Novel DGAT1 Mutations Identified in Congenital Diarrheal Disorder 7: A Case Report with Therapeutic Experience.

Congenital diarrheal disorders (CDD) are a group of rare inherited intestinal disorders, among which CDD7 was recently identified to be associated with only 24 mutations in gene coding for diacylglycerol-acyltransferase 1 (DGAT1). We report on a female patient who presented with diarrhea, vomiting, hypoalbuminemia, and failure to thrive after birth. Two novel variants of c.1215_1216delAG and c.838C>T were found in the DGAT1 gene by whole exome sequencing, which was confirmed to be compound heterozygous by Sanger sequencing. Her symptoms and nutritional status improved significantly after 1 year of a fat-restricted enteral diet. Weight for age and weight for length increased from -5.0 SDS and -4.0 SDS at 3 months to +0.08 SDS and +1.75 SDS at 15 months, respectively. This report expanded the mutation spectrum of DGAT1-related CDD7 and enriched our knowledge of the clinical features. Moreover, early fat-restricted enteral diet intervention was suggested for the treatment of such patients.

[Effects of enriched environments on behavioral development at toddler period of preterm experienced early repeated procedural pain].

Objective: To investigate the effects of enriched environments on behavioral development at toddler period of preterm who had experienced early repeated operative pain. Methods: A cross-sectional study was conducted. A total of 80 high-risk preterm children of 2 years of age, who had experienced repeated pain stimuli in the neonatal intensive care unit (NICU), were enrolled as preterm group from the High-risk Children Clinic of Children's Hospital of Nanjing Medical University from October 2016 to March 2021. Furthermore, 39 full-term healthy children, aged 2 years, who were undergoing routine check-ups during the same period, were selected as the full-term group. The preterm group was further divided into preterm intervention group and preterm non-intervention group based on the implementation of enriched environment interventions. Data of neonatal characteristics from 3 groups were collected. Growth and development indicators at the age of 2 years were measured. Neuropsychological development evaluated by Gesell developmental scale. Behavioral development evaluated by child behavior check list. The salivary cortisol levels in response to novelty (baseline, task, end) were collected. The family environment, including maternal parenting pressure, were evaluated through a survey questionnaire. One-way ANOVA and least significant difference (LSD) tests were used to compare physical development, maternal parenting stress, Gesell neuropsychological development, and behavioral problems among the 3 groups. A repeated-ANOVA and LSD tests were employed to compare the patterns of salivary cortisol secretion. Pearson correlation analysis was used to explore the influencing factors related to neuropsychological and behavioral development and cortisol level. Results: There were 44 cases in the preterm intervention group (17 males, gestational age of (31.3±2.8) weeks), and 36 in the preterm non-intervention group (29 males, gestational age of (32.5±2.6) weeks). The full-term group consisted of 39 children (23 males, gestational age of (39.3±2.1) weeks). At 2 years of age, the height, weight, and head circumference of the preterm intervention group and non-intervention group were all lower than those of the full-term group (all P<0.05).The Gesell developmental schedule showed that the preterm non-intervention group scored all lower in gross motor, fine motor, adaptive, language and personal-social domains compared to the full-term group (91±7 vs. 97±6, 88±9 vs. 94±6, 89±8 vs. 99±8, 84±10 vs. 100±15, 89±7 vs. 95±6), with statistical significance (all P<0.01). The preterm intervention group scored all higher than the preterm non-intervention group in gross motor, fine motor, adaptive and language domains (all P<0.05), with no significant difference compared to the full-term group (all P>0.05). The number of needle painful procedures during hospitalization in NICU of the non-intervention group was negatively correlated to the adaptive development quotient (r=-0.48, P<0.05). Furthermore, the preterm non-intervention group exhibited higher scores in social withdrawal, depression, somatic complaints, aggression, and destructive behaviors compare to the full-term group and preterm intervention group (F=8.07, 5.67, 7.72, 7.90, 7.06; all P<0.05); while the preterm intervention group showed no significant difference compared to full-term group (all P>0.05). Behavioral problems (social withdrawal and depression) in the preterm non-intervention group were positively correlated with maternal parenting stress (r=0.66, 0.50; both P<0.05). In response to novel visual stimuli and cognitive challenges, the preterm non-intervention group had significantly higher salivary cortisol levels compared to the full-term group (P=0.006), which were negatively correlated with the frequency of early painful procedures (r=-0.83, -0.80; both P<0.01). There was no significant difference in cortisol secretion pattern between the intervention group and the full-term group (P=0.772). Conclusion: Enriched environmental interventions can improve neuropsychological development, decrease behavioral problems, and down-regulate consistent high cortisol response to task in preterm infants who have experienced repeated procedural pain in the NICU by the age of 2 years.

[Predictive value of neck circumference for cardiometabolic risk in children].

