Deep Sequencing of H7N9 Influenza A Viruses from 16 Infected Patients from 2013 to 2015 in Shanghai Reveals Genetic Diversity and Antigenic Drift.

Influenza A virus (IAV) infections are a major public health concern, including annual epidemics, epizootic outbreaks, and pandemics. A significant IAV epizootic outbreak was the H7N9 avian influenza A outbreak in China, which was first detected in 2013 and which has spread over 5 waves from 2013 to 2017, causing human infections in many different Chinese provinces. Here, RNA from primary clinical throat swab samples from 20 H7N9-infected local patients with different clinical outcomes, who were admitted and treated at one hospital in Shanghai, China, from April 2013 to April 2015, was analyzed. Whole-transcriptome amplification, with positive enrichment of IAV RNA, was performed, all 20 samples were subjected to deep sequencing, and data from 16 samples were analyzed in detail. Many single-nucleotide polymorphisms, including ones not previously reported, and many nonsynonymous changes that could affect hemagglutinin head and stalk antibody binding epitopes were observed. Minor populations representing viral quasispecies, including nonsynonymous hemagglutinin changes shared by antigenically variant H7N9 clades identified in the most recent wave of H7N9 infections in 2016 to 2017, were also identified.IMPORTANCE H7N9 subtype avian influenza viruses caused infections in over 1,400 humans from 2013 to 2017 and resulted in almost 600 deaths. It is important to understand how avian influenza viruses infect and cause disease in humans and to assess their potential for efficient person-to-person transmission. In this study, we used deep sequencing of primary clinical material to assess the evolution and potential for human adaptation of H7N9 influenza viruses.

Arthroscopic cyst removal and wire-guided suture for the treatment of lateral meniscal cyst of knee joint / 中国骨伤

<p><b>OBJECTIVE</b>To explore clinical effect of arthroscopic cyst removal and wire-guided suture for the treatment of lateral meniscal cyst of knee joint.</p><p><b>METHODS</b>From July 2014 to December 2017, 33 patients with lateral meniscal cyst of knee joint were treated by arthroscopic cysts removal and wire-guided suture, including 13 males and 20 females, aged from 20 to 55 years old with an average age of(36.23 ±2.30) years old, the courses of disease ranged from 3 to 14 months with an average of(4.60±0.83) months; Preoperative MRI examination was clear diagnosed. There were 14 cysts on anterior horn, 18 cysts on meniscal body and 1 cyst on posterior horn;all cysts were solitary, and 3 of them were multilocular. Lysholm score and GLASOW score of knee joint function and clinical efficacy were observed before and after operation at 6 months.</p><p><b>RESULTS</b>All patients were followed up form 6 to 24 months with an average of (7.5±1.2) months. Preoperative symptoms disappeared or significantly alleviated, and all incisions were healed by intention without complication and neurovascular injury. MRI showed meniscal tear areas and cystic defective areas healed, cyst was not recurrenced, healing time ranged form 8 to 12 weeks with an average of (9.6±1.6) weeks, and patients recovered their daily life and exercise. There was significant difference in Lysholm score before operation (61.12±4.35) and after operation at 6 momths(91.32±3.36)(=46.11, <0.01);according to GLASOW assessment, 31 patients with excellent recovery, and 2 good.</p><p><b>CONCLUSIONS</b>Arthroscopic cyst removal and wire-guided suture for the treatment of lateral meniscal cyst of knee joint could reserve meniscus, repair injury of meniscus, recover knee joint function after operation, and is worth popularizing.</p>

Incidence and risk factors of chronic kidney disease in community-based patients with diabetes / 北京大学学报(医学版)

