SARS-CoV-2 spike host cell surface exposure promoted by a COPI sorting inhibitor

Via an insufficient coat protein complex I (COPI) retrieval signal, the majority of SARS-CoV-2 spike (S) is resident in host early secretory organelles and a tiny amount is leaked out in cell surface. Only surface-exposed S can be recognized by B cell receptor (BCR) or anti-S therapeutic monoclonal antibodies (mAbs) that is the trigger step for B cell activation after S mRNA vaccination or infected cell clearance by S mAbs. Now, a drug strategy to promote S host surface exposure is absent. Here, we first combined structural and biochemical analysis to characterize S COPI sorting signals. A potent S COPI sorting inhibitor was then invented, evidently capable of promoting S surface exposure and facilitating infected cell clearance by S antibody-dependent cellular cytotoxicity (ADCC). Importantly, with the inhibitor as a probe, we revealed Omicron BA.1 S is less cell surface exposed than prototypes because of a constellation of S folding mutations, possibly corresponding to its ER chaperone association. Our findings not only suggest COPI is a druggable target against COVID-19, but also highlight SARS-CoV-2 evolution mechanism driven by S folding and trafficking mutations.

ARID1A expression in intrahepatic cholangiocarcinoma and its relationship with tumor recurrence,metastasis and tumor-specific immunomarkers / 中华普通外科杂志

Objective:To investigate the expression level between AT-Rich Interaction Domain 1A(ARID1A) in intrahepatic cholangiocarcinoma (ICC) and the correlation with tumor microenvironment.Methods:The clinicopathological and survival data of 110 ICC patients undergoing radical hepatectomy in Peking University People's Hospital from Jan 2015 to May 2021 were retrospectively analyzed. Immunohistochemical staining was used to detect the expressions of ARID1A , programmed cell death 1 ligand 1( PD-L1) in tumor tissues , programmed cell death protein 1(PD-1) and cluster of differentiation 8(CD8) in the microenvironment. The relationship between ARID1A expression and PD-L1, PD-1, CD8 protein expression was analyzed.Results:Twenty seven patients did not express ARID1A, absence of ARID1A was associated with high PD-L1, PD-1 and CD8 expression ( P<0.05). Multivariate analysis showed ARID1A expression, preoperative CEA level,preoperative CA19-9 level, lymph node metastasis and tumor number were independent risk factors. Conclusion:Absent expression of ARID1A suggests poor prognosis of ICC patients, high expression of PD-L1,PD-1 and CD8 protein in ICC tumor microenvironment with ARID1A-deficient expression suggests a possible prognosis benefit by using anti-PD-1, anti-PD-L1 and other immunotherapy regimens.

C-terminal Motifs of HIV-1 gp41 as Possible Determinants of Viral Pathogenesis / Motivos C-terminales de gp41 del VIH-1 como posibles determinantes de la patogénesis viral / Motivos C-terminais do HIV-1 gp41 como Possíveis Determinantes da Patogénese Viral

Abstract: human immunodeficiency virus type 1 (HIV-1) is the etiological agent of acquired immunodeficiency syndrome (AIDS), a pandemic with high economic and social costs. The envelope glycoprotein (ENV) of the virus mediates the infectious process by binding to and entering the host cell, one of the main target components of studies since its discovery. Its endodomain or C-terminal tail (CTT) participates in late replicative cycle processes, such as intracellular trafficking, activation, and cell death, which occurs because it interacts with multiple cellular factors through motifs or signal sequences present throughout its structure. Although these interactions have not been fully understood at specific levels, studies over more than three decades leave no doubtthatthis domain plays a fundamental role in the biology of the virus and probably the development of the disease. This review describes the studies carried out to date that demonstrate the importance of the CTT, focusing on the motifs responsible for its interactions and its possible roles in the pathogenicity of the infection.

Resumen: el virus de la inmunodeficiencia humana tipo 1 (VIH-1) es el agente etiológico del síndrome de inmunodeficiencia adguirida (SIDA), una pandemia con altos costos económicos y sociales. La glicoproteína de la envoltura (ENV) del virus media el proceso infeccioso al unirse a la célula huésped y entrar en ella, uno de los principales componentes objetivo de los estudios desde su descubrimiento. Su endodominio o cola C-terminal (CTT) participa en procesos tardíos del ciclo replicativo, como tráfico intracelular, activación y muerte celular, lo que ocurre porque interactúa con múltiples factores celulares a través de motivos o secuencias señal presentes en toda su estructura. Aunque estas interacciones no se han entendido completamente a niveles específicos, los estudios durante más de tres décadas no dejan dudas de que este campo juega un papel fundamental en la biología del virus y probablemente en el desarrollo de la enfermedad. Esta revisión describe los estudios realizados hasta la fecha que demuestran la importancia de la CTT, centrándose en los motivos responsables de sus interacciones y sus posibles roles en la patogenicidad de la infección.

