Plasmid Group Research Articles

Impact of co-carriage of IncA/C plasmids with additional plasmids on the transfer of antimicrobial resistance in Salmonella enterica isolates

BackgroundAntimicrobial resistance in Salmonella enterica is often plasmid encoded. A key resistance plasmid group is the incompatibility group (Inc) A/C plasmids that often carry multiple resistance determinants. Previous studies showed that IncA/C plasmids were often co-located with other plasmids. The current study was undertaken to evaluate the impact of plasmid co-carriage on antimicrobial resistance and plasmid transfer. MethodsA total of 1267 Salmonella isolates, representing multiple serotypes and sources were previously subjected to susceptibility testing and 251 isolates with resistance to at least 5 antimicrobial agents were identified for further study. Each isolate was subjected to PCR-based replicon typing, and those with IncA/C plasmids were selected for plasmid isolation, PCR-based mapping of IncA/C plasmid backbone genes, and conjugation assays to evaluate resistance plasmid transferability. ResultsOf the 87 identified IncA/C positive isolates, approximately 75% carried a plasmid with another identified replicon type, with the most common being I1 (39%), FIA, FIIA, FIB and HI2 (each 15%). PCR-based mapping indicated significant diversity in IncA/C backbone content, especially in regions encoding transfer-associated and hypothetical proteins. Conjugation experiments showed that nearly 68% of the isolates transferred resistance plasmids, with 90% containing additional identified plasmids or larger (>50 kb) non-typeable plasmids. ConclusionsThe majority of IncA/C-positive strains were able to conjugally transfer antimicrobial resistance to the recipient, encoded by IncA/C and/or co-carried plasmids. These findings highlight the importance of co-located plasmids for resistance dissemination either by directly transferring resistance genes or by potentially providing the needed conjugation machinery for IncA/C plasmid transfer.

MicroRNA-145 Increases the Apoptosis of Activated Hepatic Stellate Cells Induced by TRAIL through NF-κB Signaling Pathway.

During the liver fibrosis recovery stage tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can effectively induce apoptosis of activated hepatic stellate cells (HSCs). Normal hepatic stellate cells are resistant to TRAIL cytotoxicity. Therefore, enhancing the sensitivity of TRAIL-induced apoptosis of HSCs may be useful to treat hepatic fibrogenesis. Here, we demonstrated that miR-145 and TRAIL were down-regulated in both liver fibrosis tissue samples and transforming growth factor-β1 induced HSCs, concomitant with increased the expression of ZEB2. In addition, we found that mimics-mediated over-expression of miR-145 led to resistance to the ZEB2 expression and up-regulation of the TRAIL-induced apoptosis after treatment of LX-2 cells with TRAIL. Furthermore, ZEB2-siRNA transfected LX-2 cells showed the increased sensitivity to TRAIL-induced apoptosis. Whereas, opposite results were obtained in miR-145-inhibitor group or ZEB2 plasmid group. Moreover, miR-145 regulated ZEB2 gene expression by specifically interacting with the 3′-UTR of ZEB2 mRNA to inhibit the expression of ZEB2. Further studies showed that the over-expression of ZEB2 could inhibit TRAIL-induced apoptosis via inhibiting nuclear factor-κB (NF-κB) signaling pathway in LX-2 cells. Collectively, our data suggest that up-regulation of miR-145 can down-regulate ZEB2 expression, consequently promoting TRAIL-induced apoptosis in LX-2 cells through NF-κB signaling pathway, which facilitates the resolution of liver fibrosis.

Specific plasmid patterns and high rates of bacterial co-occurrence within the coral holobiont.

Despite the importance of coral microbiomes for holobiont persistence, the interactions among these are not well understood. In particular, knowledge of the co‐occurrence and taxonomic importance of specific members of the microbial core, as well as patterns of specific mobile genetic elements (MGEs), is lacking. We used seawater and mucus samples collected from Mussismilia hispida colonies on two reefs located in Bahia, Brazil, to disentangle their associated bacterial communities, intertaxa correlations, and plasmid patterns. Proxies for two broad‐host‐range (BHR) plasmid groups, IncP‐1β and PromA, were screened. Both groups were significantly (up to 252 and 100%, respectively) more abundant in coral mucus than in seawater. Notably, the PromA plasmid group was detected only in coral mucus samples. The core bacteriome of M. hispida mucus was composed primarily of members of the Proteobacteria, followed by those of Firmicutes. Significant host specificity and co‐occurrences among different groups of the dominant phyla (e.g., Bacillaceae and Pseudoalteromonadaceae and the genera Pseudomonas, Bacillus, and Vibrio) were detected. These relationships were observed for both the most abundant phyla and the bacteriome core, in which most of the operational taxonomic units showed intertaxa correlations. The observed evidence of host‐specific bacteriome and co‐occurrence (and potential symbioses or niche space co‐dominance) among the most dominant members indicates a taxonomic selection of members of the stable bacterial community. In parallel, host‐specific plasmid patterns could also be, independently, related to the assembly of members of the coral microbiome.