OBJECTIVE@#To estimate the incidence rate and effects of risk factors on chronic kidney disease (CKD) in Chinese patients with diabetes, based on Electronic Health Records (EHRs) from the Chinese Electronic health Records Research in Yinzhou (CHERRY) Study.@*METHODS@#Using the CHERRY cohort study with the individual-level information on chronic disease management; and health administrative, clinical and laboratory databases, patients with diabetes without kidney disease at baseline were enrolled and followed up from January 2009 through December 2016. CKD was defined as the estimated glomerular filtration rate(eGFR) <60 mL/(min×1.73 m2) or urine albumin/creatinine ratio (ACR)≥3 mg/mmol. Standardized incidence rates of CKD in diabetic population were calculated according to the 2010 China census data. Cox proportional hazards models were used to explore the association of risk factors on CKD in patients with diabetes.@*RESULTS@#Over a median 3.2 years of follow-up, 13 829 patients with diabetes were included in this analysis and 1 087 developed CKD. The crude and standardized incidence rate was 23.7(95%CI: 22.3-25.2) and 14.8(95%CI:12.1-17.6) per 1 000 person-years respectively. The incidence rate for developing CKD in patients with diabetes aged over 60 years was higher than those aged 60 and below (26.6 vs. 11.5 per 1 000 person-years, P<0.05). Cox proportional hazards models showed that age over 60 years(HR=1.88, 95%CI: 1.51-2.35), hypertension (HR=1.81, 95%CI: 1.56-2.10), total cholesterol (HR=1.07, 95%CI: 1.00-1.14) and duration of diabetes (HR per year increment=1.02, 95%CI: 1.00-1.03) and the level of high density lipoprotein cholesterol (HDLC, HR=0.49, 95%CI: 0.40-0.61) were significantly associated with CKD. No statistical significance was found for sex, smoking status, alcohol use and average level of fasting glucose (All P>0.05). Subgroup analysis indicated that even when the lipid levels were well-controlled, comorbidity of hypertension was still associated with CKD in the patients with diabetes.@*CONCLUSION@#Incidence rate of chronic kidney disease in this Chinese population with diabetes was high. Age and comorbidity of hypertension were the most important risk factors for CKD, suggesting the priority for CKD screening in patients with diabetes in China. Control of blood pressure and lipid were especially crucial to prevent CKD in patients with diabetes.

Study on the influence of curcumin on chemosensitivity of nephroblastoma cells

OBJECTIVE@#To study the influence of curcumin on chemosensitivity of nephroblastoma cells.@*METHODS@#Human nephroblastoma cells line SK-NEP-1 was transplanted to the nude mice subcutaneously to establish the implantation tumor model of human nephroblastoma cells. A total of 30 tumor-bearing mice were divided into three groups of ten randomly. The routine chemotherapy group was given vincristine (0.05 mg/mL·0.2 mL/d) and actinomycin D (15 ng/mL·0.2 mL/d) combined chemotherapy regime. The curcumin chemotherapy group was given the same combined chemotherapy regimens and curcumin (30 mg/kg/d) by intraperitoneal injection. The control group was given normal saline (NS) of the same volume by intraperitoneal injection. Continuous administration would be kept for 4 weeks and 3 days a week. The volumetric changes of every group were recorded. The serum of every group in different time was collected and the VEGF content was detected by ELISA. All mice were cercrificed and the tumor tissues were stripped and weighed after 4 weeks' treatment. The tumor inhibition rate was calculated. The cell proliferation activity and apoptosis rate were detected by MTT and flow cytometry method. All data were statistically analyzed by SPSS 19.0.@*RESULTS@#The tumor volume, serum VEGF content, tumor inhibition rate, cell proliferation activity and apoptosis rate of routine chemotherapy group and curcumin chemotherapy group had significant differences comparing with the control group (P < 0.05) after 4-week's treatment. The cancer growth of curcumin chemotherapy group was obviously decreased and even tended to shrink comparing with routine chemotherapy group (χ(2) = 15.732, P = 0.007). The cell proliferation activity was significantly reduced and the apoptosis rate was significantly higher, (χ(2) = 9.427, P = 0.012) which showing the effect of chemotherapy was enhanced.@*CONCLUSIONS@#The chemosensitivity of nephroblastoma cells could be improved by curcumin, then the effect of preoperative adjuvant chemotherapy scheme would be enhanced, the growth of nephroblastoma cells would be inhibited and the surgical risk of nephroblastoma would be reduced.

