To develop methods of extraction and purification of C-terminal NUDT9 homology domain of human transient receptor potential melastatin 2 (TRPM2) channel.

After sonication and centrifuge of Escherichia coli strain Rosetta (DE3) which was induced by isopropylthio-β-D-galactoside, GST-NUDT9-H was collected after the binding of supernatant with GST beads and eluted with reduced glutathione. Then the elution buffer containing fusion protein was purified by size exclusion chromatography after concentration and centrifuge. Finally, with the cleavage of thrombin and binding with the GST beads, NUDT9-H with high purity in supernatant was collected.

The GST-NUDT9-H fusion protein was stabilized with lysis buffer containing 0.5% n-dodecyl-β-d-maltoside (DDM), and wash buffer containing 0.025% DDM in size-exclusion chromatography system, and finally the NUDT9-H with high purity was obtained after cleaved by thrombin (1 U/2 mg fusion protein) for 24 h.

Due to the poor stability of NUDT9-H, it is necessary to add DDM in extraction and purification buffer to stabilize the conformation of NUDT9-H, so as to increase its yields and purity.

To investigate the mechanism of congenital paramyotonia caused by human skeletal muscle voltage-gated sodium channel hNav1.4 mutant I1363T.

The conservation of the mutant site were detecled by using amino acid sequence alignment; the C-terminal mCherry fusion hNav1.4 was constructed, and the expression and distribution of wild type and hNav1.4 mutant I1363T were determined by confocal microscopy; the steady-state activation, fast inactivation and window current of wild type and hNav1.4 mutant I1363T were examined by whole-cell patch clamp.

Alignment of the amino acid sequences revealed that Ile1363 is highly conserved in human sodium channels. There was no significant difference in expression level and distribution between wild type and I1363T. Although no significant differences were observed between I1363T mutant and wild type in the activation upon channel gating, the V_0.5 of voltage-dependence of fast inactivation of I1363T mutant [(-59.01±0.26) mV] shifted 9 mV towards depolarization as compared with wild type [(-68.03±0.34) mV], and the slope factor of voltage-dependence curve increased to (5.24±0.23) mV, compared with (4.55±0.21) mV of the wild type. Moreover, I1363T showed the larger window current than that of the wild type.

I1363T causes the defect in fast inactivation of hNav1.4, which may increase the excitability of muscle cells and be responsible for myotonia. The increased window current of I1363T may result in an increase of inward Na⁺ current, could subsequently inactivate the channels and lead to loss of excitability and paralysis.

To construct a three-dimensional structural model for the selectivity filter in the transient receptor pontential melastatin 8 (TRPM8) ion channel.

In the Rosetta computational structural biology suite, multiple rounds of de novo modeling with the kinematic loop closure algorithm were performed.

After nine rounds of computational modeling, we obtained the models of the selectivity filter within the TRPM8 channel with the lowest energy and high convergence. The model showed that the sidechain of D918 points were away from the central ion permeation pathway, while the sidechains of Q914, D920 and T923 pointed towards it.The glycosylation site N934 was located outside the pore region and its side chain directed to the extracellular water environment.

A three-dimensional structural model for the selectivity filter in the TRPM8 ion channel was constructed, which provides reliable structural information for exploring the mechanism of ion selectivity.

To investigate the expression of Wilms’ tumor 1 (WT1) gene in patients with acute myeloid leukemia (AML), and to explore its application in predicting prognosis of AML in patients with wild or mutated nucleophosmin 1(NPM1) and Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD).

One hundred and sixty-seven newly diagnosed AML patients（exclued M3 type）were enrolled in this study. The survival of patients were analyzed with Kaplan-Meier method. The clinical data, laboratory findings and the survival of patients were analyzed and compared between patients with high WT1 expression (high-WT1 group) and those with low WT1 expression (low-WT1 group), as well as among the patients with NPM1 or FLT3-ITD wild type and mutants.

The overall response rates (ORR) in high-WT1 and low-WT1 groups were 65.9% (83/126) and 95.1% (39/41), respectively (P＜0.01). Compared with the low-WT1 group, the high-WT1 group had lower 2-y overall survival (OS) rate (46.1% vs. 75.2%，P＜0.05) and 2-y disease free survival (DFS) rate (43.5% vs. 68.5%，P＜0.05). After induction chemotherapy, the patients with decreased WT1 gene expression ≥1log was associated with higher ORR and 2-y OS rate (all P＜0.05), but the advantage of 2-y DFS rate was not shown (P＞0.05). In patients with NPM1 wild type, the high-WT1 group had inferior ORR and 2-y OS rate (all P＜0.05), while in the patients with FLT3-ITD wild type, the high-WT1 group had inferior ORR, 2-y OS rate and 2-y DFS rate (all P＜0.05). In patients with NPM1 or FLT3-ITD mutations, the WT1 expression had no significantly predicting values in treatment efficacy and survival (all P＞0.05).

