Bordetella pertussis autotransporter Vag8 binds human C1 esterase inhibitor and confers serum resistance

Bordetella pertussis employs numerous strategies to evade the immune system, including the ability to resist killing via complement. Previously we have shown that B. pertussis binds a complement regulatory protein, C1 esterase inhibitor (C1inh) to its surface in a Bvg-regulated manner (i.e. during its virulence phase), but the B. pertussis factor was not identified. Here we set out to identify the B. pertussis C1inh-binding factor. Using a serum overlay assay, we found that this factor migrates at approximately 100 kDa on an SDS-PAGE gel. To identify this factor, we isolated proteins of approximately 100 kDa from wild type strain BP338 and from BP347, an isogenic Bvg mutant that does not bind C1inh. Using mass spectrometry and bioinformatics, we identified the autotransporter protein Vag8 as the putative C1inh binding protein. To prove that Vag8 binds C1inh, vag8 was disrupted in two different B. pertussis strains, namely BP338 and 18-323, and the mutants were tested for their ability to bind C1inh in a surface-binding assay. Neither mutant strain was capable of binding C1inh, whereas a complemented strain successfully bound C1inh. In addition, the passenger domain of Vag8 was expressed and purified as a histidine-tagged fusion protein and tested for C1inh-binding in an ELISA assay. Whereas the purified Vag8 passenger bound C1inh, the passenger domain of BrkA (a related autotransporter protein) failed to do so. Finally, serum assays were conducted to compare wild type and vag8 mutants. We determined that vag8 mutants from both strains were more susceptible to killing compared to their isogenic wild type counterparts. In conclusion, we have discovered a novel role for the previously uncharacterized protein Vag8 in the immune evasion of B. pertussis. Vag8 binds C1inh to the surface of the bacterium and confers serum resistance.     

A Novel IgM‐capture enzyme‐linked immunosorbent assay using recombinant Vag8 fusion protein for the accurate and early diagnosis of Bordetella pertussis infection

An ELISA that measures anti‐PT IgG antibody has been used widely for the serodiagnosis of pertussis; however, the IgG‐based ELISA is inadequate for patients during the acute phase of the disease because of the slow response of anti‐PT IgG antibodies. To solve this problem, we developed a novel IgM‐capture ELISA that measures serum anti‐Bordetella pertussis Vag8 IgM levels for the accurate and early diagnosis of pertussis. First, we confirmed that Vag8 was highly expressed in all B. pertussis isolates tested (n = 30), but little or none in other Bordetella species, and that DTaP vaccines did not induce anti‐Vag8 IgG antibodies in mice (i.e. the antibody level could be unaffected by the vaccination). To determine the immune response to Vag8 in B. pertussis infection, anti‐Vag8 IgM levels were compared between 38 patients (acute phase of pertussis) and 29 healthy individuals using the anti‐Vag8 IgM‐capture ELISA. The results revealed that the anti‐Vag8 IgM levels were significantly higher in the patients compared with the healthy individuals (P < 0.001). ROC analysis also showed that the anti‐Vag8 IgM‐capture ELISA has higher diagnostic accuracy (AUC, 0.92) than a commercial anti‐PT IgG ELISA kit. Moreover, it was shown that anti‐Vag8 IgM antibodies were induced earlier than anti‐PT IgG antibodies on sequential patients’ sera. These data indicate that our novel anti‐Vag8 IgM‐capture ELISA is a potentially useful tool for making the accurate and early diagnosis of B. pertussis infection.     

Degradation of C1-inhibitor by plasmin: implications for the control of inflammatory processes.

