Effects of prenatal nicotine exposure on enamel formation of offspring mice / 中华口腔医学杂志

Objective: To investigate the effects of nicotine on the morphology, structure of offspring's dental germ, enamel organ and other dental tissues and the further potential epigenetic mechanisms by establishing prenatal nicotine exposure mouse model. Methods: Ten C57BL/6 pregnant mice were randomly divided into control group (physiological saline subcutaneous injection) and prenatal nicotine exposure (PNE) group (nicotine subcutaneous injection) by using a random number table. Postnatal day 0 (P0), postnatal day 14 (P14) and postnatal day 25 (P25) offspring mice were collected for subsequent experiments. The offspring mice were divided into offspring control group and offspring PNE group according to the maternal group respectively. Weights of P0 and P25 offspring mice were recorded. Micro-CT, scanning electron microscope (SEM) and Vickers hardness test were performed to analyze the related parameters of hard tissues including alveolar bones and mandibular incisors. Total RNAs were extracted from mandible tissues and the third generation of dental epithelial stem cells (DESC) in P25 mice. The relative expression levels of osteogenic and ameloblastic differentiation related genes were measured by real-time quantitative PCR (RT-qPCR). Immunohistochemical stainings of paraffin sections were then performed to observe the distribution and expression level of proliferating cell nuclear antigen (Pcna), amelogenin (Amelx), histone H3 trimethylated at lysine 27 (H3K27me3) and enhancer of zeste homolog 2 (Ezh2). Cell counting kit-8 (CCK-8) assays were used to detect the cell viabilities of DESCs after administrations of different concentrations of nicotine (0.01, 0.1, 1 mmol/L) and GSK126 (an inhibitor of histone methyltransferase Ezh2). Results: Compared with the control group, pregnant mice in PNE group were more likely to have adverse pregnancy outcomes, such as significantly lower offspring body weight [P0: offspring control (1.20±0.04) g, offspring PNE (0.99±0.02) g, P<0.001; P25: offspring control (15.26±1.70) g, offspring PNE (9.65±1.32) g, P<0.001] and increased stillbirths rate [offspring control (0), offspring PNE (46.40±9.30) %, P<0.001]. At P14 and P25, the distance parameters between the enamel mineralized deposits of mandibular incisors and the mesial surface of the first molar in offspring PNE group [P14: (-1 349±45) μm; P25: (-1 192±147) μm] was significantly decreased compared with the control group [P14: (-506±380) μm, P25: (504±198) μm] (P<0.05, P<0.001). The enamel column and enamel column stroma of incisors in offspring PNE group were blurred, arranged loosely and disorderly than those in the control group, while the microhardness of incisor enamel in offspring PNE group [(245.7±18.4) MPa] was significantly lower compared to the control group [(371.9±28.7) MPa] (P<0.001). HE staining showed disordered pre-ameloblast (Pre-Am) arrangement and delayed mineralization deposition point in offspring PNE group compared with the control group, while the length of transit-amplifying cell (TA) and Pre-Am region were prolonged as well. Immunohistochemical staining results displayed that the overall Pcna (P<0.05), H3K27me3 (P<0.01), Ezh2 (P<0.01) expression of labial cervical loop (LaCL) in PNE group were increased, while the positive signal of Amelx in ameloblast cytoplasm was impaired. In vitro, the addition of 1 mmol/L nicotine could significantly upregulate the expression level of Pcna (P<0.01) and downregulate the expression levels of B lymphoma Mo-MLV insertion region 1 (P<0.05), leucine rich repeats and immunoglobulin like domains 1 (P<0.05), Amelx (P<0.01). In addition, 1 mmol/L nicotine could also significantly enhance the proliferation activity of DESCs (P<0.001). Addition of 10 μmol/L GSK126, could rescue the proliferation activation effect of 1 mmol/L nicotine on DESCs. Conclusions: PNE may delay the process of enamel formation and lineage differentiation, leading to the abnormal proliferation of DESCs and changes of epigenetic modification state in H3K27me3, which affect the development of enamel in offspring mice,suggesting PNE might be one of risk environmental factor for tooth development.

