Artificial intelligence,such as deep learning technology,has advanced the study of facial expression recognition since facial expression carries rich emotional information and is significant for many naturalistic situ...Artificial intelligence,such as deep learning technology,has advanced the study of facial expression recognition since facial expression carries rich emotional information and is significant for many naturalistic situations.To pursue a high facial expression recognition accuracy,the network model of deep learning is generally designed to be very deep while the model’s real-time performance is typically constrained and limited.With MobileNetV3,a lightweight model with a good accuracy,a further study is conducted by adding a basic ResNet module to each of its existing modules and an SSH(Single Stage Headless Face Detector)context module to expand the model’s perceptual field.In this article,the enhanced model named Res-MobileNetV3,could alleviate the subpar of real-time performance and compress the size of large network models,which can process information at a rate of up to 33 frames per second.Although the improved model has been verified to be slightly inferior to the current state-of-the-art method in aspect of accuracy rate on the publically available face expression datasets,it can bring a good balance on accuracy,real-time performance,model size and model complexity in practical applications.展开更多
Objective To investigate the effect of electroacupuncture(EA)on microRNA(miRNA)expression spectrum and PI3K/Akt/mTOR signaling pathway in uterine tissue of rats with primary dysmenorrhea(PDM),and to explore the potent...Objective To investigate the effect of electroacupuncture(EA)on microRNA(miRNA)expression spectrum and PI3K/Akt/mTOR signaling pathway in uterine tissue of rats with primary dysmenorrhea(PDM),and to explore the potential mechanism of EA in the treatment of PDM.Methods Thirty female SD rats,weighted(200±20)g were randomly divided into control group,model group and EA group,10 rats in each group.By using subcutaneous injection of estradiol diphenhydrate combined with intraperitoneal injection of oxytocin,PDM models were established.Rats in the EA group received EA at“Sanyinjiao”(SP6)and“Guanyuan”(CV4)at dense waves and a frequency of 50 Hz,once a day,20 min each time,for 10 consecutive days.After the 10-day intervention,samples were collected and transmission electron microscopy was used to observe the ultrastructural changes of the cells in uterine tissue in each group.With RNA-seq method,the changes of miRNA expression spectrum in rat uterine tissue were detected.Bioinformatics analysis such as GO functional annotation and KEGG pathway was performed according to differentially expressed miRNAs.Differentially expressed miRNAs were verified by qRT-PCR.Endometrial stromal cells were selected as the target cells and transfected;and they were divided into control group,NC mimics group,mimic miR-144–3p group,NC inhibitor group and inhibitor miR-144–3p group.The apoptosis was determined by using flow cytometrydetect apoptosis,the miRNA and protein expression of PI3K/Akt/mTOR signaling pathway were detected by qRT-PCR and Western blot in each group separately.Results 1.Transmission electron microscope.(1)Control group:no obvious morphological changes in the uterine tissue.(2)Model group:fibroblasts in uterine tissue were irregular,the edema was presented in cellular cytoplasm,the nuclei were irregular and mitochondria swollen seriously;the rough endoplasmic reticulum was expanded moderately.(3)EA group:fibroblasts were spindle-shaped and pyknotic,the cytoplasm increased in electron density,the nuclei were slightly irregular and pyknotic,mitochondria were oval in shape,with little swelling and vacuolation;the rough endoplasmic reticulum was expanded slightly and retained,with a small amount of degranulation.2.Compared with the control group,there were 26 differentially expressed miRNAs in the uterine tissue of rats with PDM.After EA intervention,the expression of miR-144–3p was significantly up-regulated.GO functional analysis of differentially expressed miRNAs in PDM rats after EA showed that the biological functions involved calcium transmembrane transporter activity,mitogen-activated protein kinase binding,epithelial cell migration,tissue migration,etc.3.KEGG pathway analysis showed that PI3K/Akt signaling pathway,MAPK signaling pathway and calcium signaling pathway were enriched.Mimic miR-144-3p increased the apotosis of endometrial stromal cells,and decreased the mRNA and protein expression of PI3K,Akt,and mTOR(P<0.01).Conclusion EA can optimize the cell morphology in the uterine tissue of rats with PDM and affect the miRNA expression spectrum,which may be associated with the effect of EA for up-regulating miR-144–3p expression in endometrial stromal cells,suppressing PI3K/Akt/mTOR signaling pathway and causing apoptosis.展开更多
BACKGROUND Laryngeal squamous cell carcinoma(LSCC)is a prevalent head and neck malignancy with suboptimal survival rates due to late detection and therapeutic resistance.AIM To investigate chaperonin-containing TCP1 s...BACKGROUND Laryngeal squamous cell carcinoma(LSCC)is a prevalent head and neck malignancy with suboptimal survival rates due to late detection and therapeutic resistance.AIM To investigate chaperonin-containing TCP1 subunit 3(CCT3)expression and its clinical implications,and its effects on LSCC cell growth.METHODS Systematic data on CCT3 mRNA expression were collected from biomedical databases,and integrated further based on the standardized mean difference and the summary receiver operating characteristic curve.Single-cell RNA-seq data were mined to validate the expression level of CCT3 mRNA.In-house immunohistochemistry was performed to explore the CCT3 protein levels of clinical LSCC samples and their relationship with clinical parameters.The growth function of LSCC cell was analyzed using CRISPR knockout screening.CCT3-related signaling pathway analyses were conducted using gene set enrichment analysis.Protein-protein interaction network construction was performed to identify hub genes.RESULTS CCT3 mRNA was significantly overexpressed in 269 LSCC tissues cases across multiple independent datasets(standardized mean difference=32,area under the curve=0.93);At the translational level,the in-house immunohistochemical analysis further demonstrated the consistent upregulation of CCT3 protein in 88 cases of LSCC samples(58 non-LSCC samples vs 30 LSCC samples,P=1.4e^(-14)).Analysis of clinical parameters showed no significant differences among subgroup.Functional characterization with clustered regularly interspaced short palindromic repeats--mediated gene knockout revealed that depletion of CCT3 potently suppressed LSCC cell viability in vitro.Gene set enrichment analysis indicated that CCT3 was markedly associated with several key oncogenic pathways,including extracellular matrix receptor interaction and cell cycle regulation pathways.CONCLUSION CCT3 upregulation in LSCC may influence cellular growth by regulating related pathways,indicating its potential as a biomarker and therapeutic target for LSCC.展开更多
