Design Space Development for the Extraction Process of Danhong Injection Using a Monte Carlo Simulation Method

A design space approach was applied to optimize the extraction process of Danhong injection. Dry matter yield and the yields of five active ingredients were selected as process critical quality attributes (CQAs). Extraction number, extraction time, and the mass ratio of water and material (W/M ratio) were selected as critical process parameters (CPPs). Quadratic models between CPPs and CQAs were developed with determination coefficients higher than 0.94. Active ingredient yields and dry matter yield increased as the extraction number increased. Monte-Carlo simulation with models established using a stepwise regression method was applied to calculate the probability-based design space. Step length showed little effect on the calculation results. Higher simulation number led to results with lower dispersion. Data generated in a Monte Carlo simulation following a normal distribution led to a design space with a smaller size. An optimized calculation condition was obtained with 10000 simulation times, 0.01 calculation step length, a significance level value of 0.35 for adding or removing terms in a stepwise regression, and a normal distribution for data generation. The design space with a probability higher than 0.95 to attain the CQA criteria was calculated and verified successfully. Normal operating ranges of 8.2-10 g/g of W/M ratio, 1.25-1.63 h of extraction time, and two extractions were recommended. The optimized calculation conditions can conveniently be used in design space development for other pharmaceutical processes.     

Genomic analysis of Asian honeybee populations in China reveals evolutionary relationships and adaptation to abiotic stress

The geographic and biological diversity of China has resulted in the differential adaptation of the eastern honeybee, Apis cerana, to these varied habitats. A. cerana were collected from 14 locations in China. Their genomes were sequenced, and nucleotide polymorphisms were identified at more than 9 million sites. Both STRUCTURE and principal component analysis placed the bees into seven groups. Phylogenomic analysis groups the honeybees into many of the same clusters with high bootstrap values (91%–100%). Populations from Tibet and South Yunnan are sister taxa and together represent the earliest diverging lineage included in this study. We propose that the evolutionary origin of A. cerana in China was in the southern region of Yunnan Province and expanded from there into the southeastern regions and into the northeastern mountain regions. The Cold‐Temperate West Sichuan Plateau and Tropical Diannan populations were compared to identify genes under adaptive selection in these two habitats. Pathway enrichment analysis showing genes under selection, including the Hippo signaling pathway, GABAergic pathway, and trehalose‐phosphate synthase, indicates that most genes under selection pressure are involved in the process of signal transduction and energy metabolism. qRT‐PCR analysis reveals that one gene under selection, the AcVIAAT gene, involved in the GABAergic pathway, is responding to cold temperature stress. Through homologous recombination, we show that the AcVIAAT gene is able to replace the CNAG_01904 gene in the fungus Cryptococcus neoformans and that it makes the fungus less sensitive to conditions of oxidative stress and variations in temperature. Our results contribute to our understanding of the evolutionary origin of A. cerana in China and the molecular basis of environmental adaptation.     

不同时期根施钾肥对沿黄灌区‘骏枣’光合特性及其产量和品质的影响

在沿黄灌区选择8年生‘骏枣’植株,以不施钾肥作为对照,分别在果树开花期、幼果期、果实膨大期、转色期每株沟施300 g硫酸钾,测定各处理果实品质、产量、叶片光合以及荧光参数的变化,以明确沿黄灌区‘骏枣’的最佳施钾时期,为制定枣树合理施肥措施提供理论依据。结果表明:(1)与对照相比,施用钾肥可促进骏枣的光合作用、改善果实品质,且不同生育期效应不同,并以果实膨大期施用钾肥对提高骏枣果实品质、产量、叶片光合效果最为显著。(2)果实膨大期施用钾肥后,‘骏枣’的果实纵径、横径、果形指数、单果重、单株产量比对照分别显著提高了22.64%、39.33%、13.62%、19.52%和19.58%,果实维生素C(Vc)、可溶性糖和果糖的含量、糖酸比分别比对照显著提高了37.98%、22.82%、31.37%和29.95%,叶片叶绿素含量、净光合速率(P_n)、气孔导度(G_s)和蒸腾速率(T_r)比对照分别显著提高了28.91%、38.44%、36.75%和27.25%,叶片初始荧光(F_o)、最大荧光(F_m)、电子传递速率(ETR)比对照分别显著提高了28.18%、29.15%和37.75%,而胞间CO _2浓度(C_i)和裂果率在果实膨大期达到最低值,比对照分别显著降低了28.34%和35.50%。研究发现,在‘骏枣’果实膨大期株施300 g钾肥,可显著增加植株叶片叶绿素含量、光合作用效率和光能吸收、电子传递效率,提高树体光合能力,显著降低裂果率,有效改善骏枣果实外在和内在品质,显著提高单株产量。     

Survival analysis for long noncoding RNAs identifies TP53TG1 as an antioncogenic target for the breast cancer

Breast carcinoma is one of the most commonly diagnosed tumors and also one of the deadliest cancers in the female. Long noncoding RNAs (lncRNAs) are emerging as novel targets and biomarkers for breast cancer diagnosis and treatment. In this study, we aimed to study the lncRNAs associated with the outcomes in patients using the breast invasive carcinoma datasets from The Cancer Genome Atlas. The Cox proportional hazards regression model was fitted to each lncRNA. Hierarchy clustering was carried out using these survival-related lncRNAs and the log-rank test was carried out for the clustered groups. DNA methylation status was utilized to identify the lncRNAs regulated by epigenetics. Finally, the coexpressed messenger RNA with the potential lncRNAs were utilized to study the possible functions and mechanisms of lncRNAs. In total, 182 lncRNAs had an impact on the survival time of the patients with a cutoff <0.01. The patients were clustered into three groups using these survival-related genes, which performed significantly different prognosis. Two lncRNAs, which were significantly correlated with the outcomes of breast cancer and were regulated by methylation status, were obtained. These two lncRNAs were TP53TG1 and RP5-1061H20.4. We proposed that TP53TG1 was activated by the wild-type TP53 and performed an impact on the PI3Ks family by binding YBX2 in breast cancer.     