Objective: To investigate the predictive value of neck circumference on cardiometabolic risk in children. Methods: This was a cross-sectional study of natural sources. As the prediction cohort, clinical data were collected from 3 443 children aged 5-14 years who underwent physical examination in the Department of Child Healthcare, Children's Hospital of Nanjing Medical University from July 2021 to September 2022. As the validation cohort for external validation, clinical data were collected from 604 children aged 5-14 years who underwent physical examination in the Department of Child Healthcare, Children's Hospital of Nanjing Medical University from October 2022 to March 2023. Height, weight, neck circumference, waist circumference and body composition were measured in both groups, and body mass index, neck circumference to height ratio (NHtR), waist circumference to height ratio, body fat percentage and skeletal muscle percentage were calculated. Systolic blood pressure, diastolic blood pressure, fasting blood glucose, blood lipid and uric acid and other cardiovascular and metabolic risk indicators were collected in both groups. The prediction cohort was further stratified into clustered and non-clustered groups based on the clustering of cardiometabolic risk factors (CCRF). Various variables between these 2 groups were compared using the Mann-Whitney U test. Pearson correlation and binary Logistic regression were conducted to investigate the correlations between neck circumference and cardiovascular metabolic risk factors. The accuracy of NHtR in predicting the CCRF was evaluated using the area under the curve (AUC) of receiver operating characteristic (ROC). The cutoff value was determined using the Youden index. The validation cohort was then divided into groups above and below the cutoff value, and the detection rate of CCRF between the 2 groups was compared using the χ2 test for validation. Results: In the prediction cohort of 3 443 children (2 316 boys and 1 127 girls), 1 395 (40.5%) children were overweight or obese, and 1 157 (33.6%) children had CCRF. Pearson correlation analysis revealed all significant positive correlations (all P<0.01) between neck circumference and systolic blood pressure (r=0.47, 0.39), diastolic blood pressure (r=0.27, 0.21), uric acid (r=0.36, 0.30), and triglycerides (r=0.20, 0.20) after adjusting for age in both males and females. Among both males and females, neck circumference both showed significant negative correlation (both P<0.01) with high-density lipoprotein cholesterol (r=-0.27, -0.28), and no correlation with fasting glucose levels (r=0.03, -0.03, both P>0.05). After adjusting for gender, age, and body fat percentage, increased body mass index, neck circumference, or waist circumference increased the risks of hypertension (OR=1.23, 1.39, 1.07, all P<0.001), hyperuricemia (OR=1.16, 1.23, 1.05, all P<0.001), hypertriglyceridemia (OR=1.08, 1.16, 1.02, all P<0.01), low high-density lipoprotein cholesterol (OR=1.10, 1.27, 1.03, all P<0.01), and the CCRF (OR=1.51, 1.73, 1.15, all P<0.01). The areas under the ROC curves of NHtR in predicting CCRF was 0.73, with sensitivity and specificity at 0.66 and 0.71, respectively. The corresponding optimal cut-off value was 0.21. Validation with 604 children confirmed that the detection of CCRF in the NHtR≥0.21 group was 3.29 times (60.5% (112/185) vs. 18.7% (79/422),χ2=107.82, P<0.01) higher compared to the NHtR <0.21 group. Conclusions: Neck circumference is associated with cardiovascular metabolic risks such as hypertension, hyperlipidemia, hyperglycemia, and hyperuricemia in children. When the NHtR is ≥0.21, there is an increased likelihood of CCRF.

[Reference values of skeletal muscle mass for children in Nanjing area].

Objective: To establish the reference values and growth curves of skeletal muscle mass among children in the Nanjing area. Methods: A cross-sectional study was conducted with children who underwent physical examination at the Department of Child Health Care, Children's Hospital of Nanjing Medical University from 2020 January to 2022 September. Their height, weight, body fat mass and skeletal muscle mass were measured. Body mass index, percentage of body fat mass, percentage of skeletal muscle mass, relative skeletal muscle mass index and the ratio of skeletal muscle to body fat were calculated. The associations between skeletal muscle mass indices and physical measurements index were analyzed through the Spearman correlation test. The Mann-Kendall test was used to assess the trend for skeletal muscle mass. Generalized additive models for location, scale and shape were used to construct percentile reference values and growth curves of male and female skeletal muscle mass indices at different ages. Results: A total of 32 690 children aged 4-14 years were enrolled in this study, including 19 912 boys (60.91%). Skeletal muscle mass, percentage of skeletal muscle mass, relative skeletal muscle mass index and the ratio of skeletal muscle to body fat of boys and girls was 11.10 (8.40, 14.90) and 10.30 (7.90, 13.20) kg, 40.36% (37.01%, 43.13%) and 39.38% (36.43%, 41.88%), 6.70 (6.07, 7.52) and 6.33 (5.79, 7.00), 2.39 (1.46, 3.47) and 2.14 (1.45, 3.00) kg/m2, respectively. Skeletal muscle mass of both boys and girls was all positively associated with weight (r=0.97, 0.96), body mass index (r=0.68, 0.63) and percentage of body fat mass (r=0.40, 0.43) (all P<0.01). The reference values and growth curves showed that the percentage of skeletal muscle mass P50 ranged from 37.75%-44.61% in boys and from 36.22%-40.55% in girls. The relative skeletal muscle mass index P50 ranged from 5.80-9.68 kg/m2 in boys and from 5.57-7.98 kg/m2 in girls. The ratio of skeletal muscle to body fat P50 ranged from 1.86-2.67 in boys and from 1.29-2.41 in girls. There was an increasing trend with age for both boys and girls in the growth of skeletal muscle mass (Z=4.20, 3.75, both Ptrend<0.01), and increased slightly before 9 years of age and then increased rapidly until 14 years of age in both boys and girls. Conclusions: The skeletal muscle mass indices change with age and gender during childhood. Percentile reference values for pediatric skeletal muscle mass indices can be used to evaluate the muscular growth and development in children in the Nanjing area.

