Chemical reference material(CRM)is an important material basis in the process of chemical drug research and development and quality control.This paper introduces the definition and classification of CRMs;the domestic and international regulations and guidelines for the research and development,production,management and use of CRMs by pharmaceutical companies and authoritative CRM issuing organizations;the common preparation methods and key technologies of CRM raw materials;and the technical requirements for the selection of raw materials for different types of CRMs.In addition,this paper also introduces the routine development process and data requirements for the candidate raw material to become a CRM after chemical structure verification,physical and chemical property analysis,homogeneity assessment,stability monitoring,and assignment.It also introduces the classical assignment method,mass balance method,in detail,to provide users of CRMs and the developers of new drugs with some technical references related to the development,application and management of CRMs in China.

Objective:To analyze the gene mutation type and clinical phenotype of patients with PRRT2 mutation, and explore their relations with prognosis. Methods:A total of 18 patients with PRRT2 gene mutation (1 patient with novel mutation in PRRT2 gene, and 17 probands in 17 families with PRRT2 gene mutation) were enrolled in Department of Neurology, First Affiliated Hospital of Air Force Medical University from January 2018 to July 2023. Serum of the patients was collected for whole exon sequencing, and mutation sites and types of PRRT2 gene were analyzed. SWISS-MODEL website was used to predict the changes in protein structure caused by PRRT2 gene mutation. The relations of gene mutation type and clinical phenotype with prognosis of these patients were analyzed. Results:(1) All 18 patients with PRRT2 gene mutation were heterozygous mutation, including 12 frameshift mutations, 5 missense mutations, and 1 integer mutation. The clinical phenotype included benign familial infantile epilepsy (BFIE) in 5 patients, epilepsy in 6 patients, exercise-induced paroxysmal kinesigenic dyskinesia (PKD) in 5 patients, and infantile convulsion and choreoathetosis (ICCA) in 2 patients. A total of 8 mutation sites were found in 18 patients with PRRT2 gene mutation, of which 3 mutation sites have been reported, and 5 mutation sites have not been reported, including c.647(exon2)C>A, c.647(exon2)C>G, c.170(exon2)delC, c.981(exon3)C>G, and lossl(EXON: 2)(all). (2) Eighteen patients mainly accepted oxcarbazepine, levetiracetam, and sodium valproate in combination or monotherapy. Among them, 5 BFIE patients, 2 ICCA patients and 3 epilepsy patients were seizure-free after treatment. PKD patients did not respond well to oxcarbazepine. (3) Three frameshift mutations (mutation sites: c.649 [exon2]_c.650 [exon2] insC, c.640 [exon2]_c.641 [exon2] insC, and c.170 [exon2] delC) led to premature termination of protein translation, resulting in significant changes in protein structure. Four missense mutations (mutation sites: c.640[exo2]G>C, c.647[exon2]C>A, c.647[exon2]C>G, and c.981[exon3]C>G) had little effect on protein structure changes. No relation was found between changes of protein structure caused by different mutation types and prognosis. Conclusion:PRRT2 gene mutation patients with clinical phenotypes of BFIE and ICCA have good prognosis, but the mutation type is not related with the prognosis of patients.

Accurately assessing and positioning the current development status of disciplines can help hospitals formulate targeted disciplinary development strategies and achieve disciplinary development goals. In 2022, a large tertiary public hospital established a discipline-demand-oriented " pyramid" hierarchical discipline evaluation system based on Maslow′s need-hierarchy theory. The discipline evaluation system set six levels of evaluation indicators from low to high, including medical quality and safety, department operation, sub specialty construction, scientific and educational achievements, platform construction, and talent cultivation(including 6 primary indicators, 21 secondary indicators, and 44 tertiary indicators). By applying the evaluation system and providing feedback on the evaluation results in the form of " disciplinary diagnostic reports" and " disciplinary evaluation conferences, " the hospital′s discipline construction had been improved at six levels. From 2022 to 2023, the hospital added five national clinical key specialties; The number of sub major construction disciplines increased from 18 in 2021 to 61 in 2023, and patient satisfaction increased from 94.64% to 96.25%. This evaluation system could objectively reflect the level of discipline development, lead the high-quality development of disciplines, and provide references for other public hospitals to promote discipline development.