BACKGROUND: A correct balance between protease and inhibitor activity is critical in the maintenance of homoeostasis; excessive activation of enzyme pathways is frequently associated with inflammatory disorders. Plasmin is an enzyme ubiquitously activated in inflammatory disorder, and C1-inhibitor (C1-Inh) is a pivotal inhibitor of protease activity, which is particularly important in the regulation of enzyme cascades generated in plasma. The nature of the interaction between plasmin and C1-Inh is poorly understood. MATERIALS AND METHODS: C1-Inh was immunoadsorbed from the plasma of normal individuals (n = 21), from that of patients with systemic lupus erythematosus (n = 18) or adult respiratory distress syndrome (n = 9), and from the plasma and synovial fluid of patients with rheumatoid arthritis (n = 18). As plasmin is a putative enzyme responsible for C1-Inh was examined using SDS-PAGE. In addition, peptides cleaved from C1-Inh by plasmin were isolated and sequenced and the precise cleavage sites determined from the known primary sequence of C1-Inh. Homology models of C1-Inh were then constructed. RESULTS: Increased levels of cleaved and inactivated C1-Inh were found in each of the inflammatory disorders examined. Through SDS-PAGE analysis it was shown that plasmin rapidly degraded C1-Inh in vitro. The pattern of C1-Inh cleavage seen in vivo in patients with inflammatory disorders and that produced in vitro following incubation with plasmin were very similar. Homology models of C1-Inh indicate that the majority of the plasmin cleavage sites are adjacent to the reactive site of the inhibitor. CONCLUSIONS: This study suggests that local C1-Inh degradation by plasmin may be a central and critical event in the loss of protease inhibition during inflammation. These findings have important implications for our understanding of pathogenic mechanisms in inflammation and for the development of more effectively targeted therapeutic regimes. These findings may also explain the efficacy of anti-plasmin agents in the treatment of C1-Inh deficiency states, as they may diminish plasmin-mediated C1-Inh degradation.     

Inactivation of the proteolytic activity of mouse nerve growth factor by human C1(activated)-inhibitor

The interaction between the serine protease gamma subunit of NGF (gamma-NGF) and human C1(activated)-inhibitor (C1-Inh) has been studied. C1-Inh inactivates the protease activity of gamma-NGF as measured by its ability to cleave the synthetic substrate benzoyl-arginine-p-nitroanilide (L-BAPNA). Experiments in which gamma-NGF and C1-Inh were mixed at differing molar ratios indicated that inhibition was due to the formation of a 1:1 stoichiometric complex. Analysis of the interaction of 125I-labeled gamma-NGF with C1-Inh by SDS-PAGE and autoradiography indicated that a covalent bond was formed between gamma-NGF and C1-Inh. The covalent bond was hydrolyzed by hydroxylamine, which suggested that the two proteins were linked via an acyl linkage. The formation of this complex was time dependent and required the proteolytic activity of the gamma-NGF.     

Recombinant human C1-inhibitor produced in Pichia pastoris has the same inhibitory capacity as plasma C1-inhibitor

Therapeutic application of the serpin C1-inhibitor (C1-Inh) in inflammatory diseases like sepsis, acute myocardial infarction and vascular leakage syndrome seems promising, but large doses may be required. Therefore, a high-yield recombinant expression system for C1-Inh is very interesting. Earlier attempts to produce high levels of C1-Inh resulted in predominantly inactive C1-Inh. We describe the high yield expression of rhC1-Inh in Pichia pastoris, with 180 mg/l active C1-Inh at maximum. On average, 30 mg/l of 80-100% active C1-Inh was obtained. Progress curves were used to study the interaction with C1s, kallikrein, coagulation factor XIIa and XIa, and demonstrated that rhC1-Inh had the same inhibitory capacity as plasma C1-Inh. Structural integrity, as monitored via heat stability, was comparable despite differences in extent and nature of glycosylation. We conclude that the P. pastoris system is capable of high-level production of functionally and structurally intact human C1 inhibitor.     