[Differential diagnosis model of benign and malignant breast BI-RADS category 4 nodules based on serum SP70 and conventional laboratory indicators].

Objective: To develop a nomogram model for the differential diagnosis of benign and malignant breast BI-RADS (Breast Imaging Reporting and Data System) category 4 nodules based on serum tumor specific protein 70 (SP70) and conventional laboratory indicators and validate its predictive efficacy. Methods: A case-control study design was used to retrospectively analyze the data of 429 female patients diagnosed with BI-RADS category 4 breast nodules by breast color doppler flow imaging at the First Affiliated Hospital of Nanjing Medical University from January 2021 to April 2022 with an age range of 16 to 91 years and a median age of 50 years, and the patients were divided into a training cohort (314 patients) and a validation cohort (115 patients) according to the inclusion time successively. Using postoperative pathological findings as the"gold standard", univariate and multivariate logistic regression analyses were used to identify the predictor variables used for the model. The nomogram, receiver operating characteristic (ROC) curves and calibration curves were drawn for the prediction model, and the discrimination and calibration of the model were evaluated using the consistency index (C-index) and calibration plots. Results: The postoperative pathological results showed that 286 (66.7%) were malignant nodules and 143 (33.3%) were benign nodules of 429 breast BI-RADS category 4 nodules. The serum SP70 (OR=1.227,95%CI: 1.033-1.458,P=0.020), NLR (OR=1.545,95%CI: 1.047-2.280,P=0.028), LDL-C (OR=2.215, 95%CI: 1.354-3.622, P=0.002), GLU (OR=2.050,95%CI:1.222-3.438,P=0.007), PT (OR=1.383,95%CI: 1.046-1.828,P=0.023), nodule diameter (OR=1.042, 95%CI: 1.008-1.076, P=0.015) and age (OR=1.062,95%CI: 1.011-1.116,P=0.016) were independent risk factors which could be used to distinguish benign and malignant breast BI-RADS category 4 nodules (P<0.05). The nomogram was plotted by the above seven independent variables, and the concordance index (C-index) for the training cohort and validation cohort were 0.842 (95%CI:0.786-0.898) and 0.787 (95%CI:0.687-0.886), respectively. The sensitivity and specificity of using this model to identify benign and malignant breast BI-RADS category 4 nodules in the training and validation cohort were 83.5%, 72.5% and 79.2%, 73.6%, respectively. The calibration curves showed good agreement between the predicted and actual values in the nomogram. Conclusions: This study combined serum SP70, conventional laboratory indicators and breast color doppler flow imaging to develop a nomogram model for the differential diagnosis of benign and malignant breast BI-RADS category 4 nodules. The model may have good predictive efficacy and may provide a basis for clinical treatment options, which is beneficial for guiding breast cancer screening and prevention.

[Study on the consistency of IGF-1 and IGFBP-3 of peripheral whole blood and venous serum in children].

This study aimed to explore the consistency of insulin-like growth factor-1 (IGF-1) and insulin-like growth factor-binding protein-3 (IGFBP-3) by detecting peripheral whole blood and venous serum among children. As a cross-sectional study, children who were aged 0-14 as well as received physical examinations in the Child Healthcare Department of the Children's Hospital of Nanjing Medical University during January 2022 to April 2022 were enrolled in this study. Meanwhile, both of peripheral whole blood and venous serum samples were collected, and the levels of IGF-1 and IGFBP-3 were assayed individually via chemiluminescence immunoassay (CLIA). Additionally, linear regression equation was used to analyze the correlation of results between two categories of samples, while Inter-class Correlation Coefficient (ICC) was used to evaluate the consistency of test results among two types of samples. The change trends of IGF-1 and IGFBP-3 with age were analyzed at the same time. A total of 203 valid matched samples were collected, including 117 boys and 86 girls. Peripheral whole blood was well correlated with serum IGF-1 (r=0.986, P<0.001) and IGFBP-3 (r=0.974, P<0.001), and the linear regression equation is shown as follows: (IGF-1) venous serum =1.047×(IGF-1) peripheral whole blood-6.840; (IGFBP-3) venous serum=0.924×(IGFBP-3) peripheral whole blood+0.396. The correlation and consistency were still persisted after being stratified by sex and age. ICC of IGF-1 and IGFBP-3 were 0.983 and 0.967, respectively which provided an excellent strength of agreement. The levels of IGF-1 or IGFBP-3 in boys' and girls' peripheral whole blood and serum showed significant statistical differences among various age groups (all P<0.001), and also increased significantly with age (all P trend<0.001). In conclusion, the results of IGF-1 and IGFBP-3 in peripheral whole blood and venous serum had positive comparability that could be mutually recognized. The detection of IGF-1 and IGFBP-3 in peripheral whole blood had great potential for young age children by providing guidance for nutritional intervention, growth and development assessment.

[Standardization of clinical application of mass spectrometry method for measurement of vitamin D in capillary blood of children: a multicenter study].

