Abstract: It is well-known that crystal form of a drug is a key factor impacting the physicochemical properties of the drug, which in turn affects its in vivo efficacy, safety and stability. The study on crystal forms of solid-state drugs is crucial for drug quality control, selection of production process and evaluation of clinical efficacy. The combination of chemometric and analytical techniques exhibited its great ability to analyze a large amount of multidimensional data, providing the possibility for quantification of trace amount of crystals (< 1%). Meanwhile, using the process analytical technology (PAT) to monitor the crystal content real-time during prescription preparation process can further realize the control on formulation quality and serve as a core technology to support the patent protection of crystalline forms. In this review, the combined application of crystal analytical techniques and chemometric methods for the quantitative analysis of trace crystals were summarized, aiming to provide guidance for the manufacturing of pharmaceutical preparations and their quality control.

Objective:To investigate the value of SHOX2 and RASSF1A gene promoter region methylation detection for screening and diagnosis of early-stage lung adenocarcinoma.Methods:The mRNA sequencing data of 471 lung adenocarcinoma patients and corresponding methylation data of 413 cases were downloaded from The Cancer Genome Atlas (TCGA) database, the methylation levels of SHOX2 and RASSF1A gene promoter regions were calculated, and the difference in methy lation level between normal lung tissues and tumor tissues was analyzed. The clinical data of 54 patients with early-stage lung adenocarcinoma and 31 patients with benign lung tumors who underwent surgery at Drum Tower Hospital Affiliated to Nanjing University Medical School from January 2018 to January 2019 were retrospectively analyzed. The methylation status of SHOX2 and RASSF1A in tumor tissues and normal lung tissues (>5 cm from the edge of the tumor foci) (called clinical samples) was detect, and a positive methylation in the promoter region of either gene was considered as a combination of two genes methylation positivity. Using pathological diagnosis as the gold standard, the efficacy of gene methylation positivity in diagnosing early-stage lung adenocarcinoma was analyzed by receiver operating characteristic (ROC) curve. Patients with >80% of tumor cells in paraffin samples were screened, and mRNA high-throughput sequencing was performed in their tumor tissues and normal lung tissues. The relationship between positive methylation of the two genes and clinicopathological features was analyzed, and the correlation between the promoter region methylation level of the two genes and mRNA expression levels in clinical samples and TCGA database samples was analyzed by Spearman method. Gene set variance analysis (GSVA) method was used to analyze the differences in Kyoto Encyclopedia of Genes and Genomes enrichment pathways between two-gene methylation-positive clinical lung adenocarcinoma samples and corresponding methylation-negative lung adenocarcinoma.Results:In TCGA database, the SHOX2 promoter region methylation island contained 6 sequenced methylation sites, of which sites cg04532033 and cg01557547 methylation levels were higher in lung adenocarcinoma tissues than in normal lung tissues (both P < 0.05); the RASSF1A gene promoter region methylation island contained 11 sequenced methylation sites, and the methylation levels of 6 of these sites in lung adenocarcinoma tissues were higher than those in normal lung tissues (all P < 0.05). Compared with normal lung tissues, the methylation level of SHOX2 promoter region was higher in stage Ⅰ and Ⅱ lung adenocarcinoma tissues (both P < 0.05); the methylation level of RASSF1A promoter region was higher in all stages of lung adenocarcinoma ( P < 0.001). Among 54 patients with early-stage lung adenocarcinoma, 28 were positive for SHOX2 promoter region methylation in tumor tissues, 21 were positive for RASSF1A promoter region methylation, and 40 were positive for combined methylation of both genes; 31 benign lung nodules were negative for SHOX2 and RASSF1A methylation. ROC curve analysis showed that the sensitivity of positive SHOX2 promoter region methylation for diagnosing early-stage lung adenocarcinoma was higher than that of RASSF1A promoter region methylation positivity (51.8% vs. 38.9%), and the area under the curve (AUC) for diagnosis by two-gene methylation positivity was larger than that for diagnosis by SHOX2 or RASSF1A gene methylation