Resumo: o vírus da imunodeficiência humana tipo 1 (HIV-1) é o agente etiológico da síndrome da imunodeficiência adquirida (AUXILIA), urna pandemia com elevados custos económicos e sociais. A glicoproteína do envelope (ENV) do vírus media o processo infeccioso ligando-se e entrando na célula hospedeira, um dos principais componentes alvo dos estudos desde sua descoberta. Seu endo domínio ou cauda C-terminal (CTT) participa de processos do ciclo replicativo tardio, como tráfego intracelular, ativação e morte celular, que ocorre porque interage com múltiplos fatores celulares por meio de motivos ou sequências-sinal presentes em toda a sua estrutura. Embora essas interações não tenham sido totalmente compreendidas em níveis específicos, estudos ao longo de mais de três décadas não deixam dúvidas de que esse domínio desempenha um papel fundamental na biologia do vírus e provavelmente no desenvolvimento da doença. Esta revisão descreve os estudos realizados até o momento que demonstram a importância da CTT, com foco nos motivos responsáveis por suas interações e seus possíveis papéis na patogenicidade da infecção.

Eukaryotic Multi-subunit DNA dependent RNA Polymerases: An Insight into Their Active Sites and Catalytic Mechanism

Aim:To analyze the most complex multi-subunit (MSU) DNA dependent RNA polymerases (RNAPs) of eukaryotic organisms and find out conserved motifs, metal binding sites and catalytic regions and propose a plausible mechanism of action for these complex eukaryoticMSU RNAPs, using yeast (Saccharomyces cerevisiae) RNAP II, as a model enzyme.Study Design: Bioinformatics, Biochemical, Site-directed mutagenesis and X-ray crystallographic data were analyzed.Place and Duration of Study: School of Biotechnology, MaduraiKamaraj University, Madurai, India, between 2007-2013. Methodology:Bioinformatics, Biochemical, Site-directed mutagenesis (SDM) and X-ray crystallographic data of the enzyme were analyzed. The advanced version of Clustal Omega was used for protein sequence analysis of the MSU DNA dependent RNAPs from various eukaryotic sources. Along with the conserved motifs identified by the bioinformatics analysis, the data already available by biochemical and SDM experiments and X-ray crystallographic analysis of these enzymes were used to confirm the possible amino acids involved in the active sites and catalysis. Results:Multiple sequence alignment (MSA) of RNAPs from different eukaryotic organisms showed a large number of highly conserved motifs among them. Possible catalytic regions in the catalytic subunits of the yeast Rpb2 (= β in eubacteria) and Rpb1 (= β’ in eubacteria) consist of an absolutely conserved amino acid R, in contrast to a K that was reported for DNA polymerases and single subunit (SSU) RNAPs. However, the invariant ‘gatekeeper/DNA template binding’ YG pair that was reported in all SSU RNAPs, prokaryotic MSU RNAPs and DNA polymerases is also highly conserved in eukaryotic Rpb2 initiation subunits, but unusually a KG pair is found in higher eukaryotes including the human RNAPs. Like the eubacterial initiation subunits of MSU RNAPs, the eukaryotic initiation subunits, viz. Rpb2, exhibit very similar active site and catalytic regions but slightly different distance conservations between the templatebinding YG/KG pair and the catalytic R. In the eukaryotic initiation subunits, the proposed catalytic R is placed at the -9thposition from the YG/KG pair and an invariant R is placed at -5 which are implicated to play a role in nucleoside triphosphate (NTP) selection as reported for SSU RNAPs (viral family) and DNA polymerases. Similarly, the eukaryotic elongation subunits (Rpb1) are also found to be very much homologous to the elongation subunits (β’) of prokaryotes. Interestingly, the catalytic regionsare highly conserved, and the metal binding sites are absolutely conserved as in prokaryotic MSU RNAPs. In eukaryotes, the template binding YG pair is replaced with an FG pair. Another interesting observation is, similar to the prokaryotic β’ subunits, inthe eukaryotic Rpb1 elongation subunits also, the proposed catalytic R is placed double the distance, i.e., -18 amino acids downstream from the FG pair unlike in the SSU RNAPs and DNA polymerases where the distance is only -8 amino acids downstream from the YG pair. Thus, the completely conserved FG pair, catalytic R with an invariant R, at -6thposition are proposed to play a crucial role in template binding, NTP selection and polymerization reactions in the elongation subunits of eukaryotic MSU RNAPs. Moreover, the Zn binding motif with the three completely conserved Cs is also highly conserved in the eukaryotic elongation subunits. Another important difference is that the catalytic region is placed very close to the N-terminal region in eukaryotes.Conclusions: Unlike reported for the DNA polymerases and SSU RNA polymerases, the of eukaryotic MSU RNAPs use an R as the catalytic amino acid and exhibit a different distance conservation in the initiation and elongation subunits. An invariant Zn2+binding motif found in the Rpb1 elongation subunits is proposed to participate in proof-reading function. Differences in the active sites of bacterial and human RNA polymerases may pave the way for the design of new and effective drugs for many bacterial infections, including the multidrug resistant strains which are a global crisis at present