Objective: To establish the norms and clinical application standards of mass spectrometry method to measure vitamin D in capillary blood. Methods: Following the "Province-City-Hospital" sampling procedure, a cross-sectional sample of 1 655 healthy children under 7 years of age were recruited from 12 provinces, autonomous regions, or municipalities in China from November 2020 to December 2021. Both venous and capillary blood samples from the same individual were collected, for which serum 25(OH)D levels were measured by high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) method. Pearson correlation analysis and linear regression analysis were used to detect the correlation and determine a correction algorithm. The agreement was analyzed using Bland-Altman plot and Kappa statistic. The sensitivity and specificity were evaluated using receiver operating characteristic (ROC) curve method. Results: Venous and capillary 25(OH)D levels of 1 655 healthy children under 7 years of age were 74.25 (59.50, 92.00) and 68.75 (54.44, 86.25) nmol/L, respectively, showed a significant difference(Z=22.14, P<0.001) as well as a highly significant correlation between venous and capillary 25(OH)D levels(r=0.95, P<0.001). Linear regression analysis was then performed to determine the correction algorithm: lg(corrected capillary 25(OH)D)=0.13+0.95×lg(capillary 25(OH)D)(R2=0.90,P<0.001). The deviation between venous and corrected capillary 25(OH)D levels was (0.50±17.50) nmol/L, a difference value that did not reach statistical significance (P>0.05). The cut-off values of capillary blood 25(OH)D values 30.00, 50.00, 75.00 nmol/L corresponding to venous blood 25(OH)D values were 26.59, 45.56, and 69.84 nmol/L, respectively. Good consistency was observed between venous and corrected capillary 25(OH)D levels in clinical diagnosis (Kappa value 0.68-0.81). Corrected capillary 25(OH)D showed a high clinically predictive value (area under curve 0.97-0.99,sensitivity 0.72-0.92,specificity 0.89-0.99). Conclusion: The standardized capillary HPLC-MS/MS method can be used to detect 25(OH)D levels in children clinically.

[The correlations of abdominal adipose tissue with anthropometric and metabolic parameters in obese children by magnetic resonance imaging].

Objective: To explore abdominal fat mass distribution and contents among obese children via magnetic resonance imaging (MRI), and analyze the correlations of abdominal adipose tissue with anthropometric and metabolic parameters. Methods: Cross-sectional study. There were 60 obese children admitted to the Children's Health Care Department and Endocrinology Department at Children's Hospital of Nanjing Medical University from July 2016 to December 2018. Children's gender, age, height, weight, body composition, waist circumference and blood pressure were recorded. The levels of fasting blood glucose, lipids, insulin were measured, and liver ultrasound was performed, and the body mass index Z score (BMI-Z), waist-to-height ratio (WHtR) and homeostasis model assessment of insulin resistance (HOMA-IR) were calculated. In addition, contents of subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT) and total abdominal adipose tissue (TAAT) were calculated according to feedback of abdominal MRI scan images. The associations between the contents of abdominal adipose tissue, physical examination status and metabolic disorders among obese children were analyzed through correlation analysis and regression analysis. Receiver operating characteristic (ROC) curve was used to compare the accuracy of fat mass in different parts of the abdomen in predicting their metabolic disorders. Results: A total of 60 children were enrolled in the study, included 44 boys and 16 girls, with age of (9.2±1.4) years. The contents of SAT, VAT and TAAT among the 60 children were positively associated with BMI-Z (r=0.60, 0.46, 0.59), body fat percentage (r=0.64, 0.67, 0.68) and waist-to-height ratio (r=0.60, 0.57, 0.61) (all P<0.01). Meanwhile, contents of SAT and TAAT were also positively correlated with systolic blood pressure (r=0.47, 0.49), triglyceride (r=0.33, 0.35) and HOMA-IR (r=0.33, 0.28)(all P<0.05). In order to adjust the confounding effects among various variables, regression analysis was applied and the result showed that the body fat percentage (ß=0.59, 0.66, 0.65) and waist-to-height ratio (ß=0.53, 0.63, 0.59) were most related to abdominal fat contents (all P<0.01), including SAT, VAT and TAAT among obese children. According to ROC, SAT had outstanding evaluation performances for the diagnosis of insulin resistance and metabolic syndrome, while VAT had excellent evaluation performances for non-alcoholic fatty liver disease (area under curve=0.68, 0.69, 0.69, 95%CI 0.54-0.82, 0.55-0.84, 0.53-0.85, P=0.017, 0.014, 0.019). Conclusions: As one of the best indexes, body fat percentage and WHtR can be used to predict the contents of SAT, VAT and TAAT among obese children. With the increase of abdominal SAT or VAT, the risks for insulin resistance, metabolic syndrome and non-alcoholic fatty liver disease would increase. Assessment of abdominal fat and metabolic risks in obese children should combine BMI-Z with waist circumference and body composition analysis.

[Analysis of vitamin D status among children under 7 years of age in some regions of China].

