Objective:To investigate the signaling pathway of inhibiting macrophage phagocytosis of TIR domain-containing protein encoded by Escherichia coli (TcpC) N-terminal ubiquitin ligase active fragments of uropathogenic Escherichia coli. Methods:Bioinformatics software was used to analyze the amino acid sequences and the function of TcpC N-terminal ubiquitin ligase active fragments as well as the functional sites. PCR was performed to amplify tcpc-330, tcpc-450 and tcpc-510 genes and a prokaryotic expression system was constructed to express the target proteins. The recombinant proteins rTcpC-N110, rTcpC-N150 and rTcpC-N170 were purified by Ni-NTA affinity chromatography. LPS in the recombinant proteins was removed by Detoxi-gel chromatography. The expression of MyD88 at protein and mRNA levels in macrophages incubated with rTcpC-N110, rTcpC-N150, rTcpC-N170 or rTcpC-TIR was detected by Western blot and qRT-PCR. The activation of NF-κB signal pathway and the levels of proinflammatory factors in macrophages incubated with the above TcpC protein fragments were measured by Western blot and ELISA, respectively. Results:Cys12, Trp104 and Trp106 in the N-terminal fragment of TcpC were crucial amino acids in maintaining its ubiquitin ligase activity. The target recombinant proteins rTcpC-N110, rTcpC-N150 and rTcpC-N170 were successfully expressed and purified. After Detoxi-gel chromatography, rTcpC-N110, rTcpC-N150 and rTcpC-N170 extracts were undetectable for LPS. TcpC ubiquitin ligase fragments inhibited the expression of MyD88 at protein level, but not affect its expression at mRNA level in macrophages. LPS-induced phosphorylation of NF-κB signaling pathway-related proteins p50 and p65 was significantly inhibited in macrophages treated with TcpC ubiquitin ligase fragments. Moreover, LPS-induced production of pro-inflammatory factors was also significantly inhibited.Conclusions:The recombinant proteins rTcpC-N110, rTcpC-N150 and rTcpC-N170 could inhibit the expression of MyD88 at protein level and suppress the activation of NF-κB signaling pathway, suggesting that they were closely related to the inhibition of innate immune activity of macrophages.

Objective:To investigate whether the hypervirulent Klebsiella pneumoniae (hvKP) induces liver abscess through activating NLRP3 inflammasome. Methods:K1-hvKP and K35-non-hvKP bacterial suspensions were intraperitoneally injected into C57BL/6 mice to establish the models of liver abscess. Human peripheral blood neutrophils were sorted by immunomagnetic beads with CD45 + and Gr-1 + , and the purity was detected by flow cytometry. The concentrations of capsular polysaccharide of K1-hvKP and K35-non-hvKP were detected by total carbohydrate assay kit. The expression of IL-18 and IL-33 by neutrophils at mRNA and protein levels was detected by real-time fluorescence quantitative PCR and ELISA, respectively. The activation of NLRP3 inflammasome in neutrophils was detected by Western blot. Neutrophil extracellular trap formation (NETosis) was observed under confocal laser scanning microscope. Results:The C57BL/6 mice with K1-hvKP infection had significantly serious liver abscess as compared with the K35-non-hvKP-infected mice. The purity of human neutrophils was more than 95%. The concentration of capsular polysaccharide in K1-hvKP was significantly higher than that in K35-non-hvKP. Compared with K35-non-hvKP, K1-hvKP significantly promoted the neutrophils to express IL-18 and IL-33 at both mRNA and protein levels, enhanced the activation of NLRP3 and induced NETosis.Conclusions:This study suggested that hvKP could promote NETosis by activating NLRP3 inflammasome to cause liver abscess.

