Chloroplast-based expression system is promising for the hyper-expression of plant-derived recombinant therapeutic proteins and vaccines. To verify the feasibility of obtaining high-level expression of the SARS subunit vaccine and to provide a suitable plant-derived vaccine production platform against the severe acute respiratory syndrome coronavirus (SARS-CoV), a 193-amino acid fragment of SARS CoV spike protein receptor-binding domain (RBD), fused with the peptide vector cholera toxin B subunit (CTB), was expressed in tobacco chloroplasts. Codon-optimized CTB-RBD sequence was integrated into the chloroplast genome and homoplasmy was obtained, as confirmed by PCR and Southern blot analysis. Western blot showed expression of the recombinant fusion protein mostly in soluble monomeric form. Quantification of the recombinant fusion protein CTB-RBD was conducted by ELISA analysis from the transplastomic leaves at different developmental stages, attachment positions and time points in a day and the different expression levels of the CTB-RBD were observed with the highest expression of 10.2% total soluble protein obtained from mature transplastomic leaves. Taken together, our results demonstrate the feasibility of highly expressing SARS subunit vaccine RBD, indicating its potential in subsequent development of a plant-derived recombinant subunit vaccine and reagents production for antibody detection in SARS serological tests.
Chloroplasts ; metabolism ; Cholera Toxin ; Protein Interaction Domains and Motifs ; Recombinant Fusion Proteins ; biosynthesis ; SARS Virus ; Spike Glycoprotein, Coronavirus ; biosynthesis ; Tobacco ; metabolism ; Vaccines, Subunit ; biosynthesis

Objective:Taking Chongyang county of Hubei Province as an example, the paper describes the in-tegration of the maternal and child health ( MCH) and family planning ( FP) service system and analyses the key ele-ments to provide reference for promoting integration. Methods: qualitative interviews and quantitative questionnaire were used to collect data. The software QSR Nvivo 8. 0 and SPSS 17. 0 were used for qualitative and quantitative data analysis. Results:Based on the correct understanding of integration, Chongyang implements the supporting policies actively, maintains the original compensation mode unchanged, follows the principle of“no reducing headcounts and no downsizing” strictly, promotes the merger of institutions rapidly, adjusts the service contents and methods reasona-bly, and integrates the maternal and child and family planning information platform. Conclusion:The correct concept of integration, appropriate staffing, and stable funding in Chongyang provide rich experiences for future research. However, there is room for improvement in staffing, incentive mechanisms, service content, and forms.

OBJECTIVE To study the molecular mechanism of cisplatin(DDP)by which HeLa cell growth and proliferation are inhibited. METHODS Cultured HeLa cells were treated with DDP 0.02-75 μmol · L-1 for 24 or 48 h. CCK-8 assay was used to determine the cell proliferation. The wound scratch assay was used to detect the cell migration and invasion. Flow cytometry was used to detect the cell cycle arresting. q-PCR was used to test the expression of metastasis suppressor gene 1 (MTSS1)mRNA. Western blot was used to determine protein levels of MTSS1,phosphorylated-extra?cellular signal-regulated kinase(p-ERK) and phosphorylated-serine-threonine kinase(p-AKT). RESULTS Following the treatment with DDP for 24 or 48 h,the proliferation of HeLa cells was inhibited significantly (P<0.05),the value of the half inhibitory concentration (IC50) of cells was 4.14 and 11.82 μmol · L-1. Migration and invasion activity of HeLa cells were reduced according to the wound scratch assay(P<0.05). Flow cytometry results showed that the cell cycle was arrested at S phase. q-PCR results showed that MTSS1 mRNA expression changed with DDP in a concentration-dependent manner (r24 h=-0.965,P<0.01;r48 h=-0.953,P<0.01). Western blot showed that the protein levels of MTSS1,p-ERK and p-AKT expression declined significantly with the increase in DDP concentrations(p-ERK:r24 h=-0.875,P<0.01;r48 h=-0.966,P<0.01. p-AKT:r24 h=-0.831,P<0.01;r48 h=-0.863,P<0.01. MTSS1:r24 h=-0.969,P<0.01;r48 h=-0.988,P<0.01). CONCLUSION DDP treatment inhibits HeLa growth and proliferation by interfering with the MTSS1 expression and disturbing the activation of ERK and AKT signaling pathways.