CEA-EPO质粒的构建及诱导造血干细胞定向生成红细胞疫苗抗结肠癌的研究

目的：研究CEA-EPO质粒的构建及诱导造血干细胞定向生成红细胞疫苗抗结肠癌的研究。方法：将EPO-cDNA插入pcDNA3.1质粒的多克隆位点，用CEA基因启动子替代pcDNA3.1质粒的CMV启动子，构建成CEA-EPO重组质粒。用含抗体CD117磁珠分离纯化和收集小鼠骨髓造血干细胞，用重组腺病毒法将CEA-EPO基因体外转染CEA-EPOmRNA基因到造血干细胞内，检测CEA-EPO基因和蛋白表达，将疫苗注入小鼠体内，进行扩增，使造血干细胞定向生成携带CEA-EPO双基因红细胞，经过提取和纯化获得CEA-EPO基因共转染红细胞疫苗，再进行双基因共转染红细胞疫苗的体外和体内实验。结果：成功收集造血干细胞，流式细胞仪分析结果表明：纯化前后有明显差异(P < 0.05)；检测CEA-EPOmRNA和蛋白表达的结果表明：造血干细胞体外转染CEA-EPOmRNA基因成功，获得CEA-EPO双基因共转染红细胞疫苗；应用细胞红系相关标志抗体GPA和CD71标记，利用流式细胞证实红细胞疫苗培养成功；研究相关疫苗激活的单核细胞对结肠癌细胞株CT26细胞杀伤作用表明：在比例为40:1时，CEA-EPO疫苗组杀伤作用为57.34 ± 1.13，高于空载组、CEA疫苗组和EPO疫苗组(P < 0.05)，体内试验的成瘤性研究表明：CEA-EPO疫苗组肿瘤的生长的大小为0.29 ± 0.11 (cm3)；高于其他各组的大小(P < 0.05)，CEA-EPO疫苗组的成活期分别为：69 ± 6.91天，高于其他各组的大小(P < 0.05)；CEA-EPO疫苗组在肿瘤大小和小鼠的生存时间上，与空载组、CEA疫苗组，EPO疫苗组之间差异有统计学意义(P < 0.05)。结论：携带CEA-EPO双基因肿瘤抗原的红细胞疫苗对结肠癌有明显的抗肿瘤作用，既可以诱导造血干细胞定向分化成红细胞疫苗，又可以合成肿瘤抗原的红细胞疫苗，是临床治疗结肠的重要疫苗。 Objective: To investigate the effects of recombinant adenovirus-mediated co-transfection of CEA gene and EPO gene on promoting hematopoietic stem cell directly producing erythrocyte vaccine against colon cancer. Method: The expression adenovirus vectors carrying CEA and EPO or green fluorescent protein (GFP) gene were constructed respectively, and recombinant adenovirus car-rying CEA, EPO or GFP were packaged and produced respectively. The bone marrow-derived MSCs of mice were isolated and cultured in vitro by anti-CD117magnetic bead separation, and were transfected with CEA (CEA group), EPO (EPO group) or GFP(GFP group), co-transfected with CEA and EPO (CEA and EPO group), or transfected with no virus (controlgroup). The expression of CEA and EPO gene and protein after transfection in supernatant fluid of culture was detected by RT-PCR and western blot in each group. We have checked and obtained the vaccine with co-transfection of CEA gene and EPO gene by cell red line marker antibody CD71 and GPA, then we carried on experiments with the vaccine in vitro and in vivo. Result: We have successfully gathered the hematopoietic stem cells; flow cytometry analysis result showed that there were significant differences before and after purification (P < 0.05). The expression of double genes (CEA-EPO gene) and protein shows CEA-EPO gene were successfully transfected into the hematopoietic stem cells. We have confirmed erythrocyte vaccine with co-transfection of CEA and EPO gene by antibody CD71 and GPA with flow cytometry. The monocytes cytotoxicity on colon cancer cell line CT26 showed that CEA-EPO group was 57.34 ± 1.13% in proportion of 40:1. Compared with CEAg group and EPO group, there was significant difference between them (P < 0.05). In the experimentation of neoplasma format, the size of murine growth of tumor was 0.29 ± 0.11 cm3 differently in the group of CEA-EPO group; the number of murine live time on day was 69 ± 6.91 days. Compared with the group of CEA and plasmid, EPO, the group of CEA-EPO was significant differences in size and live time (P < 0.05). Conclusion: The erythrocyte vaccine with co-transfection of CEA gene and EPO gene has efficient anti-tumor effects on colon cancer. It not only can promote hematopoietic stem cell directly producing erythrocyte vaccine, but also can produce tumor Antigen vaccine against stomach cancer.

The Role for the Small Cryptic Plasmids As Moldable Vectors for Genetic Innovation in Aeromonas salmonicida subsp. salmonicida.

In Aeromonas salmonicida subsp. salmonicida, a bacterium that causes fish disease, there are two types of small plasmids (<15 kbp): plasmids without known function, called cryptic plasmids, and plasmids that bear beneficial genes for the bacterium. Four among them are frequently detected in strains of A. salmonicida subsp. salmonicida: pAsa1, pAsa2, pAsa3, and pAsal1. The latter harbors a gene which codes for an effector of the type three secretion system, while the three others are cryptic. It is currently unclear why these cryptic plasmids are so highly conserved throughout strains of A. salmonicida subsp. salmonicida. In this study, three small plasmids, named pAsa10, pAsaXI and pAsaXII, are described. Linked to tetracycline resistance, a partial Tn1721 occupies half of pAsa10. A whole Tn1721 is also present in pAsa8, another plasmid previously described in A. salmonicida subsp. salmonicida. The backbone of pAsa10 has no relation with other plasmids described in this bacterium. However, the pAsaXI and pAsaXII plasmids are derivatives of cryptic plasmids pAsa3 and pAsa2, respectively. pAsaXI is identical to pAsa3, but bears a transposon with a gene that encodes for a putative virulence factor. pAsaXII, also found in Aeromonas bivalvium, has a 95% nucleotide identity with the backbone of pAsa2. Like pAsa7, another pAsa2-like plasmid recently described, orf2 and orf3 are missing and are replaced in pAsaXII by genes that encode a formaldehyde detoxification system. These new observations suggest that transposons and particularly Tn1721 are frequent and diversified in A. salmonicida subsp. salmonicida. Moreover, the discovery of pAsaXI and pAsaXII expands the group of small plasmids that are derived from cryptic plasmids and have a function. Although their precise roles remain to be determined, the present study shows that cryptic plasmids could serve as moldable vectors to acquire mobile elements such as transposons. Consequently, they could act as key agents of the diversification of virulence and adaptive traits of Aeromonas salmonicida subsp. salmonicida.

