[Application of Bionano-DNA Full-Length Optical Mapping Technology in Genetic Diagnosis of Hemophilia A Carriers].

OBJECTIVE: To apply Bionano Saphyr visual full-length DNA optical mapping technology to the precise genetic diagnosis of hemophilia A carriers. METHODS: For 2 suspected F8 gene deficiency female carriers who could not be diagnosed by conventional next-generation sequencing technology, the full-length DNA optical mapping technology was used to detect and scan the sample X chromosome full-length visual haplotype characteristic map, which was compared with the normal haplotype. The gene structure variation information of the samples was obtained by compare with DNA atlas library. RESULTS: The average fluorescent marker length of the X chromosome DNA molecular where the F8 gene was located in the two samples was greater than 2.5 Mbp, and the average copy number was greater than 20×. After comparative analysis, one of the samples was a proximal inversion of intron 22 of the F8 gene, and another was an inversion of intron 22 accompanied by multiple deletions of large fragments. CONCLUSIONS: Bionano technology has a good detection rate for gene defects with large length and complex variation. In the absence of a proband or accurate genetic diagnosis results of the proband, the application of this technology to detect the heterozygous complex variant of the F8 gene is of great significance for the prenatal diagnosis and pre-pregnancy diagnosis of hemophilia carriers.

[Research Progress on the Mechanism of Tubulin in Megakaryopoiesis and Regulation of Platelet Count--Review].

Tubulin affects platelets count through the control of mitosis and the formation of pro-platelets during the maturation of megakaryoblast to platelets. Tubulin is involved in maintaining the integrity of platelet skeleton, and also participates in the change of platelet morphology during platelet activation. Some new anti-tumor drugs targeting cell mitosis are trying to reduce the effect on tubulin in order to reduce the side effect of drugs on platelet formation. In some patients with thrombocytopenia, the variation and polymorphism of the tubulin gene affect the structure of microtubule multimers, which leads to the decrease of platelet formation. This review summarized the latest progresses of tubulin in the regulation of megakaryopoiesis and thrombopoiesis.

[Genetic and Clinical Characteristics of A Family with Combined PROC and PROS1 Genetic Variants].

OBJECTIVE: To test the anticoagulation functions, perform the genetic diagnosis and analyze the clinical characteristics in a family with combined heterozygous genetic variants of PROC and PROS1. METHODS: Peripheral blood was collected from all the family members. Hematological phenotypes and activity of anticoagulant factors were analyzed. Target genes were amplified by PCR from DNA isolated from peripheral blood, and then were analyzed by Sanger DNA sequencing. RESULTS: Many members in the family displayed the combined genetic variants in protein C and protein S, and six family members accompanied by deep venous thrombosis (DVT). The influences of genetic and secondary factors on the incidence of venous thrombosis in the family members were analyzed. The results showed that in this family, carriers of combined protein C and protein S gene defects had a higher incidence of VTE, but acquired factors still played a key role in the eventual thrombotic symptoms. CONCLUSION: Venous thromboembolism (VTE) is a multifactorial disease, the combined genetic heterozygous mutations of protein C and S is an important genetic factor, and the clinical phenotype show a high heterogenicity, the secondary factors contribute to the VTE incidence.

A novel BODIPY-based fluorescent probe for selective detection of hydrogen sulfide in living cells and tissues.

Hydrogen sulfide (H2S) is a critical biological messenger in numerous physiological and pathophysiological processes. Small-molecule fluorescent probes combined with fluorescent microscopy have been developed for the sensitive detection of H2S. Here, we designed and synthesized a long-wavelength BODIPY-based probe (TMSDNPOB) based on the thiolysis of dinitrophenyl ether according to photo-induced electron transfer theory and the results of computational calculation. 4,4-Difluoro-8-phenyl-1,5,7-trimethyl-3-(4-(2,4-dinitrophenoxy)styryl- 4-bora-3a,4a-diaza-s-indacene (TMSDNPOB) is nearly non-fluorescent, but the reaction product (TMSHOB) emits strong red fluorescence at 592nm when excited at 574nm. The long wavelengths of the designed probe indicate low background interference from biological matrix and little photo damage on fluorescence imaging of H2S. With the advantages of the turn-on probe for H2S including high sensitivity, high selectivity, good biocompatibility and low toxicity, the probe has been used for imaging H2S in living cells and liver tissues.

Protective efficacy of recombinant canine adenovirus type-2 expressing TgROP18 (CAV-2-ROP18) against acute and chronic Toxoplasma gondii infection in mice.