Expressions of Versican and ADAMTS-1 in polycysticovary syndrome patients and their clincal significances / 吉林大学学报(医学版)

Objective: To determine the expressions of Versican and a distintegrin and metalloprotease with thrombospondin type l motifs (ADAMTS-1) in serum in the polycystic ovary syndrome (PCOS) patients, and to clarify their roles in the pathogenesis of PCOS. Methods: A total of 80 patients with PCOS ( PCOS group) and 100 healthy women (control group) were selected. The heights and body weights of the subjects in two groups were measured; and the body mass index (BMI) was calculated. The levels of serum Versican and ADAMTS-1 of the subjects in two groups were measured by enzyme-linked immunosorbent assay (ELISA) method. The levels of serum follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), fasting blood glucose (FBG), fasting insulin of the subjects in two groups were detected; insulin resistance was evaluated according to Homeostasis Model Assessment of Insulin Resistance (HOMA-IR). The correlations between Versican, ADAMTS-1 and the metabolic indexes were analyzed by Pearson linear correlation analysis. Results: The serum Versican level of the patients in PCOS group was significantly decreased compared with control group (P=0.004); the serum ADAMTS-1 level was also decreased (P<0.01). The sensitivities, the specificities and the area under receiver operating characteristic (ROC) curve （AUC）of Versican and ADAMTS-1 in prediction of PCOS were 76.74%?vs 63.64% (P=0.018), 52.94% vs 40.73% (P=0.009) and 0675 (0.550-0.795) vs 0.714 (0.601-0.827) (P=0.032), respectively. The correlation analysis showed that in PCOS group the serum level of Versican of the patients was negatively correlated with FBG (r=-0.738, P=0.022), and ADAMTS-1 was negatively correlated with FBG (r=-0.524, P=0.043). Conclusion； Versican and ADAMTS-1 lowly express in the patients with PCOS. They are negatively correlated with the insulin resistance. They may play inhibitory effects in the occurrence of PCOS.

Expressions of Versican and ADAMTS-1 in polycysticovary syndrome patients and their clincal significances / 吉林大学学报(医学版)

Objective: To determine the expressions of Versican and a distintegrin and metalloprotease with thrombospondin type 1 motifs (ADAMTS-1) in serum in the polycystic ovary syndrome (PCOS) patients, and to clarify their roles in the pathogenesis of PCOS. Methods: A total of 80 patients with PCOS (PCOS group) and 100 healthy women (control group) were selected The heights and body weights of the subjects in two groups were measured; and the body mass index (BMI) was calculated The levels of serum Versican and ADAMTS-1 of the subjects in two groups were measured by enzyme-linked immunosorbent assay (ELISA) method The levels of serum follicle stimulating hormone (FSH), luteinizing hormone (L H), testosterone (T), fasting blood glucose (FBG), fasting insulin of the subjects in two groups were detected; insulin resistance was evaluated according to Homeostasis Model Assessment of Insulin Resistance (HOMA-IR). The correlations between Versican, ADAMTS-1 and the metabolic indexes were analyzed by Pearson linear correlation analysis Results: The serum Versican level of the patients in PCOS group was significantly decreased compared with control group (P = 0 . 004); the serum ADAMTS-1 level was also decreased (P < 0 . 01). The sensitivities, the specificities and the area under receiver operating characteristic (ROC) curve (AUC) of Versican and ADAMTS-1 in prediction of PCOS were 76.74% vs 63. 64% (P = 0 . 018), 52.94% 1)5 40.73% (P = 0 . 009) and 0. 675 (0.550-0.795) vs 0.714 (0 . 6 0 1 - 0. 827) (P = 0 . 032), respectively. The correlation analysis showed that in PCOS group the serum level of Versican of the patients was negatively correlated with FBG (r = - 0 . 7 3 8, P= 0.022), and ADAMTS-1 was negatively correlated with FBG (r = -0.524, P = 0.043). Conclusion: Versican and ADAMTS-1 lowly express in the patients with PCOS. They are negatively correlated with the insulin resistance. They may play inhibitory effects in the occurrence of PCOS.