A helicase-containing module defines a family of pCD630-like plasmids in Clostridium difficile

Clostridium difficile is a Gram-positive and sporulating enteropathogen that is a major cause of healthcare-associated infections. Even though a large number of genomes of this species have been sequenced, only a few plasmids have been described in the literature. Here, we use a combination of in silico analyses and laboratory experiments to show that plasmids are common in C. difficile. We focus on a group of plasmids that share similarity with the plasmid pCD630, from the reference strain 630. The family of pCD630-like plasmids is defined by the presence of a conserved putative helicase that is likely part of the plasmid replicon. This replicon is compatible with at least some other C. difficile replicons, as strains can carry pCD630-like plasmids in addition to other plasmids. We find two distinct sub-groups of pCD630-like plasmids that differ in size and accessory modules. This study is the first to describe a family of plasmids in C. difficile.

Differentiation of rat adipose-derived mesenchymal stem cells into corneal-like epithelial cells driven by PAX6.

Corneal integrity, transparency and vision acuity are maintained by corneal epithelial cells (CECs), which are continuously renewed by corneal limbal stem cells (LSCs). Deficiency of CECs and/or LSCs is associated with numerous ocular diseases. Paired box (PAX)6 is an eye development-associated transcription factor that is necessary for cell fate determination and differentiation of LSCs and CECs. In the present study, the PAX6 gene was introduced into adipose-derived rat mesenchymal stem cells (ADMSCs) to investigate whether PAX6-transfected cells were able to transdifferentiate into corneal-like epithelial cells and to further verify whether the cells were suitable as a cell source for corneal transplantation. The ADMSCs were isolated from the bilateral inguinal region of healthy Sprague Dawley rats. The characteristics of ADMSCs were identified using flow cytometric analysis. After subculture, ADMSCs underwent transfection with recombinant plasmid containing either PAX6-enhanced green fluorescent protein (EGFP) complementary (c)DNA or EGFP cDNA (blank plasmid group), followed by selection with G418 and determination of the transfection efficiency. Subsequently, the morphology of the ADMSCs and the expression profiles of corneal-specific markers CK3/12 and epithelial-specific adhesion protein were determined. E-cadherin was detected using immunofluorescence staining and western blot analysis at 21 days following transfection. An MTT cell proliferation and a colony formation assay were performed to assess the proliferative activity and clonogenicity of PAX6-transfected ADMSCs. Finally, the PAX6-expressing ADMSCs were transplanted onto the cornea of a rabbits with limbal stem cell deficiency (LSCD). At 21 days after transfection, the ADMSCs with PAX6 transfection exhibited a characteristic flagstone-like appearance with assembled corneal-like epithelial cells, and concomitant prominent expression of the corneal-specific markers cytokeratin 3/12 and E-cadherin. Furthermore, the proliferation and colony formation ability of PAX6-overexpressing ADMSCs was significantly retarded. The transplantation experiment indicated that PAX6-reprogramed ADMSCs attached to and replenished the damaged cornea via formation of stratified corneal epithelium. Taken together, these results suggested that conversion of ADMSCs into corneal-like epithelium may be driven by PAX6 transfection, which makes ADMSCs a promising cell candidate for the treatment of LSCD.

Direct reprogramming of mouse fibroblasts into neural cells via Porphyra yezoensis polysaccharide based high efficient gene co-delivery

BackgroundThe cell source for transplantation therapy is always a prerequisite question to be solved in clinical applications. Neural cells are considered non-regenerable, which highly restrict their application in the treatment for nerve injury. Therefore, neural trans-differentiation based on gene transfection provides a new solution to this issue. Compared to viral strategy, non-viral gene delivery systems are considered as a more promising way to achieve this aim. This study centers on a novel application of Porphyra yezoensis polysaccharide as a non-viral gene carrier for the neural trans-differentiation of mouse fibroblasts.ResultsEthanediamine modified P. yezoensis polysaccharide (Ed-PYP) served as a gene carrier and a group of plasmids that encode Ascl1, Brn4, and Tcf3 (pABT) self-assembled into nanoparticles. Results demonstrated that Ed-PYP–pABT nanoparticles at Ed-PYP: pABT weight ratio of 40:1 was the optimal candidate for gene delivery. ELISA assay revealed the highest expression levels of NGF, BDNF and SHH at 14 days after last transfection. Immunofluorescence and western blot assays also showed robust expression of neural markers including Nestin, GFAP, β-3tubulin, NF200, GAP43 and MAP2, in induced 3T6 cells at this time point.ConclusionOverall, these findings indicated that the P. yezoensis polysaccharide-based non-viral gene co-delivery system is a promising strategy for the generation of neural cells, which might facilitate the developments in the recovery of neural injuries.