Study on the influence of curcumin on chemosensitivity of nephroblastoma cells

Objective To study the influence of curcumin on chemosensitivity of nephroblastoma cells. Methods Human nephroblastoma cells line SK-NEP-1 was transplanted to the nude mice subcutaneously to establish the implantation tumor model of human nephroblastoma cells. A total of 30 tumor-bearing mice were divided into three groups of ten randomly. The routine chemotherapy group was given vincristine (0.05 mg/mL·0.2 mL/d) and actinomycin D (15 ng/mL·0.2 mL/d) combined chemotherapy regime. The curcumin chemotherapy group was given the same combined chemotherapy regimens and curcumin (30 mg/kg/d) by intraperitoneal injection. The control group was given normal saline (NS) of the same volume by intraperitoneal injection. Continuous administration would be kept for 4 weeks and 3 days a week. The volumetric changes of every group were recorded. The serum of every group in different time was collected and the VEGF content was detected by ELISA. All mice were cercrificed and the tumor tissues were stripped and weighed after 4 week's treatment. The tumor inhibition rate was calculated. The cell proliferation activity and apoptosis rate were detected by MTT and flow cytometry method. All data were statistically analyzed by SPSS 19.0. Results The tumor volume, serum VEGF content, tumor inhibition rate, cell proliferation activity and apoptosis rate of routine chemotherapy group and curcumin chemotherapy group had significant differences comparing with the control group (P < 0.05) after 4-week's treatment. The cancer growth of curcumin chemotherapy group was obviously decreased and even tended to shrink comparing with routine chemotherapy group (χ

Cytoplasmic male sterility-associated chimeric open reading frames identified by mitochondrial genome sequencing of four Cajanus genotypes.

The hybrid pigeonpea (Cajanus cajan) breeding technology based on cytoplasmic male sterility (CMS) is currently unique among legumes and displays major potential for yield increase. CMS is defined as a condition in which a plant is unable to produce functional pollen grains. The novel chimeric open reading frames (ORFs) produced as a results of mitochondrial genome rearrangements are considered to be the main cause of CMS. To identify these CMS-related ORFs in pigeonpea, we sequenced the mitochondrial genomes of three C. cajan lines (the male-sterile line ICPA 2039, the maintainer line ICPB 2039, and the hybrid line ICPH 2433) and of the wild relative (Cajanus cajanifolius ICPW 29). A single, circular-mapping molecule of length 545.7 kb was assembled and annotated for the ICPA 2039 line. Sequence annotation predicted 51 genes, including 34 protein-coding and 17 RNA genes. Comparison of the mitochondrial genomes from different Cajanus genotypes identified 31 ORFs, which differ between lines within which CMS is present or absent. Among these chimeric ORFs, 13 were identified by comparison of the related male-sterile and maintainer lines. These ORFs display features that are known to trigger CMS in other plant species and to represent the most promising candidates for CMS-related mitochondrial rearrangements in pigeonpea.

High-throughput RNA sequencing of a formalin-fixed, paraffin-embedded autopsy lung tissue sample from the 1918 influenza pandemic.

Most biopsy and autopsy tissues are formalin-fixed and paraffin-embedded (FFPE), but this process leads to RNA degradation that limits gene expression analysis. The RNA genome of the 1918 pandemic influenza virus was previously determined in a 9-year effort by overlapping RT-PCR from post-mortem samples. Here, the full genome of the 1918 virus at 3000× coverage was determined in one high-throughput sequencing run of a library derived from total RNA of a 1918 FFPE sample after duplex-specific nuclease treatments. Bacterial sequences associated with secondary bacterial pneumonias were also detected. Host transcripts were well represented in the library. Compared to a 2009 pandemic influenza virus FFPE post-mortem library, the 1918 sample showed significant enrichment for host defence and cell death response genes, concordant with prior animal studies. This methodological approach should assist in the analysis of FFPE tissue samples isolated over the past century from a variety of diseases.

High throughput generation of promoter reporter (GFP) transgenic lines of low expressing genes in Arabidopsis and analysis of their expression patterns.