Primary structure of the reactive site of human C1-inhibitor

Human C1-inhibitor (C1-Inh) forms an equimolar complex with complement proteinase C1s that is resistant to dissociation by sodium dodecyl sulfate. The formation of this stable complex results in the cleavage of a peptide bond near the carboxyl terminus of the inhibitor and, whereas the bulk of C1-Inh remains covalently bound to the light chain of C1s, the postcomplex inhibitor peptide can be isolated under denaturing conditions. We have sequenced the amino-terminal region of this peptide and deduced that it represents the carboxyl-terminal side of the reactive site of C1-Inh. Limited proteolysis of C1-Inh by Crotalus atrox protease results in an active derivative lacking an amino-terminal peptide of 36 residues. Further proteolysis of this derivative with Pseudomonas aeruginosa elastase inactivates the inhibitor and a peptide is released. The amino-terminal sequence of this peptide overlaps with that of the postcomplex peptide and indicates that the residue imparting primary specificity to the inhibitor is arginine.     

Changes in protein conformation and stability accompany complex formation between human C1 inhibitor and C1-s

The fluorescence spectrum of C1 inhibitor (C1-Inh) in aqueous buffer has a maximum at 324 nm which shifts to 358 nm in 6.0 M guanidinium chloride (GdmC1), indicating that fluorescent tryptophans are buried in the native protein. When titrated with GdmC1, the fluorescence intensity, polarization, and emission maximum of C1-Inh and C1-s exhibited clear transitions which were more prominent than those of the enzyme-inhibitor complex. Two of the variables (intensity and emission maximum) suggest biphasic unfolding of C1-Inh. Differential absorption measurements and sodium iodide quenching of intrinsic fluorescence were consistent with a net increase in the exposure of tryptophans and tyrosines upon complex formation. This reaction, i.e., complex formation, was also accompanied by an increase in the ability to enhance the fluorescence of the hydrophobic probe 8-anilino-1-naphthalenesulfonate. Fluorescence assays of heat denaturation showed transitions at 40 and 52 degrees C for C1-s and at 60 degrees C for C1-Inh whereas there was no detectable melting transition for the complex. Similarly, differential scanning calorimetric measurements revealed transitions at 42, 52, and 62 degrees C for C1-s and one transition at 60 degrees C for C1-Inh, with no major transitions detectable for the complex. The ratio of the calorimetric enthalpy to the apparent van't Hoff enthalpy for thermal unfolding of C1-Inh was 1.6. Taken together, these results suggest that C1-Inh and C1-s are each composed of at least two independently unfolding domains and that complex formation, which involves conformational change, yields a protein substantially more stable than either component alone.     

The functional integrity of the serpin domain of C1-inhibitor depends on the unique N-terminal domain,as revealed by a pathological mutant

C1-inhibitor (C1-Inh) is a serine protease inhibitor (serpin) with a unique, non-conserved N-terminal domain of unknown function. Genetic deficiency of C1-Inh causes hereditary angioedema. A novel type of mutation (Delta 3) in exon 3 of the C1-Inh gene, resulting in deletion of Asp62-Thr116 in this unique domain, was encountered in a hereditary angioedema pedigree. Because the domain is supposedly not essential for inhibitory activity, the unexpected loss-of-function of this deletion mutant was further investigated. The Delta 3 mutant and three additional mutants starting at Pro76, Gly98, and Ser115, lacking increasing parts of the N-terminal domain, were produced recombinantly. C1-Inh76 and C1-Inh98 retained normal conformation and interaction kinetics with target proteases. In contrast, C1-Inh115 and Delta 3, which both lack the connection between the serpin and the non-serpin domain via two disulfide bridges, were completely non-functional because of a complex-like and multimeric conformation, as demonstrated by several criteria. The Delta 3 mutant also circulated in multimeric form in plasma from affected family members. The C1-Inh mutant reported here is unique in that deletion of an entire amino acid stretch from a domain not shared by other serpins leads to a loss-of-function. The deletion in the unique N-terminal domain results in a "multimerization phenotype" of C1-Inh, because of diminished stability of the central beta-sheet. This phenotype, as well as the location of the disulfide bridges between the serpin and the non-serpin domain of C1-Inh, suggests that the function of the N-terminal region may be similar to one of the effects of heparin in antithrombin III, maintenance of the metastable serpin conformation.     

