Targeting neuronal PAS domain protein 2 and KN motif/ankyrin repeat domains 1:Advances in type 2 diabetes therapy

This editorial summarizes the latest literature on the roles of neuronal PAS domain protein 2 and KN motif/ankyrin repeat domain 1 in type 2 diabetes(T2D).We highlight their involvement inβ-cell dysfunction,explore their potential as therapeutic targets,and discuss the implications for new treatment strategies.We offer valuable insights into relevant gene regulation and cellular mechanisms relevant for the targeted management of T2D.

GATA binding protein 2 mediated ankyrin repeat domain containing 26 high expression in myeloid-derived cell lines

BACKGROUND Thrombocytopenia 2,an autosomal dominant inherited disease characterized by moderate thrombocytopenia,predisposition to myeloid malignancies and normal platelet size and function,can be caused by 5’-untranslated region(UTR)point mutations in ankyrin repeat domain containing 26(ANKRD26).Runt related transcription factor 1(RUNX1)and friend leukemia integration 1(FLI1)have been identified as negative regulators of ANKRD26.However,the positive regulators of ANKRD26 are still unknown.AIM To prove the positive regulatory effect of GATA binding protein 2(GATA2)on ANKRD26 transcription.METHODS Human induced pluripotent stem cells derived from bone marrow(hiPSC-BM)INTRODUCTION Ankyrin repeat domain containing protein 26(ANKRD26)acts as a regulator of adipogenesis and is involved in the regulation of feeding behavior[1-3].The ANKRD26 gene is located on chromosome 10 and shares regions of homology with the primate-specific gene family POTE.According to the Human Protein Atlas database,the ANKRD26 protein is localized to the Golgi apparatus and vesicles,and its expression can be detected in nearly all human tissues[4].Moreover,UniProt annotation revealed that ANKRD26 is localized in the centrosome and contains coiled-coil domains formed by spectrin helices and ankyrin repeats[5,6].The most common disease related to ANKRD26 is thrombocytopenia 2(THC2),which is a rare autosomal dominant inherited disease characterized by lifelong mild-to-moderate thrombocytopenia and mild bleeding[7-9].Caused by the variants in the 5’-untranslated region(UTR)of ANKRD26,THC2 is defined by a decrease in the number of platelets in circulating blood and results in increased bleeding and decreased clotting ability[8,10].Due to the point mutations that occur in the 5’-UTR of ANKRD26,its negative transcription factors(TFs),Runt related transcription factor 1(RUNX1)and friend leukemia integration 1(FLI1),lose their repression effect[11].The persistent expression of ANKRD26 increases the activity of the mitogen activated protein kinase and extracellular signal regulated kinase 1/2 signaling pathways,which are potentially involved in the regulation of thrombopoietin-dependent signaling and further impair proplatelet formation by megakaryocytes(MKs)[11].However,the positive regulators of ANKRD26,which might be associated with THC2 pathology,are still unknown.

Eukaryotic expression, purification and activity characterization of human soluble DSG2 extracellular domain protein

Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.Methods:The DSG2 extracellular domain fragment gene(DSG2ex),was amplified by PCR,and was inserted into the eukaryotic expression plasmid pCMV3-IgG1 to construct the recombinant eukaryotic expression plasmid-pCMV3-DSG2ex-IgG1.The successfully constructed eukaryotic expression plasmid was transfected into 293T cells to express and secrete DSG2 extracellular domain protein.The targeted protein was purified from the cell culture supernatant by Protein A affinity chromatography and confirmed by Western Blotting and ELISA.Results:The pCMV3-DSG2ex-IgG1 eukaryotic expression plasmid was successfully constructed.The highest protein expression level was obtained with 293T cells after 96 h of transfection.The relative molecular mass of the purified product was between 100 and 130 kDa was estimated by SDS-PAGE,which was consistent with the expectation.The yield of the purified protein reached 0.8 mg/ml with a purity over 90%.The purified DSG2 extracellular domain protein with IgG1 tag was recognized by IgG monoclonal antibodies by Western blotting.Moreover,the ELISA results showed that the prepared DSG2 extracellular domain protein had significant binding activity to human type 55 adenovirus Fiber Knob protein(HAdV-55).Conclusion:A simple and efficient method for eukaryotic expression and purification of human soluble DSG2 extracellular domain protein was successfully established,and biologically active DSG2 extracellular domain protein was purified,which laid the foundation for the later study of its protein function and anti-adenovirus drugs.

