Proteomic analysis of HeLa cells after stable transfection with the Chlamydia trachomatis CT143 gene.

BACKGROUND: The CT143 protein of Chlamydia trachomatis (Ct) is a key immunodominant antigen and candidate type-III secretion substrate. Although CT143 expression has not been detected in the cytosol of infected cells, it is known to interfere with the physiological behavior of HeLa cells. This study aims to investigate how the CT143 protein affects the protein expression profile of HeLa cells, providing a basis for further research into Ct's pathogenic mechanisms. METHODS: We constructed a stably transfected HeLa cell line, pCD513B-1-CT143-HeLa, and a control cell line, pCD513B-1-HeLa. Protein expression profiles of these cell lines were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Differentially expressed proteins were identified, constructed into a database, and verified using parallel reaction monitoring (PRM). Bioinformatics software facilitated the preliminary analysis of the biological functions of these differential proteins. RESULTS: A total of 221 host proteins were differentially expressed, with 68 upregulated and 153 downregulated. These variations influence the regulation of peptidase activity and are crucial in biological processes such as cell secretion and protease activity. Significant changes were noted in protein processing, alcohol dehydrogenase activity, Aldo-Keto reductase activity, and peptidase regulator activity. Furthermore, alterations were observed in cellular components like the plasma membrane and cell periphery. Pathways involving the hematopoietic system, glycosaminoglycan degradation, retinol metabolism, and cytochrome P450-mediated exogenous drug metabolism were notably affected. Indirect interactions among differentially expressed proteins included three key nodal proteins: C3, IFIT3, and IFIT1. CONCLUSION: The successful construction of a host differential protein expression profile was achieved through stable transfection of HeLa cells with the CT143 gene. The differential proteins identified are implicated in regulating various biological processes such as intracellular signal transduction, cell secretion, protein processing, hydrolysis, and enzyme activity. These findings suggest that the CT143 protein may influence the host cell's biological behavior by altering host protein expression, potentially hindering Ct growth and development.

RET c.1901G>A and Novel SLC12A3 Mutations in Familial Pheochromocytomas

Familial PHEOs (pheochromocytomas) are inherited as an autosomal dominant trait, and inherited PHEOs can be one clinical phenotype of clinical syndromes, such as multiple endocrine neoplasia type 2A (MEN2A). In recent years, there has been a lot of controversy about the factors affecting the penetrance of PHEOs in MEN2A, of which the effects of RET (rearranged during transfection) proto-oncogene mutations are the primary concern. In this report, we performed genetic screening of patients in one family presenting with PHEOs and found they carried a RET c.1901G>A mutation. They were ultimately diagnosed with familial MEN2A. We found that MEN2A patients with the RET c.1901G>A mutation tended to have bilateral PHEOs that appeared earlier than medullary thyroid carcinoma. Genetic analysis showed that the patients also carried novel SLC12A3 (solute carrier family 12 member 3) variants, which are highly associated with Giteman syndrome. The results of protein structure prediction models suggest this SLC12A3 mutant has altered both the protein structure and the interaction with surrounding amino acids. Further studies of the phenotypes and related mechanisms of the gene mutations are required to guide individual assessment and treatment.

PDGF-BB induces conversion, proliferation, migration, and collagen synthesis of oral mucosal fibroblasts through PDGFR-ß/PI3K/ AKT signaling pathway.

OBJECTIVE: To explore the pathogenesis of oral submucosal fibrosis (OSF) by analyzing the impact of Platelet Derived Growth Factor (PDGF)-BB on oral mucosal fibroblasts (FB) and PDGFR-ß/Phosphoinositide 3-kinase (PI3K)/serine/threonine protein kinase (AKT) signaling pathway. METHODS: The isolated and purified oral mucosal fibroblasts were divided into four groups: the control group (CON, 10% FBS DMEM), the PDGF-BB group (40 ng/ml PDGF-BB), the PDGF-BB+IMA group (40 ng/ml PDGF-BB and 60 µmol/L IMA), and the PDGF-BB+LY294002 group (40 ng/ml PDGF-BB and 48 µmol/L LY294002). Primary human FB cells were isolated and cultured for detecting the effects of PDGF-BB on α-smooth muscle actin (α-SMA) by indirect immunofluorescence. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, Thiazolyl Blue Tetrazolium Bromide (MTT) method and scratch test were used to detect the proliferation and migration of FB. Western blots were used to detect the synthesis of type I collagen (Col I) and the expression of PDGFR-ß/PI3K/AKT signaling pathway-related proteins. The effects of PDGFR-ß inhibitor and PI3K inhibitor were observed. RESULTS: Compared with group CON, group IMA, and group LY294002, α-SMA was upregulated in group PDGF-BB (p< 0.05), with higher OD490 nm value (p< 0.05), narrower average scratch width, and higher relative cell migration rate (p< 0.05). The expression levels of Col I, p-PDGFR-ß, p-PI3K, and p-AKT were higher in group PDGF-BB (p< 0.05). CONCLUSIONS: PDGF-BB induces FB to transform into myofibroblasts (MFB) through the PDGFR-ß/PI3K/AKT signaling pathway, and promotes the proliferation, migration, and collagen synthesis.

