dGlcNAcβ1 → 6dGal and/or dGalβ1 → 4dGlcNAcβ1 → 6dGalNAc determinants at nonreducing ends. The second Smith degraded material precipitated well with Ricinus communis hemagglutinin, Arachis hypogaea, Geodia cydonium, Maclura pomifera, and Helix pomatia lectins showing that dGalNAc, dGalβ1 → 3dGalNAc, dGalβ1 → 4dGlcNAc residues at terminal nonreducing ends could be involved. Monoclonal anti-I Ma (group 1) serum reacted strongly with the first Smith degraded product indicating large numbers of anti-I Ma determinants, dGalβ1 → 4dGlcNAcβ1 → d 6dGal and/or dGalβ1 → 4dGlcNAcβ1 → 6dGalNAc at nonreducing ends. The comparable activities of the native and Smith degraded products with wheat germ lectin indicate capacity to react with DGlcNAc residues at nonreducing ends and/or at positions in the interior of the chain. The totality of lectin reactivities indicates heterogeneity of the carbohydrate side chains. Oligosaccharides with ³H at their reducing ends released from the protein core of the first and second Smith degraded products were obtained by treatment with 0.05 m NaOH and 1 M NaB³H₄ at 50 °C for 16 h (Carlson degradation). The liberated reduced oligosaccharides were fractionated by dialysis, followed by retardion, Bio-Gel P-2, P-4, and P-6 columns. They were further purified on charcoal-celite columns, and by preparative paper chromatography and high-pressure liquid chromatography. Their distribution by size was estimated by the yields on dialysis, Bio-Gel P-2, and Bio-Gel P-6 chromatography, and from the radioactivity of the reduced sugars. Of the oligosaccharide fractions from the first Smith degraded product, about 77% of the carbohydrate side chain residues contained from 1 to 6 sugars, 13% from 7 to perhaps 12 sugars, and 10% was nondialyzable (polysaccharides and glycopeptide fragments). Of the second Smith degraded product, approximately 82% of carbohydrate residues had from 1 to 6 sugars, 14% from 7 to perhaps 20 sugars and 4% was nondialyzable. The biological activity profile of the two Smith degraded products together with the size distributions of the oligosaccharides indicated that their carbohydrate side chains, comprised a heterogeneous population ranging in size from 1 to about 12 sugars. When most of these chains that are shorter than hexasaccharides are fully characterized it may be possible to reconstruct the overall structure of the carbohydrate moiety of the blood group substances and account for their biological activities.     

Stem cell research in China

In the past 5 years, China has increased its efforts in the field of stem cell research and practice. Basic research mainly focuses on bone marrow and embryonic stem cells. Clinical applications of stem cells in the treatment of acute heart failure, acute liver failure and lower limb ischaemia have been reported by many hospitals. China enacted its 'Ethical Guidelines for Human Embryonic Stem Cell Research' in 2003. At present, China has the most liberal and favourable environments for human embryonic stem cell research.     

Discovery of genomic regions and candidate genes controlling shelling percentage using QTL‐seq approach in cultivated peanut (Arachis hypogaea L.)

Cultivated peanut (Arachis hypogaea L.) is an important grain legume providing high‐quality cooking oil, rich proteins and other nutrients. Shelling percentage (SP) is the 2nd most important agronomic trait after pod yield and this trait significantly affects the economic value of peanut in the market. Deployment of diagnostic markers through genomics‐assisted breeding (GAB) can accelerate the process of developing improved varieties with enhanced SP. In this context, we deployed the QTL‐seq approach to identify genomic regions and candidate genes controlling SP in a recombinant inbred line population (Yuanza 9102 × Xuzhou 68‐4). Four libraries (two parents and two extreme bulks) were constructed and sequenced, generating 456.89–790.32 million reads and achieving 91.85%–93.18% genome coverage and 14.04–21.37 mean read depth. Comprehensive analysis of two sets of data (Yuanza 9102/two bulks and Xuzhou 68‐4/two bulks) using the QTL‐seq pipeline resulted in discovery of two overlapped genomic regions (2.75 Mb on A09 and 1.1 Mb on B02). Nine candidate genes affected by 10 SNPs with non‐synonymous effects or in UTRs were identified in these regions for SP. Cost‐effective KASP (Kompetitive Allele‐Specific PCR) markers were developed for one SNP from A09 and three SNPs from B02 chromosome. Genotyping of the mapping population with these newly developed KASP markers confirmed the major control and stable expressions of these genomic regions across five environments. The identified candidate genomic regions and genes for SP further provide opportunity for gene cloning and deployment of diagnostic markers in molecular breeding for achieving high SP in improved varieties.     

