Objective To perfect the purification method of recombinant fusion protein of Hespintor (rHespintor) for increasing the protein extraction efficiency,and to investigate its effects on the proliferation,migration and invasion of hepatoblastoma cell line HepG2.Methods In the recombinant protein extraction,the inclusion body washing process was added and the protein purification buffer system was changed.BAPNA was used as the substrate.The inhibitory effect tof purified rHespintor on trypsin hydrolysis was detected.The blank group served as the control group.The MTT test,cell scratch wound healing test and tumor cell invasion test were performed to detect the effect of rHespintor on growth of hepatoblastoma HepG2 cells and its effect.Results The urea gradient washing on the inclusion body protein could effectively remove the vast majority of impure proteins from the targeted protein.After one-step purification,the target protein rHespintor exhibited a high inhibition effect of trypsin hydrolysis,and the inhibitory effect was exhibited a dose-dependent manner.After acting on hepatoblastoma HepG2 cells with rHespintor,the cell proliferation ability was inhibited,the migration ability was reduced and the number of invaded cells were significantly decreased.Conclusion rHespintor can significantly inhibit the proliferation,migration and invasion of hepatoblastoma cell line HepG2 cells in vitro.

Objective To explore the value of prenatal MRI in the diagnosis of fetal simple expansion of lateral ventricle(ventriculomegaly), and follow up the nervous system development status after birth. Methods Simple expansion of the lateral ventricle fetus by prenatal MRI examination were collected in Huzhou Maternal and Child Care Hospital from May 2013 to June 2015, 126 cases of live births in expansion group, 50 normal cases were recruited in the same period as the control group. In expansion group, fetal subgroup analysis was done:(1) unilateral or bilateral lateral ventricle expasion:one group was 98 cases was lateral ventricle expansion (77.8%, 98/126), expansion of bilateral ventricle group was 28 cases (22.2%, 28/126). (2) Prenatal MRI in the diagnosis of the lateral ventricle of expansion: expansion of the lateral ventricle width was greater than 10.0 mm, if both sides were expanding, the expand width was the heavier one side, divided into 3 subgroups: ①Expansion in group A (lateral ventricle width 10.0-12.0 mm) were 88 cases (69.8%, 88/126).②Expansion in group B (lateral ventricle width 12.1-15.0 mm) were 29 cases (23.0%, 29/126). ③Expansion of group C (lateral ventricle width> 15.0 mm) were 9 cases (7.12%, 9/126). All 176 cases were followed up after birth at the 3rd, 6th, 12th, 18th month (corrected age was used for premature babies), and Gesell developmental schedules (GDS) were used to evaluate the neurobehavioral development. Results (1) The MRI results after birth:21 cases were followed up by MRI after birth. In group A, 11 cases had MRI and 9 were normal (the ventricular width<10.0 mm after birth) , the other 2 cases were stable (the ventricular width measured first time after birth was ≥10.0 mm, but the difference was within 2.0 mm from the MRI before birth). In group B, 4 cases had MRI, 1 was normal, 1 was stable, and 2 cases were getting better (the ventricular width measured first time after birth was ≥10.0 mm, but the width decreased more than 2.0 mm from the MRI before birth). In group C, 6 cases had MRI. 3 cases were getting better and 3 cases were stable. (2) Overall GDS results:expansion group after the birth of the 3rd, 6th, 12th, 18th month GDS evaluation results compared with control group, respectively, the differences were not statistically significant (all P>0.05). (3) The GDS results among the subgroups:in each evaluation after birth, there were no statistically significant differences between group A and the control group (all P>0.05). The GDS results of group B at the 3rd and 6th month were lower than those of the control group (P<0.05); while there were no statistically significant differences between the 2 goups at the 12th and 18th month (P>0.05). And for group C, statistically significant differences were found compared to the control group at each follow-up time (all P<0.05). (4) GDS results at different times after birth in the expansion group:there was no statistically significant difference between the results at the 3rd and 6th month (P>0.05). But when the result at the 3rd month was compared to the results of the 12th or 18th month, the differences were statistically significant (P<0.05). GDS result of 6th months after birth compared with 12th and 18th months, respectively, there were no statistically significant differences (P>0.05). There was no statistically significant difference between the results at the 12th and 18th month (P>0.05). (5) The GDS results in unilateral and bilateral ventricle expansion:at the 18th month, among the 98 unilateral cases, 86 (87.8%, 86/98) had normal GDS results(>85 scores);8 (8.2%, 8/98) had borderline results (75-85 scores);4 (4.1%, 4/98) had delayed results (<75 scores). Among the 28 bilateral cases, 23 (82.1%, 23/28) had normal GDS results;3 (10.7%, 3/28) had borderline results; 2 (7.1%, 2/28) had delayed results. There was no statistically significant difference (P>0.05). Conclusions Among the simple expansion of lateral ventricle, those whose ventricular width are≤12.0 mm may not need clinical treatment. If the width is between 12.1 to 15.0 mm, closely follow-up and targeted rehabilitation training after birth are recommended. When the width is more than 15.0 mm, the risk of the central nervous system function delay is significantly increased, and early intervention might improve the prognosis.