Chlamydia trachomatis ct143 stimulates secretion of proinflammatory cytokines via activating the p38/MAPK signal pathway in THP-1 cells.

Chlamydia trachomatis (Ct) infections can cause bacterial sexually-transmitted and preventable blindness. The Ct infections induced excessive cytokines generation which attributed to pathologic changes in host cells. However, the precise mechanisms of Ct-induced cytokines production are still unclear.CT143 protein was identified as a novel Ct specific protein with high immunogenicity. In the present study. The CT143 fusion protein was recombined and purified. The mice immune serum was prepared by immunizing BALB/c mice with the purified fusion protein. The specificity of the antibody was confirmed using Immunoblotting. Indirect immunoflurescence assay (IFA) and Immunoblotting assays were performed to detect the temporal and spatial characteristics of CT143 in Ct infected cells. ELISA was performed to analyze the secretion of proinflammatory cytokines IL-1ß, IL-8 and TNF-α by human macrophages under the stimulation of CT143 protein. Finally, the involvement of p38 signaling in CT143-induced cytokine secretion was validated. CT143 protein was located in the inclusion body and represented an Elementary body (EB)-related protein, which may be encoded by the mid- and late-stage expressing genes. CT143 protein could stimulate the secretion of inflammatory cytokines in macrophages which differentiated from THP-1 This induction may be mediated by the activation of p38 signaling. In summary, CT143 protein is involved in inflammatory processes during Ct infection.

Dysfunction of mechanical heart valve prosthesis: experience with surgical management in 48 patients.

BACKGROUND: Dysfunction of mechanical heart valve prostheses is an unusual but potentially lethal complication after mechanical prosthetic valve replacement. We seek to report our experience with mechanical valve dysfunction regarding etiology, surgical techniques and early outcomes. METHODS: Clinical data of 48 patients with mechanical valve dysfunction surgically treated between October 1996 and June 2011 were analyzed. RESULTS: Mean age was 43.7±10.9 years and 34 were female (70.8%). The median interval from primary valve implantation to dysfunction was 44.5 months (range, 1 hour to 20 years). There were 21 emergent and 27 elective reoperations. The etiology was thrombosis in 19 cases (39.6%), pannus in 12 (25%), thrombosis and pannus in 11 (22.9%), improper disc orientation in 2 (4.1%), missing leaflet in 1 (2.1%), excessively long knot end in 1 (2.1%), endogenous factor in 1 (2.1%) and unidentified in 1 (2.1%). Surgical procedure was mechanical valve replacement in 37 cases (77.1%), bioprosthetic valve replacement in 7 (14.9%), disc rotation in 2 (4.2%) and excision of excessive knot end in 1 (2.1%). Early deaths occurred in 7 patients (14.6%), due to low cardiac output in 3 (6.3%), multi-organ failure in 2 (4.2%) and refractory ventricular fibrillation in 2 (4.2%). Complications occurred in 10 patients (20.8%). CONCLUSIONS: Surgical management of mechanical valve dysfunction is associated with significant mortality and morbidity. Earlier identification and prompt reoperation are vital to achieving better clinical outcomes. The high incidence of thrombosis in this series highlights the need for adequate anticoagulation and regular follow-up after mechanical valve replacement.

Effect of mitochondrial aldehyde dehydrogenase-2 genotype on cardioprotection in patients with congenital heart disease.

AIMS: About 40% of East Asians carry an aldehyde dehydrogenase-2*2 (ALDH2*2) allele, and the influence of the ALDH2*2 allele on human cardioprotection has not been studied. This study was designed to evaluate the effect of ALDH2*2 allele on cardioprotection of patients with congenital heart diseases after open-heart surgery. METHODS AND RESULTS: The right atrial appendage was harvested before performing cardiopulmonary bypass in cyanotic and acyanotic congenital heart disease groups (n = 20 per group). Tissues were assayed to determine the impact of cyanosis on metabolic remodelling. A prospective cohort of Tetralogy of Fallot (TOF) patients (n = 118) was recruited to investigate the influence of the ALDH2*2 allele on cardioprotection after surgical repair. Myocardium samples were dissected after cardioplegia. ALDH2 activity, oxidative stress and glutathione (GSH) levels, and activating transcription factor-4 (ATF4) were analysed. After genotyping and grouping, all of the experimental and clinical results were compared between ALDH2*2 carriers and non-carriers. Cyanosis inhibited ALDH2 activity and led to aldehyde accumulation in ALDH2*2 carriers. This accumulation in turn increased expression of ATF4 and resulted in larger myocardium GSH pools. The differences in ALDH2 activity and GSH level between carriers and non-carriers disappeared during cardioplegic arrest, and more aldehydes accumulated in the non-carriers. Consequently, ALDH2*2 carriers showed lower postoperative troponin I, inotrope score, and shorter postoperative length of ICU and hospital stay. CONCLUSIONS: ALDH2*2 carriers with cyanotic congenital heart disease were associated with an induced metabolic remodelling phenotype and a compensatory myocardium GSH pool. When ALDH2 activity was impaired during open-heart surgery, this larger GSH pool could lead to unexpectedly better cardioprotection. This may aid in the prediction of cardioprotection outcomes and identification of individualized cardioprotective strategies.