Identification and validation of key genes associated with non-small-cell lung cancer

Non-small-cell lung cancer (NSCLC) is one of the main causes of death induced by cancer globally. However, the molecular aberrations in NSCLC patients remain unclearly. In the present study, four messenger RNA microarray datasets (GSE18842, GSE40275, GSE43458, and GSE102287) were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) between NSCLC tissues and adjacent lung tissues were obtained from GEO2R and the overlapping DEGs were identified. Moreover, functional and pathway enrichment were performed by Funrich, while the protein–protein interaction (PPI) network construction were obtained from STRING and hub genes were visualized and identified by Cytoscape software. Furthermore, validation, overall survival (OS) and tumor staging analysis of selected hub genes were performed by GEPIA. A total of 367 DEGs (95 upregulated and 272 downregulated) were obtained through gene integration analysis. The PPI network consisted of 94 nodes and 1036 edges in the upregulated DEGs and 272 nodes and 464 edges in the downregulated DEGs, respectively. The PPI network identified 46 upregulated and 27 downregulated hub genes among the DEGs, and six (such as CENPE, NCAPH, MYH11, LRRK2, HSD17B6, and A2M) of that have not been identified to be associated with NSCLC so far. Moreover, the expression differences of the mentioned hub genes were consistent with that in lung adenocarcinoma and lung squamous cell carcinoma in the TCGA database. Further analysis showed that all the six hub genes were associated with tumor staging except MYH11, while only the upregulated DEG CENPE was associated with the worse OS of patients with NSCLC. In conclusion, the current study showed that CENPE, NCAPH, MYH11, LRRK2, HSD17B6, and A2M might be the key genes contributed to tumorigenesis or tumor progression in NSCLC, further functional study is needed to explore the involved mechanisms.     

生物复苏——大绝灭后生物演化历史的第一幕

生命史是一部生物界短期,快速剧变与长期,慢速稳定相互交替的历史。大绝灭（即集群绝灭）事件反映了全球环境的大突变,点断了地质历史中的生命记录及其发展历程,预示着生物界的演化出现了最有意义的飞跃,近年来尝试研究大绝灭后全球生物界的残存－复苏及其基本型式,并探索复苏的控制因素,标志着地质科学中一个重心的转移（即从大绝灭转向其后的生物残存与复苏的研究）。生物复苏揭示了大绝灭后生物演化历史的第一幕,其研究的     

青海湖裸鲤寄生舌状绦虫的空间格局研究

舌状绦虫裂头蚴只寄生在体长小于２２０ｍｍ的青海湖裸鲤中,其种群在宿主种群中呈聚集分布。其聚集分布的强度随寄生虫种群平均密度的增加而降低。由于舌状绦虫在宿主体腔生长,产生明显的空间拥挤效应,可能导致宿主死亡或被食鱼鸟类捕食而转移到终末宿主中。在体长小于１２０ｍｍ的宿主中,密度依赖的死亡过程可能是使聚集强度降低的原因;而体长１４０－２００ｍｍ的鱼中,则是非密度依赖的全死过程使聚集强度增加．     

Functional Roles of the Non-Catalytic Calcium-Binding Sites in the N-Terminal Domain of Human Peptidylarginine Deiminase 4

This study investigated the functional roles of the N-terminal Ca²⁺ ion-binding sites, in terms of enzyme catalysis and stability, of peptidylarginine deiminase 4 (PAD4). Amino acid residues located in the N-terminal Ca²⁺-binding site of PAD4 were mutated to disrupt the binding of Ca²⁺ ions. Kinetic data suggest that Asp155, Asp157 and Asp179, which directly coordinate Ca3 and Ca4, are essential for catalysis in PAD4. For D155A, D157A and D179A, the k _cat/K _m,BAEE values were 0.02, 0.63 and 0.01 s⁻¹mM⁻¹ (20.8 s⁻¹mM⁻¹ for WT), respectively. Asn153 and Asp176 are directly coordinated with Ca3 and indirectly coordinated with Ca5 via a water molecule. However, N153A displayed low enzymatic activity with a k _cat value of 0.3 s⁻¹ (13.3 s⁻¹ for wild-type), whereas D176A retained some catalytic power with a k _cat of 9.7 s⁻¹. Asp168 is the direct ligand for Ca5, and Ca5 coordination by Glu252 is mediated by two water molecules. However, mutation of these two residues to Ala did not cause a reduction in the k _cat/K _m,BAEE values, which indicates that the binding of Ca5 may not be required for PAD4 enzymatic activity. The possible conformational changes of these PAD4 mutants were examined. Thermal stability analysis of the PAD4 mutants in the absence or presence of Ca²⁺ indicated that the conformational stability of the enzyme is highly dependent on Ca²⁺ ions. In addition, the results of urea-induced denaturation for the N153, D155, D157 and D179 series mutants further suggest that the binding of Ca²⁺ ions in the N-terminal Ca²⁺-binding site stabilizes the overall conformational stability of PAD4. Therefore, our data strongly suggest that the N-terminal Ca²⁺ ions play critical roles in the full activation of the PAD4 enzyme.     

