OBJECTIVE To investigate the effect of Guanxinning tablets(GXN) on early heart failure model rats, and to explore the protective mechanism of GXN on heart failure rats from the perspective of intestinal flora. METHODS Six rats who underwent sham operation were set as sham operation group. Took 80 SD rats to undergo aortic arch stenosis and established a heart failure rat model. The surviving rats were divided into 4 groups, namely the model control group, the positive control group(captopril tablets 12.5 mg·kg–1), high-dose and low-dose of GXN group(600, 1 200 mg·kg–1). The 4 groups were administered continuously for 8 weeks. Cardiac ultrasonography was performed every 4 week. Serum NT-proBNP, hs-CRP, IL-6, TNF-α, SOD and MDA levels were measured. The effects of GXN on the structure and function of intestinal flora were observed based on the high-throughput sequencing technology and bioinformatics analysis of 16S gut microbiome. RESULTS Compared to the model control group, after giving different doses of GXN, the survival rate of rats increased, and the thickness of the ventricular wall decreased to varying degrees. The weight of the heart and coefficient of the heart were all reduced. GXN could also reduce the level of inflammatory factors, inhibit the level increase of NT-proBNP in rats, and increase the activity of serum SOD. In addition, GXN intervention could significantly improve the intestinal flora diversity of rats with heart failure, the possible target genera of GXN were Akkermansia genera, Phascolarctobacterium genera and Oxalobacter genera. The effect of GXN on intestinal function in rats with heart failure might be concentrated in non-homologous end-joining, influenza A, carotenoid synthesis, indole alkaloids biosynthesis, betalain biosynthesis, renin-angiotensin system and other biological pathways. CONCLUSION The protective effect of GXN on early heart failure rats may be related to the regulation of intestinal flora pathway.

Objective:To explore the pathogenicity and genotype-phenotype correlation of a c. 158G>A variant of phenylalanine hydroxylase ( PAH) gene among patients with PAH deficiency. Methods:Thirty seven children diagnosed with PAH deficiency at the Obstetrics and Gynecology Hospital Affiliated to Nanjing Medical University between July 2016 and June 2021 were selected as the study subjects. Clinical data and results of genetic testing were retrospectively analyzed.Results:Among the 37 patients, mild hyperphenylalaninemia (HPA) was observed in 34 cases, two PAH variants (including c. 158G>A), which formed a compound heterozygous mutation genotype, were detected in 33 patients, and the remainder one was found to harbor three PAH variants, including homozygous c. 158G>A variants and a heterozygous c. 842+ 2T>A variant. Classical phenylketonuria (PKU) was observed in 3 patients, and three PAH variants were detected in each of them, including two with c. [158G>A, 842+ 2T>A]/c.728G>A and c. [158G>A, 842+ 2T>A]/c.611A>G, respectively, and one with c. [158G>A, c. 722G>A]/c.728G>A. The c. 158G>A variant has a minimal influence on the PAH activity and is associated with a mild HPA phenotype. The variant should thereby be classified as likely benign. Conclusion:When the c. 158G>A variant and other pathogenic variants are arranged in cis position, the ultimate phenotype will be determined by the pathogenicity of other variants.