Diabetic cardiomyopathy:Importance of direct evidence to support the roles of NOD-like receptor protein 3 inflammasome and pyroptosis

Recently,the roles of pyroptosis,a form of cell death induced by activated NODlike receptor protein 3(NLRP3)inflammasome,in the pathogenesis of diabetic cardiomyopathy(DCM)have been extensively investigated.However,most studies have focused mainly on whether diabetes increases the NLRP3 inflammasome and associated pyroptosis in the heart of type 1 or type 2 diabetic rodent models,and whether various medications and natural products prevent the development of DCM,associated with decreased levels of cardiac NLRP3 inflammasome and pyroptosis.The direct link of NLRP3 inflammasome and associated pyroptosis to the pathogenesis of DCM remains unclear based on the limited evidence derived from the available studies,with the approaches of NLRP3 gene silencing or pharmaceutical application of NLRP3 specific inhibitors.We thus emphasize the requirement for more systematic studies that are designed to provide direct evidence to support the link,given that several studies have provided both direct and indirect evidence under specific conditions.This editorial emphasizes that the current investigation should be circumspect in its conclusion,i.e.,not overemphasizing its role in the pathogenesis of DCM with the fact of only significantly increased expression or activation of NLRP3 inflammasome and pyroptosis in the heart of diabetic rodent models.Only clear-cut evidence-based causative roles of NLRP3 inflammasome and pyroptosis in the pathogenesis of DCM can help to develop effective and safe medications for the clinical management of DCM,targeting these biomarkers.

Modeling the Cysteine Rich Domain of Plant Metallothionein-like Protein

With the progress of plant genome research, more than 50 plant metallothionein_like (MT_L) genes have been found, but only several MT_L proteins have been detected and no experimental structural information for MT_L proteins has been reported so far. Since detailed knowledge of the protein tertiary structure is required to understand its biological function, a method is needed to determine the structure of these proteins. In this study, the structural data of known mammal MT was used to determine the interatomic distance constraints of the CXC and CXXC motifs and the metal_sulfur chelating cluster. Then several possible MT conformations were predicted using a distance geometry algorithm. The statistical analysis was used to select those with much lower target function values and lower conformation energies as the predicted tertiary structural models of the cysteine_rich (CR) domains of these proteins. A suitable prediction method for modeling the CR domain of the plant MT_L protein was constructed. The accurately predicted result for the known structure of an MT protein from blue crab suggests that this method is practicable. The tertiary structures of CR domains of rape MT_L protein LSC54 was then modeled with this method.

Bioinformatic identification of genes encoding C1q-domain-containing proteins in zebrafish

C1q is the first subcomponent of classical pathway in the complement system and a major link between innate and acquired immunities. The globular （gC1q） domain similar with C1q was also found in many non-complement C1q-domain-containing （C1qDC） proteins which have similar crystal structure to that of the multifunctional tumor necrosis factor （TNF） ligand family, and also have diverse functions. In this study, we identified a total of 52 independent gene sequences encoding C1q-domain-containing proteins through comprehensive searches of zebrafish genome, cDNA and EST databases. In comparison to 31 orthologous genes in human and different numbers in other species, a significant selective pressure was suggested during vertebrate evolution. Domain organization of C1q-domain-containing （C1qDC） proteins mainly includes a leading signal peptide, a collagen-like region of variable length, and a C-terminal C1q domain. There are 11 highly conserved residues within the C1q domain, among which 2 are invariant within the zebrafish gene set. A more extensive database searches also revealed homologous C1qDC proteins in other vertebrates, invertebrates and even bacterium, but no homologous sequences for encoding C1qDC proteins were found in many species that have a more recent evolutionary history with zebrafish. Therefore, further studies on C1q-domain-containing genes among different species will help us understand evolutionary mechanism of innate and acquired immunities.