Objective: To explore current vitamin D status and influential factors of vitamin D deficiency and insufficiency among children under 7 years of age in 11 provinces, autonomous regions or municipalities of China. Methods: According to the "province-city-hospital" sampling technical route, a total of 1 531 healthy children under 7 years of age were sampled from 11 provinces, autonomous regions or municipalities in China by the cluster random sampling method from November 2020 to November 2021. The demographic information, family conditions, behavior and living habits and feeding behaviors were collected using unified questionnaire. Serum 25-hydroxyvitamin D(25(OH)D) levels were measured by liquid chromatography-tandem mass spectrometry. Serum 25(OH)D<30 nmol/L was considered deficient and 30-50 nmol/L was considered insufficient. With 25(OH)D≤50 nmol/L as the dependent variable, multivariate Logistic regression was applied to analyze the association between vitamin D deficiency and insufficiency and potential influential factors. Results: The prevalence of vitamin D deficiency and insufficiency among children under 7 years of age in 11 provinces, autonomous regions or municipalities of China was 14.0% (215/1 531), 3.8% (25/664) and 21.9% (190/867) in 0-<3 and 3-<7 of age years, respectively. Compared to children aged 0-<3 years, children aged 3-<7 years had a 2.6-fold increased risk of vitamin D deficiency and insufficiency (OR=3.60, 95%CI 1.93-6.72, P<0.001). Frequent sunlight exposure (OR=0.46, 95%CI 0.29-0.73, P=0.001), vitamin D supplementation (sometimes, OR=0.33, 95%CI 0.21-0.51, P<0.001; daily, OR=0.20, 95%CI 0.11-0.36, P<0.001) and infant formula intake(4-7 times per weeks, OR=0.43, 95%CI 0.28-0.68, P<0.001) were protective factors for vitamin D deficiency and insufficiency. Conclusion: Vitamin D deficiency and insufficiency are common among children under 7 years of age in 11 provinces, autonomous regions or municipalities of China, which is affected by age, sunlight exposure, vitamin D supplementation and infant formula intake.

[Rapamycin activates autophagy by inhibiting mTOR pathway to alleviate early osteoporosis in rats with skeletal fluorosis].

Objective: To investigate the effects of rapamycin target protein (mTOR) pathway and autophagy on bone formation and bone resorption in fluorosis osteoporosis in rats. Methods: In September 2018, a rat model of skeletal fluorosis was established by intragastric administration of fluorine. The experimental animals were divided into control group, 10 mgF(-)/kg group, 20 mgF(-)/kg group, 2 mg/kg rapamycin (RAPA) +10 mgF(-)/kg group and 2 mg/kg RAPA+20 mgF(-)/kg group, 20 per group. The experiment lasted for 3 months. The changes of bone tissue in rats were observed by hematoxylin-eosin (HE) staining. Bone mineral density (BMD) and biomechanical indexes, such as Modulus of elasticity, Stiffness, Maximum stress and Maximum load, were measured by BMD and biomechanical biometer. Serum levels of alkaline phosphatase (ALP) , osteocalcin (BGP) , osteoprotectin (OPG) , type I procollagen amino-terminal peptide (PINP) , tartrate-resistant acid phosphatase (TRACP) and nuclear factor kappa B receptor activator ligand (RANKL) were determined by enzymatic linked immunosorbent assay (ELISA) . Bone tissue phosphorylated mTOR (p-mTOR) , autophagy-related index selective autophagy adaptor protein p62, microtubule associated protein II (LC3-II) , ALP, osteoblastic transcription factor (Osterix) , and RNT Expression of related transcription factor 2 (Runx2) and bone resorption indicator RANKL were detected by Western blotting. Results: Compared with the control group, dental fluorosis in the 10 mgF(-)/kg and 20 mgF(-)/kg groups was significantly increased, periosteum thickness and absorption lacunae appeared, and BGP, OPG, PINP, TRACP and RANKL in serum contents were increased (P<0.05) , BMD, Modulus of elasticity, Stiffness, Maximum stress and Maximum load of bone tissue decreased significantly (P<0.05) , and the expressions of p-mTOR and p62 were decreased (P<0.05) , also the expressions of ALP, Osterix, Runx2 and RANKL were increased (P<0.05) . Compared with 10 mgF(-)/kg and 20 mgF(-)/kg groups, there were no obvious dental fluorosis symptoms in 2 mg/kg RAPA+10 mgF(-)/kg group and 2 mg/kg RAPA+20 mgF(-)/kg group, and serum ALP, BGP and OPG levels were significantly increased (P<0.05) . TRACP and RANKL contents were significantly decreased (P<0.05) . BMD, Modulus of elasticity, Stiffness, Maximum stress and Maximum load were significantly increased (P<0.05) . The levels of p-mTOR, p62 and RANKL in bone tissues were decreased (P<0.05) , and the expressions of LC3-II, LC3-II/LC3-I, ALP, Osterix and Runx2 were increased (P<0.05) . Conclusion: RAPA may activate autophagy by inhibiting mTOR phosphorylation, and inhibit bone resorption while promoting bone formation, thus alleviating early osteoporosis in skeletal fluorosis rats.

[Effects of duodenal ligation on gastroesophageal reflux and bleomycin-induced pulmonary fibrosis in rats].