Electroacupuncture down-regulates expression of ADAMTS-4 of intervertebral disc in rats with lumbar intervertebral disc degeneration / 针刺研究

OBJECTIVE: To observe the effect of electroacupuncture (EA) stimulation on the levels of a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) protein in the lumbar intervertebral disc tissue and serum in prolapsed lumbar intervertebral disc degeneration (PLIDD) rats, so as to explore its mechanism underlying improvement of intervertebral disc degeneration (IDD). METHODS: A total of 48 Sprague-Dawley rats were divided into sham operation (n＝12), model (n＝18) and EA (n＝18) groups. The PLIDD model was established by puncturing the lumbar discs (L4-L5, L5-L6) with a gauge-22 syringe needle. After modeling, EA stimulation was applied to "Pangguangshu"(BL28), "Zusanli"(ST36) and "Zhijian"for 20 min, 6 times per week for 4 weeks. The lumbar intervertebral disc tissue and blood samples were collected at the 4th, 6th and 8th week after modeling, respectively. The expression of ADAMTS-4 in the lumbar intervertebral disc tissue was detected by immunohistochemistry and Western blot (WB), separately. The content of ADAMTS-4 in the serum was detected by ELISA. RESULTS: Both immunohistochemical stain and WB showed that the expression levels of lumbar ADAMTS-4 at the 4th, 6th and 8th week were significantly up-regulated in the model group relevant to the sham operation group (P<0.01). Following EA treatment, the expression levels of ADAMTS-4 on day 14 and 28 were notably lower in the EA group than in the model group (P<0.05). The serum ADAMTS-4 contents were significantly increased in the model group than in the sham operation group at the 3 time-points (P<0.05, P<0.01), and considerably decreased in the EA group than in the model group on day 28 after EA intervention(P<0.05)．. CONCLUSION: EA can down-regulate the ADAMTS-4 expression of lumbar intervertebral disc tissue in PLIDD rats, which may contribute to its effect in relieving lumbar intervertebral disc degeneration.

The correlation of leptin and a disintegrin and metalloproteinase with thrombospondin-like motifs 4 with joint destruction in patients with rheumatoid arthritis / 中华风湿病学杂志

Objective To explore how leptin affects RA,especially those with joint erosion.Methods The study recruited 48 consecutive patients with RA (14 patients with knee joint effusion) and 23 age and sex matched healthy people.RA patients were grouped into low and moderate activity group [2.6＜28-joint disease activity score (DAS28) ≤5.1,n =5] and high activity group (DAS28 ＞5.1,n =43) according DAS28-ESR;Meanwhile,they were grouped into bone erosive group (n=20) and non-erosive group (n=28) according to X-ray of both hands.Demographic data of RA patients were recorded.ELISA was applied to assess leptin and a disintegrin and metalloproteinase with thrombospondin-like motifs (ADAMTS4) in serum and synovial fluid of RA group.Sharp/van der Heijde scores were used to assess bone erosion and joint space narrowing.Leptin and ADAMTS4 from serum and synovial fluid were compared between different groups using t test,Rank sum test,Chi-square test and Analysis of Variance,and we did Pearson and Spearman's Corre-lation analyses between these values and clinical features,lab indicators and radiological scores.Moreover,we did single factor and logistic regression analyses,which facilitated screening risk factors of joint destruction.Results Serum leptin in RA group was significantly higher than that of the control group [8.06(6.24) ng/ml vs 4.62(7.13),Z=-2.113,P=0.035],and leptin was positively correlated with Shar/van der Heijde score (r=0.347,P=0.016).Serum leptin in erosive RA patients was higher than that of the non-erosive patients (Z=-2.070,P=0.038),and there was a positive correlation between leptin and ADAMTS4 only in synovial fluid of RA patients with erosion (r=0.900,P=0.037).It was found in logistic regression results that RA patients with more tender joint counts and elevated leptin were more likely to develop bone erosion [OR=1.229,95％CI (1.007,1.500),P=0.043;OR=1.159,95％CI (1.015,1.324),P=0.030].Conclusion Leptin participates RA joint destruction probably by modulating expression of ADAMTS4.Leptin and tender joint count are independent risk factors for RA with joint destruction.