BACKGROUND:Santoni put forward the cortical bone trajectory technology by changing the traditional pedicle screw placement for lumbar internal fixation in order to obtain better control of the screw and bone in 2009. OBJECTIVE:To analyze biomechanical stability of cortical bone trajectory system in the lumbar fusion. METHODS:Twenty fresh newborn calf L3/4, L5/6 motion segment specimens were obtained, and their ranges of motion were detected under different states, as normal controls. Subsequently, twenty samples were divided into cortical bone trajectory screw group and traditional pedicle screw group, which underwent cortical bone trajectory screw fixation combined with posterior lumbar fusion and traditional pedicle screw fixation combined with posterior lumbar fusion, respectively. Without destruction, ranges of motion were detected under different states in both groups. In the revision group, after the test in the traditional pedicle screw group, screw was withdrawn, and cortical bone trajectory screw was used to detect its range of motion under different states. RESULTS AND CONCLUSION:Ranges of motion at bending to the left and right, anteflexion, posterior extension and axial rotation were significantly lower in the cortical bone trajectory screw group and traditional pedicle screw group than in the normal control group (P<0.05). No significant difference in bending to the left and right, anteflexion, posterior extension and axial rotation was detected between the cortical bone trajectory screw and revision groups and traditional pedicle screw group (P>0.05). These results confirmed that cortical bone trajectory technology combined with posterior lumbar fusion can obtain identical stability as the traditional pedicle screw fixation combined with posterior lumbar fusion. Simultaneously, it is a new choice for revision after traditional pedicle screw fixation.

Objective To investigate the effects of cyclopamine (CYP) on endometrial carcinoma (HEC-1A) cell survival and on induction of cell apoptosis .Methods HEC-1A cells were treated with various doses of CYP (0, 5,10, 20 and 40 μmol/L) for 24 h respectively .Then,the inverted microscope was used to observe cell morphology .Cell proliferation and apoptosis were tested by CCK-8 assay and AO/EB bi-labelling assay.The apoptosis rate of HEC-1A was analyzed using flow cytometric analysis , and the key gene expression of Bax and Bcl-2 was detected by quantitative PCR .Results The HEC-1A cells exhibited dramatic morphological changes after treatment with CYP and in a dose-dependent manner .CYP significantly inhibited HEC-1A cell proliferation using CCK8 assays(P<0.05), and induced cell death by AO/EB bi-labelling assay.Moreover,flow cytometry analysis showed that CYP treatment resulted in HEC-1A cell apoptosis, and that a higher concentration of CYP induced severer cell apoptosis (P<0.05).Meanwhile, CYP treated HEC-1A cells exhibited up-regulated expression of Bax and down-regulated expression of Bcl-2 according to Q-PCR.Conclusion Our findings indicatee that CYP can inhibit HEC-1A cell proliferation and induce cell apoptosis .

Objective To analyze the impact of Helicobacter pylori standard strain (Hp P12) and its virulence factor vacuolating cytotoxin A ( VacA) on DNA damage and homologous recombination ( HR) repair in a human gastric epithelial cell line (GES-1). Methods Strains of Hp P12 and vacA gene knock-out Hp P12 ( Hp P12 ΔvacA) were respectively used to infect GES-1 cells at a multiplicity of infection of 100. GES-1 cells treated with etoposide (50μmol/L) or mitomycin (0. 5μg/ml) for 2 h were used as posi-tive control. Western blot and immunofluorescence were performed to detect the expression of DNA damage marker protein γH2AX and key HR repair proteins (Rad51, pMRE11, CtIP and pCtIP) and the recruitment of them at DNA damage sites. Human embryonic kidney HEK-293 ( DR-GFP) cells were infected with Hp P12 and Hp P12 ΔvacA strains to verify the impact of VacA on HR repair efficiency. Results The expres-sion and recruitment of γH2AX and key HR repair proteins ( Rad51, pMRE11, CtIP and pCtIP) were in-creased in Hp P12-infected cells as compared with that in uninfected and Hp P12 ΔvacA-infected cells ( all P<0. 05). To evaluate the HR repair efficiency, I-SceⅠ plasmid-transfected HEK-293 (DR-GFP) cells were infected with Hp P12 and Hp P12 ΔvacA and the results showed that green fluorescent protein ( GFP)-positive cells were decreased after infection, especially in Hp P12 ΔvacA-infected cells (both P<0. 05). Conclusions Hp P12 infection could cause DNA damage and promote HR repair in GES-1 cells, in which the virulence factor VacA played an important role.

