Investigation of the frequency of kdr mutation of voltage-gated sodium channel in Anopheles sinensis in Yunnan Province / 中国热带医学

@#Abstract: Objective　To understand the kdr (knockdown resistance, kdr) gene mutation of the voltage-gated sodium channel (VGSC) of Anopheles sinensis in Yunnan Province. Methods　From 2018 to 2019, mosquitoes were collected in Luoping County, Suijiang County, Tengchong City, Yingjiang County, Yuanjiang County and Mengla County in Yunnan Province. The collected mosquitoes were morphologically identified as Anopheles sinensis and genomic DNA was extracted by kits. The DNA templates were sequenced after PCR amplification and the sequencing results were identify as Anopheles sinensis by homology alignment in NCBI. After the ⅡS5 and ⅡS6 fragments of the sodium channels in Anopheles sinensis were amplified and sequenced, the sequencing results were multiple aligned by DNAMAN software, and the mutations were analyzed one by one with BioEdit software to determine the kdr allele types and genotypes, and the frequencies were calculated. Results This survey amplified 287 sequences, and the sequence maps showed that 1014 loci had three alleles, including wild type TTG/L (89.20%), mutant type TTT/F (9.76%) and TCG/S (1.04%). Five genotypes: homozygous wildtype L/L (85.02%), homozygous mutant F/F (6.27%) and S/S (0.35%), heterozygous mutant L/F (6.97%) and L/S (1.39%). The wild type allele TTG/L was the main allele in six sampling sites except Suijiang County. The frequency of wild type allele in Tengchong City was the highest (100.00%). That is, no mutation was detected, while the rest of counties occurred different degrees of mutation at 1014 loci. The frequency of mutant allele in Suijiang County was the highest, reaching 55.68%. Luoping County, Mengla County and Suijiang County had two mutant types. Yingjiang County and Yuanjiang County had one heterozygous mutant L/F. Conclusion　Wild type L1014 (TTG/L) is still dominant in most areas of Yunnan Province. The kdr mutation type is mainly L1014F, followed by L1014S, and the mutation frequency is lower than that in central provinces of China.

Color characterization and its correlation with quality index during ripening of Rubus chingii / 中国中药杂志

The color of Rubus chingii was characterized by digital method, and the content of water extract, alcohol extract, total flavonoids, total polysaccharides, total polyphenols, ellagic acid, linden glycoside, kaophenol-3-O-rutin were determined. Correlation regression was used to analyze the correlation between color and composition. The results showed that L~* was positively correlated with total polyphenols, kaophenol-3-O-rutin and tilide, and moderately positively correlated with total flavones, ellagic acid and aqueous extracts. The a~* value was negatively correlated with total polyphenols, kaophenol-3-O-rutin, and linden glycosides, while was moderately correlated with total flavones, aqueous extracts, and ellagic acid. The b~* value was negatively correlated with the water extract, and moderately correlated with the content of total polyphenols, total polysaccharides, alcohol extract and kaophenol-3-O-rutin, which showed that R. chingii mature color had a significant correlation with material composition in the process of dynamic change. According to the law of dynamic change in the color and quality indexes, it is determined that the appropriate harvest time is in late April to May 1, while the fruit is not turn yellow. The agronomic traits related to fruit was(12.49±0.56) mm in diameter,(14.25±1.19)mm in height,(1.20±0.14) g in weight, the chroma L~* value was 52.87±3.14,a~* value was 2.01±1.58, b~* values was 28.31±3.88. The results lay a foundation for establishing an objective quantitative evaluation model of R. chingii color from experience.