Role of α2-macroglobulins and a disintegrin and metallo-proteinase with throm-bospondin motifs in pathogenesis of osteoarthritis:a review / 中华地方病学杂志

Osteoarthritis is a chronic disabling disease that tends to occur in middle-aged and elderly population, the main pathological characteristics are the dynamic imbalance between the synthesis and degradation of articular chondrocytes, extracellular matrix and subchondral bone. The occurrence and development is related to many factors. This article reviews the structure, the function and the interaction between α2-macroglobulins and a disintegrin and metallo-proteinase with throm-bospondin motifs in the development of osteoarthritis.

Computational identification and systematic classification of novel GRAS genes in Isatis indigotica / 中国天然药物

Isatis indigotica Fort., belonging to Cruciferae, is one of the most commonly used plants in traditional Chinese medicine. The accumulation of the effective components of I. indigotica is related with its growth conditions. The GRAS genes are members of a multigene family of transcriptional regulators that play a crucial role in plant growth. Although the activities of many GRAS genes have long been recognized, only in recent years were some of them identified and functionally characterized in detail. In the present study, 41 GRAS genes were identified from I. indigotica through bioinformatics methods for the first time. They were classified into ten groups according to the classification of Arabidopsis and rice. The characterization, gene structure, conserved motifs, disordered N-terminal domains, and phylogenetic reconstruction of these GRASs were analyzed. Forty-three orthologous gene pairs were shared by I. indigotica and Arabidopsis, and interaction networks of these orthologous genes were constructed. Furthermore, gene expression patterns were investigated by analysis in methyl jasmonate (MeJA)-treated I. indigotica hairy roots based on RNA-seq data. In conclusion, this comprehensive analysis would provide rich resources for further studies of GRAS protein functions in this plant.

Role of metalloproteinases and their inhibitors in intervertebral disc degeneration: Research progress / 第二军医大学学报

Intervertebral disc degeneration (IVDD) is characterized by the excessive degradation of extracellular matrix (ECM), which underlies many spine-related disorders. Matrix metalloproteinases (MMPs) and a disintegrin metalloproteinases with thrombospondin motifs (ADAMTSs) are believed to be the major proteolytic enzymes responsible for ECM degradation. This review summarizes the current literatures on gene expression and regulation of MMPs, ADAMTSs, and tissue inhibitors of metalloproteinases (TIMPs) in IVDD. Reports have showen that specific MMPs (MMP-1, -2, -3, -7, -8, -10, and -13) and ADAMTS (ADAMTS-1, -4, and -15) are upregulated in human degenerated intervertebral discs. Tissue inhibitor of metalloproteinase-3 is downregulated and TIMP-1 is upregulated in human degenerated intervertebral discs relative to nondegenerated intervertebral discs. Regulation of the MMP and ADAMTS expression is affected by many factors including mechanical, inflammatory, oxidative stress, and so on. Genetic predisposition also plays an important role in expression of MMP-1, -2, -3, and -9. Upregulation of MMP and ADAMTS expression is implicated in ECM destruction, which can lead to the development of IVDD. Future IVDD therapeutics may target specific MMPs and ADAMTSs which is essential in the pathological proteolysis of ECM.

Bioinformatics analysis of biomarkers and transcriptional factor motifs in Down syndrome

In this study, biomarkers and transcriptional factor motifs were identified in order to investigate the etiology and phenotypic severity of Down syndrome. GSE 1281, GSE 1611, and GSE 5390 were downloaded from the gene expression ominibus (GEO). A robust multiarray analysis (RMA) algorithm was applied to detect differentially expressed genes (DEGs). In order to screen for biological pathways and to interrogate the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, the database for annotation, visualization, and integrated discovery (DAVID) was used to carry out a gene ontology (GO) function enrichment for DEGs. Finally, a transcriptional regulatory network was constructed, and a hypergeometric distribution test was applied to select for significantly enriched transcriptional factor motifs. CBR1, DYRK1A, HMGN1, ITSN1, RCAN1, SON, TMEM50B, and TTC3 were each up-regulated two-fold in Down syndrome samples compared to normal samples; of these, SON and TTC3 were newly reported. CBR1, DYRK1A, HMGN1, ITSN1, RCAN1, SON, TMEM50B, and TTC3 were located on human chromosome 21 (mouse chromosome 16). The DEGs were significantly enriched in macromolecular complex subunit organization and focal adhesion pathways. Eleven significantly enriched transcription factor motifs (PAX5, EGR1, XBP1, SREBP1, OLF1, MZF1, NFY, NFKAPPAB, MYCMAX, NFE2, and RP58) were identified. The DEGs and transcription factor motifs identified in our study provide biomarkers for the understanding of Down syndrome pathogenesis and progression.

