RESUMO
Soluble inorganic pyrophosphatases (PPases) are essential for facilitating the growth and development of organisms, making them attractive functional proteins. To provide insight into the molecular basis of PPases in Schistosoma japonicum (SjPPase), we expressed the recombinant SjPPase, analyzed the hydrolysis mechanism of inorganic pyrophosphate (PPi), and measured its activity. Moreover, we solved the crystal structure of SjPPase in complex with orthophosphate (Pi) and performed PPi and methylene diphosphonic acid (MDP) docking into the active site. Our results suggest that the SjPPase possesses PPi hydrolysis activity, and the activity declines with increased MDP or NaF concentration. However, the enzyme shows unexpected substrate inhibition properties. Through PPi metabolic pathway analysis, the physiological action of substrate inhibition might be energy saving, adaptably cytoprotective, and biosynthetic rate regulating. Furthermore, the structure of apo-SjPPase and SjPPase with Pi has been solved at 2.6 and 2.3 Å, respectively. The docking of PPi into the active site of the SjPPase-Pi complex revealed that substrate inhibition might result from blocking Pi exit due to excess PPi in the SjPPase-Pi complex of the catalytic cycle. Our results revealed the structural features of apo-SjPPase and the SjPPase-Pi complex by X-ray crystallography, providing novel insights into the physiological functions of PPase in S. japonicum without the PPi transporter and the mechanism of its substrate inhibition.
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BACKGROUND: Babesiosis is caused by the invasion of erythrocytes by parasites of the Babesia spp. Babesia microti is one of the primary causative agents of human babesiosis. To better understand the status of the disease, discovering key biomarkers of the different infection stages is crucial. RESULTS: This study investigated B. microti infection in the mouse model from 0 to 270 days post-infection (dpi), using blood smears, PCR assays and ELISA. PCR assays showed a higher sensitivity when compared to microscopic examination. Specific IgG antibodies could be detected from 7 days to 270 dpi. Two-dimensional electrophoresis was combined with western blotting and mass spectrometric analysis to screen for specific reactive antigens during both the peak parasitaemia period (7 dpi) and IgG antibody response peak period (30 dpi) by the infected mice plasma. The 87 positive reactive proteins were identified and then expressed with the wheat germ cell-free system. Protein microarrays of all 87 targeted proteins were produced and hybridized with the serial plasma of infected mice model. Based on the antigen reaction profile during the infection procedure, 6 antigens were selected and expressed in Escherichia coli. Due to an early response to IgM, lower immunoreactivity levels of IgG after two months and higher immunoreactivity level IgG during nine months, four recombinant proteins were selected for further characterization, namely rBm2D97(CCF75281.1), rBm2D33(CCF74637.1), rBm2D41(CCF75408.1) and rBm7(CCF73510.1). The diagnostic efficacy of the four recombinant protein candidates was evaluated in a clinical setting using babesiosis patient plasma. The rBm2D33 showed the highest sensitivity with a positive rate of 62.5%. Additional characterization of the two candidate proteins using a mouse vaccination assay, demonstrated that rBm2D41 could reduce peak parasitaemia by 37.4%, indicating its efficacy in preventing severe babesiosis. CONCLUSIONS: The detection technologies of microscopic examination, PCR assays and antibody tests showed different sensitivities and accuracy during the different stages of B. microti infection. Antibody detection has a unique significance for B. microti infection in the asymptomatic stages. Using immunoreactivity profiles, biomarkers for disease progression were identified and represent useful information for future the diagnosis and vaccine development for this serious disease of public health significance.