Objective： To investigate the expression of Glypican-3 gene in （α-fetoprotein （AFP）-negative hepatocellular carcinoma （HCC） and clarify whether Glypican-3 expression is a useful parameter for the diagnosis of h...Objective： To investigate the expression of Glypican-3 gene in （α-fetoprotein （AFP）-negative hepatocellular carcinoma （HCC） and clarify whether Glypican-3 expression is a useful parameter for the diagnosis of hepatocelhllar carcinoma （HCC）, especially for AFP-negative ones. Methods： Forty-one specimens of AFP-negative hepatocellular carcinoma and para-carcimoma tissue were studied for the expression of Glypican-3 by Northern blot. The expression of Glypican-3 protein was detected immunohistochemically with specific polyclonal antibody. Results： Northern blot analysis indicated that the expression of Glypican-3 mRNA was intensively detected in 30 of 41 AFP-negative HCC （73.17%）. In contrast, Glypican-3 mRNA was only weakly detected in 4 of the surrounding non-tumor liver tissues. There was significant difference in the Glypican-3 mRNA expression in large tumors （〉5 cm） （79.31%） and in small tumors （〈5 cm） （41.67%） （P〈0.01）. Gypican-3 mRNA was more frequently overexpressed in poorly differentiated HCC than in well differentiated ones （76.47% vs. 42.86%, P〈0.05）. The Glypican-3 expression was not correlated with age. sex. ttbsAg seropositivity, fibroeapsule, portal venous embolus and intrahepatic metastasis. The overexpression of Glypican-3 protein in HCC was targeted in tumor cells, not in bile duct cells and other interstitial cells. Conclusion： Glypican-3 was specially overexpressed in AFP-negative HCC, and its expression was closely correlated with the tumor size and tumor grade. Glypican-3 gene may play important roles in hepatocareinogenesis, and can be used as a new biochemical marker of HCC, especially for AFP-negative HCC.展开更多
Zymography and in situ hybridization were used to investigate matrixmetalloproteinase -2, -9 (MMP -2, MMP-9) activities and expressions of MMP -2, -9 and TIMP1, -2, -3 (tissue inhibitors of matrix metallo-proteinases)...Zymography and in situ hybridization were used to investigate matrixmetalloproteinase -2, -9 (MMP -2, MMP-9) activities and expressions of MMP -2, -9 and TIMP1, -2, -3 (tissue inhibitors of matrix metallo-proteinases) mRNA in the rat uterus during estrouscycle. The relative activity was semiquanted by using densitometric analysis. The MMP-2(67 kDa) activity in every stage during estrpus cycle was detected by zymography. MMP-2activity was highest at proestrus; higher at estrus and metaestrus; lowest at diestrus. Throughin situ hybridization, MMP -2, -9, TIMP -1~ -3 mRNA mainly in hasal stroma cells of uterineendometrium were detected. The positive signals of MMP -2 and -9 mRNAs in hasal stromacells were shown stronger at proestrus, estrus and metaestrus while they showed the weakest atdiestrus. The expression of MMP -2 mRNA coincided with MMP -2 activity change. MMP-2and -9 mRNAs were also highly expressed in uterine circular muscle at estrus. Weak signals ofMMP -9 mRNA were detected in uterine luminal and glandular epithelial cells at estrus.TIMP -1 mRNA in hasal stroma cells was shown as the strongest expression at estrus andmetaestrus; stronger at proestrus and the weakest at diestrus. TIMP-2 mRNA in basal stromacells was stronger at estrus and diestrus; weaker at proestrus and metaestrus. TIMP -1 and -2mRNAs were also highly expressed in uterine luminal and glandular epithelial cells at estrus.TIMP -3 mRNA in hasal stroma cells revealed the strongest expression at estrus; stronger atdiestrus and metaestrus and showed the weakest at proestrus. The mRNA was also highlyexpressed in uterine circular muscle at estrus. In short, our present results provide evidencethat MMP -2, -9 and TIMP -1~ -3 were involved in rat uterine endometrium reconstructionduring estrous cycle.展开更多
[Objective] The aim was to clone CBF3 gene from Arabidopsis thaliana and construct plant expression vector pCAMBIA1301-Rd29A-CBF3.[Method] CBF3 gene and stress-inducible promoter Rd29A were amplified from the genomic ...[Objective] The aim was to clone CBF3 gene from Arabidopsis thaliana and construct plant expression vector pCAMBIA1301-Rd29A-CBF3.[Method] CBF3 gene and stress-inducible promoter Rd29A were amplified from the genomic DNA of A.thaliana for the construction of plant expression vector.[Result] Sequencing results showed that the cloned CBF3 gene had 750 bp,and showed 100% identity with the sequence published on GenBank.The promoter Rd29A had 1 425 bp,and showed 100% identity with the sequence published on GenBank.[Conclusion] Based on the binary vector pCAMBIA1301,the plant expression vector pCAMBIA1301-Rd29A-CBF3 was constructed successfully,which could materially improve the salt resistance,drought-tolerance,cold resistance of plants.展开更多
AIM To detect the expression of caspase 3gene in primary human hepatocellular carcinoma（HCC）and investigate its relationship to p21<sup>WAF1</sup>gene expression and HCC apoptosis.METHODS In situ hybridi...AIM To detect the expression of caspase 3gene in primary human hepatocellular carcinoma（HCC）and investigate its relationship to p21<sup>WAF1</sup>gene expression and HCC apoptosis.METHODS In situ hybridization was employedto determine caspase 3 and p21<sup>WAF1</sup>expression inHCC.In situ end-labeling was used to detecthepatocytic apoptosis in HCC.RESULTS Twenty-one of 39（53.8%）cases ofHCC were found to express caspase 3transcripts,while 45.2% of HCC failed toexpress caspase 3.Non-cancerous adjacent livertissues showed more positive caspase 3（87.5%,7/8）as compared with HCC（P【0.05）.The expression of caspase 3 is correlated withHCC differentiation,72.2%（13/18）ofmoderately to highly differentiated HCC showedcaspase 3 transcripts positive,while only 38.1%of poorly differentiated HCC harbored caspase 3transcripts（P【0.05）.No relationship wasfound between caspase 3 expression and tumorsize or grade or metastasis,although 52.5%（5/8）of HCC with metastasis were caspase 3positive and a little higher than that with nometastasis（51.6%,P】0.05）.Expression of caspase 3 alone did not affect the apoptosisindex（AI）of HCC.The AI was 7.12%o in caspase3-positive tumors（n=21）,while in caspase 3-negative cases（n=18）6.59%0（P】0.05）.Expression of caspase 3 clearly segregated withp21<sup>WAF1</sup>positive tumors as compared withp21<sup>WAF1</sup>-negative cases（16 of 23,69.6% versus5 of 16,31.3%）with statistical significance（P=0.017）.In the cases with positive caspase 3and negative p21<sup>WAF1</sup>,the Al was found slightlyhigher,but with no statistical significance,thanthat with expression of p21<sup>WAF1</sup>and caspase 3（7.21‰ vs 6.98‰,P】0.05）.CONCLUSION Loss of caspase 3 expressionmay contribute to HCC carcinogenesis,althoughthe expression of caspase 3 does not correlatewell with cell apoptosis in HCC.p21<sup>WAF1</sup>may bemerely one of the inhibitors which can reducecaspase 3 mediated cell apoptosis in HCCs.展开更多