Clinical, molecular genetic analysis, and treatment of 3 children with sitosterolemia / 中华儿科杂志

Objective@#To investigate clinical, molecular genetic characteristics, and treatment outcomes of 3 children with sitosterolemia.@*Methods@#Three cases of children presented with multiple xanthomas during June 2016 to June 2017 were included. The clinical manifestations, laboratory examinations and follow-up data were retrospectively analyzed. DNA was extracted from peripheral blood and analyzed with whole exome sequencing(WES). All the detected variants were confirmed by Sanger sequencing. Plasma plant sterol concentrations were measured by gas chromatography-mass spectrometry.@*Results@#Three cases of children including 1 boy and 2 girls presented with multiple linear and intertriginous xanthomas around skin of the joint areas at the age from 15 months to 6 years and 2 months. Total cholesterol of the 3 cases was elevated to 14.45, 15.47 and 15.85 mmol/L (3.36-6.46), and low density lipoprotein cholesterol was 9.02, 13.54 and 12.47 mmol/L (< 3.36) respectively. Genetic analysis with WES revealed that 2 cases carried compound heterozygous variants in ABCG5 gene, 1 case carried compound heterozygous variants in ABCG8 gene. Two reported variants (p. N437K, p.R446X) and one novel variant (p.Q251X) of ABCG5 were identified in case 2 and 3. Two novel ABCG8 variants (p.R263Q, c.1528_1530delATC) were found in case 1. All these children had extremely high plasma plant sterol levels, thus the diagnosis of sitosterolemia was confirming. The campesterol level was 111.35, 102.86 and 58.91 μmol/L(0.01-10.00), the stigmasterol was 14.97, 29.43 and 17.79 μmol/L (0.10-8.50) and the sitosterol was 231.20, 177.66 and 114.20 μmol/L (1.00-15.00) respectively. The total serum cholesterol levels of three children decreased to nomal after the patients were placed on the low plant sterol/low cholesterol diet. The xanthomas regressed gradually, and almost disappeared after 8 months of treatment in case 1 and 3.@*Conclusions@#Children with sitosterolemia presented with skin xanthomas around the joint areas. The level of total cholesterol, low density lipoprotein cholesterol and plant sterols increased obviously. One novel variant (p.Q251X) of ABCG5 and 2 novel variants (p.R263Q, c.1528_1530delATC) of ABCG8 were identified. Children with sitosterolemia responded well to a low plant sterols/low cholesterol diet.

Clinical phenotypes and genetic study of 2 cases with 22q13 deletion syndrome / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To determine the genetic etiology and clinical characteristics of 2 boys featuring development delay (DD).</p><p><b>METHODS</b>Routine chromosomal banding was performed to analyze the karyotypes of the patients and their parents. Single nucleotide polymorphism array (SNP array) analysis was employed to identify pathogenic deletion/duplication of chromosomes, and quantitative real-time PCR (qPCR) was performed to confirm the results.</p><p><b>RESULTS</b>Patient 1 showed a global developmental delay, especially impaired language development, seizures, behavioral problems belonging to the autism spectrum and mild facial dysmorphism. Patient 2 mainly presented with severely delayed speech and moderate intellectual disability, but did not have obvious facial dysmorphism and autistic-like behavior. The diagnosis of 22q13 syndrome was established based on identification of a heterozygous microdeletion at chromosome 22q13.33 in both patients (69 kb and 587 kb, respectively) by the SNP array analysis. Both patients had deletions of SHANK3 and ACR, which are located at the end of 22q. Quantitative real-time PCR verified that the deletion of SHANK3 gene in both patients were de novo in origin.</p><p><b>CONCLUSION</b>Two cases of 22q13 deletion syndrome have been diagnosed by SNP array analysis. Deletion of SHANK3 gene may be the major contributor to the clinical manifestations of the patients. SNP array analysis can facilitate discovery of microdeletions, which has played an important role in the diagnosis and genetic counseling for the family.</p>

The clinical and genetic analysis of glycogen storage disease type IV in 5 cases / 临床儿科杂志