Transcriptome analysis of seed embryo in dormancy and dormancy release state of Thesium chinense / 中国中药杂志

We used exogenous GA_3 to break the seed dormancy of Thesium chinense. We used high-throughput sequencing technology was used to sequence the transcriptome of dormant seed embryos and dormancy breaking seed embryos of Th. chinense, and the data was analyzed bioinformatically and systematically. The results showed that exogenous GA_3 could effectively break the seed dormancy of Th. chinense; 73 794 up-regulated genes and 42 776 down regulated genes were obtained by transcriptome sequencing; 116 570 diffe-rential genes were annotated by GO function to GO items such as metabolism process, cell process, cell, cell component, binding and catalytic activity. A total of 133 metabolic pathways were found by Pathway analysis of 26 508 differentially expressed genes. In the process of dormancy release, DEGs were mainly enriched in translation, carbohydrate metabolism, folding, classification, degradation and amino acid metabolism. Based on the annotation results in KEGG database, 20 metabolic pathways related to dormancy release were found. Dormancy release of Th. chinense seeds is a complex biological process, including cell morphology construction, secondary metabolite synthesis, sugar metabolism and plant signal transduction, among which plant hormone signal transduction is one of the key factors to regulate dormancy release. The results of qRT-PCR showed that the sequencing results were consistent with the actual results.

Identification and differential expression of miRNA related to seed dormancy of Paris polyphylla var. chinensis / 中国中药杂志

The purpose of this study was to explore the expression pattern of miRNA in the process of embryo dormancy and provide a reference for the mechanism of regulating seed dormancy and germination by miRNA. We used high-throughput sequencing technology, bioinformatics analysis and real-time fluorescent quantitative PCR(qPCR) technology to sequence, screen and identify miRNAs of dormant and dormant embryos. The results showed that there were 23 811 977, 24 276 695, 20 611 876 and 20 601 811 unique sequences in the four sample libraries during the period of dormancy and dormancy release. MiRNAs are mainly distributed between 21 and 24 nt, among which the length of 24 nt occurred most frequently. A total of 31 known miRNAs were identified, belonging to 13 different families. 93 new miRNAs were predicted by bioinformatics software. Ten miRNAs(mir156 a-5 p, mir160 a-5 p, mir160 h-1, mir169 a-5 p, mir157 d, mir159 a-1, mir395-3, mir156 f-5 p, mir156-2 and mir171 a-3 p) were screened out. In this study, 10 miRNAs related to seed dormancy release were identified. The target genes mainly involved carbohydrate metabolism, plant hormone signal transduction, cell division and growth. The results of qRT-PCR showed that the sequencing results were consistent with the actual results.

Comparative analysis of active components and transcriptome between autotetraploid and diploid of Dendrobium huoshanense / 中国中药杂志

In this study, the roots, stems and leaves of diploid and autotetraploid Dendrobium huoshanense were used as materials to compare their contents of polysaccharides and alkaloids, and the transcriptome sequencing analysis was carried out. The results showed that the contents of polysaccharides and alkaloids in the roots, stems and leaves of tetraploid were 7.6%, 34.5%, 17.2%, 0.01%, 0.024% and 0.035% higher than those of diploid D. huoshanense, respectively. The contents of active components in different tissues were significantly different. There were 3 687 differentially expressed genes in diploid and tetraploid D. huoshanense, of which 2 346 genes were up-regulated and 1 341 down regulated. Go functional analysis showed that these genes were mainly involved in growth and development, stress resistance and other related functions. KEGG pathway analysis showed that most of the differential genes were concentrated in the processes of carbon metabolism, signal transduction, carbohydrate metabolism, amino acid metabolism and energy metabolism. The differential expression of key genes involved in the metabolism of polysaccharides, terpenes and polyketones, amino acid metabolism, hormone synthesis and signal transduction in diploid and tetraploid plants may be the main reason for the high energy content, the increase of active components and the growth potential of tetraploid plants.