Objective:To investigate the role of TcpC in uropathogenic Escherichia coli (UPEC)-induced cystitis in mice and to preliminarily analyze the pathogenic mechanism. Methods:C57BL/6 mice were injected with 10 9 CFU wild-type UPEC CFT073 (CFT073 wt) or tcpc gene-deleted mutant (CFT073 Δ tcpc) from urethra into bladder to construct the mouse model of cystitis. The mice were sacrificed 3 d after infection and the bladders were taken to observe the gross pathological changes. Histopathological changes in bladder tissues were observed after HE staining. Immunohistochemistry was used to detect TcpC in bladder tissues. Bacterial loads in urine samples of UPEC-infected mice were counted by tenfold dilution method, and the presence of tcpc gene in the genomic DNA of bacteria from the bladder and urine samples of CFT073 wt-infected mice was measured by PCR. Real-time quantitative RT-PCR (qRT-PCR) and Western blot were performed to detect the expression of TcpC at mRNA and protein levels in macrophages after CFT073 wt infection. The influence of UPEC strains on the activation of NF-κB signaling pathway in macrophages were determined by Western blot. The levels of proinflammatory factors and the bacterial and cell activity after infecting macrophages with UPEC strains were detected by ELISA, laser confocal microscope and fluorescence microscope, respectively. Results:Compared with the mice with CFT073 Δ tcpc infection, CFT073 wt-infected mice had significantly enlarged bladder and severe neutrophil infiltration and abundant TcpC in bladder tissues. The number of bacteria in the urine of CFT073 wt-infected mice was significantly greater than that of the CFT073 Δ tcpc group. PCR results showed that the bacteria in bladder or urine were CFT073 wt. The expression of TcpC at both mRNA and protein levels in macrophages increased significantly after CFT073 wt infection. Moreover, in CFT073 wt-infected macrophages, the expression of IκBα was promoted and the phosphorylation of p65 and the production of proinflammatory factors were suppressed. TcpC was instrumental in the survival and invasion of CFT073 wt in macrophages. Conclusions:TcpC expression increased significantly in mice with CFT073 wt-induced cystitis. TcpC inhibited the activation of NF-κB signaling pathway and the production of proinflammatory factors in macrophages to improve the survival rate of CFT073 wt, which was closely related to the pathogenesis and immune evasion of UPEC.

ObjectiveTo explore the related factors of troublemaking behaviors among patients with mental disorders induced by amphetamine-type stimulants （ATS）， and to provide references for the formulation of relevant intervention measures for ATS-induced mental disorders. MethodsA total of 105 patients who met the diagnostic criteria of International Classification of Diseases， tenth edition （ICD-10） for ATS-induced mental disorders were included， and classified into troublemaking group and non-troublemaking group. The general demographic data and clinical data of the selected individuals were collected， and all patients were assessed using Social Support Rating Scale （SSRS）. Then univariate analysis and multivariate Logistic regression model were used to screen the related factors of troublemaking behaviors. ResultsThe scores of SSRS， objective support dimension and social support utilization dimension were significantly lower in troublemaking group than those in non-troublemaking group， with statistical differences ［（24.10±6.59） vs. （28.94±5.59）， t=3.364， P=0.001； （5.50±1.96） vs. （8.20±2.13）， t=5.183， P<0.01； （4.60±2.26） vs. （6.28±1.90）， t=3.435， P=0.001］. Multivariate Logistic regression analysis showed that male （OR=6.061， P=0.014） was a risk factor， while high social support level （OR=0.873， P=0.018） was the protective factor for troublemaking behaviors among patients with ATS-induced mental disorders. ConclusionPatients with ATS-induced mental disorders of the males and with low social support level are at high risk of troublemaking behaviors.

OBJECTIVE: To assess the clinical application of single nucleotide polymorphism microarray (SNP array) in patients with intellectual disability/developmental delay(ID/DD). METHODS: SNP array was performed to detect genome-wide DNA copy number variants (CNVs) for 145 patients with ID/DD in Women's Hospital, Zhejiang University School of Medicine from January 2013 to June 2018. The CNVs were analyzed by CHAS software and related databases. RESULTS: Among 145 patients, pathogenic chromosomal abnormalities were detected in 32 cases, including 26 cases of pathogenic CNVs and 6 cases of likely pathogenic CNVs. Meanwhile, 18 cases of uncertain clinical significance and 14 cases of likely benign were identified, no significant abnormalities were found in 81 cases (including benign). CONCLUSIONS SNP array is effective for detecting chromosomal abnormalities in patients with ID/DD with high efficiency and resolution.
Chromosome Aberrations ; DNA Copy Number Variations ; Genome-Wide Association Study ; Humans ; Intellectual Disability ; diagnosis ; genetics ; Oligonucleotide Array Sequence Analysis ; standards ; Polymorphism, Single Nucleotide