Objective: To elucidate the role and prognostic significance of lymphocyte activation-gene-3(LAG-3) in soft tissue sarcoma(STS).Methods: The expression of LAG-3 in patient and matched normal blood samples was analyzed...Objective: To elucidate the role and prognostic significance of lymphocyte activation-gene-3(LAG-3) in soft tissue sarcoma(STS).Methods: The expression of LAG-3 in patient and matched normal blood samples was analyzed by flow cytometry. The localization and prognostic values of LAG-3^+ cells in 163 STS patients were analyzed by immunohistochemistry. In addition, the expression of tumor-infiltrating CD3^+ T, CD4^+ T, and CD8^+ T cells and their role in the prognosis of STS were evaluated by immunohistochemistry. The effect of LAG-3 blockade was evaluated in an immunocompetent MCA205 fibrosarcoma mouse model.Results: Peripheral CD8^+ and CD4^+ T cells from STS patients expressed higher levels of LAG-3 than those from healthy donors.LAG-3 expression in STS was significantly associated with a poor clinical outcome(P = 0.038) and was correlated with high pathological grade(P < 0.001), advanced tumor stage(P = 0.016). Additionally, LAG-3 expression was highly correlated with CD8^+ T-cell infiltration(r = 0.7034, P < 0.001). LAG-3 was expressed in murine tumor-infiltrating lymphocytes, and its blockade decreased tumor growth and enhanced secretion of interferon-gamma by CD8^+ and CD4^+ T cells.Conclusions: LAG-3 blockade may be a promising strategy to improve the effects of targeted therapy in STS.展开更多
AIM To investigate the mechanisms ofsalvianolic acid A（SA-A）against liver fibrosisin vitro.METHODS NIH/3T3 fibroblasts were culturedroutinely,and incubated with 10<sup>-4</sup>mol/L-10<sup>-7</s...AIM To investigate the mechanisms ofsalvianolic acid A（SA-A）against liver fibrosisin vitro.METHODS NIH/3T3 fibroblasts were culturedroutinely,and incubated with 10<sup>-4</sup>mol/L-10<sup>-7</sup>mol/L SA-A for 22 h.The cell viability wasassayed by[<sup>3</sup>H]proline incorporation,cellproliferation by[<sup>3</sup>H]TdR incorporation,cellcollagen synthetic rate was measured with[<sup>3</sup>H]proline impulse and collagenase digestionmethod.The total RNA was prepared from thecontrol cells and the drug treated cellsrespectively,and α（1）I pro-collagen mRNAexpression was semi-quantitatively analyzedwith RT-PCR.RESULTS 10<sup>-4</sup>mol/L SA-A decreased cellviability and exerted some cytotoxiciy,while10<sup>-5</sup>mol/L-10<sup>-7</sup>mol/L SA-A did not affect cellviability,but inhibited cell proliferationsignificantly,and 10<sup>-6</sup>mol/L SA-A had the besteffect on cell viability among theseconcentrations of drugs.10<sup>-5</sup>mol/L-10<sup>-6</sup>mol/LSA-A inhibited intracellular collagen syntheticrate,but no significant influence on extracellularcollagen secretion.Both 10<sup>-5</sup>mol/L and10<sup>-6</sup>mol/L SA-A could decrease α（1）I pro-collagen mRNA expression remarkably.CONCLUSION SA-A had potent action againstliver fibrosis.It inhibited NIH/3T3 fibroblastproliferation,intracellular collagen syntheticrate and type I pro-collagen gene expression,which may be one of the main mechanisms of thedrug.展开更多
AIM:To investigate the expression of co-stimulatorymolecule B7-H3 in gastric carcinoma and adenomatissue as well as normal gastric tissue and to explore therelationship between B7-H3 expression and pathologicalfeature...AIM:To investigate the expression of co-stimulatorymolecule B7-H3 in gastric carcinoma and adenomatissue as well as normal gastric tissue and to explore therelationship between B7-H3 expression and pathologicalfeatures and prognosis of gastric carcinoma.METHODS:B7-H3 expression was detected in 102samples of human gastric carcinoma and 10 samples ofgastric adenoma and 10 samples of normal gastric tissueby immunohistochemical assay.Correlation betweenthe expression of B7-H3 and the patients'age,sex,gastric carcinoma locus,tumor size,tissue type,tumorinfiltration depth,differentiation degree,lymph nodemetastasis,and survival time was analyzed.RESULTS:B7-H3 was expressed in all gastric adenomasamples and in 58.8% samples of gastric carcinoma.B7-H3 expression in gastric carcinoma samples wasnot related with the patients'age,sex,lymph nodemetastasis,and tumor size(P>0.05),but with thesurvival time,infiltration depth of tumor and tissue type.CONCLUSION:Detection of B7-H3 expression in gastriccarcinoma tissue is beneficial to the judgment of theprognosis of gastric carcinoma patients and the choice oftreatment.展开更多
The invertebrate parvovirus Bombyx mori Densonucleosis Virus type 3 （China isolate）, named BmDNV-3, is a kind of bidensovirus. It is a new type of virus with unique replication mechanisms. To investigate the effects...The invertebrate parvovirus Bombyx mori Densonucleosis Virus type 3 （China isolate）, named BmDNV-3, is a kind of bidensovirus. It is a new type of virus with unique replication mechanisms. To investigate the effects of the NS3 gene during viral DNA replication, a pair of primers was designed for amplifying NS3 gene of Bombyx mori densovirus （China isolate）. Gene NS3 amplified was cloned into a prokaryotic expression vector pET-30a and the donor plasmid pFastBacHTe, respectively. The NS3 protein was expressed in Escherichia coli BL21. The pFastBacHTe-NS3 was transformed to E. coli DHIOBac. The recombinant bacmid baculoviruses （rBacmid-EGFP-NS3） isolated from the white colonies were transfected into BmN-4 cells using a transfection reagent. BraN-4 cells were infected with recombinant virus to express fusion proteins. The expression of fusion protein around 30 kDa in E. coli BL21 was identified by SDS-PAGE, Western blotting, and mass spectrometry. The expressed NS3 protein by B. mori nucleopolyhedrovirus bacmid system was confirmed by Western blotting using an anti-NS3 polyclonal antibody. And about 45 kDa protein was found. The expressed fusion protein was smaller than the expected size of EGFP-NS3, 55 kDa. Western blotting analysis indicated that EGFP-NS3 protein was expressed in infected larvae with smaller molecular size.展开更多
AIM:To evaluate for the first time the protein and mRNA expression of 14-3-3εin gastric carcinogenesis.METHODS:14-3-3εprotein expression was determined by western blotting,and mRNA expression was examined by real-ti...AIM:To evaluate for the first time the protein and mRNA expression of 14-3-3εin gastric carcinogenesis.METHODS:14-3-3εprotein expression was determined by western blotting,and mRNA expression was examined by real-time quantitative RT-PCR in gastric tumors and their matched non-neoplastic gastric tissue samples.RESULTS:Authors observed a significant reduction of 14-3-3εprotein expression in gastric cancer(GC)samples compared to their matched non-neoplastic tissue.Reduced levels of 14-3-3εwere also associated with diffuse-type GC and early-onset of this pathology.Our data suggest that reduced 14-3-3εmay have a role in gastric carcinogenesis process.CONCLUSION:Our results reveal that the reduced 14-3-3εexpression in GC and investigation of 14-3-3ε interaction partners may help to elucidate the carcino-genesis process.展开更多