Objective:To explore the value of prenatal MRI in the diagnosis of isolated mild and moderate bilateral ventriculomegaly and neural development of the fetuses after birth.Methods:This is a retrospective study involving 244 singleton fetuses with isolated mild or moderate lateral ventriculomegaly diagnosed by both prenatal ultrasound and MRI in Huzhou Maternity & Child Health Care from May 2013 to June 2017, consisting of 82 cases with bilateral ventriculomegaly (BVM) and 162 with unilateral ventriculomegaly (UVM). The two groups were further divided into two subgroups: mild (lateral ventricle width: 10.0-12.0 mm, bilateral 56 cases, unilateral 120 cases) and moderate group (lateral ventricle width: >12.0-<15.0 mm, bilateral 26 cases, unilateral 42 cases). In addition, 50 singleton fetuses without any abnormality in the nervous system in prenatal check were included in the control group during the same period. All neonates were reexamined by ultrasound within one week after birth, and followed up regularly at the age of 3, 6, 12 and 18 months. Gesell Development Schedules (GDS) were used to evaluate the central nervous system's function, and postnatal changes in lateral ventriculomegaly were observed. Statistical analysis was performed by t, F, Chi-square tests (or Fisher's exact test). Results:(1) There was no difference among intervals between MRI scan and delivery in the BVM, UVM, and the control groups. The disappearance rate of lateral ventriculomegaly after birth was 80.4% (45/56) in the mild BVM group, 42.3% (11/26) in the moderate BVM group, 88.3% (106/120) in the mild UVM group, and 57.1% (24/42) in the moderate UVM group ( χ2=35.183, P<0.001). (2) The GDS evaluation results in the BVM group at 6, 12, and 18 months after birth were worse than those in the UVM group (all P<0.0167). The GDS evaluation results in the BVM group were worse than those in the control group at 3 and 6 months after birth [3 months: normal: 58.5% (48/82) vs 86.0% (43/50), borderline: 22.0% (18/82) vs 10.0% (5/50), delay: 19.5% (16/82) vs 4.0% (2/50), χ2=11.425; 6 months: normal: 63.4% (52/82) vs 88.0% (44/50), borderline: 19.5% (16/82) vs 8.0% (4/50), delay: 17.1% (14/82) vs 4.0% (2/50), χ2=9.678; all P<0.0167]. (3) The GDS evaluation results in the moderate BVM group at 6, 12, and 18 months after birth were worse than those in the moderate UVM group [6 months: normal: 30.8% (8/26) vs 69.0% (29/42), borderline: 30.8% (8/26) vs 21.4% (9/42), delay: 38.5% (10/26) vs 9.5% (4/42), χ2=11.417; 12 months: normal: 53.8% (14/26) vs 88.1% (37/42), borderline: 23.1% (6/26) vs 9.5% (4/42), delay: 23.1% (6/26) vs 2.4% (1/42), χ2=11.199; 18 months: normal: 65.4% (17/26) vs 95.2% (40/42), borderline: 15.4% (4/26) vs 2.4% (1/42), delay: 19.2% (5/26) vs 2.4% (1/42), χ2=10.568; all P<0.0167]. The GDS evaluation results of the moderate BVM group at 3, 6, 12, and 18 months after birth were worse than the control group. (4) In the BVM group, the GDS scores at 18 months of age were better than those at three months of age ( χ2=8.224, P=0.016). Conclusions:(1) Most mild BVM would disappear spontaneously after birth, while more in mild UVM cases. (2) The postnatal GDS evaluation results of the BVM group is significantly worse than that of the UBM group at months of age; (3) Fetuses with less severe isolated BVM are more likely to have improved GDS score after birth.