Effects of low-level laser irradiation on mesenchymal stem cell proliferation: a microarray analysis.

Increased proliferation after low-level laser irradiation (LLLI) has been well demonstrated in many cell types including mesenchymal stem cells (MSCs), but the exact molecular mechanisms involved remain poorly understood. The aim of this study was to investigate the change in mRNA expression in rat MSCs after LLLI and to reveal the associated molecular mechanisms. MSCs were exposed to a diode laser (635 nm) as the irradiated group. Cells undergoing the same procedure without LLLI served as the control group. Proliferation was evaluated using the MTS assay. Differences in the gene expression profiles between irradiated and control MSCs at 4 days after LLLI were analyzed using a cDNA microarray. Gene ontology and pathway analysis were used to find the key regulating genes followed by real-time PCR to validate seven representative genes from the microarray assays. This procedure identified 119 differentially expressed genes. Real-time PCR confirmed that the expression levels of v-akt murine thymoma viral oncogene homolog 1 (Akt1), the cyclin D1 gene (Ccnd1) and the phosphatidylinositol 3-kinase, catalytic alpha polypeptide gene (Pik3ca) were upregulated after LLLI, whereas those of protein tyrosine phosphatase non-receptor type 6 (Ptpn6) and serine/threonine kinase 17b (Stk17b) were downregulated. cDNA microarray analysis revealed that after LLLI the expression levels of various genes involved in cell proliferation, apoptosis and the cell cycle were affected. Five genes, including Akt1, Ptpn6, Stk17b, Ccnd1 and Pik3ca, were confirmed and the PI3K/Akt/mTOR/eIF4E pathway was identified as possibly playing an important role in mediating the effects of LLLI on the proliferation of MSCs.

Immediate repair of major abdominal wall defect after extensive tumor excision in patients with abdominal wall neoplasm: a retrospective review of 27 cases [corrected].

BACKGROUND: The treatment of abdominal wall neoplasm continues to present a challenging problem because it is not easy to repair the giant defect which is resulted from extensive tumor excision. Some techniques and materials have been reported, but most report a certain technique or material for abdominal wall reconstruction. Therefore, we retrospectively reviewed the treatment of such patients in our department and assessed the reconstruction algorithm in such a situation. METHODS: We studied 27 patients undergoing immediate abdominal wall reconstruction between 1999 and 2008 who sought care for major defects after extensive tumor excision of malignancy. We categorized the defects into three types: type I, defects involving only the loss of skin (15 cases); type II, myofascial defects with intact skin coverage (6 cases); and type III, myofascial defects without skin coverage (6 cases). Different techniques and materials were used. Postoperative morbidities, sign of herniation, and other follow-up data were recorded. RESULTS: The immediate abdominal wall reconstruction was successful in all patients. There was no severe morbidity after the operation. Only one patient developed hernia. CONCLUSIONS: Most type I defects can be corrected with primary suture. For type II defects, a prosthetic or biological mesh, or alternatively an autologous fascial substitute, may be used. For type III defects, the resulting full-thickness defect will require a myocutaneous flap, such as the tensor fascia lata flap, with or without a mesh for abdominal wall reconstruction. Human acellular dermal matrix, a biological mesh, is an ideal alternative for synthetic mesh, especially in situations of infection or contamination.

Reconstruction of the abdominal wall by using a combination of the human acellular dermal matrix implant and an interpositional omentum flap after extensive tumor resection in patients with abdominal wall neoplasm: a preliminary result.

AIM: To present our trial using a combination of the human acellular dermal matrix (HADM) implant and an interpositional omentum flap to repair giant abdominal wall defects after extensive tumor resection. METHODS: Between February and October of 2007, three patients with giant defects of the abdominal wall after extensive tumor resection underwent reconstruction with a combination of HADM and omentum flap. Postoperative morbidities and signs of herniation were monitored. RESULTS: The abdominal wall reconstruction was successful in these three patients, there was no severe morbidity and no signs of herniation in the follow-up period. CONCLUSION: The combination of HADM and omentum flap offers a new, safe and effective alternative to traditional forms in the repair of giant abdominal wall defects. Further analysis of the long-term outcome and more cases are needed to assess the reliability of this technique.