Molecular determinants of odorant receptor function in insects.

The olfactory system of Drosophila melanogaster provides a powerful model to study molecular and cellular mechanisms underlying function of a sensory system. In the 1970s Siddiqi and colleagues pioneered the application of genetics to olfactory research and isolated several mutant Drosophila with odorant-specific defects in olfactory behaviour, suggesting that odorants are detected differentially by the olfactory system. Since then basic principles of olfactory system function and development have emerged using Drosophila as a model. Nearly four decades later we can add computational methods to further our understanding of how specific odorants are detected by receptors. Using a comparative approach we identify two categories of short amino acid sequence motifs: ones that are conserved family-wide predominantly in the C-terminal half of most receptors, and ones that are present in receptors that detect a specific odorant, 4-methylphenol, found predominantly in the N-terminal half. The odorant-specific sequence motifs are predictors of phenol detection in Anopheles gambiae and other insects, suggesting they are likely to participate in odorant binding. Conversely, the family-wide motifs are expected to participate in shared functions across all receptors and a mutation in the most conserved motif leads to a reduction in odor response. These findings lay a foundation for investigating functional domains within odorant receptors that can lead to a molecular understanding of odor detection.

Transduction motif analysis of gastric cancer based on a human signaling network

To investigate signal regulation models of gastric cancer, databases and literature were used to construct the signaling network in humans. Topological characteristics of the network were analyzed by CytoScape. After marking gastric cancer-related genes extracted from the CancerResource, GeneRIF, and COSMIC databases, the FANMOD software was used for the mining of gastric cancer-related motifs in a network with three vertices. The significant motif difference method was adopted to identify significantly different motifs in the normal and cancer states. Finally, we conducted a series of analyses of the significantly different motifs, including gene ontology, function annotation of genes, and model classification. A human signaling network was constructed, with 1643 nodes and 5089 regulating interactions. The network was configured to have the characteristics of other biological networks. There were 57,942 motifs marked with gastric cancer-related genes out of a total of 69,492 motifs, and 264 motifs were selected as significantly different motifs by calculating the significant motif difference (SMD) scores. Genes in significantly different motifs were mainly enriched in functions associated with cancer genesis, such as regulation of cell death, amino acid phosphorylation of proteins, and intracellular signaling cascades. The top five significantly different motifs were mainly cascade and positive feedback types. Almost all genes in the five motifs were cancer related, including EPOR, MAPK14, BCL2L1, KRT18, PTPN6, CASP3, TGFBR2, AR, and CASP7. The development of cancer might be curbed by inhibiting signal transductions upstream and downstream of the selected motifs.

Bioinformatics analysis of differentially expressed proteins in human lung adenocarcinoma / 中国医师杂志

Objective To investigate the biological significance of differentially expressed proteins from human primary lung adenocarcinoma with lymph node metastasis adenocarcinoma (LNM AdC) and without metastasis (non-LNM AdC) according to clinical diagnosis of lymph node metastasis and distant metastasis,with bioinformatics approach.Methods Cytoscape software was used to analyze a functional enrichment analysis and a protein-protein interaction network from differentially expressed proteins from LNM AdC and non-LNM AdC.Results The top biological processes were related to glucose catabolic process,hexose catabolic process,monosaccharide catabolic process,alcohol catabolic process,and cellular carbohydrate catabolic process.The top molecular functions were related to phospholipase inhibitor activity,lipase inhibitor activity,calcium-dependent phospholipid binding,phosphlipase A2 inhibitor activity,and lipid binding.A protein-protein interaction network of differentially expressed proteins was generated with literature data.Conclusions This bioinformatics analysis demonstrated that glucose catabolic process,alcohol catabolic process,calcium-dependent phospholipid binding,phosphlipase A2 inhibitor activity,ACTB,ANXA1,ANXA2,ANXA3,VCP,NPM1,KRT1,and SUMO4 are significantly associated with a lung adenocarcinoma.These network data provide new insights into the metastasis mechanisms of human lung adenocarcinoma.