Objective To investigate the characteristics of glycogen storage disease type IV (GSD IV) clinically, in laboratory tests and in gene mutation. Methods The clinical manifestations, biochemical indexes, activity of chitotriosidase, and the follow-up of the treatment in 5 cases of GSD IV were analyzed. Results Five patients (3 boys and 2 girls) aged 4 months - 5 years presented hepatosplenomegaly and elevated liver enzyme levels for 2 months at hospital visit. Two patients had motor developmental delay and weakness but their creatine kinase (CK) level were normal. Glycogen storage and liver fibrosis were observed in the liver biopsy in 4 patients. Target sequencing found that all 5 children carried the complex heterozygous mutation of the GBE1 gene with 2 reported mutations(p.R515C,p.R524Q)and 7 novel mutations.The novel mutation contains 5 missense mutations (p.I460T, p.F76S, p.F538V, p.L650R, p.W455R), one insertion mutations (c.141_142insGCGC), and one large fragment deletion (exon 3-7). Therefore, diagnosis of liver type of GSD IV was confirmed in those children. Two patients died of liver cirrhosis. The liver transplantation was performed due to liver cirrhosis in one patient whose chitotriosidase activity increased obviously before transplantation and decreased significantly after the transplantation and liver enzyme levels were returned to normal 4 months after transplantation. In the other two patients their growth and liver enzyme levels were normal;one had not received special treatments while the other was treated with raw corn starch and level of chitotriosidase was normal. Conclusions The clinical manifestations of GSD IV are heterogeneous. Target sequencing can be used for fast and noninvasive diagnosis of GSD IV. Chitotriosidase activity is useful in the prognosis assessment for GSD IV.

Clinical and gene mutation analysis of three children with late-onset glycogen storage disease type Ⅱ with hypertrophic cardiomyopathy / 中华儿科杂志

Objective@#To investigate the clinical and laboratory features of three children with late-onset type Ⅱ glycogen storage disease(GSD) who presented with hypertrophic cardiomyopathy and to analyze the effect of five mutations identified on the acid-α-glucosidase (GAA) activity and stability.@*Method@#Three cases of children with muscle weakness were included in this study.GAA activity was analyzed in Dried Blood Spot of the patients.DNA was extracted from peripheral blood in all the patients and their parents and subjected to polymerase chain reaction and directly sequencing of GAA gene.Five mutant pcDNA3.1-myc-his-GAA expression plasmids(p.G478R, p.P361L, p.P266S, p.Q323X, p.R672Q) were constructed and transient instantaneously transfected into 293T cells to analyze the enzyme activity and stability of GAA.@*Result@#All the three children had the onset of disease at 3 years or 1.5 years of age.They presented with developmental delay, muscle weakness and hypertrophic cardiomyopathy.GAA activity of 3 patients was 2.65, 3.55 and 1.51 pmol(punch·h)(8.00-98.02)respectively. Genetic analysis found 5 mutations (p.G478R, p. P361L, p. P266S, p. Q323X, p. R672Q), and all of these 3 cases had clinical manifestations and were diagnosed as late-onset type Ⅱ glycogen storage disease.Five mutant pcDNA3.1-myc-his-GAA expression plasmids were transfected into 293T cells.Five mutant enzyme activities were found to be only 9.9%-22.5% of the wild-type enzyme activity and the protein expression of the five mutants was 32.0%-63.9% compared with the wild type.@*Conclusion@#This study reports 3 children with late-onset GSD Ⅱ accompanied by hypertrophic cardiomyopathy and compensatory stage of cardiac function in addition to limb muscle weakness.Five pathogenic mutations were identified, and these 5 mutations result in decreased GAA activity and GAA expression by in vitro functional analysis.

Inhibitory effect of curcumin on human breast cancer cell line SKBR3 and its mechanism / 中国生化药物杂志

Objective To observe the effect of curcumin on proliferation, apoptosis, Caspase-3 activity and telomerase activity of chronic granulocytic breast cancer SKBR3 cell line, and to investigate the mechanism of curcumin. Methods The cell growth curve was drawn by MTT;the apoptotic cells were detected by Annexin, V and PI double staining; the expression of Caspase-3 was detected by Westernblot; the activity of telomerase was detected by TRAP-PCR silver staining. Results Curcumin could significantly inhibit the proliferation of SKBR3 cells in a time and concentration dependent, has a certain effect on reducing the telomerase activity of SKBR3 cells can enhance the expression of Caspase-3 induced apoptosis. Conclusion Curcumin can effectively inhibit the proliferation of SKBR3 cells, increase the synthesis of Caspase-3 protein, inhibit the activity of telomerase and mediate the apoptosis of SKBR3 cells.