BACKGROUND: Although the complete genome sequence and annotation of Arabidopsis were released at the end of year 2000, it is still a great challenge to understand the function of each gene in the Arabidopsis genome. One way to understand the function of genes on a genome-wide scale is expression profiling by microarrays. However, the expression level of many genes in Arabidopsis genome cannot be detected by microarray experiments. In addition, there are many more novel genes that have been discovered by experiments or predicted by new gene prediction programs. Another way to understand the function of individual genes is to investigate their in vivo expression patterns by reporter constructs in transgenic plants which can provide basic information on the patterns of gene expression. RESULTS: A high throughput pipeline was developed to generate promoter-reporter (GFP) transgenic lines for Arabidopsis genes expressed at very low levels and to examine their expression patterns in vivo. The promoter region from a total of 627 non- or low-expressed genes in Arabidopsis based on Arabidopsis annotation release 5 were amplified and cloned into a Gateway vector. A total of 353 promoter-reporter (GFP) constructs were successfully transferred into Agrobacterium (GV3101) by triparental mating and subsequently used for Arabidopsis transformation. Kanamycin-resistant transgenic lines were obtained from 266 constructs and among them positive GFP expression was detected from 150 constructs. Of these 150 constructs, multiple transgenic lines exhibiting consistent expression patterns were obtained for 112 constructs. A total 81 different regions of expression were discovered during our screening of positive transgenic plants and assigned Plant Ontology (PO) codes. CONCLUSIONS: Many of the genes tested for which expression data were lacking previously are indeed expressed in Arabidopsis during the developmental stages screened. More importantly, our study provides plant researchers with another resource of gene expression information in Arabidopsis. The results of this study are captured in a MySQL database and can be searched at http://www.jcvi.org/arabidopsis/qpcr/index.shtml. Transgenic seeds and constructs are also available for the research community.

Relationship between hepatitis B virus DNA and hepatitis B virus large envelope protein / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the relationship between HBV DNA and hepatitis B virus large envelope protein (HBV-LP) in patients with chronic hepatitis B.</p><p><b>METHODS</b>Serum HBV DNA was detected by RT-PCR and the HBV-LP was detected by enzyme linked immunosorbent assay in 320 serum samples collected from patients with chronic hepatitis B.</p><p><b>RESULTS</b>There were no significant difference between positive rate of HBV-LP and that of HBV DNA in different HBeAg patterns (P>0.05). Serum HBV-LP levels were closely correlated with HBV DNA copies (r=0.949).</p><p><b>CONCLUSION</b>Serum HBV-LP is a reliable serological marker that can reflect the replication of HBV.</p>

Effect of tanshinone II A on the calcineurin activity in proliferating vascular smooth muscle cells of rats / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the effect of tanshinone II A (TSN) on angiotensin II (Ang II) induced proliferation of vascular smooth muscle cells (VSMCs).</p><p><b>METHODS</b>VSMCs were cultured by explant attached method, and induced to proliferative cell model with Ang II. The effect of TSN in different concentrations on calcineurin (CaN) activity was detected by enzyme reaction phosphorus measurement; the CaN mRNA expression was detected by RT-PCR; and the expression of proliferating cell nuclear antigen (PCNA) were observed by immunocytochemical method.</p><p><b>RESULTS</b>Compared with the normal control group, Ang II could significantly stimulate the proliferation of VSMCs, showing obviously elevated degree of proliferation activity (P <0. 01). After being treated with TSN, all the indexes, including CaN activity, CaN mRNA expression and PCNA expression, were obviously reduced in a dose-dependent manner (P<0.05, P<0.01).</p><p><b>CONCLUSION</b>VSMCs proliferation can be inhibited by TSN in a dose-dependent manner and the inhibiting mechanism may be related to the down-regulation of CaN activities and the inhibition on CaN mRNA and PCNA expressions.</p>

Analysis of the cDNAs of hypothetical genes on Arabidopsis chromosome 2 reveals numerous transcript variants.

In the fully sequenced Arabidopsis (Arabidopsis thaliana) genome, many gene models are annotated as "hypothetical protein," whose gene structures are predicted solely by computer algorithms with no support from either expressed sequence matches from Arabidopsis, or nucleic acid or protein homologs from other species. In order to confirm their existence and predicted gene structures, a high-throughput method of rapid amplification of cDNA ends (RACE) was used to obtain their cDNA sequences from 11 cDNA populations. Primers from all of the 797 hypothetical genes on chromosome 2 were designed, and, through 5' and 3' RACE, clones from 506 genes were sequenced and cDNA sequences from 399 target genes were recovered. The cDNA sequences were obtained by assembling their 5' and 3' RACE polymerase chain reaction products. These sequences revealed that (1) the structures of 151 hypothetical genes were different from their predictions; (2) 116 hypothetical genes had alternatively spliced transcripts and 187 genes displayed polyadenylation sites; and (3) there were transcripts arising from both strands, from the strand opposite to that of the prediction and possible dicistronic transcripts. Promoters from five randomly chosen hypothetical genes (At2g02540, At2g31270, At2g33640, At2g35550, and At2g36340) were cloned into report constructs, and their expressions are tissue or development stage specific. Our results indicate at least 50% of hypothetical genes on chromosome 2 are expressed in the cDNA populations with about 38% of the gene structures differing from their predictions. Thus, by using this targeted approach, high-throughput RACE, we revealed numerous transcripts including many uncharacterized variants from these hypothetical genes.

