Ribotrap Analysis of Proteins Associated with FHL3 3'Untranslated Region in Glioma Cells

Objective To screen the proteins associated with four-and-a-half LIM domains 3(FHL3) 3' untranslated region(3'UTR) in glioma cells. Methods Western blot was adopted to detect the regulatory effect of poly(C)-binding protein 2(PCBP2) on FHL3. Biotin pull-down and sliver staining were employed to screen and verify the candidate binding proteins of FHL3 3'UTR. Then liquid chromatography-tandem mass spectrometry(LC-MS/MS) and molecule annotation system were used to identify and analyze the candidate binding proteins. Immunoprecipitation was conducted to study the interaction between PCBP2 and polypyrimidine tract-binding protein 1(PTBP1), a binding protein identified by LC-MS/MS. Results PCBP2 could bind to FHL3 mRNA 3'UTR-A and inhibited the expression of FHL3 in T98 G glioms cells. 22 candidate binding proteins were identified. Among them, there were 11 RNA binding proteins, including PCBP2. PTBP1 associated with FHL3 mRNA 3'UTR and interacted with PCBP2 protein. Conclusion PCBP2 and PTBP1 can both associate with FHL3 mRNA 3'UTR through forming a protein complex.

A Pair of Novel Primers for Universal Detection of the NS1 Gene from Various Bluetongue Virus Serotypes

Twenty five serotypes of Bluetongue virus (BTV) have been identified worldwide. Rapid and reliable methods of virus universal detection are essential for fighting against bluetongue (BT). We have therefore developed and evaluated a pair of primers which can detect various serotypes of BTV by RT-PCR. Analysis of the viral protein 7 (VP7) and the non-structural protein (NS1) gene from different serotypes of BTV by DNAstar showed that the 5' end of the NS1 gene is the most conserved region. The primer pairs (P1 and P2) were designed based on the highly conserved region of NS1. The novel primers were evaluated by detecting BTV serotypes 1, 3, 5, 8, 10, 11, 21 and 22. The specificity of the primers was estimated by comparing to gene sequences of viruses published in GenBank, and further assessed by detecting BTV serotype 1-12 and Epizootic hemorrhagic disease virus (EHDV) serotype 1-4. The sensitivity and repeatability of PCR with the novel primers were evaluated by successfully detecting the recombinant plasmid pGEM-T121 containing the diagnosed nucleotide sequence. Our results suggest that these unique primers can be used in high throughout and universal detection of the NS1 gene from various BTV serotypes.

钠升电喷雾串联质谱技术鉴定两种髓系白血病细胞分化相关蛋白质

目的鉴定IL-6诱导小鼠髓系白血病M1细胞分化过程中出现的两种上调表达的相关蛋白质。方法将待鉴定的蛋白质斑点从双向电泳凝胶上切下,经胶内胰蛋白酶酶切后,先进行基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)肽质量指纹谱分析,再通过钠升电喷雾串联质谱(nano-ESI-MS/MS)技术获得其胰蛋白酶水解肽段的串联质谱图和肽段的全长序列,串联质谱图经Micromass专用软件MaxEnt3处理后,通过Mascot查询NCBI、SWISSPROT等数据库。结果两种未能通过肽质量指纹谱鉴定的蛋白质经Nano-ESI-MS/MS技术获得了明确鉴定,分别为蛋白质Destrin和Putative。结论Nano-ESI-MS/MS通过获得多肽序列信息在鉴定蛋白质方面具有独特的优势。

Integrative Analysis of Genome,3D Genome,and Transcriptome Alterations of Clinical Lung Cancer Samples

Genomic studies of cancer cell alterations,such as mutations,copy number variations(CNVs),and translocations,greatly promote our understanding of the genesis and development of cancers.However,the 3D genome architecture of cancers remains less studied due to the complexity of cancer genomes and technical difficulties.To explore the 3D genome structure in clinical lung cancer,we performed Hi-C experiments using paired normal and tumor cells harvested from patients with lung cancer,combining with RNA sequenceing analysis.We demonstrated the feasibility of studying 3D genome of clinical lung cancer samples with a small number of cells(1×10^(4)),compared the genome architecture between clinical samples and cell lines of lung cancer,and identified conserved and changed spatial chromatin structures between normal and cancer samples.We also showed that Hi-C data can be used to infer CNVs and point mutations in cancer.By integrating those different types of cancer alterations,we showed significant associations between CNVs,3D genome,and gene expression.We propose that 3D genome mediates the effects of cancer genomic alterations on gene expression through altering regulatory chromatin structures.Our study highlights the importance of analyzing 3D genomes of clinical cancer samples in addition to cancer cell lines and provides an integrative genomic analysis pipeline for future larger-scale studies in lung cancer and other cancers.