Inhibitory effect of curcumin on human breast cancer cell line SKBR3 and its mechanism / 中国生化药物杂志

Objective To observe the effect of curcumin on proliferation, apoptosis, Caspase-3 activity and telomerase activity of chronic granulocytic breast cancer SKBR3 cell line, and to investigate the mechanism of curcumin. Methods The cell growth curve was drawn by MTT;the apoptotic cells were detected by Annexin, V and PI double staining; the expression of Caspase-3 was detected by Westernblot; the activity of telomerase was detected by TRAP-PCR silver staining. Results Curcumin could significantly inhibit the proliferation of SKBR3 cells in a time and concentration dependent, has a certain effect on reducing the telomerase activity of SKBR3 cells can enhance the expression of Caspase-3 induced apoptosis. Conclusion Curcumin can effectively inhibit the proliferation of SKBR3 cells, increase the synthesis of Caspase-3 protein, inhibit the activity of telomerase and mediate the apoptosis of SKBR3 cells.

Clinical features and PHKG2 gene mutation analysis of 5 Chinese patients with glycogen storage disease Ⅸc / 临床儿科杂志

Objective To investigate the clinical, laboratory and genetic features of glycogen storage disease(GSD)IXc. Methods Five patients suspected as liver GSD were included in our study. DNA was extracted from peripheral blood of all the patients and diagnoses were made after target sequencing to nearly 2700 disease causing genes. All detected mutations were confirmed in the probands and their parents. Further analysis was based on clinical features, routine laboratory examinations and treatment. Results All the 5 patients manifested with severe hepatomegaly, hypoglycemia, moderately to severely elevated liver enzyme levels, hypertriglyceridemia and growth retardation. Four cases showed poor exercise tolerance but with normal creatine kinase (CK) levels. None of the patients showed liver cirrhosis. Growth velocity and hepatomegaly was improved after the uncooked corn starch treatment was initiated. In the 5 patients, 6 different pathogenic or likely pathogenic mutations in the PHKG2 gene were identified, including one reported mutation (p.E157K) and five novel mutations (p.E56X, p.R185X, c.79_88delinsTCTGGTCG, c.761delC,p.R279C). The p.E157K was the most frequently mutation identified (6/12, 50%). Conclusions The p.E157K mutation is the hot mutation in our small cohort. Main clinical features of our patients include fasting hypoglycemia, impaired liver function,short statures and poor exercise tolerance, without developing liver cirrhosis.

Strain rate imaging assessment of left atrial early function in pilots with essential hypertension / 中华医学超声杂志（电子版）

Objective To assess the left atrial early function in pilots with essential hypertension by strian rate imaging (SRI). Methods Twenty-five hypertensive pilots without left ventricular hypertrophy and expansion (LVN) were selected. Twenty-ifve healthy pilots were included as control group. At the left ventricular apical four-chamber view, three-chamber view, two-chamber view, the systolic, early and late diastolic peak strain rate (SRs, SRe, SRa) curves were acquired by SRI in the basal section, the middle section and the upper section of the wall of left atrium. The average strain rate (mSRs, mSRe, mSRa) was calculated. The LA maximal, minimal and pre-systolic volume (LAVmax , LAVmin and LAVp), LA passive ejection fraction (LAPEF), LA active ejection fraction (LAAEF) , left atrial stroke volume (LASV), E/A and E/E′ were calculated. Results The LAPEF, LAAEF, LASV, E/A, E/E′were (38.96±6.43)%, (61.11±5.86)%, (14.74±5.33)mm, 1.23±0.08, 8.63±1.77 in the control group, and were (34.18±5.72)%, (63.23±6.89)%, (10.12±3.23)mm, 1.04±0.26, 7.16±0.84 in the study group. Compared with the control group, signiifcant difference was detect in LAPEF, LAAEF, LASV, E/E′(t=4.840, 2.690, 4.801, 2.950, all P<0.05). No signiifcant difference was found in E/A between the study and control group (t=-1.424, P=0.178). There was signiifcant positive correlation between SRa and E/E′(r=0.632, P<0.05). Conclusion SRI could be applied in the evaluation of early left atrial function. In pilots with essential hypertension, the changes of strain rate in left atrial wall, which is prior to the modeling geometry of left ventricular, could be detected by SRI.