OBJECTIVE: To provide data support for the study of pathogenic mechanism of SARS-CoV-2 at the molecular level, and provide suitable candidate targets for vaccine, antibody and drug research and development through comparative analysis for structural characteristics and epitopes of S protein of SARS-CoV-2 and SARS-CoV. METHODS: Based on the reference sequences of S protein, physical and chemical properties, hydrophobicity, signal peptide, transmembrane region, domain, secondary structure, tertiary structure analysis and antigenic epitopes prediction were carried out. Meanwhile, the tissue expression, related pathways and reactome pathways of angiotensis Ⅰ converting enzyme 2 (ACE2) and C-type lectin domain family 4 member M (CLEC4M) receptors were analyzed. RESULTS: The amino acid sequence of S protein of SARS-CoV-2 and SARS-CoV has a 75.80% consistency. The structural characteristics of the two coronaviruses are highly consistent, but the secondary structure and tertiary structure of SARS-CoV-2 is not as obvious as SARS-CoV. ACE2 and CLEC4M are expressed in alimentary system, heart, kidney, lung and placenta. The main related the pathways of renin-angiotensin system, protein digestion and absorption pathway, and the reactome pathways of metabolism of angiotensinogen to angiotensins, GPCR ligand binding, are related to typical symptoms of coronavirus disease 2019 induced by SARS-CoV-2. Three pairs of highly or completely homologous epitopes of S protein were obtained. The 600-605, 695-703 and 888-896 amino acid residues in SARS-CoV-2 were highly homologous with 586-591, 677-685 and 870-878 amino acid residues in SARS-CoV, respectively. CONCLUSIONS The similarity of S protein of SARS-CoV-2 and SARS-CoV determines that they have similar infection patterns and clinical manifestations. The candidate epitopes with high reliability can provide reference for virus diagnosis and vaccine development.
Betacoronavirus ; Cell Adhesion Molecules ; Coronavirus Infections ; Epitopes ; Humans ; Lectins, C-Type ; Ligands ; Pandemics ; Peptidyl-Dipeptidase A ; Pneumonia, Viral ; Receptors, Cell Surface ; Receptors, Virus ; Reproducibility of Results ; Spike Glycoprotein, Coronavirus

【Objective】 To investigate the relationship between differential expressions of lncRNAs and mRNAs and Hespintor’s possible anti-tumor mechanism using transcriptome sequencing technology. 【Methods】 First， a nude mouse model of human hepatoma tumors was established. The tumor tissue mass was collected after 4 weeks of group treatment. The cDNA libraries of tumor tissues were established in the Hespintor treatment group and the solvent control group， and transcriptome sequencing was performed. Based on RNA-seq data， the differentially expressed lncRNA and mRNA genes were screened， and the functional enrichment and interaction analysis were performed. The network module division approach was utilized to identify the target genes of Hespintor and build its regulatory network. 【Results】 The Hespintor treatment group yielded a high-confidence differentially expressed gene collection of 2 003 lncRNAs （DEGs lncRNA） and 1 038 mRNAs （DEGs mRNA）. Target mRNAs regulated by DEGs lncRNA were mainly enriched in PIP3 to activate the Akt signal， p53 signal pathway， FOXO-mediated transcription， and cell cycle， among other things. DEGs mRNA were primarily enriched in chemokine signaling pathways， extracellular matrix receptor interactions， complement， and coagulation cascades. Both of them were related in three ways： cell behavior， transcriptional regulation， and cell cycle. 【Conclusion】 The PI3K/Akt signaling pathway may play a key role in the inhibitory effect of Hespintor on tumor， inducing G1/S phase cell cycle arrest and apoptosis.