LC-ESI/MS identification and reduction of impurities in spiramycin fermentation / 生物工程学报

There are several impurities in the spiramycin fermentation broth which leads to a lower yield and lower quality of the product. Four impurities in spiramycin broth have been simultaneously separated and identified by LC-ESI/MS. The generation of these impurities was attributed to the fluctuation of glucosylation in spiramycin biosynthesis. Nitrogen sources, ammonium in particular, were found to play an important role at the glucosylation. Aided with the information of LC-ESI/MS analysis and subsequent optimization of the culture medium, better culture medium of shake flask was designed, which leads to reduction of impurities by 22% - 88%.

Cloning and sequencing of cDNAs for hypothetical genes from chromosome 2 of Arabidopsis.

About 25% of the genes in the fully sequenced and annotated Arabidopsis genome have structures that are predicted solely by computer algorithms with no support from either nucleic acid or protein homologs from other species or expressed sequence matches from Arabidopsis. These are referred to as "hypothetical genes." On chromosome 2, sequenced by The Institute for Genomic Research, there are approximately 800 hypothetical genes among a total of approximately 4,100 genes. To test their expression under various growth conditions and in specific tissues, we used six cDNA populations prepared from cold-treated, heat-treated, and pathogen (Xanthomonas campestris pv campestris)-infected plants, callus, roots, and young seedlings. To date, 169 hypothetical genes were tested, and 138 of them are found to be expressed in one or more of the six cDNA populations. By sequencing multiple clones from each 5'- and 3'-rapid amplification of cDNA ends (RACE) product and assembling the sequences, we generated full-length sequences for 16 of these genes. For 14 genes, there was one full-length assembly that precisely supported the intron-exon boundaries of their gene predictions, adding only 5'- and 3'-untranslated region sequences. However, for three of these genes, the other assemblies represent additional exons and alternatively spliced or unspliced introns. For the remaining two genes, the cDNA sequences reveal major differences with predicted gene structures. In addition, a total of six genes displayed more than one polyadenylation site. These data will be used to update gene models in The Institute for Genomic Research annotation database ATH1.

Construction and expression of recombinant cecropin B-binding site of luteinizing hormone releasing hormone gene and its anticancer function / 中华妇产科杂志

Objective To construct and express recombinant cecropin B-binding site of luteinizing hormone releasing hormone(CB-LHRH')gene,and to evaluate the anticancer function of CB-LHRH' on human ovarian cancer cell line SKOV3 and human endometrial cancer cell line HEC-1B.Methods The sequence of the cDNA encoding CB-LHRH' was designed,artificially synthesized,verified by DNA sequence analysis and expressed by Bac-to-Bac baculovirus expression system.The expression of CB-LHRH' proteins were identified by western dot blot using rabbit polyclonal antibody against LHRH as the primary antibody.To determine the anticancer effects of the CB-LHRH' protein,ovarian cancer cell line SKOV3 and endometrial adenocarcinoma cell line HEC-1B were treated by different doses of the CB-LHRH' protein.Cell growth inhibition assay was performed using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)5[(phenylamino) carbonyl]-2H-tetrazolium hydroxide(XTT)kit at different times,and cell morphologic changes were observed under the inverted microscope.Results The inhibitory rate of proliferation by CB-LHRH' increased with the increase of dose and time respectively:SKOV3 cell,from(5.03?0.08)% to(53.24 ?1.22)%;HEC-1B cell,from(5.13?0.37)% to(56.16?1.08)%.The inhibitory effect on HEC-1B cell was stronger than that on SKOV3 cell(P