Assuntos
Babesia microti/imunologia , Babesiose/diagnóstico , Babesiose/imunologia , Progressão da Doença , Proteínas Recombinantes/isolamento & purificação , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Babesia microti/fisiologia , Babesiose/sangue , Biomarcadores/sangue , Confiabilidade dos Dados , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Eritrócitos/parasitologia , Feminino , Humanos , Imunoglobulina G/sangue , Camundongos , Parasitemia/diagnóstico , Parasitemia/parasitologia , Análise Serial de Proteínas/métodos , Proteômica , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: To explore clinical effect of modified Chevron osteotomy combined with lateral tissue loosening for the treatment of mild-moderate hallux valgus through internal signal approach. METHODS: From July 2015 to June 2016, 26 patients with mild-moderate hallux valgus treated with modified Chevron osteotomy combined with lateral tissue loosening through internal signal approach, including 2 males and 24 females aged from 45 to 65 years old with an average of(54.6±4.8) years old;the courses of diseases ranged from 1 to 5 months with an average of (7.5±3.3) months. Hallux valgus angle(HVA), inter metatarsal angle(IMA) were measured at 12 months after operation, and AOFAS score was applied to evaluate clinical effect before and after operation. RESULTS: All incisions were healed at stage I. No incision occurred infection, metatarsal necrosis and recurrence of hallux valgus deformity. Two patients occurred skin numbness caused by musculocutaneous nerve injury. Twenty-six patients were followed up from 6 to 12 months with an average of(9.12±2.06) months. HVA, IMA were(30.01±3.71)°, (14.00±1.50)° before operation and(9.41±4.16)°, (7.00±0.60)° after operation, which had significant difference. There was statistical significance in AOFAS score before operation 54.77±9.59 and after operation 92.73±5.47, and 19 cases obtained excellent results and 7 moderate. CONCLUSIONS: Modified Chevron osteotomy combined with full thread headless pressure screw fixation and lateral tissue loosening for the treatment of mild-moderate hallux valgus has advantages of excellent exposure, simple operation, stable fixation, rapid recovery. Akin osteotomy with internal capsulorrhaphy were used with lateral loosening and could recover soft tissue balance between lateral and internal, and could receive satisfied clinical effects.
Assuntos
Hallux Valgus/cirurgia , Osteotomia/métodos , Idoso , Feminino , Humanos , Masculino , Ossos do Metatarso/patologia , Pessoa de Meia-Idade , Radiografia , Recidiva , Resultado do TratamentoRESUMO
OBJECTIVES: Intrahepatic recurrence is the major cause of death among patients with hepatitis B virus (HBV)- related hepatocellular carcinoma (HCC) after curative surgical resection. Several approaches have been reported to decrease the recurrence rate. The objective of our study was to compare the clinical effects of transcatheter arterial chemoembolization (TACE) combined with interferon-alpha (IFN-α) therapy on recurrence after hepatic resection in patients with HBV-related HCC with that of TACE chemotherapy alone. METHODS: We retrospectively analyzed the data from 228 patients who were diagnosed with HBV-related HCC and underwent curative resection between January 2001 to December 2008. The patients were divided into TACE (n = 126) and TACE-IFN-α (n = 102) groups for postoperative chemotherapy. The TACE regimen consisted of 5-fluorouracil (5-FU), cisplatin (DDP) , and the emulsion mixed with mitomycin C (MMC) and lipiodol. The recurrence rates, disease-free survival (DFS), overall survival (OS), and risk of recurrence were evaluated. RESULTS: The clinicopathological parameters and adverse effects were similar between the 2 groups (P > 0.05). The median OS for the TACE- IFN-α group (36.3 months) was significantly longer than that of the TACE group (24.5 months, P < 0.05). The 3-and 5-year OS for the TACE-IFN-α group were significantly longer than those of the TACE group (P < 0.05) and the recurrence rate was significantly lower (P < 0.05). The TACE and IFN-α combination therapy, active hepatitis HBV infection, the number of tumor nodules, microvascular invasion, liver cirrhosis, and the BCLC stage were independent predictors of OS and DFS. CONCLUSIONS: The use of the TACE and IFN-α combination chemotherapy after curative hepatic resection safely and effectively improves OS and decreases recurrence in patients with HBV-related HCC who are at high risk. Our findings can serve as a guide for the selection of postoperative adjuvant chemotherapy for patients with HBV-related HCC who are at high risk of recurrence.