In vitro autotetraploid induction and analysis on DNA methylation diversity of Platycodon grandiflorum / 中国中药杂志

In order to investigate the epigenetic variations between diploid and autotetraploid of Platycodon grandiflorus. The diploid buds of P. grandiflorus were soaked in the mixture of different concentration colchicines and 0.002 g•mL ⁻¹ dimethyl sulphoxide (DMSO)．The identification of autotetraploid plants were based on morphological characteristics, chromosome number and flow cytometry. And then the level and pattern of DNA methylation explored by using the technology of methylation sensitive amplified polymorphism (MSAP)．The result demonstrated that the buds soaked in 0.2% colchicines and 0.002 g•mL ⁻¹ DMSO solution for 12 h was ideal conditions to induce autotetraploid of P. grandiflorus, with induction rate of 32.0%.The diploid and tetraploid plants existed distinctly differences in morphological indexes.Totally,1 586 bands were amplified by 20 pairs of selective primers, of which 764 and 822 bands were detected in diploid and autotetraploid respectively. The total methylation ratio,full methylation ratio and hemimethylated ratio were 91.25%,61.25% and 30.65% in diploid of P. grandiflorus,respectively.However,the total methylation ratio,full methylation ratio and hemimethylated ratio of autotetraploid of P. grandiflorus were 86.13%,54.38% and 31.75%, respectively. Compared with diploid, the genomic DNA total methylate ratio and full methylation ratio of autotetration plants decreased by 6.02% and 7.14%.But the hemimethylated ratio of autotetraploid was higher than that of diploid, which more than 1.6%. All this results indicated that DNA methylation patterns have adjusted during the polyploidy process．.

Morphology and AFLP analysis of tetraploid plantlets of Atractylodes macrocephala / 中国中药杂志

In order to investigate the genetic basis of morphological variation of tetraploid plantlets of Atractylodes macrocephala, diploid plantlets were taken as experimental material, sterile filtration colchicine was used to soak 0.5-1.0 cm long buds. The difference between morphology and stomatal of diploid and tetraploid of A. macrocephala was compared, and genome polymorphism was explored by AFLP. The results showed that the buds dipped in 0.1% colchicine solution for 36 h was optimal conditions to induce tetraploid of A. macrocephala with induction rate of 32.0%. Morphological indexes such as leaf area index, leaf length and width, the density of stomas and the number of chloroplast of tetraploid were distinctly different from diploid. Four hundred and fifty-one bands ranging with 80-500 bp were amplified with 24 pairs of primers, the rate of polymorphism was 32.59%. These amplification sites of diploid were different from tetraploid of A. macrocephala, and the differences in morphology of them were reflected in the DNA polymorphism.

FTIR fingerprint spectrograms of traditional Chinese medicine Marsdenia tenacissima / 中国中药杂志

In this paper, Fourier transform infrared spectroscopy fingerprint analysis of Marsdenia tenacissima samples was used to develop a reliable method of tracing the geographical origins. Forty-eight samples from four provinces of China were analyzed by FTIR. We analyzed and characterized the fingerprints in both the full spectrum peaks and characteristic peaks, then the principal component analysis and the cluster analysis were carried out. The results of fingerprint analysis, correlation analysis, principal component analysis and cluster analysis can identify the geographic origins correctly, which verified and supplemented each other; the identification results and the actual location showed a high degree of consistency, namely the lower the space distance, the greater the similarity of different samples. These results revealed the obvious superiority and practical value in comparison to the more tedious and time-consuming wet chemistry method normally used. Using appropriate metrology methods can trace the geographical source correctly. The M. tenacissima materials from the region of Maguan should be considered as genuine medicinal materials taking into account the good quality.

Work-related quality of life scale among Singaporean nurses.