DNA Polymerases: An Insight into Their Active Sites and Catalytic Mechanism.

To analyze the active sites of various prokaryotic and eukaryotic DNA polymerases and propose a plausible mechanism of action for the polymerases with the Escherichia coli DNA polymerase I as a model system. Study Design: Bioinformatics, Biochemical and X-ray crystallographic data were analyzed. Place and Duration of Study: Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai – 625 021, India. From 2007 to 2012. Methodology: The advanced version of T-COFFEE was used to analyze both prokaryotic and eukaryotic DNA polymerase sequences. Along with this bioinformatics data, X-ray crystallographic and biochemical data were used to confirm the possible amino acids in the active sites of different types of polymerases from various sources. Results: Multiple sequence analyses of various polymerases from different sources show only a few highly conserved motifs among these enzymes except eukaryotic epsilon polymerases where a large number of highly conserved sequences are found. Possible catalytic/active site regions in all these polymerases show a highly conserved catalytic amino acid K/R and the YG/A pair. A distance conservation is also observed between the active sites. Furthermore, two highly conserved Ds and DXD motifs are also observed. Conclusion: The highly conserved amino acid K/R acts as the proton abstractor in catalysis and the YG/A pair acts as a “steric gate” in selection of only dNTPS for polymerization reactions. The two highly conserved Ds act as the “charge shielder” of dNTPs and orient the alpha phosphate of incoming dNTPs to the 3’-OH end of the growing primer.

Fibronectin decreases its inhibiting activity on disintegrin and metalloproteinase with thrombospondin motifs-4 by citrullination / 中华风湿病学杂志

Objective To observe the inhibiting activities of fibronectin (FN) and citrullinated fibronectin (cFN) on disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4),and to explore the extracellular regulative mechanisms of ADAMTS-4.Methods FN was incubated with peptidylarginine deaminase type 4 (PADI4).Western blotting analysis was used to verify the citrullination of FN.The binding activity of FN and cFN to ADAMTS-4 were investigated by enzyme-linked immunosorbent assay (ELISA).The proteolytic ability of ADAMTS-4 after binding to FN and cFN were measured with the aggrecanase activity assay kit.One-way ANOVA,LSD-t test and t-test were used for statistical analysis.Results The immunosignal of citrulline was detected in FN after incubated with PADI4,but not in the absence of PADI4.A higher absorbance at 405 nm was detected when the full-length ADAMTS-4 protein was incubated with FN (2.182±0.042) than cFN (0.624±0.033; t=50.522,P＜0.01).Additionally,the recombinant ADAMTS-4 protein with a truncation at the C-terminus displayed low absorbance at 405 nm when the enzyme was incubated with both FN(0.971±0.024) and cFN(0.934±0.012; t=2.388,P＞0.05).Large amounts of ARGxx peptide were detected with full-length ADAMTS-4 in aggrecanase activity assay [(0.908±0.088) nmol/L],but significantly less when in the presence of FN and ADAMTS-4 [(0.573±0.000) nmol/L,P＜0.05].The production of this peptide was more when full-length ADAMTS-4 was incubated with cFN [(0.830±0.020) nmol/L,P＜0.05] than with FN.The reaction containing the truncated ADAMTS-4 without FN or cFN yielded the highest concentration of ARGxx peptide [(36.420±3.673) nmol/L],peptide production was not significantly altered when FN [(41.099±0.101) nmol/L] or eFN [(41.064±0.083) nmol/L] were added to the reaction.Conclusion FN could bind to ADAMTS-4 and inhibit its proteolytic activity.After citrullinated by PADI4,the binding activity of cFN is weakened and less inhibition to ADAMTS-4.

Expression of RECK in pancreatic carcinoma and its relationship with prognosis / 中华肝胆外科杂志