Effect of reactive site loop elongation on the inhibitory activity of C1-inhibitor

The serine protease inhibitor C1-Inhibitor (C1-Inh) inhibits several complement- and contact-system proteases, which play an important role in inflammation. C1-Inh has a short reactive site loop (RSL) compared to other serpins. RSL length determines the inhibitory activity of serpins. We investigated the effect of RSL elongation on inhibitory activity of C1-Inh by insertion of one or two alanine residues in the RSL. One of five mutants had an increased association rate with kallikrein, but was nevertheless a poor inhibitor because of a simultaneous high stoichiometry of inhibition (>10). The association rate of the other variants was lower than that of wild-type C1-Inh. These data suggest that the relatively weak inhibitory activity of C1-Inh is not the result of its short RSL. The short RSL of C1-Inh has, surprisingly, the optimal length for inhibition.     

Domain III of Bacillus thuringiensis Cry1Ie Toxin Plays an Important Role in Binding to Peritrophic Membrane of Asian Corn Borer

The insecticidal IE648 toxin is a truncated Cry1Ie protein with increased toxicity against Asian corn borer (ACB). Cry toxins are pore-forming toxins that disrupt insect midgut cells to kill the larvae. However, the peritrophic membrane (PM) is an important barrier that Cry toxins must cross before binding to midgut cells. Previously, it was shown that Cry toxins are able to bind and accumulate in the PM of several lepidopteran insects. Binding of IE648 toxin to PM of ACB was previously reported and the goal of the current work was the identification of the binding region between Cry1Ie and the PM of ACB. Homologous competition binding assays showed that this interaction was specific. Heterologous competition binding assays performed with different fragments corresponding to domain I, domain II and domain III allowed us to identify that domain III participates in the interaction of IE648 with the PM. Specifically, peptide D3-L8 (corresponding to Cry1Ie toxin residues 607 to 616), located in an exposed loop region of domain III is probably involved in this interaction. Ligand blot assays show that IE648 interact with chitin and PM proteins with sizes of 30, 32 and 80 kDa. The fact that domain III interacts with proteins of similar molecular masses supports that this region of the toxin might be involved in PM interaction. These data provide for the first time the identification of domain III as a putative binding region between PM and 3D-Cry toxin.     

Vitronectin binds to the head region of Moraxella catarrhalis ubiquitous surface protein A2 and confers complement‐inhibitory activity

The serum resistance of the common respiratory pathogen Moraxella catarrhalis is mainly dependent on ubiquitous surface proteins (Usp) A1 and A2 that interact with complement factor 3 (C3) and complement inhibitor C4b binding protein (C4BP) preventing the alternative and classical pathways of the complement system respectively. UspA2 also has the capacity to attract vitronectin that in turn binds C9 and hereby inhibits membrane attack complex (MAC) formation. We found UspA2 as a major vitronectin binding protein and hence the UspA2/vitronectin interaction was studied in detail. The affinity constant (K_D) for vitronectin binding to UspA2 was 2.3 × 10^?8 M, and the N‐terminal region encompassing residues UspA2 30–170 bound vitronectin with a K_D of 7.9 × 10^?8 M. Electron microscopy verified that the active binding domain (UspA2^30–177) was located at the head region of UspA2. Experiments with recombinantly expressed vitronectin also revealed that UspA2^30–177 bound to the C‐terminal region of vitronectin residues 312–396. Finally, when human serum was pre‐incubated with UspA2, bacteria showed significantly less serum resistance. Our study directly reveals the binding mode between the N‐terminal domain of UspA2 and the C‐terminal part of vitronectin and thus sheds light upon the mechanism of M. catarrhalis‐dependent serum resistance.     