BACKGROUND Serpin peptidase inhibitor,clade A member 3(SERPINA3)belongs to the serpin family with an inhibitory activity against proteases.Its aberrant expression has been observed in a wide range of tumor cells.Howev...BACKGROUND Serpin peptidase inhibitor,clade A member 3(SERPINA3)belongs to the serpin family with an inhibitory activity against proteases.Its aberrant expression has been observed in a wide range of tumor cells.However,its clinical significance and biological function in endometrial cancer have been rarely studied.We designed a study to determine the levels of SERPINA3 and its significance in patients with endometrial cancer.AIM To investigate the clinical significance and role of SERPINA3 expression in endometrial cancer cells.METHODS Eighty endometrial tissue samples collected from patients with endometrial cancer were included in an observation group and 80 paraffin-embedded tissues samples collected from patients with normal endometrial tissues undergoing myomectomy were employed as a control group between January 2014 and December 2018.The expression of SERPINA3 mRNA was detected by quantitative polymerase chain reaction(PCR)for all endometrial tissues included in the study.RESULTS The positive expression rate of SERPINA3 protein in endometrial cancer cells was 71.25%in the observation group,which was significantly higher than that in the control group(31.25%;P<0.05).There was no correlation between SERPINA3 protein in endometrial cancer cells and the age range at which women experienced menopause(P>0.05).However,it was associated with pathological grade,clinical stage,vascular invasion,and lymph node metastasis(P<0.05).Pathological grade,clinical stage,vascular invasion,and lymph node metastasis were independent prognostic factors for endometrial cancer.CONCLUSION The follow-up study of SERPINA3 can be used as a prognostic biomarker for endometrial cancer and as one of the targets for bio-targeted therapy for endometrial cancer.展开更多
It has been suggested that the energy required for sperm motility is produced by oxidative phosphorylation while glycolysis seems to be an important source for ATP transmission along the flagellum. Some studies have i...It has been suggested that the energy required for sperm motility is produced by oxidative phosphorylation while glycolysis seems to be an important source for ATP transmission along the flagellum. Some studies have investigated the chemical and kinetic properties of the enzyme glyceraldehyde 3-phosphate dehydrogenase to identify any changes in the regulation of glycolysis and sperm motility. In contrast, there are few studies analyzing the genetic basis of hypokinesis. For this reason, we investigated the glyceraldehyde 3-phosphate dehydrogenase gene in human sperm to evaluate whether asthenozoospermia was correlated with any changes in its expression. Semen examination and glyceraldehyde 3-phosphate dehydrogenase gene expression studies were carried out on 116 semen samples divided into two groups - Group A consisted of 58 normokinetic samples and Group B of 58 hypokinetic samples. Total RNA was extracted from spermatozoa, and real-time PCR quantification of mRNA was carried out using specific primers and probes. The expression profiles for the Groups A and B were very similar. The mean delta Ct was as follows - Group A, 5.79 ＋ 1.04; Group B, 5.47 ＋ 1.27. Our study shows that in human sperm, there is no difference in glyceraldehyde 3-phosphate dehydrogenase gene expression between samples with impaired motility and samples with normal kinetics. We believe that this study could help in the understanding of the molecular mechanisms of sperm kinetics, suggesting that hypomotility may be due to a possible posttranscriptional impairment of the control mechanism, such as mRNA splicing, or to posttranslational changes.展开更多
BACKGROUND： Studies have shown that the Repeat superfamily, could be related to neural LRRN3, a member of the Neuron Leucine-Rich development, differentiation, information transmission, and other functions, but most ...BACKGROUND： Studies have shown that the Repeat superfamily, could be related to neural LRRN3, a member of the Neuron Leucine-Rich development, differentiation, information transmission, and other functions, but most studies have focused on nucleic acid levels and few have reported on LRRN3 protein levels. OBJECTIVE： To prepare rabbit anti-rat LRRN3 polyclonal antibody and to observe protein tissue expression profiles. DESIGN, TIME AND SEI-rlNG： In vitro, molecular, biological experiments were performed from October 2007 to April 2009 in Laboratory of Neurobiology at Xiangya School of Medicine, Central South University. MATERIALS： Immunization antigen, namely rat MaI-LRRN3C-His recombinant protein, was provided by the Laboratory of Neurobiology at Xiangya School of Medicine, Central South University. METHODS： Rat Mal-LRRN3C-His recombinant protein was used to immunize male, New Zealand rabbits, and rabbit anti-rat LRRN3 polyclonal antibody was prepared. MAIN OUTCOME MEASURES： Antibody purification was conducted using Protein A affinity chromatography, and the LRRN3 anti-serum titer was identified using enzyme-linked immunosorbent assay. Immunohistochemical techniques and Western blot preliminary tests were used to determine LRRN3 protein expression profiles in adult rats. RESULTS： A highly purified rabbit anti-rat LRRN3 polyclonal antibody was obtained. Western Blot results from rat brain total protein revealed a band at 79 kD, which was consistent with the size of LRRN3. Immunohistochemistry results showed that protein was mainly expressed in the central nervous system, and no significant positive signals were observed in other tissues. Positive cells included neurons of cerebral cortex and hippocampal dentate gyrus granule cell layer, and cerebellar Purkinje cells. There was no positive expression in glial cells. CONCLUSION： Rabbit anti-rat LRRN3 polyclonal antibody was successfully prepared at a high purity from the prokaryotic-expressed MaI-LRRN3C-His recombinant protein, which served as an antigen. Rat LRRN3 protein was primarily expressed in cerebral cortex neurons, hippocampal dentate gyrus granule cell layer neurons, and cerebellar Purkinje cells.展开更多
B-3 exopolysaccharide is extracted from the Antarctic psychrophilic bacterium Psychrobacter sp. B-3. We have previously shown that it activates macrophages and affects their immunoregulatory activities. To determine w...B-3 exopolysaccharide is extracted from the Antarctic psychrophilic bacterium Psychrobacter sp. B-3. We have previously shown that it activates macrophages and affects their immunoregulatory activities. To determine what genes are affected during this process, we detected the genes differentially expressed in cells of RAW264.7 macrophages treated with B-3 exopolysaccharide by transcriptomic analysis. B-3 exopolysaccharide treatment caused differential expression of 420 genes, of which 178 were up-regulated and 242 were down-regulated. These genes were shown to be involved in many aspects of cell function, mainly metabolism and immunity. Genes were enriched in multiple immune-related pathways, and the most significantly enriched genes were involved in antigen processing and presentation pathways. The pathway in which differentially expressed genes were the most significantly enriched was the metabolic pathway; specifically, the expression of many metabolic enzyme genes was altered by B-3 exopolysaccharide treatment. Additionally, the genes involved in metabolisms of amino acids, carbohydrates, lipids and nucleotides, varied to certain degrees. B-3 exopolysaccharide, therefore, appears to directly affect the immune function of RAW264.7 macrophages as an immunostimulant, or to indirectly change intracellular metabolism. This is the first study to determine the effect of an Antarctic psychrophilic bacterial exopolysaccharide on RAW264.7 macrophages. Our findings provide an important reference for research into the regulation of macrophage immune function by different polysaccharides.展开更多