Objective: To investigate the effects of duodenal ligation on gastroesophageal reflux and bleomycin-induced pulmonary fibrosis in rats. Methods: Wistar rats were randomized into the control (Ctrl) group, bleomycin (BLM) group, duodenal ligation (GER) group and duodenal ligation plus bleomycin treatment (BLM+GER) group. At day 0 (d0), duodenum ligation was performed in the GER and the BLM+GER group through an open-abdomen surgery at 1.0 cm below the pylorus by about 30% of the circumference. Meanwhile, sham operation was performed in the Ctrl and the BLM group with similar procedures to the above without ligation of the duodenum. At day 14, bleomycin solution (5 mg/kg, for the BLM and BLM+GER groups) or saline (for the Ctrl and GER groups) was intratracheally instilled. Rats were sacrificed at d28 or at d42. HE, Masson's trichrome or TUNEL staining was performed on lung sections of the groups. The levels of hyrdoxyproline (HYP) or malondialdehyde (MDA) were measured respectively by alkaline hydrolysis or thiobarbituric acid colorimetry. The levels of pepsin and cytokines in bronchoalveolar lavage fluid (BALF) samples were assessed by ELISA. Western blot or RT-PCR was used to quantify relative lung expression of proteins or mRNA, respectively. Results: Lungs of the GER group rats were presented with mild inflammatory cell infiltration. Alveolitis and lung fibrosis was prominent in the BLM group but even more severe in the BLM+GER group. Of the Ctrl, GER, BLM and BLM+GER group, the average numbers of apoptotic cells per each magnified field (×200) on d28 lung sections was (5.6±3.0), (6.4±5.3), (15.4±5.3) and (18.4±9.1), respectively (P=0.008); the proportion (%) of blue-stained area under Masson's trichrome at d42 was (21.5±2.8), (23.4±2.5), (34.0±5.8) and (41.3±2.9) (P<0.05); the HYP contents (mg/L) at d42 of each group was (0.77±0.01), (1.26±0.01), (2.02±0.01) and (2.39±0.01) (P<0.01); the BALF levels of MDA (µmol/L) at d42 were (0.51±0.09), (0.87±0.12), (1.40±0.31) and (1.71±0.12) (P<0.001), and differences of these three indices at d42 reached statistical significance when comparing the Ctrl or GER group with the BLM or BLM+GER group (all P<0.05). The levels of pepsin, pH, interleukin (IL)-1ß, transforming growth factor (TGF)-ß1 and HYP at d28 and d42 were statistically different between the GER group and the Ctrl group (all P<0.05). As compared with the BLM group, the values of TGF-ß1, HYP, p-Smad3, vimentin, p-ERK1/2 and cleaved caspase-3 at d28 and d42 were different in the BLM+GER group (all P<0.05). At both d28 and d42, the BALF levels of pepsin and pH were statistically different between the BLM and the Ctrl group, or between the BLM+GER group and the GER group (all P<0.05). Conclusions: Gastroesophageal reflux is induced through duodenal ligation, which activates proinflammatory and profibrotic signals in the lungs and significantly aggravates bleomycin-induced lung injury and fibrosis. In addition, pulmonary fibrosis may induce or worsen the extent of reflux.

[Effects of Enterococcus faecalis supernatants on inflammatory responses of human periodontal ligament cells under pressure].

Objective: To study the effect of various concentrations of Enterococcus faecalis (Ef) supernatants on human periodontal ligament cell (hPDLC) and the inflammatory response of hPDLC under static pressure. Methods: The method of methyl thiazolyl tetrazolium (MTT) was used to detect the effect of various concentrations of Ef supernatants on the proliferation of hPDLCs and the flow cytometry was used to detect the expression of Toll-like receptor 2 (TLR-2) on the surface of hPDLC after 24-hour-stimulation of Ef supernatant. Furthermore, the hPDLCs were divided into non inducing group without Ef supernatant and inducing group with 5% Ef supernatant, and hPDLCs in each group were loaded with 0, 49 and 196 Pa static pressures respectively. The expressions of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) mRNA and protein were detected by reverse transcription-PCR (RT-PCR) and enzyme linked immunosorbent assay (ELISA) after 24 hours. Results: MTT results showed that the supernatant of Ef with concentration≥5% could significantly inhibit the proliferation activity of hPDLCs at 48 hours of cell culture (P<0.05). Flow cytometry showed that the positive cell rates of TLR-2 increased with increasing volume fractions of the Ef supernatants. The values were (2.12±0.07)%, (2.41±0.32)%, (2.65±0.27)%, (4.76±0.46)%, (9.91±0.92)% and (12.01±1.35)%, respectively. The differences were statistically significant when the concentrations≥5% (P<0.05). There were no significant differences in the expressions of IL-1ß and TNF-α mRNA between the non inducing group and the control group under the pressure of 49 Pa (P>0.05). However, there were significant differences in the expressions of IL-1ß and TNF-α mRNA between the non inducing group and the control group under the pressure of 196 Pa (P<0.05), while the expressions of IL-1ß and TNF-α in the inducing group were significantly lower than that in the control group under the pressures of 49 and 196 Pa (P<0.05). Compared with the control group, the mRNA expression was significantly increased (P<0.05). The result of ELISA was consistent with that of PCR. Conclusions: High concentration of Ef supernatant could inhibit the proliferation of hPDLC. Ef supernatant might promote the expression of TLR-2 on the surface of hPDLC. Excessive mechanical pressure induced the inflammatory response of hPDLC. The presence of inflammatory mediators could lead to the intolerance of hPDLC to pressures and small pressure could aggravate the inflammatory response.

Y-shaped airway self-expanding covered metallic stent removal via the interventional technique.