Serum resistance in bvg-regulated mutants of Bordetella pertussis

Serum resistance, or resistance to killing by antibody dependent pathway of complement, in Bordetella pertussis is bvg-regulated and the Bordetella resistance to killing (brk) locus mediates much of the resistance. Here we examined whether other bvg-regulated proteins contribute to serum resistance. We found that neither pertussis toxin, adenylate cyclase toxin, filamentous hemagglutinin, dermonecrotic toxin, tracheal colonization factor, nor Vag8 mutants were sensitive to serum killing compared to the wild-type. Filamentous hemagglutinin has been reported to bind C4 binding protein, an inhibitor of complement, but this activity does not appear to contribute to serum resistance, as evidenced by the resistant phenotype of FHA mutants. Clinical isolates were serum resistant and wild-type strains possessing an additional copy of the brk locus were 2–5-fold more resistant to serum killing.     

Human Complement Regulators C4b-Binding Protein and C1 Esterase Inhibitor Interact with a Novel Outer Surface Protein of Borrelia recurrentis

The spirochete Borrelia recurrentis is the causal agent of louse-borne relapsing fever and is transmitted to humans by the infected body louse Pediculus humanus. We have recently demonstrated that the B. recurrentis surface receptor, HcpA, specifically binds factor H, the regulator of the alternative pathway of complement activation, thereby inhibiting complement mediated bacteriolysis. Here, we show that B. recurrentis spirochetes express another potential outer membrane lipoprotein, termed CihC, and acquire C4b-binding protein (C4bp) and human C1 esterase inhibitor (C1-Inh), the major inhibitors of the classical and lectin pathway of complement activation. A highly homologous receptor for C4bp was also found in the African tick-borne relapsing fever spirochete B. duttonii. Upon its binding to B. recurrentis or recombinant CihC, C4bp retains its functional potential, i.e. facilitating the factor I-mediated degradation of C4b. The additional finding that ectopic expression of CihC in serum sensitive B. burgdorferi significantly increased spirochetal resistance against human complement suggests this receptor to substantially contribute, together with other known strategies, to immune evasion of B. recurrentis.     

Chimeric and truncated forms of human complement protein C8 alpha reveal binding sites for C8 beta and C8 gamma within the membrane attack complex/perforin region.

Human C8 is one of five components of the membrane attack complex of complement. It is an oligomeric protein composed of three subunits (C8 alpha, C8 beta, and C8 gamma) that are derived from different genes. C8 alpha and C8 beta are homologous and both contain a pair of tandemly arranged N-terminal modules [thrombospondin type 1 (TSP1) + low-density lipoprotein receptor class A (LDLRA)], an extended middle segment referred to as the membrane attack complex/perforin region (MACPF), and a pair of C-terminal modules [epidermal growth factor (EGF) + TSP1]. During biosynthetic processing, C8 alpha and C8 gamma associate to form a disulfide-linked dimer (C8 alpha-gamma) that binds to C8 beta through a site located on C8 alpha. In this study, the location of binding sites for C8 beta and C8 gamma and the importance of the modules in these interactions were investigated by use of chimeric and truncated forms of C8 alpha in which module pairs were either exchanged for those in C8 beta or deleted. Results show that exchange or deletion of one or both pairs of modules does not abrogate the ability of C8 alpha to form a disulfide-linked dimer when coexpressed with C8 gamma in COS cells. Furthermore, each chimeric and truncated form of C8 alpha-gamma retains the ability to bind C8 beta; however, only those containing the TSP1 + LDLRA modules from C8 alpha are hemolytically active. These results indicate that binding sites for C8 beta and C8 gamma reside within the MACPF region of C8 alpha and that interaction with either subunit is not dependent on the modules. They also suggest that the N-terminal modules in C8 alpha are important for C9 binding and/or expression of C8 activity.     

Structure and the evolutionary implication of the triplicated complement factor B genes of a urochordate ascidian, Ciona intestinalis