positivity alone (0.870 vs. 0.759 and 0.694). The circulating thresholds (Ct) of SHOX2 and RASSF1A methylation tested by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) in stage Ⅰ and Ⅱ lung adenocarcinoma were lower than those in normal lung tissues (all P < 0.05); patients with two-gene methylation positivity were characterized by older age, longer tumor longest diameter and more advanced pathological stage compared with patients with two-gene methylation negativity (all P < 0.05). In clinical stage Ⅰ-Ⅱ lung adenocarcinoma samples, the Ct of SHOX2 and RASSF1A promoter region methylation tested by qRT-PCR was negatively correlated with their mRNA relative expression levels ( r=-0.43, P = 0.003; r = -0.48, P = 0.001); in TCGA database stage Ⅰ-Ⅱ lung adenocarcinoma samples, the level of SHOX2 promoter region methylation was negatively correlated with its mRNA relative expression level ( r = -0.23, P < 0.001), and the level of RASSF1A promoter region methylation was also negatively correlated with its mRNA relative expression level, but without statistical difference ( r = -0.05, P = 0.310). In two-gene promoter methylation-positive lung adenocarcinoma samples, the pathways related to folate metabolism and DNA stability were upregulated, and the pathways related to vasoconstriction and cell growth and differentiation were downregulated. Conclusions:The combined detection of SHOX2 and RASSF1A promoter region methylation can be used as an indicator for screening and diagnosis of early-stage lung adenocarcinoma. Abnormal promoter region methylation of the two genes may affect multiple tumor-related pathways and promote the occurrence and progression of early-stage lung adenocarcinoma.

ObjectiveTo reveal the targets and molecular mechanisms of the action of Qiangxin Decoction （强心汤） for the treatment of chronic heart failure based on the combination of network pharmacology and molecular docking. MethodsThe active ingredients of Qiangxin Decoction were retrieved from TCMSP database， and the targets of chronic heart failure were screened by searching GeneCards， OMIM， TTD， PharmGkb， and DrugBank databases， and the intersections were taken to obtain the intersecting targets of Qiangxin Decoction for the treatment of chronic heart failure. STRING platform was used to construct the protein-protein interaction network （PPI）， Cytoscape 3.8.0 software was used to calculate the network topology to screen the core targets， and R 4.2.3 was used to construct the “active ingredient-target” network by analyzing the GO enrichment analysis and KEGG pathway enrichment analysis. AutoDock 1.5.7 was used for molecular docking to predict the binding performance of active ingredients and core targets. ResultsSeventy-five intersecting targets were identified for the treatment of chronic heart failure with Qiangxin Decoction， among which the core targets were estrogen receptor 1 （ESR1， degree value=7）， nuclear receptor coactivator 1 （NCOA1， degree value=8）， glucocorticoid receptor （NR3C1， degree value=7）， and nuclear receptor coactivator 2 （NCOA2， degree value=7）. GO enrichment analysis showed that the top 3 items with the smallest P value in molecular function were G protein-coupled amine receptor activity， postsynaptic neurotransmitter receptor activity， and neurotransmitter receptor activity （P＜0.01）； the top 3 items with the smallest P value in biological process were adenylyl cyclase-activated adrenergic receptor signaling pathway， adrenergic receptor signaling pathway， and adenylyl cyclase-regulated G protein-coupled receptor signaling pathway （P＜0.01）； the top 3 items with the smallest P values in cellular composition were components of the postsynaptic membrane， synaptic membrane， and presynaptic membrane （P＜0.01）. KEGG enrichment analysis showed that the top 5 key signaling pathways were neuroactive ligand-receptor interactions， calcium signaling pathway， dopaminergic synapses， cocaine addiction， and cyclic guanosine monophosphate-protein kinase G （cGMP-PKG） signaling pathway. The molecular docking results showed that lignans and isoflavones had lower binding energies and more structural stability with the four core targets （ESR1， NCOA1， NR3C1， NCOA2）. ConclusionThe treatment of chronic heart failure by Qiangxin Decoction was associated with neuroactive ligand-receptor interactions， calcium signaling pathway， dopaminergic synapses， chemoattractant-receptor activation， cGMP-PKG signaling pathway， lipids and atherosclerosis， and cAMP signaling pathway， and lignans and isoflavones may be the core active compounds in its treatment of chronic heart failure.