As a member of the Frizzled family, Frizzled3 (FZD3) is a receptor of the canonical Wnt signaling pathway and plays a vital role in mammalian hair follicle developmental processes. However, its effects on wool traits ...As a member of the Frizzled family, Frizzled3 (FZD3) is a receptor of the canonical Wnt signaling pathway and plays a vital role in mammalian hair follicle developmental processes. However, its effects on wool traits are not clear. The objectives of this study were to identify the single nucleotide polymorphisms (SNPs) and the expression patterns of FZD3 gene, and then to determine whether it affected wool traits of Chinese Merino sheep (Xinjiang Type) or not. PCR-single stranded conformational polymorphism (PCR-SSCP) and sequencing were used to identify mutation loci, and general linear model (GLM) with SAS 9.1 was used for the association analysis between wool traits and SNPs. Quantitative real-time PCR (qRT-PCR) was used to investigate FZD3 gene expression levels. The results showed that six exons of FZD3 gene were amplified and two mutation loci were identified in exon 1 (NC_019459.2: g.101771685 T>C (SNP1)) and exon 3 (NC_019459.2: g.101810848, A>C (SNP2)), respectively. Association analysis showed that SNP1 was significantly associated with mean fiber diameter (MFD)(P=0.04) and live weight (LW)(P=0.0004), SNP2 was significantly associated with greasy fleece weight (GFW)(P=0.04). The expression level of FZD3 gene in skin tissues of the superfine wool (SF) group was significantly lower (P<0.05) than that of the fine wool (F) group. Moreover, it had a higher expression level (P<0.01) in skin tissues than in other tissues of Chinese Merino ewes. While, its expression level had a fluctuant expression in skin tissues at different developmental stages of embryos and born lambs, with the highest expression levels (P<0.01) at the 65th day of embryos. Our study revealed the genetic relationship between FZD3 variants and wool traits and two identified SNPs might serve as potential and valuable genetic markers for sheep breeding and lay a molecular genetic foundation for sheep marker-assisted selection (MAS).展开更多
AIM To obtain greater antigenicity of HCV NS3 protein. METHODS The HCV NS3 cDNA fragment was amplified by reverse transcription polymerase chain reaction from the sera of the HCV infected patients. The DNA sequence...AIM To obtain greater antigenicity of HCV NS3 protein. METHODS The HCV NS3 cDNA fragment was amplified by reverse transcription polymerase chain reaction from the sera of the HCV infected patients. The DNA sequence was determined by dideoxy mediated chain termination method using T7 polymerase. HCV NS3 protein was expressed in E. coli . RESULTS Sequence analysis indicated that the HCV isolate of this study belongs to HCV Ⅱ; SDS PAGE demonstrated an M r 23800 and an M r 22000 recombinant protein band which amount to 14% and 11% of the total bacterial proteins separately. Western blotting and ELISA showed NS3 protein possessed greater antigenicity. CONCLUSION Recombinant HCV NS3 protein was expressed successfully, which provided the basis for developing HCV diagnostic reagents.展开更多
To find optimal conditions for expressing the soluble form of sFv-2F3 and to study the purification and property of its derivative Se-sFv-2F3,the preferred expression conditions were investigated by means of orthogona...To find optimal conditions for expressing the soluble form of sFv-2F3 and to study the purification and property of its derivative Se-sFv-2F3,the preferred expression conditions were investigated by means of orthogonal design. These culture conditions included incubation temperature,inducer concentration, induction time and cell concentration. The evaluation of expression was accomplished by the analysis of whole cell lysates and the yield of soluble sFv-2F3 was calculated according to the analysis of Profinder(FTI-500,Pharmacia). The purification procedure was carried out via a two-step purification procedure consisting of ion-exchange chromatography,followed by immobilized metal affinity chromatography(IMAC). The antioxidant efficacy of Se-sFv-2F3 was demonstrated by the determination of the content of the main product of lipid peroxidation,MDA,the viability of cells and the activity of LDH. We obtained the preferred culture conditions to grow the engineered bacteria and the procedure for preparing soluble sFv-2F3 and confirmed the antioxidant efficacy of Se-sFv-2F3.展开更多
Circadian clock genes are crucial for generating and sustaining most rhythmic daily functions in the animal kingdom,which entrain the rhythms of biochemical,physiological,and behavioural processes.To better understand...Circadian clock genes are crucial for generating and sustaining most rhythmic daily functions in the animal kingdom,which entrain the rhythms of biochemical,physiological,and behavioural processes.To better understand the molecular oscillations of the circadian rhythms in darkbarbel catfish(Pelteobagrus vachellii),we isolated and characterized two circadian clock genes in P.vachellii,period 1(per1),and period 3(per3).The circadian clock gene per1 was found to encode a 1428-amino acid polypeptide,including PER-ARNT-SIM(PAS)dimerisation domains,a PAS-associated C-terminal motif(PAC),a short mutable domain(S/M),and a nuclear export signal(NES).The 4902-bp per3 cDNA includes an open reading frame encoding a 1292-amino acid residue polypeptide with a PER-ARNT-SIM(PAS)domain,cytoplasmic localisation domain(CLD),interaction site(TIS),and a nuclear localisation signal(NLS).The per1 and per3 gene was constitutively expressed in all examined tissues.Moreover,per1 expression within a light/dark cycles showed rhythmic expression in the diencephalon,brain,liver and intestine,with the acrophase at 15:15,12:52,7:51 and 12:55,respectively.Daily expression of per3 was rhythmic in the diencephalonbrain,liver and intestine,with the acrophase at 8:15,9:54,10:39,and 10:25 h,respectively.These findings expand our understanding of circadian mechanism at the molecular level in this species.展开更多
基金supported by China Academy of Railway Sciences Corporation Limited(No.2021YJ127).
文摘Artificial intelligence,such as deep learning technology,has advanced the study of facial expression recognition since facial expression carries rich emotional information and is significant for many naturalistic situations.To pursue a high facial expression recognition accuracy,the network model of deep learning is generally designed to be very deep while the model’s real-time performance is typically constrained and limited.With MobileNetV3,a lightweight model with a good accuracy,a further study is conducted by adding a basic ResNet module to each of its existing modules and an SSH(Single Stage Headless Face Detector)context module to expand the model’s perceptual field.In this article,the enhanced model named Res-MobileNetV3,could alleviate the subpar of real-time performance and compress the size of large network models,which can process information at a rate of up to 33 frames per second.Although the improved model has been verified to be slightly inferior to the current state-of-the-art method in aspect of accuracy rate on the publically available face expression datasets,it can bring a good balance on accuracy,real-time performance,model size and model complexity in practical applications.