Background: The Work-Related Quality of Life Scale (WRQLS) was developed for health-care workers in England but might be useful, if valid, in other parts of the world or for other professions. Objective: We test the construct validity and reliability of the WRQLS as applied to nurses in Singapore. Methods: A descriptive study was undertaken in 2009 on a sample of 811 nurses at the National University Hospital (NUH) of Singapore who had at least one year’s work experience. The WRQLS was used in order to test its construct validity. Exploratory factor analysis was performed to reduce the factors used to determine WRQLS variance. The Pearson’s correlation was used to evaluate the relationship between the WRQLS and the 12-item short form health survey (SF-12) in order to substantiate conclusions regarding construct validity while Cronbach’s alpha was calculated to test WRQLS reliability. Results: The median age of the respondents was 31 (IQR=12) and the majority were female (96.9%). The median duration of work experience was seven years (IQR=10). Following the exploratory factor analysis, a five-factor model with 22 items was selected, including; 1) job and career satisfaction, 2) general well-being, 3) home-work interface, 4) stress at work, and 5) working conditions. The correlation coefficient showed a moderate relationship between the WRQLS and mental component scale (MCS-12) (r=0.495); and a weak relationship between the WRQLS and physical component scale (PCS-12) (r=0.149). The Cronbach’s alpha revealed good reliability (r=0.92). Conclusion: The WRQLS test among nurses in Singapore showed good construct validity and reliability. It could be useful in a working environment in Asia but further testing might be warranted.

Cannabinoid receptor-2 selective antagonist negatively regulates receptor activator of nuclear factor kappa B ligand mediated osteoclastogenesis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The cannabinoid receptor-2 (CB2) is important for bone remodeling. In this study, we investigated the effects of CB2 selective antagonist (AM630) on receptor activator of nuclear factor kappa B (RANK) ligand (RANKL) induced osteoclast differentiation and the underlying signaling pathway using a monocyte-macrophage cell line-RAW264.7.</p><p><b>METHODS</b>RAW264.7 was cultured with RANKL for 6 days and then treated with AM630 for 24 hours. Mature osteoclasts were measured by tartrate-resistant acid phosphatase (TRAP) staining using a commercial kit. Total ribonucleic acid (RNA) was isolated and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was done to examine the expression of RANK, cathepsin K (CPK) and nuclear factor kappa B (NF-κB). The extracellular signal-regulated kinase (ERK), phosphorylation of ERK (P-ERK) and NF-κB production were tested by Western blotting. The effect of AM630 on RAW264.7 viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay.</p><p><b>RESULTS</b>AM630 did not affect the viability of RAW264.7. However, this CB2 selective antagonist markedly inhibited osteoclast formation and the inhibition rate was dose-dependent. The dose of ≥ 100 nmol/L could reduce TRAP positive cells to the levels that were significantly lower than the control. AM630 suppressed the expression of genes associated with osteoclast differentiation and activation, such as RANK and CPK. An analysis of a signaling pathway showed that AM630 inhibited the RANKL-induced activation of ERK, but not NF-κB.</p><p><b>CONCLUSION</b>AM630 could inhibit the osteoclastogenesis from RAW264.7 induced with RANKL.</p>

Application and study of HPLC fingerprint of Lonicera japonica for identification of geo-authenticity / 中国中药杂志

<p><b>OBJECTIVE</b>To establish a chromatographic fingerprint of Lonicera japonica and apply it to identify geo-authentic of L. japonica.</p><p><b>METHOD</b>The HPLC was applied in chromatographic separation and data were analysed by "computed aided similarity evaluation" software.</p><p><b>RESULT</b>There exited distinct difference of chemical components group between and non-authentic samples.</p><p><b>CONCLUSION</b>The established HPLC fingerprint can be used for the identification of geo-authentic of L. japonica.</p>

Determination of 18 organophosphate pesticide residues in Flos Lonicerae / 中国中药杂志

<p><b>OBJECTIVE</b>To establish an analysical method for the quantitative determination of 18 kinds of organophosphate pesticide residues in Flos Lonicerae.</p><p><b>METHOD</b>The pesticides in samples were extracted with acetone by Ultrasonic wave Assistant Extraction (UAE), purified by Solid-Phase Extraction (SPE). Using PFPD detecter gas chromatography with HP-1701 column.</p><p><b>RESULT AND CONCLUSION</b>All 18 pesticides were separated well in 30 minutes. The detection limits was 4.0-18.0 microg x kg(-1). The range of recovery and RSD were 83.64%-88.65% and 2.8%-6.0%, respectively. And it met the requirements of pesticide multi-residues analysis.</p>