Objective:To report the clinical phenotype and mutation site of a patient with grey matter heterotopia caused by a de novo heterozygous missense mutation in the TUBB2B gene, and to expand the phenotypic and mutational spectrum of TUBB2B mutations. Methods:One patient with TUBB2B mutation who presented to the Department of Neurology, the First Affiliated Hospital of Air Force Medical University in July 2017 was collected and analyzed for clinical features and mutation site, and a review of previous studies was performed. Results:The male patient started at the age of 18 and presented mainly with seizures, poor left-handed fine motor skills and poor spatial imagination. Magnetic resonance imaging showed nodular grey matter heterotopia in the right cerebral hemisphere, right frontoparietal-temporal localized cerebral gyrus, and cerebral sulcus shallow flat.The whole exon gene test suggested a heterozygous missense mutation in the TUBB2B gene: c.776 C>T (p.Pro259Leu), which was wild-type in both of his parents. The mutation site was located between the tubulin and tubulin-c structural domains and did not affect the function of the essential structural domain. After treatment with magnesium valproate in combination with levetiracetam, the patient′s seizure symptoms were significantly controlled and he has been seizure-free for 3 years now. Conclusions:The TUBB2B gene c.776 C>T (p.Pro259Leu) heterozygous missense mutation is a novel missense mutation causing grey matter heterotopia. The patient had a good prognosis, and the combination of two antiepileptic drugs resulted in complete seizure control.

Aristolochic acid (AA), extracted from Aristolochiaceae plants, plays an essential role in traditional herbal medicines and is used for different diseases. However, AA has been found to be nephrotoxic and is known to cause aristolochic acid nephropathy (AAN).AA-induced acute kidney injury (AKI) is a syndrome in AAN with a high morbidity that manifests mitochondrial damage as a key part of its pathological progression. Melatonin primarily serves as a mitochondria-targeted antioxidant. However, its mitochondrial protective role in AA-induced AKI is barely reported. In this study, mice were administrated 2.5 mg/kg AA to induce AKI. Melatonin reduced the increase in Upro and Scr and attenuated the necrosis and atrophy of renal proximal tubules in mice exposed to AA. Melatonin suppressed ROS generation, MDA levels and iNOS expression and increased SOD activities in vivo and in vitro. Intriguingly, the in vivo study revealed that melatonin decreased mitochondrial fragmentation in renal proximal tubular cells and increased ATP levels in kidney tissues in response to AA. In vitro, melatonin restored the mitochondrial membrane potential (MMP) in NRK-52E and HK-2 cells and led to an elevation in ATP levels. Confocal immunofluorescence data showed that puncta containing Mito-tracker and GFP-LC3A/B were reduced, thereby impeding the mitophagy of tubular epithelial cells. Furthermore, melatonin decreased LC3A/B-II expression and increased p62 expression. The apoptosis of tubular epithelial cells induced by AA was decreased. Therefore, our findings revealed that melatonin could prevent AA-induced AKI by attenuating mitochondrial damage, which may provide a potential therapeutic method for renal AA toxicity.

ObjectiveTo explore the characteristics of oral sensory-motor function in children with functional articulation disorders (FAD). MethodsFrom June, 2021 to January, 2022, 61 children with FAD in the Department of Children's Health of the Affiliated Hospital of Qingdao University were as case group, 90 normal healthy children were as control group. They were assessed with Oral Sensory-Motor Assessment, and were compared among different genders and different ages. Hyperactivity problems were assessed using Conners Parent Symptoms Questionnaire, and their oral sensory-motor function were compared between patients with hyperactivity problem (n = 13) and without hyperactivity problem (n = 48). ResultsThe total score and the scores of oral sensation, mandibular motion, lip motion and tongue motion were significantly lower in the case group than in the control group (t > 4.471, P < 0.001). There were significant differences in the total score and motor function scores among different ages in the control group (H > 17.015, P < 0.001), and they increased with age. There were significant differences in the total score, oral sensory and motor function scores among different ages in the case group (H > 10.567, P < 0.01), and they increased with age. The total scores, and the scores of mandibular, lip and tongue movements were lower in the boys and girls of the case group than in the same gender of the control group (t > 2.49, P < 0.05). The total score and the scores of the lip and tongue movements were lower in boys than in girls in the case group (|Z| > 2.409, P < 0.05). There was no significant difference in the total score and other scores between the patients with hyperactivity problem and without hyperactivity problem (P > 0.05). ConclusionThe oral sensory-motor function is poor for children with FAD, and can develop with age in both normal and FAD children.