Objective To investigate the impact of lncRNA DSCAM-AS1 on the malignant biological behaviors of thyroid cancer(TC)cells by regulating the miR-150-5p/BRAF axis.Methods The expression of DSCAM-AS1 in TC cells was detected by qRT-PCR,and the best intervention cell line was screened.;the relationship between DSCAM-AS1,BRAF and miR-150-5p targeting regulation was verified by FISH,pull down and double Luciferase reporter gene experiment;The proliferation,migration and invasion of SW579 cells were detected;Western blot was applied to detect the expression of BRAF,E-Cadherin,and vimentin proteins;the tumor formation experiment in mice was applied to verify the effect of DSCAM-AS1 on TC tumor growth.Results DSCAM-AS1 was highly expressed in TC tissue and cells(P<0.05);There is a targeted regulation relationship between DSCAM-AS1,BRAF and miR-150-5p;inhibition of DSCAM-AS1 expression or overexpression of miR-150-5p obviously inhibited the proliferation,migration,invasion,and EMT of SW579 cells(P<0.05);Inhibition of miR-150-5p expression or overexpression of BRAF reversed the inhibitory effect of inhibition of DSCAM-AS1 expression or overexpression of miR-150-5p on the malignant behavior of SW579 cells(P<0.05);in vivo experiments showed that inhibiting the expression of DSCAM-AS1 obviously inhibited the growth of transplanted tumors in mice(P<0.05).Conclusion DSCAM-AS1 is up-regulated in TC cells,inhibiting the expression of LncRNA DSCAM-AS1 can inhibit the malignant progression of TC by regulating the miR-150-5p/BRAF signaling axis.

Objective:To analyze the difference in immune microenvironment between primary tumor tissues and metastatic tumor tissues of metastatic colorectal cancer, and to screen specific immune-related differentially expressed genes (DEG) related to prognosis of metastatic colorectal cancer via bioinformatics methods.Methods:The GSE131418 microarray dataset of colorectal cancer and metastases was downloaded from gene expression omnibus (GEO) database, including 517 samples from the MCC cohort and 618 samples from the Consortium cohort in Moffitt Cancer Center. Immune-related gene sets were downloaded from immunology database and analysis portal IMMPORT, including 2 483 immune-related genes. A total of 695 cases of RNA sequencing data and 627 cases of clinical information of colorectal cancer tumors and adjacent tissues were downloaded from Cancer Genome Atlas (TCGA) data. The stroma cell score, immune cell score and stromal immune total score of metastatic tumor tissues and primary tumor tissues were calculated by using ESTIMATE algorithm, and 22 kinds of immune cell infiltration in primary tumor and metastatic tumor tissues of colorectal cancer were compared and analyzed by using CIBERSORT deconvolution algorithm. Immune-related DEG were screened to make Kyoto Encyclopedia of Genes and Gnomes (KEGG) signaling pathway enrichment analysis. The patients were divided into high and low expression groups according to the median expression levels of immune-related DEG. The Kaplan-Meier method and Cox regression risk model were used to analyze immune-related DEG, and the genes significantly related to prognosis in the results of the two methods were screened (all P < 0.01), and multivariate analysis was performed by using Cox regression method. The expression differences of each gene in tumor tissues, adjacent tissues, primary tumor tissues and metastatic tissues in GSE131418 data sets of TCGA database and GEO database were compared, and survival analysis was also performed. Results:The stroma cell score, immune cell score and stromal immune total score of colorectal cancer metastatic tissues were lower than those of primary tumor tissues (all P < 0.001). Compared with primary tumor tissues, the proportion of activated natural killer (NK) cells, monocytes, CD8 + T cells, T cells, activated dendritic cells in metastatic colorectal cancer tissues was increased, while the proportion of inactive mast cells, inactive dendritic cells, inactive NK cells, activated memory CD4 + T cells, M1 macrophages, and neutrophils was decreased. There were 289 immune-related