BACKGROUND: The use of recombinant viral vectors expressing T. gondii antigens is a safe and efficient approach to induce immune responses against the parasite, as well as a valuable tool for vaccine development. We have previously prolonged the survival time of mice challenged with the RH strain of T. gondii by immunizing the mice with a eukaryotic vector expressing the protein ROP18 of T. gondii. We are now looking for ways to improve this vaccination strategy and enhance protection. METHODS: In this study, we constructed and characterized a novel recombinant canine adenovirus type 2 expressing ROP18 (CAV-2-ROP18) of T. gondii by cytopathic effect (CPE) and indirect immunofluorescence assay (IFA) following transfection into MDCK cells. Intramuscular immunization of Kunming mice with CAV-2-ROP18 was carried out to evaluate humoral and cellular immune responses. RESULTS: The vaccination of experimental mice with CAV-2-ROP18 elicited antibody production against ROP18, including high levels of a mixed IgG1/IgG2a and significant production of IFN-γ or IL-2, and displayed a significant bias towards a helper T cell type 1 (Th1) profile. Furthermore, the presence of T. gondii-specific IFN-γ-production and TNF-α-production T cells was elicited in both CD4+ and CD8+ T cell compartments. Significantly higher survival rates (40%) occurred in the experimental group, and a reduction in brain cyst burden was detected in vaccinated mice. CONCLUSION: These results demonstrate the potential use of a CAV vector harboring the ROP18 gene in the development of a vaccine against acute and chronic toxoplasmosis.

Multicenter study of creatinine- and/or cystatin C-based equations for estimation of glomerular filtration rates in Chinese patients with chronic kidney disease.

OBJECTIVE: To establish equations for the estimation of glomerular filtration rates (eGFRs) based on serum creatinine (SCr) and/or serum cystatin C (SCysC) in Chinese patients with chronic kidney disease (CKD), and to compare the new equations with both the reference GFR (rGFR) and the literature equations to evaluate their applicability. METHODS: The 788 Chinese CKD patients were randomly divided into two groups, the training group and the testing group, to establish new eGFR-formulas based on serum CysC and to validate the established formulas, respectively. (99m)Tc-DTPA clearance (as the rGFR), serum Cr, and serum CysC were determined for all patients, and GFR was calculated using the Cockcroft-Gault equation (eGFR1), the MDRD formula (eGFR2), the CKD-EPI formulas (eGFR3, eGFR4), and the Chinese eGFR Investigation Collaboration formulas (eGFR5, eGFR6). The accuracy of each eGFR was compared with the rGFR. RESULTS: The training and testing groups' mean GFRs were 50.84±31.36 mL/min/1.73 m(2) and 54.16±29.45 mL/min/1.73 m(2), respectively. The two newly developed eGFR formulas were fitted using iterative computation: [Formula: see text] and [Formula: see text]. Significant correlation was observed between each eGFR and the rGFR. However, proportional errors and constant errors were observed between rGFR and eGFR1, eGFR2, eGFR4, eGFR5 or eGFR6, and constant errors were observed between eGFR3 and rGFR, as revealed by the Passing & Bablok plot analysis. The Bland-Altman analysis illustrated that the 95% limits of agreement of all equations exceeded the previously accepted limits of <60 mL/min •1.73 m(2), except the equations of eGFR7 and eGFR8. CONCLUSION: The newly developed formulas, eGFR7 and eGFR8, provide precise and accurate GFR estimation using serum CysC detection alone or in combination with serum Cr detection. Differences in detection methods should be carefully considered when choosing literature eGFR equations to avoid misdiagnosis and mistreatment.

[The effects of recombinant human beta-defensin-3 on expression of interleukin-17A and interleukin-22 in BEAS-2B cell].

OBJECTIVE: To research the effects of recombinant human beta-defensin-3 (hBD-3) on expression of interleukin-17A (IL-17A) and interleukin-22 (IL-22) in BEAS-2B cell. METHODS: The BEAS-2B cells were stimulated with different concentrations of hBD-3 for 6 hours and 24 hours, respectively. Toll-like receptor 2 (TLR2), IL-17A and IL-22 mRNA expression levels were determined by real-time PCR, and the expression levels of IL-17A and IL-22 protein were examined by enzyme linked immune-sorbent assay. RESULTS: TLR2 mRNA in BEAS-2B cells were significantly increased in a concentration-and time-dependent manner after stimulating by hBD-3 for 24 hours compared to 6 hours. The IL-17A has significantly increased in mRNA and protein levels stimulated 24 hours in a concentration of 100 ng/ml, however, IL-17A mRNA expression has increased while protein didn't change stimulated 6 hours in a concentration of 50 ng/ml. The IL-22 mRNA and protein expression reached peak levels after stimulating in a concentration of 50 ng/ml of hBD-3 while IL-22 expression declined in mRNA and protein levels as the concentration of hBD-3 increased. CONCLUSIONS: Recombinant hBD-3 can up-regulated the expression of TLR2, IL-17A and IL-22, lower concentration of hBD-3 mainly increased the expression of IL-22 while higher concentration of hBD-3 mainly increased the expression of IL-17A. These results show that different concentrations of hBD-3 maybe activate different transcription factors which was mediated by TLR2, initiating host immune response.

[Etiology and epidemic characteristics of hospital acquired pneumonia in children].