To elucidate the evolution of the complement system and MHC class III region, we analyzed the complement factor B (Bf) genes of a urochordate ascidian, Ciona intestinalis. Three different cDNA species, termed CiBf-1, CiBf-2 and CiBf-3, were identified. The deduced amino-acid sequences all contained the usual domains of vertebrate Bf and, in addition, three extra domains at the N-terminus. Furthermore, the serine protease domain of these CiBfs shared unique features with vertebrate complement components C1r/s and mannose-binding lectin-associated serine protease (MASP)-2/3, the absence of the disulfide bond designated histidine loop, and the usage of the AGY codon for the catalytic serine residue. These results indicate that complement genes have evolved through extensive exon shuffling events in the early stage of chordate evolution. Overall deduced amino-acid identity between CiBf-1 and -2 was 88%, whereas CiBf-3 showed 49% identity to both CiBf-1 and CiBf-2. These three CiBf genes were located within an approximately 50-kb genomic region, and exons 3 and 5 of all the three Bf genes showed an extremely high degree of nucleotide identity, indicating that the CiBf genes experienced extensive reorganization, such as duplication and gene conversion, since its divergence from the vertebrate Bf/C2 gene. Fluorescent in situ hybridization (FISH) to the chromosomes showed that genetic loci for the CiBfs, CiC3-1 and CiC3-2 genes are present on three different chromosomes, suggesting the possibility that the linkage among the MHC class III complement genes was established in the vertebrate lineage after its divergence from urochordates.The sequences reported in this paper have been deposited in the DDBJ database (accession nos. AB180044–AB180051).     

Changes in the activity of the multifunctional β-glycosyl hydrolase (Cel44C-Man26A) from Paenibacillus polymyxa by removal of the C-terminal region to minimum size

Paenibacillus polymyxa GS01 secretes Cel44C-Man26A as a multifunctional enzyme with cellulase, xylanase, lichenase, and mannanase activities. Cel44C-Man26A consists of 1,352 amino acids in which present a catalytic domain (CD) of the glycosyl hydrolase family 44 (GH44), fibronectin domain type 3 (Fn3), catalytic domain of glycosyl hydrolase family 26 (GH26), and a cellulose-binding module type 3 (CBM3). A truncated Cel44C-Man26A protein, consisting of 549 amino acid residues, reacted as a multifunctional mature enzyme despite the absence of the 10 amino acids containing GH44, Fn3, GH26, and CBM3. However, the multifunctional activity was not found in the mature Cel44C-Man26A protein truncated to less than 548 amino acids. The truncated Cel44C-Man26A proteins showed the optimum pH for the lichenase activity was pH 7.0, pH 6.0 for the xylanase and mannanase, and pH 5.0 for the cellulase. The truncated Cel44C-Man26A proteins exhibited enzymatic activity 40–120% higher than the full-length Cel44C.     

Identification and antigenic epitope mapping of immunodominant region amino residues 510 to 672 on the spike protein of the severe acute respiratory syndrome coronavirus

The severe acute respiratory syndrome (SARS) is a newly emerging human infectious disease caused by the severe acute respiratory syndrome coronavirus (SARS-CoV). The spike (S) protein of SARS-CoV is a major virion structural protein. It plays an important role in the interaction with receptors and neutralizing antibodies. In this study, the S1 domain of the spike protein and three truncated fragments were expressed by fusion with GST in a pGEX-6p-1 vector. Western blot results demonstrated that the 510-672 fragment of the S1 domain is a linear epitope dominant region. To map the antigenic epitope of this linear epitope dominant region, a set of 16 partially overlapping fragments spanning the fragment were fused with GST and expressed. Four antigenic epitopes S1C3 (539-559), S1C4 (548-567), S1C7/8 (583-606), and S1C10/11 (607-630) were identified. Immunization of mice with each of the four antigenic epitope-fused proteins revealed that all four proteins could elicit spike protein specific antisera. All of them were able to bind to the surface domain of the whole spike protein expressed by recombinant baculovirus in insect cells. Identification of antigenic epitopes of the spike protein of SARS-CoV may provide the basis for the development of immunity-based prophylactic, therapeutic, and diagnostic clinical techniques for the severe acute respiratory syndrome.     