DEG in metastatic tissues and primary tumor tissues of metastatic colorectal cancer, including 101 up-regulated genes and 188 down-regulated genes. KEGG signaling pathway enrichment analysis showed that in the immune microenvironment of metastatic tissues in metastatic colorectal cancer, vascular endothelial growth factor (VEGF) signaling pathway, programmed death ligand 1 (PD-L1) expression and programmed death 1 (PD-1) checkpoint pathway, T helper cell (Th) 1, Th2 and Th17 cell differentiation, NF-kappa B signaling pathway, interleukin 17 (IL-17) signaling pathway, chemokine signaling pathway, T cell receptor signaling pathway, MAPK signaling pathway, and NK cell-mediated cytotoxicity pathways enrichment were detected. Immune-related DEG related to prognosis including ANGPTL5, FPR1, HSPA8, NR2E3, PSMD2, PSMD8 and SBDS were screened out. Cox regression multivariate analysis showed that immune-related DEG ANGPTL5 ( HR = 2.69, 95% CI 1.22-5.92, P < 0.05), HSPA8 ( HR = 0.57, 95% CI 0.33-0.97, P < 0.05), and SBDS ( HR = 2.23, 95% CI 1.18-4.21, P < 0.05) were independent prognostic factors for metastatic colorectal cancer. The expression of ANGPTL5 in tumor tissues was lower than that in normal tissues, and the expression of ANGPTL5 in metastatic tissues was higher than that in primary tumor tissues. Patients with high expression of ANGPTL5 in tumor tissues had worse prognosis. The expression of HSPA8 in tumor tissues was higher than that in normal tissues, and the expression of HSPA8 in metastatic tissues was lower than that in primary tumor tissues. Patients with high expression of HSPA8 in tumor tissues had a better prognosis. The expression of SBDS in tumor tissues was lower than that in normal tissues, and the expression of SBDS in metastatic tissues was lower than that in primary tumor tissues. Patients with high expression of SBDS in tumor tissues had worse prognosis. Conclusions:Immune microenvironment of metastatic colorectal cancer is quite different from that of primary tumor. The degree of immune cell infiltration is reduced and the whole is immunosuppressed. The specific immune-related DEG related to prognosis of metastatic colorectal cancer may be new therapeutic targets of metastatic colorectal cancer.

Objective:To observe the morphological changes of the sylvian fissure on the transthalamic section of fetal brain at 20-32 weeks, and grade the fetal sylvian fissure development by means of a simple scoring system and explore its clinical feasibility.Methods:From September 2018 to June 2020, 487 normal single fetuses of 20-32 weeks were examined in Shenzhen Maternal and Child Health Hospital Affiliated to Southern Medical University. The sylvian fissure maturation was analyzed on the transthalamic section of fetal brain at 20-32 weeks and was graded from 0 to 5: un-visualized (grade 0), shallow arc (grade 1), obtuse-angled platform (grade 2), right-angled platform (grade 3), acute-angled platform (grade 4), and closed operculum (grade 5). The pregnancy outcomes and gestational age were recorded.Statistical analysis was performed by SPSS 20.0 software using box plot, Mann-Whitney U test, Weighted Kappa coefficient. Results:Left sylvian fissuer grades were obtained in 280 fetuses and right sylvian fissure grades were obtained in 247 fetuses. The fetal sylvian fissure maturation at 20-32 weeks was graded from 0 to 5, which increased with advancing gestation. Grade 0 only appeared in 3 fetuses at 20 weeks, and 99.4% fetuses at 20 weeks had grade ≥1. Grade 1 appeared in 20-22 weeks, grade 2 in 20-25 weeks, grade 3 in 22-26 weeks, grade 4 in 25-32 weeks, and grade 5 in 27-32 weeks. Box-plot and Mann-Whitney U test showed that gestational week distribution of sylvian fissure at all grades was symmetric on both sides ( P>0.05). The Weighted Kappa coefficients were 0.857(95% CI=0.750-0.957) and 0.939 (95% CI=0.859-1.000), respectively, with strong consistency regarding inter- and intra-observer agreements. Conclusions:Fetal sylvian fissure maturation at 20-32 weeks can be evaluated by means of a simple scoring system with symmetrical grading of both sides.