OBJECTIVE: To summarize the genetic diagnosis, low-depth copy number variation sequencing (CNV-seq) and prenatal finding in 7 fetuses with 2p16.3 deletions only involving the NRXN1 gene. METHODS: The 7 fetuses have all been found to have loss of heterozygosity at 2p16.3 by CNV-seq, which were verified by quantitative real-time PCR (qPCR). Specific regions of NRXN1 gene deletions were identified, and the CNVs were verified in their parents. Outcome of the pregnancies were followed up. RESULTS: Among 16 502 prenatal samples, 7 fetuses were found to harbor a 120 kb ~ 900 kb microdeletion in the 2p16.3 region, which yielded a prevalence of 0.424‰. The deleted region mainly involved 50 200 000-51 880 000 positions of chromosome 2 and involved only the NRXN1 gene. All of the 7 fetal CNVs were confirmed by qPCR, including 2 cases with heterozygous deletion of exons 1 to 6, 1 with heterozygous deletion of exons 1 to 19, 1 with heterozygous deletion of exons 19 to 22, and 3 with heterozygous deletion of introns 6 to 7 of the NRXN1 gene. Verification in the parents had found that one deletion was inherited from the father, 1 was from the mother, 2 cases were de novo in origin, whilst the remaining 3 had refused parental verification. After genetic counseling, one couple had elected induced abortion, 1 case has not been born yet, whilst the other 5 cases were born healthy. Follow up had identified no mental abnormalities among the children. CONCLUSION Seven fetuses with heterozygous 2p16.3 deletions only involving the NRXN1 gene were detected by CNV-seq. The specific deletion of the NRXN1 gene was verified by qPCR. Prenatal genetic counseling and fertility guidance has been provided to the particular family by combining the results of CNV testing, pedigree analysis and pregnancy outcome.
Female ; Humans ; Pregnancy ; Calcium-Binding Proteins/genetics* ; Cell Adhesion Molecules, Neuronal/genetics* ; DNA Copy Number Variations ; Nerve Tissue Proteins/genetics* ; Neural Cell Adhesion Molecules/genetics* ; Prenatal Diagnosis ; Real-Time Polymerase Chain Reaction ; Infant, Newborn

In order to explore the regulation mechanisms of follicular helper T cell (Tfh Cell) differentiation,optimized conditions of in vitro induction from both peripheral blood mononuclear cells and MAC sorted Na(i)ve CD4 + T cells to human Tfh cells were developed.Induction efficiency difference of TCR signal anti-hCD3e stimulation between coated on solid phase and in soluble phase was also determined.Differentiation efficiency of CD4 + CXCR5 + ICOS+PD-1 + Tfh cell was determined by FACS while the expression level of IL-21 in cell supernatant was determined by ELISA tests.An ultimate induction condition that 5 μg/mL coated overnight anti-hCD3e stimulated na(i)ve CD4 + T cells to differentiate into Tfh at an up to 20.4％ percentage was finally determined.The optimization of in vitro induction protocol of human Tfh provided an effective examine platform for the studies on Tfh differentiation mechanisms and related pharmacology,toxicity and metabolic experiments.