OBJECTIVE: To assess the clinical application of single nucleotide polymorphism microarray (SNP array) in prenatal genetic diagnosis for fetuses with absent nasal bone. METHODS: Seventy four fetuses with absent nasal bone detected by prenatal ultrasound scanning were recruited from Women's Hospital, Zhejiang University School of Medicine during June 2015 and October 2018. The chromosome karyotypes analysis and SNP array were performed. The correlation between absent fetal nasal bone and chromosome copy number variants was analyzed. RESULTS: Among 74 fetuses, 19 were detected to have chromosomal abnormalities, including 16 cases of trisomy-21, 1 case of trisomy-18 and two cases of micro-deletion/duplication. Among 46 cases with isolated absence of nasal bone, 3 had trisomy-21, and 1 had a micro-duplication. Absence of nasal bone in association with nuchal translucency thickening had a higher rate of abnormal karyotypes compared with isolated absence of nasal bone (=32.27,<0.01). CONCLUSIONS Fetuses with absent nasal bone and nuchal translucency thickening are likely to have chromosome abnormalities, and SNP array testing is recommended to exclude the chromosome abnormalities.
Chromosome Aberrations ; Female ; Fetus ; Humans ; Nasal Bone ; abnormalities ; Oligonucleotide Array Sequence Analysis ; standards ; Polymorphism, Single Nucleotide ; genetics ; Pregnancy ; Pregnancy Trimester, First ; Prenatal Diagnosis ; methods

OBJECTIVE: To conduct genetic analysis in a fetus with complex translocation of four chromosomes. METHODS: G-banded chromosome karyotype analysis, single nucleotide polymorphism array (SNP array) and fluorescence hybridization (FISH) were performed in a fetus with multiple malformations. Peripheral blood chromosome karyotype and FISH were also carried out for the parents. RESULTS: The fetal amniotic fluid karyotype was 46, XY, t(12; 13)(q22; q32). SNP array analysis showed that there were 20 192 kb duplication at 1q42.13q44 and 13 293 kb deletion at 15q26.1q26.3 in the fetus. The results of karyotype and SNP array were inconsistent. FISH analyses on the parental peripheral blood samples demonstrated that the mother was a cryptic 46, XX, t(1; 15)(q42.1; q26.1) translocation. The fetus had inherited 46, XY, t(12; 13)(q22; q32) from his father and der(15)t(1; 15)(q42.1; q26.1) from his mother. CONCLUSIONS The 1q42.13q44 duplication and 15q26.1q26.3 deletion may have contributed to the abnormal sonographic features of the fetus. The combination of cytogenetic, SNP array and FISH techniques was beneficial for providing an accurate genetic counseling.
Chromosome Aberrations ; Female ; Fetus ; abnormalities ; Humans ; In Situ Hybridization, Fluorescence ; Karyotyping ; Male ; Polymorphism, Single Nucleotide ; Translocation, Genetic

Objective: To explore the relationship between HCM pathogenic gene mutations and clinical phenotypes by analyzing the prenatal diagnosis and genetic characteristics of a pregnant woman from a family with hypertrophic cardiomyopathy (HCM). Methods: The clinical data of the proband and her family members was collected. The DNA was extracted from the peripheral blood, amniotic fluid cells and cultured amniotic fluid cells of proband. Next generation sequencing (NGS) was utilized for screening pathogenetic loci of the proband. The suspected mutation sequences of HCM pathogenic candidate genes MYH7 and MYBPC3 were directly sequenced after PCR. Pathogenicity prediction of amniotic fluid cells was performed by using genetic data and bioinformatics software, such as Mutation taster, PolyPhen-2 and ANTHEPROT. Results: The sequencing results showed that heterozygous mutations of MYH7 c.1988G＞A (p.Arg663His) and MYBPC3 c.151G＞A (p.Ala51Thr) were found in the proband. The phenotype of her father was normal, and no abnormal mutations were detectable. Her mother also showed normal phenotype but carried MYBPC3 c.151G＞A heterozygous mutation. Only MYH7 c.1988G＞A heterozygous mutation was found in the fetus and no abnormal variation of MYBPC3 was showed. The prediction of mutation effect and analysis of protein structure and function revealed that the two missense mutations could affect the hydrophobicity and antigenicity of the protein. The genetic data demonstrated MYH7 c.1988G＞A was defined as a pathogenic mutation. Conclusion MYH7 c.1988G＞A should be a newly generated pathogenic mutation in the proband, or caused by reproductive chimerism of her parents. MYBPC3 c.151G＞A mutation may promote the occurrence of HCM. Although the fetus only carries MYH7 c.1988G＞A, her phenotype may still display as HCM.