Effects of SMP-30 overexpression on apoptosis of human lens epithelial cells induced by ultraviolet B irradiation

Objective: This study was to observe the effect of SMP-30 on ultraviolet B (UVB)-induced apoptosis of human lens epithelial cells(HLE-B3) in vitro. Methods: Experimental study. The SMP-30 cDNA was amplified by PCR and inserted into the pRFP-N1 expressing vector which had been double digested by XhoI/HindIII. HLE-B3 cells were cultured and divided into three groups: normal group, pRFP-N1 vector plasmid group and pRFP-N1-SMP-30 plasmid group (SMP-30). Then cells were exposed to UVB and the survival rate of cells was detected by MTT assay. The effects of SMP-30 on UVB-induced HLE-B3 apoptosis were measured by the Cell Death Detection ELISA kit. Meanwhile, the influence of SMP-30 on UVB-induced apoptosis-relative protein expression in HLE-B3 cells was tested by Western blots. Moreover, 2', 7'-Dichlorofluorescin diacetate staining was performed to monitor the protective effects of SMP-30 on UVB-induced HLE-B3 reactive oxygen species(ROS). One-way analysis of variance combined with Dunnett's statistical method were performed to analyze the data. Results: The full length of PSF cDNA fragment was correctly inserted into the pRFP-N1 vector, which was confirmed by DNA sequencing. The SMP-30 fragment was inserted to the plasmid pRFP-N1 correctly, which was also confirmed by DNA sequencing. The PRFP-N1-SMP-30 plasmid was transfected into HLE-B3 successfully. SMP-30 expression was up-regulated in the transfection group, compared with the control group. Data showed that the survival rate of HLE-B3 after the pRFP-N1-SMP-30 plasmid transfection was 0.90±0.14, while the apoptosis rate was 0.43±0.06 and the ROS production was 0.52±0.02, showing significant difference in comparison with the vector plasmid group and the normal group(t=5.830, 9.934, 12.19, P<0.05). In the meantime, SMP-30 overexpression down-regulated the levels of Bax and cleav-caspase-3, but up-regulated the Bcl-2 and Pro-caspase-3 expression levels under UVB irradiation. Conclusion: SMP-30 overexpression plays a protective role in UVB-induced apoptosis via regulating the expression of apoptosis-related proteins and inhibiting the production of ROS in HLE-B3 cells. (Chin J Ophthalmol, 2017, 53: 835-841).

Calcitonin gene-related peptide-transfected bone marrow mesenchymal stem cells combined with autologous bone grafting to treat osteonecrosis of the femoral head in rabbit model

Objective To investigate the histopathological changes of calcitonin peptide-transfected bone marrow mesenchymal stem cells (BMSCs) combined with bone transplantation in the treatment of osteonecrosis of the femoral head (ONFH) in rabbit model. Methods The osteonecrosis models on one side femoral head of rabbits were established by liquid nitrogen. Forty-eight rabbit ONFH models were randomly divided into 3 groups. In human calcitonin gene-related peptide α (hCGRPα)-transfected BMSCs group (A), the BMSCs transfected with hCGRPα in combination with autologous bone graft were implanted into the femoral head. In empty plasmid group (B), the BMSCs transfected with empty plasmid (only pLNCX2 plasmid) and autologous bone graft were implanted. In irrelated sequence group (C), BMSCs transfected with irrelated gene [only pLNCX2-green fluorescent protein (GFP)] and autologous bone graft were implanted. Animals were sacrificed at 3rd and 6th month respectively to observe the histopathological changes in the femoral heads of rabbits. Results At 3rd month, the bone trabecular area fraction respectively was (40.89±1.12)%, (36.12±1.17)%, (36.33±1.53)% and (42.11±1.91)% in groups A, B and C and normal femoral head. The percentage of empty osteocyte lacunae respectively was (11.34±1.58)%, (16.33±1.45)%, (17.21±1.23)% and (10.47±1.41)% in groups A, B and C and normal femoral head. In group A, there were proliferation and growth of a large number of osteocytes, more mature bone trabeculae and higher area fraction, less empty osteocyte lacunae, and stronger biomechanical strength in the femoral head, which were nearly close to those in the normal control group and obviously superior to those in groups B and C. In groups B and C, there were thin and irregular bone trabeculae, more empty osteocyte lacunae, lower area fraction and imperfect bone repair. At 6th month, the bone trabeculae were mature, densification and satiation, and neatly arranged rules were seen in the femoral head of group A. The area fraction of bone trabeculae and the number of empty bone lacunae in group A were superior to those in groups B and C. Conclusion The implantation of hCGRPα-transfected BMSCs combined with autologous bone graft into the femoral head of ONFH animal models can significantly promote cells proliferation, accelerate osteogenesis, enhance bone tissue repair, and obtain good effect in the treatment of ONFH. Key words: Calcitonin gene related peptide; Bone marrow mesenchymal stem cells; Osteonecrosis of the femoral head necrosis; Model, animal

Convergence of plasmid architectures drives emergence of multi-drug resistance in a clonally diverse Escherichia coli population from a veterinary clinical care setting

The purpose of this study was to determine the plasmid architecture and context of resistance genes in multi-drug resistant (MDR) Escherichia coli strains isolated from urinary tract infections in dogs. Illumina and single-molecule real-time (SMRT) sequencing were applied to assemble the complete genomes of E. coli strains associated with clinical urinary tract infections, which were either phenotypically MDR or drug susceptible. This revealed that multiple distinct families of plasmids were associated with building an MDR phenotype. Plasmid-mediated AmpC (CMY-2) beta-lactamase resistance was associated with a clonal group of IncI1 plasmids that has remained stable in isolates collected up to a decade apart. Other plasmids, in particular those with an IncF replicon type, contained other resistance gene markers, so that the emergence of these MDR strains was driven by the accumulation of multiple plasmids, up to 5 replicons in specific cases. This study indicates that vulnerable patients, often with complex clinical histories provide a setting leading to the emergence of MDR E. coli strains in clonally distinct commensal backgrounds. While it is known that horizontally-transferred resistance supplements uropathogenic strains of E. coli such as ST131, our study demonstrates that the selection of an MDR phenotype in commensal E. coli strains can result in opportunistic infections in vulnerable patient populations. These strains provide a reservoir for the onward transfer of resistance alleles into more typically pathogenic strains and provide opportunities for the coalition of resistance and virulence determinants on plasmids as evidenced by the IncF replicons characterised in this study.