Polyclonal Antibodies in Microplates to Predict the Maximum Adsorption Activities of Enzyme/Mutants from Cell Lysates

With microplate-immobilized polyclonal antibodies against a starting enzyme or its active mutant bearing consistent accessible epitopes, the maximum activity of an adsorbed enzyme/mutant (Vs) was predicted for comparison to recognize weakly-positive mutants. Rabbit antisera against Escherichia coli alkaline phosphatase (ECAP) were fractionated with 33% ammonium sulfate to yield crude polyclonal antibodies for conventional immobilization in 96-well microplates. The response curve of the activities of ECAP/mutant adsorbed by the immobilized polyclonal antibodies to protein quantities from a cell lysate was fit to an approximation model to predict Vs. With 0.4 μg crude polyclonal antibody for immobilization, Vs was consistent for ECAP in cell lysates bearing fourfold differences in its apparent specific activities when its abundance was greater than 0.9%. The ratio of Vs of the mutant R168K to that of ECAP was 1.5?±?0.1 (n?=?2), consistent with that of their specific activities after affinity purification. Unfortunately, the prediction of Vs with polyclonal antibodies that saturated microplate wells was ineffective to Pseudomonas aeruginosa arylsulfatase bearing less than 2% specific activity of ECAP. Therefore, with microplate-immobilized polyclonal antibodies to adsorb enzyme/mutants from cell lysates, high-throughput prediction of Vs was practical to recognize weakly-positive mutants of starting enzymes bearing fairly-high activities.     

Proteasome Inhibition-Induced Downregulation of Akt/GSK-3β Pathway Contributes to Abnormality of Tau in Hippocampal Slice

Proteasome inhibition can induce abnormal accumulation and phosphorylation of microtubule-associated protein tau. The major function of tau protein is to promote microtubules assembly and stabilization, and abnormal tau protein would disturb its microtubule-binding function. In this study, proteasome inhibitor MG132 was used to treat hippocampal slices to explore the role and mechanism of Akt/glycogen synthase kinase-3β (GSK-3β) in proteasome inhibition-induced tau abnormality. During the culture period, we measure the lactate dehydrogenase (LDH) content to assay the viability of hippocampal slices. Following 2.5 and 5 μM MG132 treatment for 6 h, we detected the expression, phosphorylation modification, and microtubule-binding function of tau protein of slices. We also analyzed the changed activities of glycogen synthase kinase-3β (GSK-3β) and protein kinase B (PKB/Akt) and the level of heat shock protein 90 (Hsp90) in the process. In addition, co-immunoprecipitation was used to investigate the interaction between Akt and Hsp90, Akt and protein phosphatase-2A (PP2A) in the MG132-treated organotypic hippocampal slices. Our results indicated that proteasome inhibition led to degradation obstacles and abnormal phosphorylation of tau protein. The downregulated Akt/GSK-3β signaling pathway might be responsible for the abnormal phosphorylation of tau protein at multiple sites which further reduced the microtubule-binding function of tau protein. Furthermore, proteasome inhibition decreased the binding capacity of Akt-Hsp90 while increased the Akt-PP2A binding ability which mediated Akt inactivity. This current study establishes a hippocampal slice model targeting Akt/GSK-3β signaling pathway to explore the pivotal role of proteasome inhibition in tau pathology.     

Risk Factors of Pneumothorax after Endobronchial Ultrasound-Guided Transbronchial Biopsy for Peripheral Lung Lesions

Background

The risk of endobronchial ultrasound-guided transbronchial biopsy-related pneumothorax is a major concern and warrants further studies. The aim of our study was to estimate the risk of pneumothorax after this procedure and identify its risk factors.

Methods

From 2007 to 2011, 399 patients who underwent endobronchial ultrasound-guided transbronchial biopsy for peripheral lung lesions were included in this study. The variables analyzed included patient factors, lesion factors and procedure factors. Multivariate logistic regression analysis was used to identify independent risk factors for pneumothorax.

Results

The incidence of pneumothorax was 3.3% (13/399). Chest tube placement was required for 31% (4/13) of pneumothoraces. Independent risk factors for pneumothorax included pulmonary emphysema (OR, 55.09; 95% CI, 9.37–324.03; p<0.001) and probe position adjacent to the lesion (OR, 17.01; 95% CI, 2.85–101.64; p = 0.002). The number of biopsy specimens, age, sex, history of prior lung surgery and lesion size, location and character did not influence the risk of pneumothorax in our analyses.

Conclusions

The risk of pneumothorax after endobronchial ultrasound-guided transbronchial biopsy is low. To further reduce the risk of pneumothorax, every effort should be made to advance the endobronchial ultrasound probe into the bronchus where it is imaged within the target lesion before embarking on transbronchial biopsy.