【Objective】 To establish and evaluate a nephelometric assay for the determination of immunoglobulin A (IgA) residues in human intravenous immunoglobulin(IVIG). 【Methods】 BN ProSpec^© automatic protein analyzer and its supporting immunoglobulin A determination kit (nephelometry) produced by German Siemens and the national standard of human IgA were used to establish the nephelometric assay to determine IgA residue in test products and verify the methodology. The test products include IVIG (pH4) prepared by low-temperature ethanol protein separation process and a novel IVIG prepared by chromatography. 【Results】 The average deviation of three calibration curves for IgA residues determination by the nephelometric assay were 1.08%, 0.95% and 1.54%,, and the three deviations of the quality control were 4.00%, -2.30% and -0.20%, respectively, which indicated good calibration and quality control. In the specificity test, the average recovery rates of IgA for reference substance 1 containing 100g/L maltose and reference substance 2 containing 20g/L glycine were 102.7% and 105.8%, respectively. The relative standard deviation (RSD) values of the repeatability tests of the two test products were 3.9% and 1.9%, and the RSD values of the intermediate precision test were 3.6% and 2.3%, respectively.The difference values at each time point in the durability test of test products′ storage time were all less than 10%, and the RSD values of the two test products in the durability test of kits of different batches were 2.8% and 2.2%, respectively. In the accuracy test, the average recovery rates of IVIG (pH4) added to the standard were 94.2%, 101.7% and 96.2%, respectively, and the average recovery rates of the novel IVIG added to the standard were 102.8%, 106.3% and 99.7%, respectively. The average recovery rate of the limit quantification test was 101.0%, and the RSD was 4.0%. 【Conclusion】 Nephelometric assay has the advantages of strong specificity, high precision and accuracy, good repeatability, simple and rapid operation, and automation, and can be used for the determination of IgA residue in IVIG (pH4) and novel IVIG products.

Objective:To explore the current status and the influencing factors of knowledge, attitude and behavior of radiodermatitis in patients with nasopharyngeal carcinoma undergoing radiotherapy, so as to provide a scientific basis for nursing staff to formulate effective health education programs.Methods:A self-designed questionnaire was used to investigate 220 radiotherapy patients with nasopharyngeal carcinoma in the radiotherapy department of 4 tertiary A hospitals in Hunan Province.Results:The scores of knowledge, attitude, and behavior of radiodermatitis patients with nasopharyngeal carcinoma were (61.58±19.93), (75.70 ±15.64), (65.87±14.21) points, respectively. The main factors influencing of behavior are knowledge, attitude, radiodermatitis grade, radiotherapy frequency, and family personal monthly income level ( t values were 1.978-8.081, P<0.05). Conclusion:At present, patients with nasopharyngeal carcinoma undergoing radiotherapy have a partial understanding of radiodermatitis and poor self-observation of radiodermatitis. Nursing staff should pay special attention to the patients with incomplete knowledge, negative attitudes, low family personal monthly income, low frequency of radiotherapy, and low grade of radiodermatitis.