WT1 gene overexpression indicated poor prognosis of AML patients; the patients with decreased WT1 gene expression ≥1 log after the first induction therapy show better prognosis than those with＜1 log. The WT1 gene expression level at diagnosis can be used as an unfavorable prognostic factor for AML patients with NPM1 or FLT3-ITD wild types.

To explore the effects of intrauterine infection on early growth and neurobehavioral development in neonatal rats.

Escherichia coli (E. coli) was inoculated into uterine cervix of pregnant rats with gestation of 15 d to establish the intrauterine infection model, and the effect on the delivery of pregnant rats was observed. The neonatal rat brain tissue was stained with Hematoxylin-Eosin and the cerebral white matter damage was assessed. Immunohistochemical staining and Western blot analysis were performed to evaluate the expression of glial fibrillary acidic protein (GFAP), 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and neurofilament (NF) in pup brains. Birth weight and early growth development indices were monitored， and neurobehavioral tests were performed to access the change of neurobehavioral development in neonatal rats.

The white blood cell count increased significantly in the uterus and placenta of the pregnant rats after intrauterine E. coli infection and no significant impact was observed on the delivery of pregnant rats. Weak staining and focal rarefaction of cerebral white matter from rats at P7 in intrauterine infection group were observed. The expression of GFAP markedly increased (P＜0.05) in infection group, while the level of CNPase and NF in pup brains at P7 significantly decreased (P＜0.05 or P＜0.01). Compared with control group, the neonatal rats in infection group had lower birth weight and slower weight gain during the suckling period (P＜0.05 or P＜0.01), and the completion times of ear opening, eye opening, surface righting, negative geotaxis, acoustic startle and swimming test in infection group were significantly delayed (P＜0.05 or P＜0.01). Conclussion: Intrauterine infection in pregnant rats can induce cerebral white matter damage and retardation of early growth and neurobehavioral development in neonatal rats.

To investigate the developmental characteristics of resilience in children aged 3-5, and to explore the relationship between temperament, parenting style and resilience.

A total of 570 preschoolers aged 3-5 years in Hangzhou participated in this study. The children’s teachers completed the assessment of the resilience scale of DECA-P2 (Devereux Early Childhood Assessment for Preschoolers Second Edition); the children’s parents completed assessment of temperament questionnaire CBQ (Children’s Behavior Questionnaire) and parenting style questionnaire PSDQ (Parenting Styles and Dimensions Questionnaire).

Totally 432 valid questionnaires were retrieved with a recovery rate of 75.79%. The levels of initiative and self-regulation of 5-y children were higher than those of children aged 3 or 4 (all P＜0.01); the level of attachment/relationship of 5-y children was higher than that of children aged 4 (P＜0.01); the levels of initiative and self-regulation of girls were higher than those of boys (P＜0.05 or P＜0.01). The negative affect dimension of temperament was negatively correlated with resilience (all P＜0.05), while the effortful control and authoritative parenting styles were positively correlated with resilience (all P＜0.05). The negative affect and effortful control were able to partially predict resilience of children through authoritative parenting style (mediating effect were -0.0143 and 0.0363).

Preschoolers aged 3-5 years with different age and gender show differences in resilience, and parenting styles may play a mediating effect between temperament and resilience.

To investigate risk factors of death in newborns with congenital diaphragmatic hernia (CDH).

A total of 126 newborns with CDH from June 2012 to September 2018 were enrolled. Concomitant malformations were recorded by descriptive analysis. Newborns received surgical treatment (n=120) for CDH were divided into survival group and fatal group. The risk factors of death were analyzed by univariate and multivariate logistic regression and the ROC curve with generated with relevant variables.

There were 55 CDH newborns with concomitant malformations (43.7%), including 20 cases (15.9%) with multi-malformation. Logistic regression analysis showed that premature rupture of membranes (PROM), postoperative atelectasis, long duration of postoperative mechanical ventilation, postoperative high oxygenation index (OI) were related to death (all P＜0.05), and the delayed surgery was a protective factor (P＜0.05). In ROC analysis of postoperative OI in predicting death, the area under the curve (AUC) was 0.841, with the cutoff value of 5.74, the sensibility and specificity of OI was 81.0% and 75.0%, respectively(P＜0.01).

Newborns with CDH have a high rate of malformations. The risk factors of death were PROM, postoperative atelectasis, postoperative long duration of mechanical ventilation and higher postoperative OI, and delayed surgery may reduce mortality.