基金supported by the National Natural Science Foundation of China(no.82004490)Hunan Natural Science Foundation Youth Project:2021JJ40402the Innovation Platform Open Fund project of the Hunan Education Department(no.20K091).
文摘Objective To investigate the effect of electroacupuncture(EA)on microRNA(miRNA)expression spectrum and PI3K/Akt/mTOR signaling pathway in uterine tissue of rats with primary dysmenorrhea(PDM),and to explore the potential mechanism of EA in the treatment of PDM.Methods Thirty female SD rats,weighted(200±20)g were randomly divided into control group,model group and EA group,10 rats in each group.By using subcutaneous injection of estradiol diphenhydrate combined with intraperitoneal injection of oxytocin,PDM models were established.Rats in the EA group received EA at“Sanyinjiao”(SP6)and“Guanyuan”(CV4)at dense waves and a frequency of 50 Hz,once a day,20 min each time,for 10 consecutive days.After the 10-day intervention,samples were collected and transmission electron microscopy was used to observe the ultrastructural changes of the cells in uterine tissue in each group.With RNA-seq method,the changes of miRNA expression spectrum in rat uterine tissue were detected.Bioinformatics analysis such as GO functional annotation and KEGG pathway was performed according to differentially expressed miRNAs.Differentially expressed miRNAs were verified by qRT-PCR.Endometrial stromal cells were selected as the target cells and transfected;and they were divided into control group,NC mimics group,mimic miR-144–3p group,NC inhibitor group and inhibitor miR-144–3p group.The apoptosis was determined by using flow cytometrydetect apoptosis,the miRNA and protein expression of PI3K/Akt/mTOR signaling pathway were detected by qRT-PCR and Western blot in each group separately.Results 1.Transmission electron microscope.(1)Control group:no obvious morphological changes in the uterine tissue.(2)Model group:fibroblasts in uterine tissue were irregular,the edema was presented in cellular cytoplasm,the nuclei were irregular and mitochondria swollen seriously;the rough endoplasmic reticulum was expanded moderately.(3)EA group:fibroblasts were spindle-shaped and pyknotic,the cytoplasm increased in electron density,the nuclei were slightly irregular and pyknotic,mitochondria were oval in shape,with little swelling and vacuolation;the rough endoplasmic reticulum was expanded slightly and retained,with a small amount of degranulation.2.Compared with the control group,there were 26 differentially expressed miRNAs in the uterine tissue of rats with PDM.After EA intervention,the expression of miR-144–3p was significantly up-regulated.GO functional analysis of differentially expressed miRNAs in PDM rats after EA showed that the biological functions involved calcium transmembrane transporter activity,mitogen-activated protein kinase binding,epithelial cell migration,tissue migration,etc.3.KEGG pathway analysis showed that PI3K/Akt signaling pathway,MAPK signaling pathway and calcium signaling pathway were enriched.Mimic miR-144-3p increased the apotosis of endometrial stromal cells,and decreased the mRNA and protein expression of PI3K,Akt,and mTOR(P<0.01).Conclusion EA can optimize the cell morphology in the uterine tissue of rats with PDM and affect the miRNA expression spectrum,which may be associated with the effect of EA for up-regulating miR-144–3p expression in endometrial stromal cells,suppressing PI3K/Akt/mTOR signaling pathway and causing apoptosis.
基金Supported by the National Natural Science Foundation of China,No.82160213 and No.U22A2022the Guangxi Natural Science Foundation,No.2023GXNSFAA026029,No.2024GXNSFBA010059,and No.2024GXNSFAA010079。
文摘BACKGROUND Laryngeal squamous cell carcinoma(LSCC)is a prevalent head and neck malignancy with suboptimal survival rates due to late detection and therapeutic resistance.AIM To investigate chaperonin-containing TCP1 subunit 3(CCT3)expression and its clinical implications,and its effects on LSCC cell growth.METHODS Systematic data on CCT3 mRNA expression were collected from biomedical databases,and integrated further based on the standardized mean difference and the summary receiver operating characteristic curve.Single-cell RNA-seq data were mined to validate the expression level of CCT3 mRNA.In-house immunohistochemistry was performed to explore the CCT3 protein levels of clinical LSCC samples and their relationship with clinical parameters.The growth function of LSCC cell was analyzed using CRISPR knockout screening.CCT3-related signaling pathway analyses were conducted using gene set enrichment analysis.Protein-protein interaction network construction was performed to identify hub genes.RESULTS CCT3 mRNA was significantly overexpressed in 269 LSCC tissues cases across multiple independent datasets(standardized mean difference=32,area under the curve=0.93);At the translational level,the in-house immunohistochemical analysis further demonstrated the consistent upregulation of CCT3 protein in 88 cases of LSCC samples(58 non-LSCC samples vs 30 LSCC samples,P=1.4e^(-14)).Analysis of clinical parameters showed no significant differences among subgroup.Functional characterization with clustered regularly interspaced short palindromic repeats--mediated gene knockout revealed that depletion of CCT3 potently suppressed LSCC cell viability in vitro.Gene set enrichment analysis indicated that CCT3 was markedly associated with several key oncogenic pathways,including extracellular matrix receptor interaction and cell cycle regulation pathways.CONCLUSION CCT3 upregulation in LSCC may influence cellular growth by regulating related pathways,indicating its potential as a biomarker and therapeutic target for LSCC.
基金This work was supported in part by grants from the State Key Basic Research Program (No.39825114) and the NationalNatural Science Foundation of China (No.39770379).
文摘Objective： To investigate the expression of Glypican-3 gene in （α-fetoprotein （AFP）-negative hepatocellular carcinoma （HCC） and clarify whether Glypican-3 expression is a useful parameter for the diagnosis of hepatocelhllar carcinoma （HCC）, especially for AFP-negative ones. Methods： Forty-one specimens of AFP-negative hepatocellular carcinoma and para-carcimoma tissue were studied for the expression of Glypican-3 by Northern blot. The expression of Glypican-3 protein was detected immunohistochemically with specific polyclonal antibody. Results： Northern blot analysis indicated that the expression of Glypican-3 mRNA was intensively detected in 30 of 41 AFP-negative HCC （73.17%）. In contrast, Glypican-3 mRNA was only weakly detected in 4 of the surrounding non-tumor liver tissues. There was significant difference in the Glypican-3 mRNA expression in large tumors （〉5 cm） （79.31%） and in small tumors （〈5 cm） （41.67%） （P〈0.01）. Gypican-3 mRNA was more frequently overexpressed in poorly differentiated HCC than in well differentiated ones （76.47% vs. 42.86%, P〈0.05）. The Glypican-3 expression was not correlated with age. sex. ttbsAg seropositivity, fibroeapsule, portal venous embolus and intrahepatic metastasis. The overexpression of Glypican-3 protein in HCC was targeted in tumor cells, not in bile duct cells and other interstitial cells. Conclusion： Glypican-3 was specially overexpressed in AFP-negative HCC, and its expression was closely correlated with the tumor size and tumor grade. Glypican-3 gene may play important roles in hepatocareinogenesis, and can be used as a new biochemical marker of HCC, especially for AFP-negative HCC.