Objective:To preliminarily investigate the effect of blue light on the biological activity of human skin keratinocytes, fibroblasts and melanocytes.Methods:Discarded foreskin tissues were collected from 10 healthy children aged from 3 to 12 years after circumcision surgery in the First Affiliated Hospital of Kunming Medical University from June 2021 to December 2021. After epidermis-dermis separation, selective culture was performed to isolate keratinocytes, fibroblasts, and melanocytes. According to the pre-experiment results, the above three types of cells were irradiated with 440 - 450 nm blue light at doses of 0, 5, 10, 20, 30, and 40 J/cm 2, and then continued to be cultured for 0, 6, 24, and 48 hours. Cell counting kit 8 (CCK8) assay was performed to evaluate cellular proliferative activity at each time point, enzyme-linked immunosorbent assay (ELISA) to detect levels of interleukin (IL) -18, IL-33, nerve growth factor (NGF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) secreted by keratinocytes, as well as levels of IL-33 and keratinocyte growth factor (KGF) secreted by fibroblasts, NaOH lysis method to determine melanin synthesis rates in melanocytes, and Western blot analysis to determine the relative expression of tyrosinase (TYR), tyrosine-related protease 1 (TRP-1) and dopachrome isomerase (DCT) in melanocytes. Two-way analysis of variance was used to analyze group effects, time effects and interaction effects. Results:After irradiation with blue light, the cellular proliferative activity significantly differed among different doses of blue light irradiation groups and different time points in keratinocytes ( Ftime = 516.20, Fdose = 421.20, Finteraction = 25.05, all P < 0.003), fibroblasts ( Ftime = 129.30, Fdose = 477.80, Finteraction = 10.91, all P < 0.003), and melanocytes ( Ftime = 77.61, Fdose = 138.70, Finteraction = 3.50, all P < 0.003) ; immediately after irradiation, the proliferative activity of keratinocytes and fibroblasts was significantly lower in the 20 - 40 J/cm 2 blue light group than in the 0 J/cm 2 blue light group (all P < 0.003), and the proliferative activity of melanocytes was significantly higher in the 5 J/cm 2 blue light group than in the 0 J/cm 2 blue light group ( P < 0.003) ; the proliferative activity of the 3 types of cells showed decreasing trends with the increase of blue light irradiation doses and culture time. ELISA showed that the concentrations of IL-18, IL-33, NGF, and GM-CSF secreted by keratinocytes, as well as the concentrations of IL-33 and KGF secreted by fibroblasts, tended to increase with the increase of blue light irradiation doses and culture time. The melanin synthesis rates in melanocytes significantly differed among different doses of blue light irradiation groups and different time points ( Ftime = 833.50, Fdose = 249.40, Finteraction = 81.38, all P < 0.003) ; during 0 - 24 hours after blue light irradiation, the melanin synthesis rates tended to increase with the increase of blue light irradiation doses and time; during 24 - 48 hours, the melanin synthesis rates showed decreasing trends with the increase of blue light irradiation doses and culture time compared with that at 24 hours after irradiation; 24 hours after irradiation, the melanin synthesis rates were significantly higher in the 5, 10, 20, 30 and 40 J/cm 2 blue light groups (159.50% ± 10.88%, 218.76% ± 8.49%, 333.72% ± 7.72%, 393.29% ± 6.00%, 427.21% ± 8.39%, respectively) than in the 0 J/cm 2 blue light group (102.29% ± 6.57%, all P < 0.003). The relative expression of TYR ( Ftime = 67.94, Fdose = 28.99, Finteraction = 3.71, all P < 0.003), TRP-1 ( Ftime = 21.73, Fdose = 8.38, both P < 0.003) and DCT ( Ftime = 34.51, Fdose = 11.79, both P < 0.003) in melanocytes significantly differed among different doses of blue light irradiation groups and different time points, and tended to increase with the increase of blue light irradiation doses and culture time. Conclusion:Blue light irradiation at doses of 5 - 40 J/cm 2 could inhibit the proliferative activity of human skin keratinocytes, fibroblasts, and melanocytes, and the inhibitory effect tended to increase with the increase of blue light irradiation doses, except an enhancing effect on the proliferative activity of melanocytes observed immediately after irradiation with blue light at 5 J/cm 2; additionally, blue light irradiation at 5 - 40 J/cm 2 could enhance the expression of melanin synthesis-related enzymes in melanocytes, and increase the melanin synthesis rate in melanocytes over a short period of time.