Liver kinase B1 gene enhances radiosensitivity of lung cancer H460 cells: an in vivo study

Objective To investigate the effect of liver kinase B1(LKB1) on the radiosensitivity of subcutaneous xenograft tumor of lung cancer H460 cells in nude mice. Methods Human lung cancer H460 cells were implanted into female nude mice (BALB/c-nu) to establish a subcutaneous xenograft tumor model of lung cancer. A total of 24 female nude mice in which the model was successfully established were equally and randomly divided into four groups: pEGFP-Ctrl plasmid (empty vector plasmid) group, irradiation (IR)+ pEGFP-Ctrl plasmid group, pEGFP-LKB1 plasmid (overexpressing LKB1) group, and IR+ pEGFP-LKB1 plasmid group. The growth of xenograft tumors was observed and the tumor inhibition rate and enhancement factor (EF) were calculated. The expression of LKB1 in each group was measured by immunohistochemistry and Western blot to analyze the relationship between LKB1 and radiosensitivity. Results Compared with the pEGFP-Ctrl plasmid group, the IR+ pEGFP-Ctrl plasmid group, pEGFP-LKB1 plasmid group, and IR+ pEGFP-LKB1 plasmid group showed varying degrees of inhibition of tumor growth, particularly in the IR+ pEGFP-LKB1 plasmid group, and the tumor inhibition rates were 31.30%, 14.78%, and 43.48%, respectively. The EF of LKB1 in the IR+ pEGFP-LKB1 plasmid group was 1.18.The immunohistochemistry and Western blot showed that LKB1 could be effectively expressed in the pEGFP-LKB1 plasmid group and IR+ pEGFP-LKB1 plasmid group, but not in the other two groups. Conclusions The subcutaneous xenograft tumor model of human lung cancer H460 cells has been successfully established in nude mice. LKB1 has a radiosensitizing effect on the subcutaneous xenograft tumor of lung cancer H460 cells in nude mice. Key words: Lung cancer cell line; Liver kinase B1; Radiosensitivity; Xenograft

Bam35 Tectivirus Intraviral Interaction Map Unveils New Function and Localization of Phage ORFan Proteins.

The family Tectiviridae comprises a group of tailless, icosahedral, membrane-containing bacteriophages that can be divided into two groups by their hosts, either Gram-negative or Gram-positive bacteria. While the first group is composed of PRD1 and nearly identical well-characterized lytic viruses, the second one includes more variable temperate phages, like GIL16 or Bam35, whose hosts are Bacillus cereus and related Gram-positive bacteria. In the genome of Bam35, nearly half of the 32 annotated open reading frames (ORFs) have no homologs in databases (ORFans), being putative proteins of unknown function, which hinders the understanding of their biology. With the aim of increasing knowledge about the viral proteome, we carried out a comprehensive yeast two-hybrid analysis of all the putative proteins encoded by the Bam35 genome. The resulting protein interactome comprised 76 unique interactions among 24 proteins, of which 12 have an unknown function. These results suggest that the P17 protein is the minor capsid protein of Bam35 and P24 is the penton protein, with the latter finding also being supported by iterative threading protein modeling. Moreover, the inner membrane transglycosylase protein P26 could have an additional structural role. We also detected interactions involving nonstructural proteins, such as the DNA-binding protein P1 and the genome terminal protein (P4), which was confirmed by coimmunoprecipitation of recombinant proteins. Altogether, our results provide a functional view of the Bam35 viral proteome, with a focus on the composition and organization of the viral particle.IMPORTANCE Tailless viruses of the family Tectiviridae can infect commensal and pathogenic Gram-positive and Gram-negative bacteria. Moreover, they have been proposed to be at the evolutionary origin of several groups of large eukaryotic DNA viruses and self-replicating plasmids. However, due to their ancient origin and complex diversity, many tectiviral proteins are ORFans of unknown function. Comprehensive protein-protein interaction (PPI) analysis of viral proteins can eventually disclose biological mechanisms and thus provide new insights into protein function unattainable by studying proteins one by one. Here we comprehensively describe intraviral PPIs among tectivirus Bam35 proteins determined using multivector yeast two-hybrid screening, and these PPIs were further supported by the results of coimmunoprecipitation assays and protein structural models. This approach allowed us to propose new functions for known proteins and hypothesize about the biological role of the localization of some viral ORFan proteins within the viral particle that will be helpful for understanding the biology of tectiviruses infecting Gram-positive bacteria.

MicroRNA-7-5p regulates the proliferation and migration of intestinal epithelial cells by targeting trefoil factor 3 via inhibiting the phosphoinositide 3-kinase/Akt signalling pathway.