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Hepatocelular/terapia , Neoplasias Hepáticas/terapia , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Carcinoma Hepatocelular/virologia , Quimioembolização Terapêutica/efeitos adversos , Quimioembolização Terapêutica/métodos , Cisplatino/administração & dosagem , Terapia Combinada/efeitos adversos , Terapia Combinada/métodos , Intervalo Livre de Doença , Óleo Etiodado/administração & dosagem , Feminino , Fluoruracila/administração & dosagem , Hepatite B/virologia , Vírus da Hepatite B , Humanos , Interferon-alfa/administração & dosagem , Interferon-alfa/efeitos adversos , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , Mitomicina/administração & dosagem , Recidiva Local de Neoplasia/terapia , Recidiva Local de Neoplasia/virologia , Estudos Retrospectivos , Adulto JovemRESUMO
OBJECTIVE: To clone and express Echinococcus granulosus pyruvate dehydrogenase (EgPDH) gene and analyze EgPDH protein with bioinformatics tools and online database. METHODS: The total RNAs of E. granulosus was extracted and reversely transcribed into cDNA. The EgPDH gene was cloned into pET28b to construct the recombinant vector and expressed in E. coli BL21 (DE3) system subsequently. The signal peptide, transmembrane helices and subcellular location in EgPDH sequence were analyzed by the online software SignalP4.1, TMHMM sever v.2.0 and TargetP1.1, respectively. Subsequently, the structure of EgPDH was predicted by SMART. Finally, the homologue sequence and conserved sites were aligned by using BLASTP and GeneDoc among the homologous sequences of EgPDH. Based on the alignment of PDH sequence, an evolutionary tree of E. granulosus and other species were constructed by the neighbor joining method of MEGA6 software. RESULTS: The EgPDH gene was successfully amplified from cDNA of E. granulosus and expressed in the soluble fractions. The bioinformatics analysis revealed that EgPDH was a classical secreted protein and contained transketolase domain. The homology analysis revealed that the amino acid sequence of EgPDH was highly conserved in catalytic sites Glu57, Leu72, Ile86 and Phe114. The phylogenetic tree analysis of PDH proteins showed the closest relationship between E. granulosus and E. multilocularis. CONCLUSION: An EgPDH gene is cloned and expressed successfully, and the recombinant protein is analyzed by the bioinformatics approaches and structure predication. The study provides useful information for further functional study of the EgPDH protein.
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Biologia Computacional , Echinococcus granulosus/enzimologia , Cetona Oxirredutases/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cetona Oxirredutases/química , Cetona Oxirredutases/fisiologia , Dados de Sequência MolecularRESUMO
OBJECTIVE: To investigate the treatment of femoral neck fractures after the fixation of ipsilateral femoral shaft fracture by antegrade intramedullary nail. METHODS: A retrospective study on 12 patients with femoral neck fractures after the fixation of ipsilateral femoral shaft fracture by antegrade intramedullary nail, which were identified intraoperatively or postoperatively from January 2000 to January 2010. All the patients were treated with 2 supplemental screws placed anteriorly and posteriorly to intramedullary nail seperately. All the patients were periodic followed-up, fractures union and functional recovery were evaluated. RESULTS: All the patients were followed up, and the duration ranged from 10 to 36 months (averaged 16.5 months). The mean healing time was 3.6 months in femoral neck fractures and 5.4 months in femoral shaft fractures. No osteonecrosis of femoral head was found. According to Harris scoring system for hip function, 7 patients got an excellent result, 3 good, 2 fair. CONCLUSION: Treatment of femoral neck after the fixation of ipsilateral femoral shaft by antegrade intramedullary nail with 2 screws placed anteriorly and posteriorly to intramedullary nail separately is feasible, and has the advantages of reliable fixation, less trauma and high rate of fracture healing.