AIM: To review the removal of Y-shaped airway self-expanding covered metallic stents using the interventional technique under fluoroscopy. MATERIALS AND METHODS: The clinical data of 33 patients who underwent removal of Y-shaped airway self-expanding covered metallic stents from March 2011 to August 2019 were analysed retrospectively. RESULTS: A total of 35 Y-shaped stents were removed. The average indwelling duration of the tracheal stents was 101.7 ± 105.4 days. Four cases were removed via the conventional method (grasping the upper tip of the stent to collapse and adduct the proximal end of the stent), whereas 31 cases were removed using the reversal method (grasping the distal end of the stent to invert and strip out the stent). The duration of stent removal was 24.3 ± 12.4 minutes (median time, 20 minutes). CONCLUSION: The interventional radiology technique is a feasible, safe, and effective method for removing Y-shaped airway self-expanding covered metallic stents, and can be considered for use in the clinical setting.

[Guidance of magnetic resonance imaging for target area delineation of postoperative presacral recurrence of rectal cancer].

Objective: Presacral recurrence of rectal cancer have altered the adjacent structures of original pelvic organs due to the previous radical surgery of rectal cancer, and the boundary between recurrent tumor tissues and pelvic internal structures is not clear. Conventional CT examination has poor soft tissue resolution, which makes it difficult to accurately delineate the target area of radiotherapy. This study aimed to explore the guiding role of magnetic resonance imaging (MRI) in delineating the target area of presacral recurrence after radical resection of rectal cancer. Methods: A descriptive case series research method was adopted. From May 2014 to May 2019, the clinical data of 30 patients with presacral recurrence after radical resection of rectal cancer were collected, who were admitted to Peking University People's Hospital, confirmed by pathology or discussed by multidisciplinary team (MDT), with complete MRI, CT and case information. According to the gross tumor volume (GTV) with presacral recurrence outlined in CT and MRI images, including presacral recurrent lesions (GTVT) and metastatic lymph nodes (GTVN), the GTV volume was calculated, and the tumor boundary and diameter were measured. The differences between MRI and CT were compared. Results: The volume of GTVT-CT was larger than that of GTVT-MR in all the 30 patients. The median volume of GTVT-CT was 67.86 (range 5.12-234.10) cm(3), which was significantly larger than 43.02 (range 3.42-142.50) cm(3) of GTVT-MR with statistically significant difference (Z=-4.288, P<0.001). The mean volume of GTVN outlined by CT and MRI was (0.43±0.11) cm(3) and (0.40±0.10) cm(3) respectively without statistically significant difference (t=1.550, P=0.132). The mean values of boundary and radial line of the presacral lesions on CT images were all longer than those on MRI images. The vertical diameter of GTVT on CT and MRI images was (6.66±2.92) cm and (5.17±2.40) cm (t=5.466, P<0.001); the anterior boundary was (3.24±2.51) cm and (2.69±2.48) cm (t=4.685, P<0.001); the anteroposterior diameter was (4.92±2.02) cm and (4.04±1.57) cm (t=6.210, P<0.001); the left boundary was (3.05±1.00) cm and (2.64±0.78) cm (t=2.561, P=0.016); the right boundary was 2.66 (0.00-4.23) cm and 1.82 (-1.10-3.59) cm (Z=-3.950, P<0.001); the transverse diameter was (5.01±1.78) cm and (3.82±1.29) cm (t=4.648, P<0.001), respectively, whose differences were all statistically significant. MRI was superior to CT in judging the involvement of anterior organs, such as intestine, prostate, bladder and the posterior sacrum. Fifteen patients received radiotherapy according to the target area guided by MRI and 10 patients obtained clinical symptom relief. Conclusion: Compared with CT, the GTV of postoperative presacral recurrence of rectal cancer outlined in MRI images is smaller, and MRI can determine the boundary between tumor and surrounding normal tissues more precisely, so it can show the invasion range of tumor more accurately and guide the accurate implementation of radiotherapy.

ISOC1 promotes the proliferation of gastric cancer cells by positively regulating CDK19.

OBJECTIVE: To explore the role and potential mechanism of isochorismatase domain-containing 1 (ISOC1) in gastric cancer. PATIENTS AND METHODS: The expression levels of ISOC1 in gastric cancer (GC) tissues, as well as corresponding cell lines, was evaluated by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). A cell line stably expressing ISOC1 was constructed by vector construction and cell transfection, and the proliferation ability of the stably transfected cells was examined. Subsequently, the ISOC1 target database was screened, which suggested that CDK19 may be the potential target. The correlation between ISOC1 and CDK19 mRNA and protein expressions in clinical tissue specimens and cell lines was evaluated by qRT-PCR and Western blot, and the Luciferase reporter gene experiment was applied to verify the regulatory effect of ISOC1 on CDK19. RESULTS: ISOC1 was shown to be markedly increased in GC tissues compared to adjacent cancer tissues by qRT-PCR. In addition, compared with patients with low ISOC1 expression, the pathological stage and tumor size of gastric cancer patients with high ISOC1 expression were remarkably larger. Then, the ISOC1 knockdown cell line was established, and it was found through cell proliferation function experiments that the proliferation rate of gastric cancer cells was remarkably slower than the control group after knocking down ISOC1. Subsequently, bioinformatics and Luciferase reporter gene experiments suggested that ISOC1 had a direct regulatory effect on CDK19. In addition, recovery experiments also demonstrated that CDK19 overexpression could reverse the effect of ISOC1 silencing on cell proliferation. CONCLUSIONS: ISOC1 was markedly upregulated in GC tissues. It could positively regulate its downstream target CDK19, which in turn promoted the proliferation of GC cells. Therefore, our study may provide new ideas for understanding the progression of GC.