Objective To investigate the value of next-generation sequencing (NGS) technique for genetic analysis of spontaneous abortion. Methods From January to June 2017, 154 patients who visited the First Affiliated Hospital of Zhengzhou University for spontaneous abortion were enrolled. All abortion tissue samples were analyzed by both NGS combined with short tandem repeat (STR) and single nucleotide polymorphism array (SNP-array). Results of the two methods were compared by Chi-square or Fisher's exact test. Results (1) Chromosomal abnormalities were detected in 109 of the 154 cases (70.7%), including 52 (47.7%) of numerical chromosomal abnormalities, 49 (45.0%) of structural chromosomal abnormalities, six (5.5%) of mosaicism, and two (1.8%) of uniparental disomy (UPD). In those 52 cases of numerical chromosome abnormalities, there were 45 of chromosome aneuploidy and seven of polyploidy. The top three numerical chromosomal abnormalities were 45,X (27.0%, 14/52), trisomy 22 (9.6%, 5/52) and trisomy 16 (7.7%, 4/52). Forty-nine structural abnormality cases carried 67 copy number variations (CNV), including 13 pathogenic CNV (pCNV, 19.4%), 24 variants of unknown clinical significance (35.8%) and 30 benign CNV (44.8%). In those 13 pCNVs, two were responsible for microdeletion and microduplication syndromes. (2) SNP-array was successful in 152 cases, but failed in two (1.3%) due to genomic DNA <200 ng. However, NGS technology was successful in all 154 cases and identified chromosomal abnormalities in the two cases that SNP-array had failed. No statistically significant difference was shown in the detection rate of chromosomal abnormalities between SNP-array and NGS technology [70.4% (107/152) vs 67.5% (104/154), χ2=0.293, P=0.588]. (3) No significant difference in the detection of chromosome aneuploidy (six cases in each group, 3.9% vs 3.9%) and mosaicism (45 cases in each group, 29.2% vs 29.6%) was found between NGS technology and SNP-array. Three cases of polyploidy (69, XXX) and two of UPD were identified by SNP-array, but not by NGS. When combined with STR, NGS was able to detect all three cases of polyploidy (69, XXX). (4) Forty-seven structural abnormality cases detected by SNP-array carried 53 CNVs, and 49 detected by NGS carried 67 CNVs. (5) NGS detected ten, three and one more CNVs than SNP-array did when the genome lengths were 100-<500, 500-<1 000 and ≥1 000 kb, respectively. Conclusions NGS can be used to detect chromosomal aneuploidy and mosaicism that can be identified by SNP-array with fewer limitations on total amount of genome. Moreover, CNVs that fail to be identified by SNP-array can also be detected by NGS. When combined with STR, NGS can effectively detect chromosomal polyploidy. Therefore, NGS could be a potential genetic analysis method for spontaneous abortion and of importance for genetic counseling.

OBJECTIVE: To explore the prevalence and characteristics of chromosomal abnormalities in abortuses during early pregnancy with single nucleotide polymorphism microarray (SNP-array). METHODS: For 520 abortuses, copy number variations (CNVs) in chorionic villi were analyzed with SNP-array. RESULTS: In 510 (98.1%) of the samples, the analysis was successful. Among these, 57.6% (294/510) of the samples were found to harbor clinically significant chromosomal abnormalities. 38.8% of the samples (198/510) had a normal result. 2.4% (12/510) of the samples harbored benign CNVs, and 1.2% (6/510) harbored variants of uncertain significance (VOUS). Aneuploidies, polyploidies, pathogenic CNVs and uniparental disomies (UPD) had accounted for 75.2% (221/294), 13.9% (41/294), 8.2% (24/294), and 2.7% (8/294) of the samples, respectively. 45,XO was the most common finding, which was followed by trisomy 16 and trisomy 22. 69,XXY was the most common polyploidy. CONCLUSION Chromosomal abnormalities are the main cause for early miscarriage, among which aneuploidies are most common. The prevalence of aneuploidies is significantly increased among women over 35. SNP-array analysis has the advantage of high success rate, high resolution and great accuracy, but the clinical significance of microdeletions/microduplications found by SNP-array can be difficult for interpretation.
Chorionic Villi ; Chromosome Aberrations ; Chromosome Disorders ; DNA Copy Number Variations ; Female ; Genetic Testing ; Humans ; Karyotyping ; Polymorphism, Single Nucleotide ; Pregnancy