Objective To study the expression of reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) in human pancreatic carcinoma tissues and pancreatic carcinoma cell lines; the effects of recombinant lentiviruses carrying RECK gene(LV-RECK) therapy on human pancreatic carcinoma xenograft in nude mice; and to find out the relationship between the expression of RECK and the prognosis of pancreatic carcinoma.Methods Immunohistochemical method was used to detect the expression of RECK in the resected specimens of pancreatic carcinoma and their corresponding normal pancreatic tissues in 42 patients.Western blotting was used to examine the expression of RECK in human pancreatic carcinoma cell lines (PANC-1,MIAPaCa-2,AsPC-1).Statistical analyses were performed to determine the relationship between RECK expression and the clinicopathological characteristics and prognosis in pancreatic carcinoma.Subcutaneous xenograft tumor models of human pancreatic carcinoma were established in nude mice.These nude mice were then divided into the experimental group,the negative control group and the blank control group randomly.The three groups of nude mice were intratumorally injected with LV-RECK,LV-EGFP and normal saline (NS) respectively.The antitumor effect was studied.Immunohistochemical method was used to detect the expression of RECK and microvessel density (MVD).Terminal deoxynucleotidyl transferase mediated dUTP-DIG nick end labeling (TUNEL) was used to detect the apoptosis of tumor cells.Survival analysis was performed.Results All three pancreatic carcinoma cell lines did not express RECK.The overall positive rate of RECK expression was 45.2 ％ (19/42) in pancreatic carcinoma,and 88.1 ％ (37/42) in normal pancreatic tissue.The expression level of RECK was significantly lower in the tumor tissues than in the normal tissues (P＜0.01).The expression of RECK was significantly associated with TNM stage,lymph node metastasis and local infiltration of pancreatic carcinoma (P＜0.05).Kaplan-Meier survival analysis revealed that the survival time was significantly longer in the RECK positive patient group than in the RECK negative patient group.Univariate Cox regression analysis revealed that RECK expression,TNM stage,lymph node metastasis and local infiltration were significantly related with prognosis for pancreatic carcinoma (P＜0.05).Multivariate Cox regression analysis revealed that only RECK expression remained as an independent significant factor in predicting the prognosis of pancreatic carcinoma (P ＜ 0.001).When compared with the negative control and the blank control groups,the volume of subcutaneous xenograft tumor in the experimental group was significantly decreased (P＜0.05).RECK protein in the experimental group was re-expressed.MVD of the experimental group was significantly less than those of the control groups (P＜0.05).Apoptotic index (AI)of the experimental group was significantly higher than those of the control groups (P＜0.05).The survival time of nude mice in the experimental group was significantly longer than those in the control groups (P＜0.05).Conclusions RECK expression was closely related to invasion,metastasis and prognosis of pancreatic carcinoma and it was an independent prognostic marker.RECK gene over-expression inhibited neovascularization of pancreatic carcinoma,induced apoptosis of tumor cells,inhibited the growth of tumor xenograft and improved the prognosis of tumor-bearing mice.These results suggest a possible new treatment for pancreatic carcinoma.

Probing the evolutionary conserved regions within functional site of drug-resistant target proteins of Staphylococcus aureus: In silico phylogenetic motif profiling approach.

Staphylococcus aureus is one of the major causes of clinical infections and increasing mortality due to multi-drug resistance. In this study, eight drug-resistant genes, beta-lactamase, metallo-beta-lactamase, vanB, mecA, norA, qacA, qacB and qacC of S. aureus strain Mu50 (vancomycin resistant) were studied to predict the evolutionary conserved functional site residues in their protein sequences. It was found that in beta-lactamase, Tyr, Gly, Thr, Asn and in metallo-beta-lactamase, Thr, His, Gly, Leu, Arg and Asp residues were highly conserved. In vanB, Gly, His and Asp residues were highly conserved. Whereas in mecA, His, Val, Phe, Gln, Lys and in norA, Ser, Leu and Ala residues showed conservedness at moderate level. In the multi-drug efflux pump (corresponding to qacA, qacB and qacC), Gly residue was found to be highly conserved. The homology clustering of target proteins through SCI-PHY algorithm and homologues identified through PSI-BLAST were compared to identify the degree of conservation of functional residues. The phylogenetic motifs identified using homologues of target proteins were validated through domain search to locate their site and functionality in the protein sequences. Interactome analysis was performed to understand the possible mode of interaction of target proteins with their functional partners.

Roles of SUMOylation and de-SUMOylation in carcinogenesis / 国际肿瘤学杂志

SUMO (small ubiquitin-related modifier) is involved in the post-translational modifications of proteins,and this process is referred to as SUMOylation.SUMOylation plays an important role in the regulation of cellular activities such as strengthening the stability of the protein,nucleocytoplasmic transport,DNA repair,DNA replication,mitotic and meiotic chromosome behavior,et al.SUMOylation is a dynamic process and can be reversed by SUMO-specific proteases (SENP).Once the balance between SUMOylation and de-SUMOylation is broken,there will be an aberrant expression of SUMO or SENPs in cells to happen,which may lead to the tumor occturtrence.