Secretory/releasing proteome-based identification of plasma biomarkers in HBV-associated hepatocellular carcinoma

For successful therapy, hepatocellular carcinoma (HCC) must be detected at an early stage. Herein, we used a proteomic approach to analyze the secretory/releasing proteome of HCC tissues to identify plasma biomarkers. Serum-free conditioned media (CM) were collected from primary cultures of cancerous tissues and surrounding noncancerous tissues. Proteomic analysis of the CM proteins permitted the identification of 1365 proteins. The enriched molecular functions and biological processes of the CM proteins, such as hydrolase activity and catabolic processes, were consistent with the liver being the most important metabolic organ. Moreover, 19% of the proteins were characterized as extracellular or membrane-bound. For validation, secretory proteins involved in transforming growth factor-β signaling pathways were validated in plasma samples. Alphafetoprotein (AFP), metalloproteinase (MMP)1, osteopontin (OPN), and pregnancy-specific beta-1-glycoprotein (PSG)9 were significantly increased in HCC patients. The overall performance of MMP1 and OPN in the diagnosis of HCC remained greater than that of AFP. In addition, this study represents the first report of MMP1 as a biomarker with a higher sensitivity and specificity than AFP. Thus, this study provides a valuable resource of the HCC secretome with the potential to investigate serological biomarkers. MMP1 and OPN could be used as novel biomarkers for the early detection of HCC and to improve the sensitivity of biomarkers compared with AFP.

Whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for rapid identification of bacteria cultured in liquid media

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used for many years to rapidly identify whole bacteria. However, no consistent methodology exists for the rapid identification of bacteria cultured in liquid media. Thus, in this study we explored the use of MALDI-TOF MS analysis for rapid identification of cells cultured in liquid media. We determined that 2,5-dihydroxybenzoic acid (50 mg mL-1, 50% acetonitrile, 0.1% trifluoroacetic acid) was the best matrix solution for MALDI-TOF MS for this type of study. Moreover, the tested strains were successfully differentiated by principal component analysis, and the main characteristics of the mass peaks for each species were found in mixed culture samples. In addition, we found that the minimum number of cells for detection was 1.8×103. In conclusion, our findings suggest that MS-based techniques can be developed as an auxiliary method for rapidly and accurately identifying bacteria cultured in liquid media.

Breast cancer-specific serum peptide profiles

Early detection of breast cancer is paramount to successful clinical therapy. Yet, early-stage breast cancer lacks specific symptoms or biomarkers. With the emerging of the mass spectrometric (MS) -based signatures as biomarkers, we investigated breast cancer-related serum profile pattern through class prediction and independent validation, and used Fourier transfer MS to identify breast cancer signature. We now show a distinctive serum peptide pattern that discriminates breast cancer from healthy controls with 93.2% sensitivity and 95.4% specificity. m/z 5901.70 and 4465.74 of ion fragment of FPA and alpha1-antichymotrypsin are found in the signatures that predominantly discriminate breast cancer from healthy individuals. These novel findings identify an MS-based serum peptide pattern of breast cancer that may have direct clinical utility in future.

Morphological and electrophysiological evidence for regeneration of transected spinal cord fibers and restoration of motor functions in adult rats

After 2/3 transection of the right ninth thoracic spinal cord of an adult rat, a chitosan tube seeded with L-poly-lysine was implanted between the rostral and caudal end of the lesioned cord. Twelve months after the operation, regeneration of myeli-nated and non-myelinated axons and new blood vessels were observed along the wall of the chitosan tube implanted under an electron microscope. Somatosensory evoked potentials (SEP) could be consistently recorded from the left somatosensory cortex following electrical stimulation of the right hind limb, while transcranial magnetic stimulation of the left motor cortex could also evoke motor activity from the right hind limb. The present result suggests that implanted chitosan tube might be useful in regenera-tion of injured nerve fibers of the spinal cord resulting in a long-term restoration of motor functions.