Objective: To explore the genetic basis for a pedigree affected with Marfan syndrome (MFS). Methods: Clinical data of the patients was collected.With genomic DNA extracted from peripheral blood samples, potential mutation was detected by targeted exome sequencing.Candidate variants were validated by Sanger sequencing and bioinformatic analysis. Results: Targeted exome sequencing and Sanger sequencing revealed a missense c. 649T>C(p.Trp217Arg) variant in the exon 7 of FBN1 gene, which was unreported previously.Bioinformatics analysis suggested that the variant can cause amino acid replacement and affect the structure and function of fibrillin-1. Conclusion A novel missense variant of the FBN1 gene was identified, which probably underlies the autosomal dominant MFS in this pedigree.

Objective To assess the longitudinal mitral annular plane systolic excursion ( M APSE) of different directions in normal fetuses during mid‐late pregnancy based on two‐dimensional speckle tracking imaging ( ST I) . Methods Seventy‐six normal fetuses during middle and late pregnancy were selected at 26－32 weeks of gestation . T he peak M APSE was measured by free angle M‐mode echocardiography ( FAM ) perpendicular to the lateral annulus in the mitral annular plane . The time‐displacement curves of interventricular septal mitral annulus in three different directions including points A ,B and C through transverse level of apex were recorded by STI . T he peak M APSE of interventricular septal mitral annulus ( SEPT‐M APSE‐A ,SEPT‐M APSE‐B ,SEPT‐M APSE‐C) in three different directions including points A ,B and C and the time to peak ( T T P :SEPT‐T T P‐A ,SEPT‐T T P‐B ,SEPT‐T T P‐C) were recorded respectively . T he time‐displacement curves of lateral mitral annulus in three different directions including points A ,B and C through transverse level of apex were recorded by STI . T he peak M APSE of lateral mitral annulus ( LAT‐M APSE‐A ,LAT‐MAPSE‐B ,LAT‐MAPSE‐C) in three different directions including points A ,B and C ,the time to peak( LA T‐T T P‐A ,LA T‐T T P‐B ,LA T‐T T P‐C) were recorded respectively . Finally ,the data were analyzed statistically . Results T he peak M APSE of the lateral mitral annulus in 3 different directions including points A ,B and C[ LA T‐M APSE‐A ( 3 .62 ± 1 .01) mm ,LA T‐M APSE‐B ( 3 .95 ± 1 .04) mm ,LAT‐M APSE‐C ( 4 .45 ± 1 .05) mm ] were greater than those of the interventricular septum mitral annulus[ SEPT‐MAPSE‐A (3 .41 ± 0 .63)mm ,SEPT‐MAPSE‐B (3 .07 ± 0 .50) mm ,SEPT‐MAPSE‐C (2 .82 ± 0 .51) mm] . LAT‐M APSE‐C and SEPT‐M APSE‐A were the largest longitudinal excursions of mitral annulus . T he differences were statistically significant in points B and C ( P ＜0 .05) . T here was no significant difference in point A ( P ＞0 .05) . LA T‐M APSE‐C was less than FAM‐M APSE [ ( 6 .06 ± 1 .35 ) mm ] . T here was a significant difference between them ( P ＜0 .05 ) . Strong correlation was found between them ( r ＝0 .896 , P＜0 .05) . T here were no significant differences in the time to peak of interventricular septal mitral annulus [ SEPT‐T T P‐A ( 0 .210 ± 0 .008 ) s ,SEPT‐T T P‐B ( 0 .213 ± 0 .008 ) s ,SEPT‐T T P‐C ( 0 .210 ± 0 .005 ) s] in directions including points A ,B ,C ( P＞ 0 .05 ) . T here were no significant differences in time to peak of lateral mitral annulus [ LAT‐T T P‐A ( 0 .210 ± 0 .008 ) s , LAT‐T T P‐B ( 0 .213 ± 0 .006 ) s , LAT‐T T P‐C ( 0 .210 ± 0 .007) s] in directions inclucling points A ,B ,C ( P ＞0 .05) . Conclusions Longitudinal systolic motion of fetal left ventricular wall during mid‐late pregnancy has good synchronization . Longitudinal motion of fetal mitral annulus is a comprehensive movement of multiple directions and different degrees of displacement ,with the movement perpendicular to the annulus as the maximum displacement direction . T he displacement parameters of mitral annulus measured by ST I can reflect the left ventricular longitudinal systolic function and have clinical application value in evaluating the left ventricular longitudinal systolic function of fetuses .