文摘Zymography and in situ hybridization were used to investigate matrixmetalloproteinase -2, -9 (MMP -2, MMP-9) activities and expressions of MMP -2, -9 and TIMP1, -2, -3 (tissue inhibitors of matrix metallo-proteinases) mRNA in the rat uterus during estrouscycle. The relative activity was semiquanted by using densitometric analysis. The MMP-2(67 kDa) activity in every stage during estrpus cycle was detected by zymography. MMP-2activity was highest at proestrus; higher at estrus and metaestrus; lowest at diestrus. Throughin situ hybridization, MMP -2, -9, TIMP -1~ -3 mRNA mainly in hasal stroma cells of uterineendometrium were detected. The positive signals of MMP -2 and -9 mRNAs in hasal stromacells were shown stronger at proestrus, estrus and metaestrus while they showed the weakest atdiestrus. The expression of MMP -2 mRNA coincided with MMP -2 activity change. MMP-2and -9 mRNAs were also highly expressed in uterine circular muscle at estrus. Weak signals ofMMP -9 mRNA were detected in uterine luminal and glandular epithelial cells at estrus.TIMP -1 mRNA in hasal stroma cells was shown as the strongest expression at estrus andmetaestrus; stronger at proestrus and the weakest at diestrus. TIMP-2 mRNA in basal stromacells was stronger at estrus and diestrus; weaker at proestrus and metaestrus. TIMP -1 and -2mRNAs were also highly expressed in uterine luminal and glandular epithelial cells at estrus.TIMP -3 mRNA in hasal stroma cells revealed the strongest expression at estrus; stronger atdiestrus and metaestrus and showed the weakest at proestrus. The mRNA was also highlyexpressed in uterine circular muscle at estrus. In short, our present results provide evidencethat MMP -2, -9 and TIMP -1~ -3 were involved in rat uterine endometrium reconstructionduring estrous cycle.
基金Supported by Cultivation for New Varieties of Genetically Modified Organisms Technology Projects(2008ZX08001-004)Key Projects of Nanjing Xiaozhuang University(2007NXY01)Natural ScienceFoundation for Jiangsu Province Universities(08KJD180011)~~
文摘[Objective] The aim was to clone CBF3 gene from Arabidopsis thaliana and construct plant expression vector pCAMBIA1301-Rd29A-CBF3.[Method] CBF3 gene and stress-inducible promoter Rd29A were amplified from the genomic DNA of A.thaliana for the construction of plant expression vector.[Result] Sequencing results showed that the cloned CBF3 gene had 750 bp,and showed 100% identity with the sequence published on GenBank.The promoter Rd29A had 1 425 bp,and showed 100% identity with the sequence published on GenBank.[Conclusion] Based on the binary vector pCAMBIA1301,the plant expression vector pCAMBIA1301-Rd29A-CBF3 was constructed successfully,which could materially improve the salt resistance,drought-tolerance,cold resistance of plants.
文摘AIM To detect the expression of caspase 3gene in primary human hepatocellular carcinoma（HCC）and investigate its relationship to p21<sup>WAF1</sup>gene expression and HCC apoptosis.METHODS In situ hybridization was employedto determine caspase 3 and p21<sup>WAF1</sup>expression inHCC.In situ end-labeling was used to detecthepatocytic apoptosis in HCC.RESULTS Twenty-one of 39（53.8%）cases ofHCC were found to express caspase 3transcripts,while 45.2% of HCC failed toexpress caspase 3.Non-cancerous adjacent livertissues showed more positive caspase 3（87.5%,7/8）as compared with HCC（P【0.05）.The expression of caspase 3 is correlated withHCC differentiation,72.2%（13/18）ofmoderately to highly differentiated HCC showedcaspase 3 transcripts positive,while only 38.1%of poorly differentiated HCC harbored caspase 3transcripts（P【0.05）.No relationship wasfound between caspase 3 expression and tumorsize or grade or metastasis,although 52.5%（5/8）of HCC with metastasis were caspase 3positive and a little higher than that with nometastasis（51.6%,P】0.05）.Expression of caspase 3 alone did not affect the apoptosisindex（AI）of HCC.The AI was 7.12%o in caspase3-positive tumors（n=21）,while in caspase 3-negative cases（n=18）6.59%0（P】0.05）.Expression of caspase 3 clearly segregated withp21<sup>WAF1</sup>positive tumors as compared withp21<sup>WAF1</sup>-negative cases（16 of 23,69.6% versus5 of 16,31.3%）with statistical significance（P=0.017）.In the cases with positive caspase 3and negative p21<sup>WAF1</sup>,the Al was found slightlyhigher,but with no statistical significance,thanthat with expression of p21<sup>WAF1</sup>and caspase 3（7.21‰ vs 6.98‰,P】0.05）.CONCLUSION Loss of caspase 3 expressionmay contribute to HCC carcinogenesis,althoughthe expression of caspase 3 does not correlatewell with cell apoptosis in HCC.p21<sup>WAF1</sup>may bemerely one of the inhibitors which can reducecaspase 3 mediated cell apoptosis in HCCs.
基金supported by grants from the National Key R & D Program of China (Grant No. 2017YFA0505600-04)National Natural Science Foundation of China (Grant No. 81372887, 81572403, and 81772863)
文摘Objective: To elucidate the role and prognostic significance of lymphocyte activation-gene-3(LAG-3) in soft tissue sarcoma(STS).Methods: The expression of LAG-3 in patient and matched normal blood samples was analyzed by flow cytometry. The localization and prognostic values of LAG-3^+ cells in 163 STS patients were analyzed by immunohistochemistry. In addition, the expression of tumor-infiltrating CD3^+ T, CD4^+ T, and CD8^+ T cells and their role in the prognosis of STS were evaluated by immunohistochemistry. The effect of LAG-3 blockade was evaluated in an immunocompetent MCA205 fibrosarcoma mouse model.Results: Peripheral CD8^+ and CD4^+ T cells from STS patients expressed higher levels of LAG-3 than those from healthy donors.LAG-3 expression in STS was significantly associated with a poor clinical outcome(P = 0.038) and was correlated with high pathological grade(P < 0.001), advanced tumor stage(P = 0.016). Additionally, LAG-3 expression was highly correlated with CD8^+ T-cell infiltration(r = 0.7034, P < 0.001). LAG-3 was expressed in murine tumor-infiltrating lymphocytes, and its blockade decreased tumor growth and enhanced secretion of interferon-gamma by CD8^+ and CD4^+ T cells.Conclusions: LAG-3 blockade may be a promising strategy to improve the effects of targeted therapy in STS.