Methylation State of the EDA Gene Promoter in Chinese X-Linked Hypohidrotic Ectodermal Dysplasia Carriers

Introduction

Hypodontia, hypohidrosis, sparse hair and characteristic faces are the main characters of X-linked hypohidrotic ectodermal dysplasia (XLHED) which is caused by genetic ectodysplasin A (EDA) deficiency. Heterozygous female carriers tend to have mild to moderate XLHED phenotype, even though 30% of them present no obvious symptom.

Methods

A large Chinese XLHED family was reported and the entire coding region and exon–intron boundaries of EDA gene were sequenced. To elucidate the mechanism for carriers’ tempered phenotype, we analyzed the methylation level on four sites of the promoter of EDA by the pyrosequencing system.

Results

A known frameshift mutation (c.573–574 insT) was found in this pedigree. Combined with the pedigrees we reported before, 120 samples comprised of 23 carrier females from 11 families and 97 healthy females were analyzed for the methylation state of EDA promoter. Within 95% confidence interval (CI), 18 (78.26%) carriers were hypermethylated at these 4 sites.

Conclusion

Chinese XLHED carriers often have a hypermethylated EDA promoter.     

Low Divergence of Clonorchis sinensis in China Based on Multilocus Analysis

Clonorchis sinensis, an ancient parasite that infects a number of piscivorous mammals, attracts significant public health interest due to zoonotic exposure risks in Asia. The available studies are insufficient to reflect the prevalence, geographic distribution, and intraspecific genetic diversity of C. sinensis in endemic areas. Here, a multilocus analysis based on eight genes (ITS1, act, tub, ef-1a, cox1, cox3, nad4 and nad5 [4.986 kb]) was employed to explore the intra-species genetic construction of C. sinensis in China. Two hundred and fifty-six C. sinensis isolates were obtained from environmental reservoirs from 17 provinces of China. A total of 254 recognized Multilocus Types (MSTs) showed high diversity among these isolates using multilocus analysis. The comparison analysis of nuclear and mitochondrial phylogeny supports separate clusters in a nuclear dendrogram. Genetic differentiation analysis of three clusters (A, B, and C) showed low divergence within populations. Most isolates from clusters B and C are geographically limited to central China, while cluster A is extraordinarily genetically diverse. Further genetic analyses between different geographic distributions, water bodies and hosts support the low population divergence. The latter haplotype analyses were consistent with the phylogenetic and genetic differentiation results. A recombination network based on concatenated sequences showed a concentrated linkage recombination population in cox1, cox3, nad4 and nad5, with spatial structuring in ITS1. Coupled with the history record and archaeological evidence of C. sinensis infection in mummified desiccated feces, these data point to an ancient origin of C. sinensis in China. In conclusion, we present a likely phylogenetic structure of the C. sinensis population in mainland China, highlighting its possible tendency for biogeographic expansion. Meanwhile, ITS1 was found to be an effective marker for tracking C. sinensis infection worldwide. Thus, the present study improves our understanding of the global epidemiology and evolution of C. sinensis.     

Combined Adenovirus-Mediated Artificial microRNAs Targeting mfgl2, mFas,and mTNFR1 Protect against Fulminant Hepatic Failure in Mice

Hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF) has a poor prognosis with high in-hospital mortality. Hepatic and circulating inflammatory cytokines, such as fibrinogen like protein 2 (fgl2), FasL/Fas, and TNFα/TNFR1, play a significant role in the pathophysiology of ACLF. This study aimed to investigate the therapeutic effect of recombinant adenoviral vectors carrying constructed DNA code for non-native microRNA (miRNA) targeting mouse fgl2 (mfgl2) or both mFas and mTNFR1 on murine hepatitis virus (MHV)-3-induced fulminant hepatitis in BALB/cJ mice. Artificial miRNA eukaryotic expression plasmids against mfgl2, mFas, and mTNFR1 were constructed, and their inhibitory effects on the target genes were confirmed in vitro. pcDNA6.2-mFas-mTNFR1- miRNA，which expresses miRNA against both mFas and mTNFR1 simultaneously，was constructed. To construct a miRNA adenovirus expression vector against mfgl2, pcDNA6.2-mfgl2-miRNA was cloned using Gateway technology. Ad-mFas-mTNFR1- miRNA was also constructed by the same procedure. Adenovirus vectors were delivered by tail-vein injection into MHV-3-infected BALB/cJ mice to evaluate the therapeutic effect. 8 of 18 (44.4%) mice recovered from fulminant viral hepatitis in the combined interference group treated with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA. But only 4 of 18 (22.2%) mice receiving Ad-mfgl2-miRNA and 3 of 18 (16.7%) mice receiving Ad-mFas-mTNFR1- miRNA survived. These adenovirus vectors significantly ameliorated inflammatory infiltration, fibrin deposition, hepatocyte necrosis and apoptosis, and prolonged survival time. Our data illustrated that combined interference using adenovirus-mediated artificial miRNAs targeting mfgl2, mFas, and mTNFR1 might have significant therapeutic potential for the treatment of fulminant hepatitis.