Long-term outcomes of the revision open lumbar discectomy by fenestration: a follow-up study of more than 10 years / 中华外科杂志

<p><b>OBJECTIVE</b>To evaluate the long-term clinical results and the factors that influences the outcomes of the revision open lumbar discectomy by fenestration.</p><p><b>METHODS</b>Fifty-one patients, who underwent the second open discectomy by fenestration from January 1 1988 to December 31 1994, were followed for an average of 146.8 months (range, 120 to 203 months). The long-term follow-up results were evaluated by using the MacNab classification and the Japanese Orthopaedic Association (JOA) scoring system through direct examinations and questionnaires. Radiography was also used in patients who agreed to visit the hospital, and findings were compared with those on preoperative radiographs.</p><p><b>RESULTS</b>At the final follow-up, with the MacNab classification an excellent and good outcome was achieved in 70.6% of the cases, 78.4% were satisfied with their results. The failure rate was 15.7% (8 patients). Excluding those 8 failed cases who needed another reoperation, the average improvement calculated by JOA scores was (64.6 +/- 18.2)%. The disc height of the operation site significantly decreased after surgery, nevertheless, this did not affect the long-term clinical outcome. Factors that were associated with a fair and bad outcome included smoking, isolated trauma or injury, fibrosis and the length of the remaining or recurrent primary postoperative symptoms history. Psychosociological signs were probably known as negative predictors of lumbar disc surgery outcome.</p><p><b>CONCLUSION</b>The long-term outcome of the revision open lumbar discectomy by fenestration in this series was favorable. Because the revision operation is typically associated with a higher complexity, selection of suitable surgical candidates and determination of valid indications for operative treatment are very important. JOA scores have proved to be easy to perform for patients and clinicians and standardize subjective data.</p>

Kinetogenic effects of Pericarpium Arecae on SP and VIP changes in gastrointestinal tract / 第三军医大学学报

Objective　To study the mechanisms of Pericarpium Arecae. Methods　A total of Wistar rats were randomly divided into Pericarpium Arecae group and control group, Pericarpium Arecae decoction or distilled water were given respectively. Changes of gastrointestinal motility rats were assayed by Dextran blue-2000 after 1 and 6 hours. The distributions of SP and VIP in antrum and jejunum were investigated by immunohistochemistry assay. Results　The gastrointestinal motility of rats was markedly enhanced (P<0.01 or P<0.05). The expressions of SP increased significantly (P<0.01 or P<0.05) and the expressions of VIP decreased significantly (P<0.01 or P<0.05) in antrum and jejunum of rats at 1 and 6 h after Pericarpium Arecae decoction was given. The changes were more obvious at 1 h than at 6 h. Conclusion　The kinetogenic effect of Pericarpium Arecae is closely correlated to the increase of SP expression and the decrease of VIP expression in gastrointestinal tract.

Kinetogenic effects of Pericarpium Arecae on SP and VIP changes in gastrointestinal tract / 第三军医大学学报

Objective　To study the mechanisms of Pericarpium Arecae. Methods　A total of Wistar rats were randomly divided into Pericarpium Arecae group and control group, Pericarpium Arecae decoction or distilled water were given respectively. Changes of gastrointestinal motility rats were assayed by Dextran blue-2000 after 1 and 6 hours. The distributions of SP and VIP in antrum and jejunum were investigated by immunohistochemistry assay. Results　The gastrointestinal motility of rats was markedly enhanced (P<0.01 or P<0.05). The expressions of SP increased significantly (P<0.01 or P<0.05) and the expressions of VIP decreased significantly (P<0.01 or P<0.05) in antrum and jejunum of rats at 1 and 6 h after Pericarpium Arecae decoction was given. The changes were more obvious at 1 h than at 6 h. Conclusion　The kinetogenic effect of Pericarpium Arecae is closely correlated to the increase of SP expression and the decrease of VIP expression in gastrointestinal tract.