ESI-MS study on non-covalent bond complex of rhFKBP12 and new neurogrowth promoter

An ESI-MS method for studying the non-covalent bond complex of rhFKBP12 with its nonimmunosuppressive ligands was developed. The method was used to screen out three com-pounds capable of binding to rhFKBP12 non-covalently from 52 compounds. By competing binding experiment, the binding site and the relative binding strength of these three compounds 000107, 000308 and A2B12 with rhFKBP12 were measured. All of them have the same binding site as FK506 does. X-ray crystalline diffraction experiment of non-covalent bond complex of 000107, 000308 with rhFKBP12 by Tsinghua University showed the same results. Among them 000308 has good effect on stimulating neurite to grow in chicken sensory neuronal cultures.

Dynamic Proteome Changes of Shigella flexneri 2a During Transition from Exponential Growth to Stationary Phase

Shigella flexneri is an infectious pathogen that causes dysentery to human, which remains a serious threat to public health, particularly in developing countries. In this study, the global protein expression patterns of S. flexneri during transition from exponential growth to stationary phase in vitro were analyzed by using 2-D PAGE combined with MALDI-TOF MS. In a time-course experiment with five time points, the relative abundance of 49 protein spots varied significantly. Interestingly, a putative outer membrane protein YciD （OmpW） was almost not detected in the exponential growth phase but became one of the most abundant proteins in the whole stationary-phase proteome. Some proteins regulated by the global regulator FNR were also significantly induced （such as AnsB, AspA, FrdAB, and KatG） or repressed （such as AceEF, OmpX, SodA, and SucAB） during the growth phase transition. These proteins may be the key effectors of the bacterial cell cycle or play important roles in the cellular maintenance and stress responses. Our expression profile data provide valuable information for the study of bacterial physiology and form the basis for future proteomic analyses of this pathogen.

Flavivirus-like particles in the cardiac myocyte of a patient with arrhythmogenic right ventricular cardiomyopathy

Vires infection and subsequent myocardial damage and repairing were regarded as important aspects in the pathogenesis of dilated cardiomyopathy. Enterovirus and its association with myocarditis and dilated cardiomyopathy have been investigated extensively. Arrhythmogenic right ventricular cardiomyopathy （ARVC） is a relatively uncommon kind of cardiomyopathy which could cause malignant arrhythmia and sudden death in human. Although some ARVC patients have family history and some gene defects were found to be associated with ARVC, enterovims genome was shown in endomyocardial samples from the patients with ARVC and it suggested possible association between ARVC and vires infective myocarditis. Here, we present an electron microscopic evidence for a flaviviral infection of myocytes from a case of ARVC.

Opportunities and challenges of rare and undiagnosed disease research in China

Rare and undiagnosed diseases have been recognized as major public health concerns,despite those being affected accounting for only a small percentage of the global population.Most patients cannot get precise diagnoses or effective treatments.This situation thereby causes a heavy burden on healthcare systems,posing huge challenges not only for clinicians,but also for the family members of these patients.

CUEDC2:an emerging key player in inflammation and tumorigenesis

CUE domain-containing 2(CUEDC2)is a protein involved in the regulation of the cell cycle,inflammation,and tumorigenesis and is highly expressed in many types of tumors.CUEDC2 is phosphorylated by Cdk1 during mitosis and promotes the release of anaphase-promoting complex or cyclosome(APC/C)from checkpoint inhibition.CUEDC2 is also known to interact with IkB kinaseα(IKKα)and IKKβand has an inhibitory role in the activation of transcription factor nuclear factor-κB.Moreover,CUEDC2 plays an important role in downregulating the expression of hormone receptors estrogen receptor-αand progesterone receptor,thereby impairing the responsiveness of breast cancer to endocrine therapies.In this review,current knowledge on the multi-functions of CUEDC2 in normal processes and tumorigenesis are discussed and summarized.

AI based colorectal disease detection using real-time screening colonoscopy

Colonoscopy is an effective tool for early screening of colorectal diseases.However,the application of colonoscopy in distinguishing different intestinal diseases still faces great challenges of efficiency and accuracy.Here we constructed and evaluated a deep convolution neural network(CNN)model based on 117055 images from 16004 individuals,which achieved a high accuracy of 0.933 in the validation dataset in identifying patients with polyp,colitis,colorectal cancer(CRC)from normal.The proposed approach was further validated onmulti-center real-time colonoscopy videos and images,which achieved accurate diagnostic performance on detecting colorectal diseases with high accuracy and precision to generalize across external validation datasets.The diagnostic performance of the model was further compared to the skilled endoscopists and the novices.In addition,our model has potential in diagnosis of adenomatous polyp and hyperplastic polyp with an area under the receiver operating characteristic curve of 0.975.Our proposed CNN models have potential in assisting clinicians in making clinical decisions with efficiency during application.