OBJECTIVE: To investigate the etiology and epidemic characteristics of hospital acquired pneumonia (HAP) in children. METHOD: A retrospective hospital infection study was performed in 52,639 children admitted to our hospital from January 2005 to December 2008. RESULT: Six hundred and ninety eight patients were diagnosed as HAP. The incidence of HAP was 1.33%, among which, 108 (15.47%) cases were early-onset HAP and 590 (84.53%) were late-onset HAP. The HAP patients aged 3 days to 15 years (503 male and 195 female). The proportion of patients younger than 1 year was 51.4%. Main underlying diseases were cytomegalovirus hepatitis, congenital heart disease, malignant tumor, granulocytopenia or agranulocytosis, prematurity and low birth weight. There was significant difference in the incidences among different departments with the highest one seen in ICU, followed by departments of infectious diseases, hematology and digestive diseases. Two hundred and thirty one stains of pathogens were identified from sputum of 355 cases. One hundred and fifty six (67.5%) strains were Gram-negative bacteria, which accounted for the highest proportion. There were 30 (13.0%) Gram-positive bacterial strains, and 29 (12.6%) respiratory tract virus strains, and 15 (6.5%) fungal strains, and 1 (0.4%) mycoplasma strain. The predominant bacterial pathogens were Klebsiella pneumoniae, followed by Stenotrophomonas maltophilia, Burkholderia cepacia, Escherichia coli and Acinetobacter baumannii. The isolation rates of Klebsiella pneumoniae and Escherichia coli with positive extended-spectrum beta-lactamases (ESBLs) were 94.8% and 85.7%, respectively. Those two bacteria were universally resistant to the third and forth generations cephalosporins. The main pathogens of early-onset HAP were respiratory syncytial virus (RSV), Streptococcus mitis, Streptococcus pneumonia, Haemophilus influenzae and Klebsiella pneumoniae, while the main pathogens of late-onset HAP were ESBLs-positive Klebsiella pneumonia, Stenotrophomonas maltophilia, Burkholderia cepacia, Escherichia coli and Acinetobacter baumannii. CONCLUSION: HAP in children is most common in children younger than 1 year and with underlying diseases. The main pathogens are Gram-negative bacteria. RSV was an important pathogen of HAP. The pathogens of early-onset HAP are different from those of late-onset HAP. These results may be of some help in prevention and control of HAP in children and in guiding for rational application of antibiotics, especially the empirical antibiotic choice.

[A comparative study of sentinel lymph node detection in laryngeal and hypopharyngeal carcinoma by lymphoscintigraphy method and blue dye].

OBJECTIVE: To investigate the clinical value of sentinel lymph node (SLN) detection in laryngeal and hypopharyngeal carcinoma patients with clinically negative neck (cN0) by lymphoscintigraphy method and blue dye. METHODS: Forty patients with cN0 laryngeal neoplasms and ten patients with cN0 hypopharyngeal carcinoma scheduled for tumor resection and neck dissection, were eligible for the study. single photon emission computed tomography (SPECT)/CT lymphoscintigraphy was performed with injection of radioactivity isotope 99Tc(m) labeled sulfur colloid (99Tc(m)-SC). Methylthioninium was injected into the same points as 99Tc(m)-SC during surgery, and the patients underwent lymphatic mapping with a handheld gamma-detecting probe. All removed lymph nodes were examined by routine histopathology. RESULTS: Thirty-five patients with laryngeal carcinoma and six patients with hypopharyngeal carcinoma detected SLN by radiolabeled tracer method, the detection rate of SLN was 82.0%. Twenty-nine patients with laryngeal carcinoma and 4 patients with hypopharyngeal carcinoma detected SLN by blue dye method, the detection rate of SLN was 66.0%. There were significant difference between two groups (chi² = 2.769, P < 0.05), and the number of SLN were respectively 96 and 83 by radiolabeled tracer method and blue dye (chi² = -2.098, P < 0.05), The sensitivity of SLN detection were respectively 83.3% and 66.7%. Twelve (24.0%) patients had lymph node metastasis. CONCLUSIONS: Either lymphoscintigraphy or blue dye mapping can be used to detect the SLN in cN0 laryngeal and hypopharyngeal carcinoma. The lymphoscintigraphy not only preoperatively can locate the accuracy of SLN detection, but also has higher detection rate and sensitivity than dye method.

Efficient Suzuki-Type Cross-Coupling of Enantiomerically Pure Cyclopropylboronic Acids.

Optically active cyclopropanes (e.g. 1) can be prepared by the use of tartaric acid derivatives as chiral auxiliaries in the palladium-catalyzed cross-coupling of optically active cyclopropylboronic acids with electrophiles. The absolute configuration of the chiral carbon atom is retained, and the reaction proceeds with good yields and enantiomeric excesses. R=H, p-Ph, o-CO2 CH3 , p-CO2 CH3 , p-NO2 , o-OCH3 , m-OCH3 .