Trefoil factor 3 (TFF3) reconstructs the epithelial barrier by stimulating epithelial cell migration and proliferation, and significantly contributes to intestinal mucosal damage and healing. In a previous study, TFF3 was identified as a novel target of microRNA-7-5p (miR-7-5p). The aim of the present study was to investigate the roles and mechanisms of miR-7-5p in the proliferation and migration of intestinal epithelial cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to determine the expression level of miR-7-5p in the experimental groups. In addition, western blot analysis was performed to examine the expression levels of TFF3, phosphoinositide 3-kinase (PI3K), Akt and phosphorylated (p)-AKT when miR-7-5p or TFF3 was overexpressed, and the effects of miR-7-5p and TFF3 on LS174T cell proliferation and migration were simultaneously investigated. miR-7-5p was demonstrated to decrease the expression level of TFF3, and inhibit LS174T cell proliferation and migration, which was accompanied by decreased expression levels of PI3K and p-Akt. miR-7-5p was decreased following combined treatment with the TFF3 plasmid and miR-7-5p mimics, compared with treatment with miR-7-5p mimics alone, which was accompanied by increased expression levels of TFF3, PI3K and p-Akt, and enhanced LS174T cell proliferation and migration effects. The expression levels of miR-7-5p in the miRNA negative control (NC) + LY294002 group, the miR-7-5p mimic + LY294002 group, and the miR-7-5p mimic + TFF3 plasmid + LY294002 group were higher than those in the NC group, the miR-7-5p mimic group and the miR-7-5p mimic + TFF3 plasmid group, respectively. Accordingly, the expression level of TFF3 was downregulated and the proliferation and migration ability of the cells was downregulated. The present study demonstrates that overexpressed miR-7-5p may inhibit the proliferation and migration of LS174T cells by targeting the expression of TFF3 via inhibiting the PI3K/Akt signalling pathway. The PI3K/Akt signalling pathway may exert a feedback regulation effect on miR-7-5p, inhibiting the activity of this signalling pathway, which increases the miR-7-5p expression levels and further enhances the effects of miR-7-5p on cell proliferation and migration.

Effects of Wnt5A gene overexpression on cytoskeletal proteins of melanocytes

Objective To evaluate the effect of Wnt5A gene overexpression on cytoskeletal proteins of melanocytes after the plasmid containing the Wnt5A gene is transfected into primary melanocytes. Methods In vitro cultured primary human melanocytes were divided into three groups: blank control group receiving no treatment, negative control group transfected with endotoxin-free pcDNA3.1 (+) empty vector by Lipo3000 in Opti-MEM medium, Wnt5A plasmid group transfected with endotoxin-free pcDNA3.1 (+) vector containing the Wnt5A gene by Lipo3000 in Opti-MEM medium. After the trans-fection, quantitative PCR (qPCR) was performed to measure the mRNA expression of Wnt5A, ras-related C3 botulinum toxin substrate 1 (Rac1) , filamentous actin (F-actin) and β-tubulin, Western blot analysis to determine the protein expression of Wnt5A, receptor tyrosine kinase like orphan receptor 2 (ROR2) , Rac1, F-actin and β-tubulin, and an immunofluorescence assay (IFA) to observe the expression of cytoskeletal proteins. Results qPCR showed significant differences in the mRNA expression of the Wnt5A gene and its downstream genes Rac1 and F-actin among the Wnt5A plasmid group, negative control group and blank control group (F= 1 374.179, 112.576, 66.458, respectively, all P 0.05) . Additionally, the Wnt5A plasmid group showed significantly higher mRNA expression of Wnt5A, Rac1 and F-actin compared with the blank control group and negative control group (all P 0.05) . IFA showed no obvious difference in the fluorescence intensity of β-tubulin or F-actin between the Wnt5A group and the two control groups, but melanocytes showed larger size and increased number of dendrites, and the cytoskeleton changed dramatically with varying fluorescence intensity of F-actin, fuzzy texture, fractured or locally clustered tonofilaments in the Wnt5A group. Conclusion The overexpression of the Wnt5A gene in melanocytes can regulate the mRNA and protein expression of cytoskeletal proteins, make melanocytes larger and more dendritic, and cause changes in the cytoskeleton, which may facilitate the transportation of melanosomes, and participate in the occurrence of hyperpigmented diseases. Key words: Melanocytes; Wnt signaling pathway; Cytoskeleton; Actins; Tubulin; rac1 GTP-binding protein; Wnt5A protein

Role of Yes-associated protein 1 in angiotensinⅡ-induced pulmonary fibrosis in rats

Objective: To explore the mechanism of Yes-associated protein 1 (Yap1) in angiotensinⅡ(AngⅡ)-induced pulmonary fibrosis. Methods:In vivo, 18 male Wistar rats were randomly divided into three equal groups with 6 rats in each group, including control group, bleomycin-treated group (BLM), and BLM+ AngⅡ group. 28 days later, the lung tissues in all groups were harvested for the HE and Masson staining as well as the immunohistochemical (IHC) staining for Yap1. In vitro, the isolated fibroblasts were treated with 10(-7) mmol/L AngⅡor the AngⅡ-targeted inhibitor irbesartan for the scheduled time for mRNA and protein expressions of Yap1, PDZ-binding motif (TAZ), and collagen Ⅰusing PCR and Western blot, as well as the translocation test from the nucleus to the cytoplasm of Yap1 and TAZ. Subsequently, the fibroblasts were assigned into 4 groups: the empty plasmid (vector) group, the vector+ AngⅡ group, the Yap1 shRNA group, and the Yap1 shRNA+ AngⅡ group. Western blot was used to detect the relative expressions of Yap1, TAZ, Smad3 and collagen Ⅰ. The CCK-8 and EdU assays were performed to determine the proliferative capacity. Results:In vivo, severe lung fibrosis and increased Yap1 expression of IHC staining were found in BLM group. Additionally, more severe lung fibrosis and higher Yap1 expression were detected in the BLM+ AngⅡ group than the BLM group (both P<0.05). In vitro, both the mRNA and protein relative expressions of Yap1, TAZ and collagenⅠ were markedly higher in AngⅡ-treated groups than the control group (all P<0.05). Meanwhile, the relative expression of phosphorylated Yap1 reached its peak at 2 h after AngⅡ stimulation. In the protein translocation tests, after treated with AngⅡ for 24 h, the relative protein levels of Yap1 and TAZ in the nucleus of the AngⅡ group were significantly higher than those in the control group (0.382±0.007 vs 0.031±0.001, 1.097±0.030 vs 0.357±0.015). However, the relative protein expressions in the cytoplasm of the AngⅡ group were obviously less than that in the control group (0.323±0.058 vs 0.418±0.044, 0.858±0.059 vs 1.201±0.015). Compared with the AngⅡ group, the expressions of Yap1 and TAZ in the AngⅡ+ irbesartan group were higher in cytoplasm (0.598±0.060 vs 0.323±0.058, 1.495±0.052 vs 0.858±0.059), while lower in the nucleus (0.323±0.058 vs 0.418±0.044, 0.858±0.059 vs 1.201±0.015) (all P<0.05). Furthermore, the relative protein expressions of Yap1, TAZ, Smad3 and collagenⅠin Yap1 shRNA+ AngⅡ group were distinctly lower than the vector+ AngⅡ group (all P<0.05). In the cell proliferation tests, the absorbance and the percentage of EdU positive cells of vector+ AngⅡ group exceeded that of vector group (both P<0.05). However, the absorbance and the percentage of EdU positive cells in the Yap1 shRNA+ AngⅡgroup were less than the vector+ AngⅡ group (both P<0.05). Conclusion: AngiotensinⅡ promoted the collagen synthesis and cell proliferation in primary lung fibroblasts by increasing the Yap1 activity, leading to the progress of fibrosis.