Use Chou’s 5-Steps Rule to Predict Remote Homology Proteins by Merging Grey Incidence Analysis and Domain Similarity Analysis

Detecting remote homology proteins is a challenging problem for both basic research and drug development. Although there are a couple of methods to deal with this problem, the benchmark datasets based on which the existing methods were trained and tested contain many high homologous samples as reflected by the fact that the cutoff threshold was set at 95%. In this study, we reconstructed the benchmark dataset by setting the threshold at 40%, meaning none of the proteins included in the benchmark dataset has more than 40% pairwise sequence identity with any other in the same subset. Using the new benchmark dataset, we proposed a new predictor called “dRHP-GreyFun” based on the grey modeling and functional domain approach. Rigorous cross-validations have indicated that the new predictor is superior to its counterparts in both enhancing success rates and reducing computational cost. The predictor can be downloaded from https://github.com/jcilwz/dRHP-GreyFun.

Identification of the immunogenic domains in HBsAg preS1 region using overlapping preS1 fragment fusion proteins

AIM: The incorporation of hepatitis B virus (HBV) preS1 region into epitope-based vaccines against HBV has been accepted widely, but the incorporate site and size of preS1 sequence is controversial. Therefore our purpose was to further investigate its immunogenic domains for the epitopebased hepatitis B vaccine design.METHODS: Eight GST fusion proteins containing overlapping preS1 fragments in preS1 (21-119) region were expressed in E.coli. Using these purified fusion proteins, the immunogenic domains in preS1 region were identified in detail in mice and humans by Western blot analysis and ELISA.RESULTS: The results in mice showed that the immunogenic domains mainly existed in preS1 (21-59) and preS1 (95-109). Similarly, these fragments had strong immunogenicity in humans; whereas the other parts except for preS1 (60-70) also had some immunogenicity.More importantly, a major immunogenic domain, preS1 (34-59), which has much stronger immunogenicity, was identified. Additionally, the antibodies against some preS1 fragments, especially preS1 (34-59), were speculated to be virus-neutralizing.CONCLUSION: Eight GST fusion proteins containing overlapping preS1 fragments were prepared successfully. They were used for the study on the immunogenic domains in preS1 (21-119) region. The preS1 (34-59) fragments were the major immunogenic domains in the preS1 region, and the antibodies against these fragments were speculated to be virus-neutralizing. Therefore, the incorporation of preS1 (34-59) fragments into epitopebased HBV vaccines may be efficient for enhancement of immune response. Additionally, the results also imply that there are more complex immune responses to preS1 region and more abundant immunogenic domains in humans.

Effect of Methyl-CpG Binding Domain Protein 2(MBD2) on AMD-like Lesions in ApoE-Deficient Mice

Summary： The role of methyl-CpG binding domain protein 2 （MBD2） in an ApoE-deficient mouse model of age-related macular degeneration （AMD） was investigated. Eight-week-old Mbd2/ApoE double deficient （Mbd2^-/- ApoE^-/-） mice （n=12, 24 eyes, experimental group） and MBD2 （wt） ApoE^-/- mice （n=12, 24 eyes, control group） were fed on Western-type diet for 4 months. The mice were sacrificed, and total serum cholesterol levels were analyzed and Bruch＇s membrane （BM） of the eyes was removed for ultrastructural observation by transmission electron microscopy. Moreover, intercellular adhesion molecule 1 （ICAM-1） immunoreactivities were evaluated by fluorescence microscopy in sections of the eyes in both groups for further understanding the function mechanism of MBD2. There was no significant difference in the total serum cholesterol levels between control group and experimental group （P〉0.05）. Transmission electron microscopy revealed that AMD-like lesions, various vacuoles accumulated on BM, notable outer collagenous layer deposits and dilated basal infoldings of retinal pigment epithelium （RPE） were seen in both groups, and the BM in control group was significantly thickened as compared with experimental group （P〈0.05）. Fluorescence micrographs exhibited the expression of ICAM-1 in choroid was higher in control group than in experimental group. We are led to conclude that MBD2 gene knockout may lead to accumulation of more deposits on the BM and influence the pathogenesis of AMD via triggering endothelial activation and inflammatory response in choroid, improving microcirculation, and reducing lipid deposition so as to inhibit the development of AMD-like lesions. Our study helps to provide a new therapeutic approach for the clinical treatment of AMD.