In this paper, the uncertainties of correction factors of fluconazole impurities determined by HPLC standard curve method were evaluated, and the main common factors affecting the accuracy of standard curve method were found, so as to improve the accuracy of the method.In this study, the corresponding fitting lines of fluconazole and its impurities A, B, C, D, F and I were established respectively, and the ratio of the slope of fitting lines of each impurity and its corresponding principal component was calculated as the correction factor of the impurity.Then on the basis of GUM method, the uncertainty of each impurity correction factor determined by standard curve method was evaluated according to the established uncertainty evaluation scheme of correction factor determination process.The correction factor and uncertainty of fluconazole impurities A, B, C, D, F and I were 1.068 ± 0.046, 0.102 ± 0.005, 0.0582 ± 0.0031, 1.382 ± 0.121, 0.802 ± 0.067 and 1.383 ± 0.119, respectively, and the coverage factor k was 2.Finally, the contribution rate of each uncertainty component was calculated.In the relative combined standard uncertainties urel(f) of fluconazole impurities A, B, C, D, F and I correction factors, the sum of contribution rate of slope uncertainty urel(K) of the linear equation of principal component and its impurity is more than 85%; in the slope uncertainties urel(K) of linear equation, the contribution rates of uncertainties of solution concentration in 8 of 12 data groups are more than 80%, and the contribution rates of uncertainties introduced by reference substance content in solution concentration are about 80%.It can be seen that the preparation of linear solution concentration is the most influential factor in the determination of impurity correction factor by standard curve method, followed by the linear fitting process.In the preparation process of linear solution concentration, the purity of reference substance is the most influential factor, followed by weighing and pipetting times.The conclusion can help the experimenters to better formulate experimental plans and ensure the accuracy of the results when doing similar work.

OBJECTIVE To explore the regulatory mechanism of compatibility of ginseng and gecko dispensing granule on kidney yang deficiency model rats. METHODS Male SD rats were randomly divided into normal group （no modeling ，no administration），model group （modeling，no administration ），Jinkui shenqi pill group （modeling，dose of 2.33 g/kg），ginseng group（modeling，dose of 0.53 g/kg），gecko group （modeling，dose of 0.21 g/kg）and compatibility group （modeling，ginseng 0.53 g/kg and gecko 0.21 g/kg）. The body mass and anal temperature of rats were measured at different time points ；the serum levels of cAMP ，cGMP，CRH，ACTH，CORT，T，T3，T4，E2，IgG and IgM were measured ；the pathomorphological changes of adrenal gland ，thyroid gland and testis were observed ；mRNA expression of CRH ，thyroid stimulating hormone releasing hormone （TRH）and gonadotropin releasing hormone （GnRH）in hypothalamus were detected. RESULTS Compared with model group ，the anal temperature ，the levels of cAMP ，CRH，ACTH，CORT，T3，T and cAMP/cGMP ，T/E2 in serum and mRNA expressions of TRH and GnRH in hypothalamus were significantly increased in the compatibility group （P＜0.05 or P＜0.01）；the levels of cGMP，E2 and IgG in serum and mRNA expression of CRH in hypothalamus decreased significantly （P＜0.05 or P＜0.01）； the pathological injuries of adrenal gland ，thyroid gland and testis were all improved. Compared with ginseng or gecko dispensing granules alone ，the anal temperature and T/E 2 of rats in the compatibility group increased significantly ，and mRNA expression of CRH in hypothalamus decreasedsignificantly （P＜0.05 or P＜0.01）. CONCLUSIONS Thecompatibility of ginseng and gecko dispensing granule has a synergistic regulatory effect on kidney yang deficiency model rats ， the mechanism of which may be associated with hypothalamus-pituitary-adrenal axis ， hypothalamus-pituitary-thyroid axis ， hypothalamus-pituitary-gonad axis and neuroendocrine immune network formed by immune function. Compatible drugs are better than single drugs.