STAT3 influences the characteristics of stem cells in cervical carcinoma.

The purpose of the study was to investigate the role of the signal transducer and activator of transcription 3 (STAT3), signal transduction protein in regulating the biological characteristics of stem cells in cervical carcinoma. Overexpressed plasmid of STAT3 was constructed and used to transfect SiHa into cervical carcinoma cells. STAT3-targeted specific siRNA was designed and produced. The effects of STAT3 upregulation (or inhibition) on the expression of NANOG, OCT4 and SOX2 markers of stem cells were measured, using western blot analysis and RT-qPCR. In addition, the tumor sphere experiment was also conducted to detect the formation of tumor spheres after the intervention of expression of STAT3 and the expression of STAT3, NANOG, OCT4 and SOX2 was detected in 35 cases of cervical carcinoma tissues and 31 cases of normal cervical tissues using immunohistochemistry. We determined whether the STAT3 overexpression plasmid was successfully constructed using enzyme digestion, PCR for bacterium solution, western blot analysis and RT-qPCR and found that the plasmid met the requirements of subsequent procedures. Compared with the empty plasmid group and STAT3 low expression group, the mRNA and protein expression of markers of stem cells, OCT4, SOX2 and NANOG were significantly elevated in the STAT3 overexpression group with statistically significant differences (P<0.05), the formation ratio of tumor spheres in the STAT3 overexpression group was also significantly higher than those in the other two groups (P<0.05). The positive expression of STAT3, OCT4, NANOG and SOX2 in the cervical squamous carcinoma group was also markedly higher than that in the chronic cervicitis group (P<0.05). This study led us to a conclusion that STAT3 can regulate the characteristics of stem cells in cervical carcinoma, and STAT3, NANOG, OCT4 and SOX2 are highly expressed in cervical squamous carcinoma, thus able to promote the progression of cervical carcinoma.

RACK1 Silencing Induces Cell Apoptosis and Inhibits Cell Proliferation in Hepatocellular Carcinoma MHCC97-H Cells.

This study aimed to explore the effects of RACK1 gene silencing on the apoptosis and proliferation of hepatocellular carcinoma (HCC) MHCC97-H cells. After transfecting MHCC97-H cells with siRNA, RACK1 gene silencing model was established. The cells were divided into blank group, siRNA group and empty plasmid group, respectively. The mRNA and protein expressions of RACK1, cyclin D1 and BAX were determined by qRT-PCR and Western blotting. CCK-8 assay, flow cytometry and FITC-Annexin V/PI staining were used to determine cell viability, cell cycle and cell apoptosis, respectively. The results of qRT-PCR and Western blotting suggested that when compared with the blank group and the empty plasmid group, the mRNA and protein expressions of RACK1 and Cyclin D1 decreased significantly while the mRNA and protein BAX expressions increased substantially in the siRNA group (all P<0.05). The results of CCK-8 assay revealed that the siRNA group exhibited significantly lower cell viability when compared with the blank group and the empty plasmid group (both P<0.05); and the cell viability in the siRNA group decreased gradually with the increase of time. The results of flow cytometry and FITC-Annexin V/PI staining indicated that when compared with the blank group and the empty plasmid group, the proportion of cells in S phase was markedly lower and the apoptosis rate was significantly higher in the siRNA group (both P<0.05). Our study suggests that inhibition of RACK1 could suppress cell proliferation and induce apoptosis in HCC MHCC97-H cells.

Effect of silencing sperm associated antigen 9 gene specially by RNA interference on human prostate carcinoma PC-3 cells