Removal of covered metallic stents with a bullet head for bronchopleural fistula using a fluoroscopy-assisted interventional technique.

AIM: To investigate the feasibility and safety of a fluoroscopy-assisted interventional technique for removal of bullet-shaped self-expanding covered metallic stents from bronchopleural fistulas (BPFs). MATERIALS AND METHODS: Clinical data for 49 consecutive patients who underwent removal of bullet-shaped self-expanding covered metal stents from October 2010 to November 2019 were analysed retrospectively. Fifty-one stents were removed in all, including 29 large Y-shaped bullet stents, 10 small Y-shaped bullet stents, and 12 branched bullet-shaped stents. The average duration for which tracheal stents were in place was 99.4±8.5 days. RESULTS: Fifty-one stents were removed successfully, of which 49 were directly removed on the first attempt. The time required for stent removal ranged from 7-60 minutes (median time, 22 minutes). In eight cases, the stent was removed by the conventional method (i.e., grasping the upper tip of the stent to collapse and adduct the proximal end), and in 43 by the eversion method (i.e., grasping the distal end of the stent to invert and peel out). CONCLUSIONS: Interventional radiology is a simple, safe, and effective method to extract self-expanding covered metallic bullet-shaped stents, with no need for general anaesthesia and tracheal intubation. It has a short operation time, is well tolerated by patients, and is worthy of clinical application.

Curcumin exerts protective effect on PC12 cells against lidocaine-induced cytotoxicity by suppressing the formation of NLRP3 inflammasome.

OBJECTIVE: To explore the protective effect of curcumin on lidocaine-insulted PC12 cells. MATERIALS AND METHODS: We first treated PC12 cells with different doses of lidocaine, and then treated the cells with curcumin or Nod-like receptor pyrin domain3 (NLRP3) inhibitor (MCC950). Subsequently, the cell viability, apoptosis, reactive oxygen species (ROS) production and NLRP3 inflammasome were detected by cell counting kit-8 (CCK8), Annexin V/PI staining, FCM and Western blot analysis, respectively, and the level of IL-1ß in PC12 cells was determined by an enzyme-linked immunosorbent assay (ELISA) kit. RESULTS: Lidocaine inhibited the viability of PC12 cells, and it induced cell apoptosis, promoted ROS release and activated NLRP3 inflammasome in PC12 cells, but its effects were reversed by the treatment of curcumin. Moreover, NLRP3 over-expression also induced cytotoxicity in PC12 cells, which was also rescued by the treatment of curcumin. CONCLUSIONS: Our study indicates that curcumin exerts protective effect against lidocaine-induced cytotoxicity on PC12 cells by suppressing the activity of NLRP3 inflammasome, which provides new ideas on screening natural product for neurological damage therapy.

[Knockdown of MTA1 inhibits the proliferation and apoptosis of esophageal cancer cells].

Objective: To investigate the effects of metastasis associated gene 1 (MTA1) expression on the proliferation and apoptosis of human esophageal cancer Eca109 cells. Methods: MTA1 siRNA was transfected into human esophageal cancer Eca109 cells, and the control group and blank group were set up. The expression of MTA1 in Eca109 cells with different treatment was detected by real-time fluorescent quantitative PCR (RT-PCR) and western blot. The proliferation of Eca109 cells was detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay. The cloning formation ability of Eca109 cells was detected by plate cloning assay. The apoptosis of Eca109 cells were detected by flow cytometry. The expression of proliferating cell nuclear antigen (PCNA) and apoptosis-related proteins, including cleaved caspase-3 and total caspase-3 protein in Eca109 cells were detected by western blot. Results: After 48 hours of transfection, RT-PCR result showed that the relative expression levels of MTA1 mRNA in Eca109 cells in the blank group, control group, and siRNA group were 1.00±0.10, 0.98±0.09 and 0.21±0.03, respectively. The expression of MTA1 mRNA in siRNA group was significantly inhibited (P<0.05), while no significant difference of MTA1 mRNA expression between the blank group and the control group has been found (P>0.05). Western blot results were consistent with those of RT-PCR. MTT array results showed that, compared with the blank group and transfection group, the absorbance values of Eca109 cells in siRNA group were dramatically reduced at 48, 72, and 96 h (all P<0.05). There were no significant differences of absorbance values between the blank group and the control group at 48, 72, and 96 h (all P>0.05). The results of the plate colony formation test showed that the number of colony formation in the blank group and control group were 58.64±6.86 and 60.02±7.04, respectively, significantly higher than 18.10±3.16 in siRNA group (P<0.05), while there was no significant difference between the blank group and control group (P>0.05). Flow cytometry results showed that the apoptosis rates in the blank group and control group were (2.13±0.54)% and (2.27±0.61)%, respectively, significantly lower than (32.61±5.28)% in siRNA group (P<0.05), while there was no significant difference between blank group and control group (P>0.05). Western blot results showed that the expression of PCNA protein was down-regulated while cleaved caspase-3 protein expression was upregulated in siRNA group, compared to the control group and blank group (P<0.05). Conclusions: Inhibition of MTA1 expression can inhibit the proliferation of Eca109 cells and induce apoptosis. This process may be related to the down-regulation of PCNA protein expression and activation of caspase-3 protein expression.