OBJECTIVE: To analyze the clinical features and genetic variants in four neonates with very long chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency. METHODS: Neonates with a tetradecenoylcarnitine (C14:1) concentration at above 0.4 μmol/L in newborn screening were recalled for re-testing. Four neonates were diagnosed with VLCAD deficiency by MS-MS and genetic testing, and their clinical features and genotypes were analyzed. RESULTS: All cases had elevated blood C14:1, and the values of first recalls were all lower than the initial test. In 2 cases, the C14:1 had dropped to the normal range. 1 case has remained at above 1 μmol/L after the reduction, and the remainder one case was slightly decreased. In total eight variants of the ADACVL genes were detected among the four neonates, which included 5 missense variants and 3 novel variants (p.Met344Val, p.Ala416Val, c.1077+6T>A). No neonate showed salient clinical manifestations. CONCLUSION Above findings have enriched the spectrum of ADACVL gene mutations and provided a valuable reference for the screening and diagnosis of VLCAD deficiency.
Acyl-CoA Dehydrogenase/genetics* ; Acyl-CoA Dehydrogenase, Long-Chain ; Congenital Bone Marrow Failure Syndromes ; Genetic Testing ; Humans ; Infant, Newborn ; Lipid Metabolism, Inborn Errors ; Mitochondrial Diseases ; Muscular Diseases ; Tandem Mass Spectrometry

Objective:To identify the possible pathogenesis of a neonate with carnitine palmitoyltransferase 1A (CPT1A) deficiency by analyzing genetic variants.Methods:Potential variants were detected with an Ion Torrent semiconductor sequencer using a gene panel for inherited diseases, and candidate variants were verified by Sanger sequencing.Results:Genetic testing indicated that the neonate has carried c. 1895T>A(p.Leu632X) and c. 1153G>A(p.Ala385Thr) compound heterozygous variants of the CPT1A gene, which were inherited from his father and mother, respectively. Both variants were verified as novel through the retrieval of HGMD database, ClinVar database and literature. According to the standards and guidelines of the American College of Medical Genetics and Genomics, the c. 1895T>A variant was predicted as pathogenic(PVS1+ PM2+ PP4) and c. 1153G>A as likely pathogenic (PM1+ PM2+ PM3+ PP3). Conclusion:The c. 1895T>A and c. 1153G>A compound heterozygous variants of the CPT1A gene might underlie the pathogenesis in this child. Above results have provided a basis for clinical diagnosis and genetic counseling, and enriched the variant spectrum of the CPT1 deficiency.

Objective:To investigate the clinical characteristics and pathogenic mutations of propionic acidemia.Methods:Clinical data of two patients with propionic acidemia admitted to the Obstetrics and Gynecology Hospital of Nanjing Medical University from May 2017 to June 2018 were collected. Genomic DNA was extracted from the peripheral blood of the patients and their parents. Inherited disease panel based on Ion Torrent semiconductor sequencing technology was performed to detect gene mutations, and those with suspected pathogenic mutations were verified by Sanger sequencing. Descriptive statistical analysis was used for data analysis.Results:Case 1 was suspected of sepsis and admitted to the Obstetrics and Gynecology Hospital of Nanjing Medical University due to "drowsiness and milk rejection" on the second day after birth. Tandem mass spectrometry suggested the level of propionyl carnitine and its ratios to acetylcarnitine and free carnitine were increased. Urine gas chromatography-mass spectrometry showed elevated 3-hydroxypropionic acid and methylcitric acid. Genetic analysis revealed that the infant carried c.331C>T (p.R111X)/c.1228C>T (p.R410W) compound heterozygous mutations in the PCCB gene. The infant was diagnosed with propionic acidemia and treated with a special diet with an L-Carnitine supplement but died of sudden coma and vomiting without precipitating factors at three months of age. Case 2 presented with sudden vomiting, drowsiness, and anergia on the admission at five-months old. Tandem mass spectrometry showed increased propionyl carnitine level and its ratios. Compound heterozygous mutations of c.146delG (p.G49EfsX16)/c.1253C>T (p.A418V) in the PCCB gene were identified in the patient, of which c.146delG (p.G49EfsX16) was a de novo mutation and was evaluated as a pathogenic mutation. The patient was on a special diet with an L-Carnitine supplement, but with disobedience. Followed up to the age of three years and eight months, the child was severely underdeveloped. Conclusions:Neonates with clinically suspected sepsis may have propionic acidemia, and tandem mass spectrometry and genetic testing should be performed as soon as possible to confirm or rule out the diagnosis. Further investigations on the pathogenesis and function of the new mutation are still needed.