Inhibition of Japanese Encephalitis Virus Infection by Flavivirus Recombinant E Protein Domain Ⅲ

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus closely related to the human pathogens including yellow fever virus, dengue virus and West Nile virus. There are currently no effective antiviral therapies for all of the flavivirus and only a few highly effective vaccines are licensed for human use. In this paper, the E protein domain III (DIII) of six heterologous flaviviruses (DENV1-4, WNV and JEV) was expressed in Escherichia coli successfully. The proteins were purified after a solubilization and refolding procedure, characterized by SDS-PAGE and Western blotting. Competitive inhibition showed that all recombinant flavivirus DIII proteins blocked the entry of JEV into BHK-21 cells. Further studies indicated that antibodies induced by the soluble recombinant flavivirus DIII partially protected mice against lethal JEV challenge. These results demonstrated that recombinant flavivirus DIII proteins could inhibit JEV infection competitively, and immunization with proper folding flavivirus DIII induced cross-protection against JEV infection in mice, implying a possible role of DIII for the cross-protection among flavivirus as well as its use in antigens for immunization in animal models.

Expression and Identification of Inclusion Forming-related Domain of NS80 Nonstructural Protein of Grass Carp Reovirus

Grass carp reovirus （GCRV）, a double stranded RNA virus that infects aquatic animals, often with disastrous effects, belongs to the genus Aquareovirus and family Reoviridea. Similar to other reoviruses, genome replication of GCRV in infected cells occurs in cytoplasmic inclusion bodies, also called viral factories. Sequences analysis revealed the nonstmctural protein NS80, encoded by GCRV segment 4, has a high similarity with μNS in MRV（Mammalian orthoreovimses）, which may be associated with viral factory formation. To understand the function of the μNS80 protein in virus replication, the initial expression and identification of the immunogenicity of the GCRV NS80 protein inclusion forming-related region （335-742） was investigated in this study. It is shown that the over-expressed fusion protein was produced by inducing with IPTG at 28℃. In addition, serum specific rabbit antibody was obtained by using super purified recombinant NS80（335-742） protein as antigen. Moreover, the expressed protein was able to bind to anti-his-tag monoclonal antibody （mouse） and NS80〈335.742） specific rabbit antibody. Further western blot analysis indicates that the antiserum could detect NS80 or NS80C protein expression in GCRV infected cells. This data provides a foundation for further investigation of the role of NS80 in viral inclusion formation and virion assembly.

Receptor-binding domain of SARS-Cov spike protein: Soluble expression in E.coli, purification and functional characterization

AIM： To find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus （SARS-Cov）, and to analyze its receptor binding ability. METHODS： Three fusion tags （glutathione S-transferase, GST; thioredoxin, Trx; maltose-binding protein, MBP）, which preferably contributes to increasing solubility and to facilitating the proper folding of heteroprotein, were used to acquire the soluble and functional expression of RBD protein in Escherichia coli （BL21（DE3） and Rosetta-gamiB （DE3） strains）. The receptor binding ability of the purified soluble RBD protein was then detected by ELISA and flow cytometry assay. RESULTS： RBD of SARS-Cov spike protein was expressed as inclusion body when fused as TrxA tag form in both BL21 （DE3） and Rosetta-gamiB （DE3） under many different cultures and induction conditions. And there was no visible expression band on SDS-PAGE when RBD was expressed as MBP tagged form. Only GST tagged RBD was soluble expressed in BL21（DE3）, and the protein was purified by AKTA Prime Chromatography system. The ELISA data showed that GST.RBD antigen had positive reaction with anti-RBD mouse monoclonal antibody 1A5. Further flow cytometry assay demonstrated the high efficiency of RBD＇s binding ability to ACE2 （angiotensin-converting enzyme 2） positive Vero E6 cell. And ACE2 was proved as a cellular receptor that meditated an initial-affinity interaction with SARS-Cov spike protein. The geometrical mean of GST and GST.RBD binding to Vero E6 cells were 77.08 and 352.73 respectively. CONCLUSION： In this paper, we get sufficient soluble N terminal GST tagged RBD protein expressed in EcoliBL21 （DE3）; data from ELISA and flow cytometry assay demonstrate that the recombinant protein is functional and binding to ACE2 positive Vero E6 cell efficiently. And the recombinant RBD derived from E.coli can be used to developing subunit vaccine to block S protein binding with receptor and to neutralizing SARS-Cov infection.