To improve the standard of quality control of tazobactam and its preparations in China, national reference standard of tazobactam impurity A was developed. After tazobactam impurity A was synthesized, its structure was validated by infrared (IR), mass spectrometry (MS) and nuclear magnetic resonance (NMR), and its content uniformity and short-term stability were measured and investigated. Then, water content and residue on ignition of impurity A were determined, and its purity was determined using high performance liquid chromatography (HPLC) with 10 mmol/L ammonium acetate solution-acetonitrile (98∶2) as the mobile phase. Mass balance method was used to determine the content of the first batch of tazobactam impurity A national standard substance. Meanwhile, nuclear magnetic quantitative method was used to calculate the content, which was mutually verified with the mass balance method. The developed reference material of tazobactam impurity A is consistent with the maximum degradation impurity in tazobactam system applicability solution and the reference material of tazobactam related substance A contained in USP41. Within the 95% confidence range, the ratio of inter- and intra-bottle variance of impurity A after separation was 0.61 (< F0.05(11,12)), proving that the uniformity was satisfying. The contents of organic impurity, water content and inorganic impurity in impurity A were 0.90%, 1.24% and 0.25%, respectively. The content of impurity A was determined to be 97.6% by mass balance method, which was basically consistent with the result of nuclear magnetic quantitative method (97.1%). Under the condition of 25 °C, the area normalized purity of impurity A was 99.1% at 0, 3, 5 and 10 days, proving that the sample was stable at room temperature for 10 days. Finally the first batch of national standard substance of tazobactam impurity A was established successfully.

Objective:The research was aimed to investigate the association between serum total homocysteine (tHcy) and subacute combined degeneration of the spinal cord (SCD).Methods:A retrospective survey of 106 newly diagnosed patients with SCD were enrolled in this research who were treated in the department of neurology of Xijing Hospital from January 2008 to February 2019, meanwhile, 121 patients with spinal cord lesion (not SCD) and 104 neurology mild outpatients were selected as controls. Serum tHcy level was determined by using the chemiluminescent immunoassay assay. A multivariate logistic regression model was used to analyze the risk factors for SCD. The area under the curve (AUC) of the receiver operating characteristic (ROC) curve, sensitivity, specificity and Youden index were used to evaluate the diagnostic efficacy of tHcy. Spearman correlation analysis was used to observe the correlation between tHcy and SCD severity. The SCD patients were categorized into normal or mild tHcy group, moderate tHcy group, and severe tHcy group based on tHcy levels. Clinical symptoms, nerve conduction velocity, magnetic resonance imaging (MRI) findings from the patients were studied.Results:The serum tHcy levels in SCD patients were 64.3(26.5, 98.8) μmol/L, while in patients with spinal cord lesion (not SCD) group were 13.7(10.8, 19.2) μmol/L, neurology mild outpatients were 10.6(8.2, 13.0) μmol/L, which was higher in SCD group ( H=112.020, P<0.001), ( H=165.525, P<0.001).The multivariate logistic regression model showed tHcy is the impact factor of SCD ( OR=1.107, 95% CI:1.077-1.139, P<0.001). At ROC analysis, tHcy showed diagnostic value with an optimal cut-off value of 24.9 μmol/L (AUC 0.913, 95% CI: 0.875-0.951, sensitivity 79.2%, specificity 91.6%). Spearman correlation analysis showed that tHcy was positively correlated with functional disability rating scale ( r=0.254, P=0.009). Conclusions:Serum tHcy is the risk factor for SCD and related to its disability. Focus on the increased level of tHcy plays a positive role in the diagnosis of SCD.