Background: Androgenetic alopecia (AGA) leads to thinning of scalp hair and affects 60%~70% of the adult population worldwide. Developing more effective treatments and studying its mechanism are of great significance. Previous clinical studies have revealed that hair growth is stimulated by 650-nm red light. Objective: This study aimed to explore the effect and mechanism of 650-nm red light on the treatment of AGA by using ex vivo hair follicle culture. Methods: Human hair follicles were obtained from hair transplant patients with AGA. Hair follicles were cultured in Williams E medium and treated with or without 650-nm red light.Real-time RT-PCR and immunofluorescence staining were used to detect the expression level of genes and proteins in hair follicles, respectively. RNA-sequencing analysis was carried out to reveal the distinct gene signatures upon 650 nm treatment. Results: Low-level 650 nm red light promoted the proliferation of human hair follicles in the experimental cultured-tissue model. Consistently, 650 nm red light significantly delayed the transition of hair cycle from anagen to catagen in vitro. RNA-seq analysis and gene clustering for the differentially expressed genes suggests that leukocyte transendothelial migration, metabolism, adherens junction and other biological process maybe involved in stimulation of hair follicles by 650-nm red light treatment. Conclusion The effect of 650-nm red light on ex vivo hair follicles and the transcriptome set which implicates the role of red light in promoting hair growth and reversing of miniaturization process of AGA were identified.

Objective:To screen new drugs for treatment of phenylalanine hydroxylase deficiency.Methods:From October 2019 to October 2020, virtual drug screening was performed in Center of Genetic Medicine, Nanjing Maternity and Child Health Care Hospital, Women's Hospital of Nanjing Medical University computer according to the characteristics of the binding ability of phenylalanine hydroxylase to drug spatial structure. Ten candidate drugs were screened from the FDA drug library (including 2 697 kinds of active pharmaceutical ingredients). A eukaryotic expression system was used to determine the effects of drugs on the activity of phenylalanine hydroxylase at the molecular level. Drug-sensitive mutants were screened.Results:Among the 10 candidate drugs, neoplasm hydrochloride, fluocinonide acetate and risperidone increased 23% [ t = 18.21, P < 0.001, vs. non-drug-treated phenylalanine hydroxylase group (i.e., only solvent and no drug added to the reaction system)], 21% ( t = 3.44, P < 0.05, vs. non-drug-treated phenylalanine hydroxylase group), 31% ( t = 19.57, P < 0.001, vs. non-drug-treated phenylalanine hydroxylase group) of the activity of phenylalanine hydroxylase. The remaining drugs exhibited weak even inhibitory effects on the activity of phenylalanine hydroxylase. 25% of p.D101N mutant could be activated by risperidone ( t = 15.86, P < 0.001, vs. non-drug-treated p.D101N mutant group). Conclusion:Neoplasm hydrochloride, fluocinonide acetate and risperidone can be used as potential therapeutic drugs for phenylalanine hydroxylase deficiency, and p.D101N mutant can be used as the drug-sensitive mutation site.