Interaction of uromodulin and complement factor H enhances C3b inactivation

Recent studies suggest that uromodulin plays an important role in chronic kidney diseases. It can interact with several complement components, various cytokines and immune system cells. Complement factor H (CFH), as a regulator of the complement alternative pathway, is also associated with various renal diseases. Thus, we have been suggested that uromodulin regulates complement activation by interacting with CFH during tubulointerstitial injury. We detected co‐localization of uromodulin and CFH in the renal tubules by using immunofluorescence. Next, we confirmed the binding of uromodulin with CFH in vitro and found that the affinity constant (K_D) of uromodulin binding to CFH was 4.07 × 10^?6M based on surface plasmon resonance results. The binding sites on CFH were defined as the short consensus repeat (SCR) units SCR1–4, SCR7 and SCR19–20. The uromodulin‐CFH interaction enhanced the cofactor activity of CFH for factor I‐mediated cleavage of C3b to iC3b. These results indicate that uromodulin plays a role via binding and enhancing the function of CFH.     

Gamma-ray-enhanced reactivation of UV-irradiated adenovirus in normal human fibroblasts.

An enhanced reactivation of UV-irradiated adenovirus type 2 (Ad 2) was detected following irradiation of the host cells with γ-rays prior to infection. Non-irradiated and γ-irradiated normal human fibroblasts were infected immediately after irradiation with either non-irradiated or UV-irradiated Ad 2. At 48h after infection, cultures were examined by indirect immunofluorescence to determine the number cells in which the viral function of viral structural antigen (Vag) was expressed. Pre-irradiation of cells with 1 krad resulted in a 2–3-fold increase in the survival of this viral function following different UV doses to the virus up to 1.75 × 10³ J/m². For a fixed UV dose of 1.0 × 10³ J/m² to the virus this enhancement increased with preirradiation dose to the cells up to a maximum factor of 2–3 for a dose of 1 krad. An examination of Vag expression at various times after infection indicates that pre-irradiation of the cells with γ-rays prior to infection with UV-irradiated virus leads to an earlier onset and/or increased rate of Vag synthesis. This enhancement of Vag production from a UV-damaged template may result from an inducible DNA-repair mechanism in human fibroblasts which may or may not be error-prone.     

Identification of messenger and long noncoding RNAs associated with gallbladder cancer via gene expression profile analysis

The present study aimed to investigate the long noncoding RNAs (lncRNAs) and messenger RNAs (mRNAs) involved in the progression of gallbladder cancer and explore the potential physiopathologic mechanisms of gallbladder cancer in terms of competing endogenous RNAs (ceRNAs). The original lncRNA and mRNA expression profile data (nine gallbladder cancer tissues samples and nine normal gallbladder samples) in GSE76633 was downloaded from the Gene Expression Omnibus database. Differentially expressed mRNAs and lncRNAs between gallbladder cancer tissue and normal control were selected and the pathways in which they are involved were analyzed using bioinformatics analyses. MicroRNAs (miRNAs) were also predicted based on the differentially expressed mRNAs. Finally, the co-expression relation between lncRNA and mRNA was analyzed and the ceRNA network was constructed by combining the lncRNA-miRNA, miRNA-mRNA, and lncRNA-mRNA pairs. Overall, 373 significantly differentially expressed mRNAs and 47 lncRNAs were identified between cancer and normal tissue samples. The upregulated genes were significantly enriched in the extracellular matrix (ECM)-receptor interaction pathway, while the downregulated genes were involved in the complement and coagulation cascades. Altogether, 128 co-expression relations between lncRNA and mRNA were obtained. In addition, 196 miRNA-mRNA regulatory relations and 145 miRNA-lncRNA relation pairs were predicted. Finally, the lncRNA-miRNA-gene ceRNA network was constructed by combining the three types of relation pairs, such as XLOC_011309-miR-548c-3p-SPOCK1 and XLOC_012588-miR-765-CEACAM6. mRNAs and lncRNAs may be involved in gallbladder cancer progression via ECM-receptor interaction pathways and the complement and coagulation cascades. Moreover, ceRNAs such as XLOC_011309-miR-548c-3p-SPOCK1 and XLOC_012588-miR-765-CEACAM6 can also be implicated in the pathogenesis of gallbladder cancer.