Mutual regulation between microRNA-373 and methyl-CpGbinding domain protein 2 in hilar cholangiocarcinoma

AIM:To investigate the reciprocal modulation between microRNA(miRNA) and DNA methylation via exploring the correlation between miR-373 and methyl-CpGbinding domain protein(MBD)2.METHODS:MiR-373 expression was examined using the TaqMan miRNA assay.Methylation of miR-373 was investigated using methylation-specific polymerase chain reaction,and recruitment of methyl binding proteins was studied using the chromatin immunoprecipitation assay.Mutation analysis was conducted using the QuikChange Site-Directed Mutagenesis kit.The activity of miR-373 gene promoter constructs and targeting at MBD2-three prime untranslated region(3'UTR) by miR-373 were evaluated by a dual-luciferase reporter gene assay.RESULTS:In hilar cholangiocarcinoma,miR-373 decreased and was closely associated with poor cell differentiation,advanced clinical stage,and shorter survival.The promoter-associated CpG island of miR-373 gene was hypermethylated and inhibited expression of miR-373.MBD2 was up-regulated and enriched at the promoter-associated CpG island of miR-373.Methylation-mediated suppression of miR-373 required MBD2 enrichment at the promoter-associated CpG island,and miR-373 negatively regulated MBD2 expression through targeting the 3'UTR.CONCLUSION:MiR-373 behaves as a direct transcriptional target and negative regulator of MBD2 activity through a feedback loop of CpG island methylation.

Protein-protein Interaction Between Domains of PDZ and BAR from PICK1

Two DNA fragments encoding PDZ domain （21-110 residues） and BAR domain （ 150-360 residues） from PICK1 （1-416 residues） were amplified by PCR and then introduced into vectors, pET-32M and pMAL-e2X respectively to generate recombinant plasmids, pE-pdz and pM-bar. Having been separately transferred into the hosts E. coli BL21 and E. coli JM109, these two strains can express fusion proteins： His-tagged PDZ（PDZ domain） and maltose binding protein-BAR（ MBP-BAR domain） respectively, as confirmed by both SDS-PAGE and Wostem blotting. The interaction between these two domains is dose-dependence, as identified by a pull-down test. Moreover, it has been shown from the ELISA analysis that the actual amount of PDZ bound to MBP-BAR-amylose beads reaches （ 16 ± 0. 5）%, as calculated by the molar ratio of PDZ to MBP-BAR. In addition, the interaction between BAR（bait） and PDZ（prey） in vivo was also examined with a yeast two-hybrid system.

Chromodomain-helicase-DNA binding protein 5, 7 and pronecrotic mixed lineage kinase domain-like protein serve as potential prognostic biomarkers in patients with resected pancreatic adenocarcinomas

Pancreatic cancer is one of the deadliest cancers with a very poor prognosis. Recently, there has been a significant increase in research directed towards identifying potential biomarkers that can be used to diagnose and provide prognostic information for pancreatic cancer. These markers can be used clinically to optimize and personalize therapy for individual patients. In this review, we focused on 3 biomarkers involved in the DNA damage response pathway and the necroptosis pathway: Chromodomainhelicase-DNA binding protein 5, chromodomain-helicaseDNA binding protein 7, and mixed lineage kinase domain-like protein. The aim of this article is to review present literature provided for these biomarkers and current studies in which their effectiveness as prognostic biomarkers are analyzed in order to determine their future use as biomarkers in clinical medicine. Based on the data presented, these biomarkers warrant further investigation,and should be validated in future studies.

Comparative analysis of protein kinases and associated domains between Ascomycota and Basidiomycota

Protein kinases play an important role in every aspect of cellular life.In this study,we systemically identified protein kinases from the predicted proteomes of 59 representative fungi from Ascomycota and Basidiomycota.Comparative analysis revealed that fungi from Ascomycota and Basidiomycota differed in the number and variety of protein kinases.Some groups of protein kinases,such as calmodulin/calcium regulated kinases(CMGC) and those with the highest group percentages are the most prevalent protein kinases among all fungal species tested.In contrast,the STE group(homologs of the yeast STE7,STE11 and STE20 genes),was more abundant in Basidiomycetes than in Ascomycetes.Importantly,the distribution of some protein kinase families appeared to be subphylum-specific.The tyrosine kinase-like(TKL) group had a higher protein kinase density in Agaricomycotina fungi.In addition,the distribution of accessory domains,which could have functional implications,demonstrated that usage bias varied between the two phyla.Principal component analysis revealed a divergence between the main functional domains and associated domains in fungi.This study provides novel insights into the variety and expansion of fungal protein kinases between Ascomycota and Basidiomycota.