Objective:To analyze the detection of neonatal inherited metabolic diseases in Nanjing.Methods:We researched the results of 175 767 newborns by tandem mass spectrometry from December 2013 to July 2018. Amino acids, acylcarnitines, and succinylacetone were detected by non-derivatized tandem mass spectrometry to screen the abnormity of newborn amino acid, organic acid, or fatty acid oxidation metabolism disease. Gene panels based on high throughput sequencing technology were carried out to detect gene mutation of positive neonates. Descriptive statistics were used to analyze all the data.Results:The positive rate of primary screening was 2.1% (3 691/175 767), 3 598 of 3 691 positive cases were recalled. At last, 62 cases of the inherited metabolic disease were diagnosed. Among them, there were 35 cases of amino acid metabolism disease, 12 cases of organic acid metabolism disorder, and 15 cases of fatty acid metabolism defect. The total incidence of neonatal inherited metabolic disease was 0.035 3%, among which amino acid metabolic diseases were 0.019 9%, organic acid metabolic diseases were 0.006 8%, and fatty acid metabolic diseases were 0.008 5%. The diseases with the highest incidence were phenylalanine hydroxylase deficiency (0.015 9%), methylmalonic acidemia (0.005 1%), and primary carnitine deficiency (0.005 1%). Among 62 children, 51 (82.2%) were diagnosed by gene diagnosis (including 17 cases of phenylalanine hydroxylase deficiency and 34 cases of other inherited metabolic diseases). Another 11 children with phenylalanine hydroxylase deficiency refused gene diagnosis. Two pathogenic mutations were found in 17 children with phenylalanine hydroxylase deficiency. Two pathogenic mutations were found in 29 of the other 34 children with inherited metabolic disease, which were from their parents, while only one pathogenic mutation was found in the other five children, of which two cases with hypermethioninemia were autosomal dominant inheritance.Conclusions:Neonatal inherited metabolic diseases with high incidence in Nanjing are phenylalanine hydroxylase deficiency, methylmalonic acidemia, and primary carnitine deficiency. Some cases screened by tandem mass spectrometry only showed abnormal screening indicators. No specific clinical symptoms were found during follow-up, and further follow-up was needed.

ObjectiveTo investigate the clinical significance of highly sensitive nucleic acid detection in precise antiviral therapy for patients with liver cirrhosis and its association with aminotransferase level. MethodsA total of 377 patients with hepatitis B cirrhosis who were hospitalized or attended the outpatient service from May 2013 to April 2019 were enrolled and tested by both domestic HBV DNA detection and highly sensitive Cobas HBV DNA detection. All patients underwent biochemical examination, four blood coagulation tests, routine blood test, and upper abdominal computed tomography or ultrasound. Sensitivity of different HBV DNA detection reagents was compared in liver cirrhosis patients with a low viral load, and the correlation between alanine aminotransferase (ALT) level and viral load was analyzed. The paired t-test was used for comparison of continuous data before and after treatment. The receiver operating characteristic (ROC) curve was used to screen out the optimal predictive values of ALT at different cut-off values of HBV DNA. ResultsAmong the 377 patients with hepatitis B cirrhosis, 215 tested positive and 162 tested negative by domestic HBV DNA, and among these 162 patients, 104 (64.2%) tested positive by Cobas HBV DNA detection, with a mean level of 267.5±42.3 IU/ml. After 24 weeks of antiviral therapy, the 104 patients with hepatitis B cirrhosis had significant improvements in viral replication level, ALT, and Child-Pugh score for liver function; HBV DNA decreased from 267.5±32.2 IU/ml before treatment to 59.6±7.7 IU/ml after treatment (t=3.486, P=0.002), ALT decreased from 871±10.8 U/L before treatment to 36.5±7.6 U/L after treatment (t=3.235, P=0.020), and the Child-Pugh score decreased from 6.5±0.7 before treatment to 5.7±0.5 after treatment (t=2.928, P=0.041). The ROC curve analysis of ALT in predicting HBV DNA decision point showed that an ALT level of 29 IU/L was the most sensitive cut-off value for predicting HBV DNA ＜20 IU/ml, with an area under the ROC curve of 0.904, a sensitivity of 1.0, and a specificity of 0.237. ConclusionPrecise detection helps to guarantee the precise clinical treatment of patients with hepatitis B cirrhosis and improve their treatment outcome and prognosis. An ALT level of 29 IU/L is a sensitive indicator for predicting patients with negative Cobas HBV DNA, so as to achieve individualized precise screening and treatment.