Objectvie:To investigate the infiltration patterns of immune cells in colorectal cancer, and to explore the correlation of immune cells infiltration with clinical characteristics and overall survival (OS) of patients.Methods:The RNA sequencing data of 615 patients with colorectal cancer were downloaded from The Cancer Genome Atlas (TCGA) database. The data was updated on July 19, 2019, and included 571 colorectal cancer tissues and 44 paracancerous tissues. There were 552 cases with clinical data, such as survival time, survival status, age, gender, clinical stage, grade, tumor location and so on. Using CIBERSORT deconvolution algorithm, the relative amounts of 22 immune cell types were calculated based on standardized gene expression data. According to the results of CIBERSORT algorithm, the samples with high accuracy of deconvolution result were selected ( P < 0.05), and they were used for analysis and graphing. The correlations between the infiltration patterns of immune cells and the clinical characteristics and OS of patients were analyzed. Results:After the CIBERSORT method was used to filter and remove samples with P ≥ 0.05, a total of 282 tumor tissue samples and 16 paracancerous tissue samples were screened. In 293 cases with clinical information, there were 277 tumor tissue samples and 16 paracancerous tissue samples. In 293 samples, M0 macrophages, M1 macrophages, M2 macrophages, CD8 + T cells and unactivated CD4 memory T cells accounted for a higher proportion of total immune cells; in tumor tissue samples, the expressions of M0 macrophages, M1 macrophages, activated CD4 memory T cells, and unactivated natural killer (NK) cells were higher; in paracancerous tissues, the expressions of naive B cells, M2 macrophages, activated NK cells, unactivated dendritic cells, unactivated mast cells and plasma cells were higher; with the increase of clinical stage, the expressions of follicular helper T cells, activated CD4 memory T cells, activated NK cells, M1 macrophages decreased, and the expressions of plasma cells and regulatory T cells increased, and the differences were statistically significant (all P < 0.05). M1 macrophages, unactivated mast cells, activated CD4 memory T cells, CD8 + T cells, and follicular helper T cells were highly expressed in right colon cancer, while M0 macrophages and activated mast cells were highly expressed in left colon and rectal cancer, and the differences were statistically significant (all P < 0.05). The patients were divided into high infiltration group and low infiltration group based on the median expression level of infiltrated immune cells, and the survival analysis was performed. The result of survival analysis showed that patients with high initial B cell infiltration had good OS; however, patients with high infiltration of M2 macrophages, activated mast cells, and neutrophils had poor OS. Conclusions:There are different types of immune cell infiltration patterns in the colorectal cancer samples of different stages and locations, which are closely related to tumor progression and OS of patients. They are expected to be applied to the development of therapeutic targets and prognosis prediction.

Objective:To study the prevalence of Akabane virus (AKAV) in Yunnan province.Methods:A group of sentinel animals including 5 goats and 10 cattles which were sero-negative for bluetongue virus and AKAV were located in Mangshi, Yunnan province, to monitor arbovirus activity from April to October 2015. The heparin-anticoagulated blood of the animals was collected weekly for arbovirus isolation. Erythrocytes were lysed in distilied water and inoculated onto BHK-21 monolayer for virus isolation. After the cells showed cytopathic effect (CPE), AKAV in the cell cuture were identified by RT-PCR amplification with AKAV S gene segment specific primers and gene sequencing.Results:The result indicated that BHK-21 cell inoculated with the blood from one sentinel goat showed CPE 48 h post inoculation. One day old suckling mice intracerebrally inoculated with the supernatant of the cell culture, they started to become sick and died after 3 days. RT-PCR identification and S gene sequencing showed that the isolate was AKAV (numbered as 16415). The full length of S segment gene of 16415 is 856 nt, encoding 233 amino acids of protein N, and 91 amino acids of protein Ns. Phylogenetic analysis showed that, among the AKAV strains isolated from China and abroad, the newly isolated 16415 was in the same branch with the other viruses in different geographical regions, and their S segment nucleotide sequence have a high homology of 83.8%-97.7%. The further study show that 16415 and the AKAV isolated from the domestic bamboo rat in Guangxi in 2013 have a closest realtionship in evolution, the nucleotide sequence homology was 97.7%, and amino acid homology is 99.6%. Compared with the Japanese OBE-1 strain, the amino acids of protein N of 16415 and the AKAVs, isolated from banboo rat in Guangxi and Anopheles vagas in Mangshi of Yunnan province of China, have two common diverse amino acids loci located at the 115th and 206th sites respectively. Conclusions:It is concluded that AKAV was newly isolated from goat in Mangshi of Yunnan province, which may have important epidemiological significance.