Objective To investigate the effect of silencing sperm associated antigen 9 (SPAG9) gene specially by RNA interference on human prostate carcinoma PC-3 cells, and the possible mechanism. Methods The plasmid vector expressing SPAG9 short hairpin RNA (shRNA) was constructed and transfected into PC-3 cells by lipofectamine 2000. Cells were collected and assayed at 48 h after transfection. The SPAG9 mRNA and protein expression level was measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting, respectively. Cell proliferation was tested by cell counting kit-8 (CCK-8) assay. Cell apoptosis was detected by fluorescence-activated cell sorter (FACS). The invasion ability of cells was examined by Transwell assay. To explore its possible mechanism, the expression of matrix metalloproteinase (MMP)-2, vascular endothelial growth factor (VEGF), Vimentin and E-cadherin proteins was measured by Western blotting after SPAG9 gene knockdown for 48 h. Results The recombinant plasmid pGPU6-green fluorescent protein (GFP)-SPAG9 expressing SPAG9 shRNA had been constructed successfully. RT-PCR and Western blotting showed the expression level of SPAG9 mRNA and proteinin in pGPU6-GFP-SPAG9 group was 1.96±0.16 and 0.39±0.05 respectively, lower than empty plasmid group (3.23±0.18 and 1.13±0.04), blank control group (3.08±0.12 and 1.25±0.05), respectively, the differences were both statistically significant (P=0.000). The absorbance (A) value was 31.9±2.6 in pGPU6-GFP-SPAG9 group, weaken than that in empty plasmid group (47.8±2.8, P=0.023), blank control group (48.1±2.8, P=0.016), the differences were statistically significant. FACS analysis showed that the apoptosis rate was (12.12±0.69)% in pGPU6-GFP-SPAG9 group, more than empty plasmid group [(6.43±0.31)%, P=0.020], blank control group [(5.19±0.33)%, P=0.014], the differences were statistically significant. Western blotting showed the expression level of MMP-2, VEGF, and Vimentin in pGPU6-GFP-SPAG9 group was lower than in the control groups, however, the average expression level of E-cadherin in pGPU6-GFP-SPAG9 group was higher than in the control groups, the differences were all statistically significant (P=0.000). Conclusion Silencing SPAG9 gene can inhibit SPAG9 gene expression, suppress proliferation, induce cell apoptosis of PC-3 cells by decreasing MMP-2, VEGF and Vimentin expression level, and increasing E-cadherin expression level. Key words: Prostate cancer; Sperm associated antigen 9 gene; RNA interference

The proliferative inhibition and apoptosis promotion of Smac on human lens epithelial cells

Background Posterior capsular opacification (PCO) is a primary complication after extracapsular cataract extraction.The mechanism of PCO is associated with proliferation, migration and epithelial-mesenchymal transition (EMT) of human lens epithelial cells (LECs). To explore the target treatment of PCO is very important. Objective This study was to investigate the biological effects of second mitochondria-derived activator of caspases (Smac) on the proliferation and apoptosis of LECs. Methods Human LECs line (HLE-B3) and Smac-overexpressed LECs line were cultured, and the cells were transfected using small interfering RNA (siRNA)-Smac3 plasmid with green fluorescent protein (GFP) for 24 hours.Different concentration of transforming growth factor -β2 (TGF-β2) (5, 10, 20 and 50 μg/ml) or 200 μmol/L H2O2 were added respectively into the culture medium to establish PCO model and oxidative stress model.Cell counting kit-8 (CCK-8) assay was used to compare the cell proliferative activity among PBS group, TGF-β2 group and Smac-hyperexpression+ TGF-β2 group.Flow cytometry was used to evaluate the apoptotic rate of the PBS group, H2O2 group and siRNA-Smac+ H2O2 group.The expressions of Smac, caspase-3 and proliferating cell nuclear antigen (PCNA) mRNA and their proteins in the cells were detected by real-time quantitative PCR (RT-PCR) and Western blot. Results The GFP+ cells were≥ 80% 12 hours after siRNA-Smac3 transfection, with the optimal plasmid of siRNA-Smac3.GFP+ cell rate was (72.32±2.31)% in the siRNA-Smac3 transfection group, which was significantly higher than that in the blank plasmid group([4.91±0.24]%) (t=116.342, P<0.001). The relevant expression levels of Smac was 35.21±4.11 in the Smac-hyperexpression group, and that in the blank plasmid group was 15.24±2.48, with a significant difference between them (t=215.47, P<0.05). The cell viability of 20 ng/ml TGF-β2 affected PBS group, TGF-β2 group and Smac-hyperepression+ TGF-β2 group was (98.4±1.7)%, (98.9±0.1)% and (64.2±3.1)%, and the cell viability of Smac-hyperepression+ TGF-β2 group was significantly lower in the Smac-hyperepression+ TGF-β2 group than that in the TGF-β2 group (P<0.05). The apoptotic rate in the PBS group, H2O2 group and siRNA-Smac+ H2O2 group were (2.9±1.2) %, (45.1±4.5)% and (27.5±1.8)%, and the apoptotic rate was evidently lower in the siRNA-Smac+ H2O2 group than that in the H2O2 group (P<0.05). RT-PCR results showed that the expression levels of caspase-3 mRNA in PBS group, H2O2 group and siRNA-Smac+ H2O2 group were 0.321±0.103, 0.715±0.112 and 0.479±0.209, respectively.Compared with the H2O2 group, the relative expression level of caspase-3 mRNA in siRNA-Smac+ H2O2 group was significantly decreased, the difference was statistically significant (P<0.05). The PCNA mRNA expression levels in PBS group, TGF-β2 group and Smac-hyperepression+ TGF-β2 group were 0.299±0.013, 0.645±0.102 and 0.490±0.209, respectively.Western blot results showed that the relative expression of caspase-3 protein in siRNA-Smac+ H2O2 group and H2O2 group was 0.712±0.012 and 0.973±0.051, with significant difference between the two groups (t=132.52, P<0.05). The relative expression of PCNA protein in Smac-hyperepression+ TGF-β2 group was 0.782±0.212, which was lower than 1.126±0.251 in the TGF-β2 group (P<0.05). Conclusions Smac may prevent and treat PCO by inhibiting the proliferation and promoting apoptosis of human LECs. Key words: Smac; Apoptsis; Proliferation; After-cataract; Human; Lens epithlial cells; Small interfering RNA