Bioinformatic Survey of S-Layer Proteins in Bifidobacteria

Surface layer (S-layer) proteins are one of the most commonly observed cell envelope components in both Archaea and Bacteria. It has versatile functions and holds considerable application potential in biotechnology. Bifidobacteria are representative probiotics conferring health promoting properties. However, there is little study of S-layer in bifidobacteria yet. The distribution and characteristics of S-layer in bifidobacteria are unknown. In this study, search for S-layer protein in the identical protein groups in NCBI yielded 49 hits belonging to bifidobacteria. These proteins were annotated as either “S-layer (domain) protein” or “putative S-layer (y) domain protein” that distributed among 26 species of Bifidobacterium genus. Multiple alignments suggest S-layer proteins are relatively conservative. Phylogenetic analysis of 24 S-layer (domain) protein sequences groups them into three distinct clusters, with the majority species in Cluster-2. S-layer (domain) protein has a universe motif DUF4381, though its function is unknown. Meanwhile, two other motifs CARDB and EphA2_TM involved in cell adhesion and cell signaling respectively, presented in most S-layer (domain) protein in bifidobacteria. All S-layer proteins have a typical N-terminal Sec-dependent signal peptide and a C-terminal trans-membrane region. Homological modeling of representative S-layer proteins from each cluster revealed a few unique structural features. All representative S-layer proteins have a plenty of β-meander motif that exclusively composed by β-barrel structural architectures linked together by hairpin loops.

Research of BH3 domain protein inducing cell apoptosis

Objective BH3 domain protein plays an important role in control mechanism of cell apoptosis.The article mainly discusses its mechanism of promoting cell apoptosis and control.Methods The article analyzed and evaluated the mechanism of BH3 domain protein promoting cell apoptosis by internal and overseas literature.Results Activation of BH3 domain protein could promote the increase of mitochondrial membrane permeability,then it would start mitochondrial apoptosis pathway,and at the last the cell apoptosis.Conclusions BH3 domain protein is the necessary condition of starting cell apoptosis.Its activation can cause cell apoptosis.

Two memory associated genes regulated by amyloid precursor protein intracellular domain Novel insights into the pathogenesis of learning and memory impairment in Alzheimer's disease

In this study, we employed chromatin immunoprecipitation, a useful method for studying the locations of transcription factors bound to specific DNA regions in specific cells, to investigate amyloid precursor protein intracellular domain binding sites in chromatin DNA from hippocampal neurons of rats, and to screen out five putative genes associated with the learning and memory functions. The promoter regions of the calcium/calmodulin-dependent protein kinase II alpha and glutamate receptor-2 genes were amplified by PCR from DNA products immunoprecipitated by amyloid precursor protein intracellular domain. An electrophoretic mobility shift assay and western blot analysis suggested that the promoter regions of these two genes associated with learning and memory were bound by amyloid precursor protein intracellular domain （in complex form）. Our experimental findings indicate that the amyloid precursor protein intracellular domain is involved in the transcriptional regulation of learning- and memory-associated genes in hippocampal neurons. These data may provide new insights into the molecular mechanism underlying the symptoms of progressive memory loss in Alzheimer＇s disease.

Evolution of a protein domain interaction network

In this paper, we attempt to understand complex network evolution from the underlying evolutionary relationship between biological organisms. Firstly, we construct a Pfam domain interaction network for each of the 470 completely sequenced organisms, and therefore each organism is correlated with a specific Pfam domain interaction network; secondly, we infer the evolutionary relationship of these organisms with the nearest neighbour joining method; thirdly, we use the evolutionary relationship between organisms constructed in the second step as the evolutionary course of the Pfam domain interaction network constructed in the first step. This analysis of the evolutionary course shows： （i） there is a conserved sub-network structure in network evolution; in this sub-network, nodes with lower degree prefer to maintain their connectivity invariant, and hubs tend to maintain their role as a hub is attached preferentially to new added nodes; （ii） few nodes are conserved as hubs; most of the other nodes are conserved as one with very low degree; （iii） in the course of network evolution, new nodes are added to the network either individually in most cases or as clusters with relative high clustering coefficients in a very few cases.