文摘AIM To investigate the mechanisms ofsalvianolic acid A（SA-A）against liver fibrosisin vitro.METHODS NIH/3T3 fibroblasts were culturedroutinely,and incubated with 10<sup>-4</sup>mol/L-10<sup>-7</sup>mol/L SA-A for 22 h.The cell viability wasassayed by[<sup>3</sup>H]proline incorporation,cellproliferation by[<sup>3</sup>H]TdR incorporation,cellcollagen synthetic rate was measured with[<sup>3</sup>H]proline impulse and collagenase digestionmethod.The total RNA was prepared from thecontrol cells and the drug treated cellsrespectively,and α（1）I pro-collagen mRNAexpression was semi-quantitatively analyzedwith RT-PCR.RESULTS 10<sup>-4</sup>mol/L SA-A decreased cellviability and exerted some cytotoxiciy,while10<sup>-5</sup>mol/L-10<sup>-7</sup>mol/L SA-A did not affect cellviability,but inhibited cell proliferationsignificantly,and 10<sup>-6</sup>mol/L SA-A had the besteffect on cell viability among theseconcentrations of drugs.10<sup>-5</sup>mol/L-10<sup>-6</sup>mol/LSA-A inhibited intracellular collagen syntheticrate,but no significant influence on extracellularcollagen secretion.Both 10<sup>-5</sup>mol/L and10<sup>-6</sup>mol/L SA-A could decrease α（1）I pro-collagen mRNA expression remarkably.CONCLUSION SA-A had potent action againstliver fibrosis.It inhibited NIH/3T3 fibroblastproliferation,intracellular collagen syntheticrate and type I pro-collagen gene expression,which may be one of the main mechanisms of thedrug.
基金Supported by the National Natural Science Foundation of China,No.300330540
文摘AIM:To investigate the expression of co-stimulatorymolecule B7-H3 in gastric carcinoma and adenomatissue as well as normal gastric tissue and to explore therelationship between B7-H3 expression and pathologicalfeatures and prognosis of gastric carcinoma.METHODS:B7-H3 expression was detected in 102samples of human gastric carcinoma and 10 samples ofgastric adenoma and 10 samples of normal gastric tissueby immunohistochemical assay.Correlation betweenthe expression of B7-H3 and the patients'age,sex,gastric carcinoma locus,tumor size,tissue type,tumorinfiltration depth,differentiation degree,lymph nodemetastasis,and survival time was analyzed.RESULTS:B7-H3 was expressed in all gastric adenomasamples and in 58.8% samples of gastric carcinoma.B7-H3 expression in gastric carcinoma samples wasnot related with the patients'age,sex,lymph nodemetastasis,and tumor size(P>0.05),but with thesurvival time,infiltration depth of tumor and tissue type.CONCLUSION:Detection of B7-H3 expression in gastriccarcinoma tissue is beneficial to the judgment of theprognosis of gastric carcinoma patients and the choice oftreatment.
文摘The invertebrate parvovirus Bombyx mori Densonucleosis Virus type 3 （China isolate）, named BmDNV-3, is a kind of bidensovirus. It is a new type of virus with unique replication mechanisms. To investigate the effects of the NS3 gene during viral DNA replication, a pair of primers was designed for amplifying NS3 gene of Bombyx mori densovirus （China isolate）. Gene NS3 amplified was cloned into a prokaryotic expression vector pET-30a and the donor plasmid pFastBacHTe, respectively. The NS3 protein was expressed in Escherichia coli BL21. The pFastBacHTe-NS3 was transformed to E. coli DHIOBac. The recombinant bacmid baculoviruses （rBacmid-EGFP-NS3） isolated from the white colonies were transfected into BmN-4 cells using a transfection reagent. BraN-4 cells were infected with recombinant virus to express fusion proteins. The expression of fusion protein around 30 kDa in E. coli BL21 was identified by SDS-PAGE, Western blotting, and mass spectrometry. The expressed NS3 protein by B. mori nucleopolyhedrovirus bacmid system was confirmed by Western blotting using an anti-NS3 polyclonal antibody. And about 45 kDa protein was found. The expressed fusion protein was smaller than the expected size of EGFP-NS3, 55 kDa. Western blotting analysis indicated that EGFP-NS3 protein was expressed in infected larvae with smaller molecular size.
基金Supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico(CNPqChammas R,Smith MAC and Burbano RR)+1 种基金Fundao de Amparoà Pesquisa do Estado de So Paulo (FAPESPLeal MF and Calcagno DQ)
文摘AIM:To evaluate for the first time the protein and mRNA expression of 14-3-3εin gastric carcinogenesis.METHODS:14-3-3εprotein expression was determined by western blotting,and mRNA expression was examined by real-time quantitative RT-PCR in gastric tumors and their matched non-neoplastic gastric tissue samples.RESULTS:Authors observed a significant reduction of 14-3-3εprotein expression in gastric cancer(GC)samples compared to their matched non-neoplastic tissue.Reduced levels of 14-3-3εwere also associated with diffuse-type GC and early-onset of this pathology.Our data suggest that reduced 14-3-3εmay have a role in gastric carcinogenesis process.CONCLUSION:Our results reveal that the reduced 14-3-3εexpression in GC and investigation of 14-3-3ε interaction partners may help to elucidate the carcino-genesis process.
文摘BACKGROUND Serpin peptidase inhibitor,clade A member 3(SERPINA3)belongs to the serpin family with an inhibitory activity against proteases.Its aberrant expression has been observed in a wide range of tumor cells.However,its clinical significance and biological function in endometrial cancer have been rarely studied.We designed a study to determine the levels of SERPINA3 and its significance in patients with endometrial cancer.AIM To investigate the clinical significance and role of SERPINA3 expression in endometrial cancer cells.METHODS Eighty endometrial tissue samples collected from patients with endometrial cancer were included in an observation group and 80 paraffin-embedded tissues samples collected from patients with normal endometrial tissues undergoing myomectomy were employed as a control group between January 2014 and December 2018.The expression of SERPINA3 mRNA was detected by quantitative polymerase chain reaction(PCR)for all endometrial tissues included in the study.RESULTS The positive expression rate of SERPINA3 protein in endometrial cancer cells was 71.25%in the observation group,which was significantly higher than that in the control group(31.25%;P<0.05).There was no correlation between SERPINA3 protein in endometrial cancer cells and the age range at which women experienced menopause(P>0.05).However,it was associated with pathological grade,clinical stage,vascular invasion,and lymph node metastasis(P<0.05).Pathological grade,clinical stage,vascular invasion,and lymph node metastasis were independent prognostic factors for endometrial cancer.CONCLUSION The follow-up study of SERPINA3 can be used as a prognostic biomarker for endometrial cancer and as one of the targets for bio-targeted therapy for endometrial cancer.
文摘It has been suggested that the energy required for sperm motility is produced by oxidative phosphorylation while glycolysis seems to be an important source for ATP transmission along the flagellum. Some studies have investigated the chemical and kinetic properties of the enzyme glyceraldehyde 3-phosphate dehydrogenase to identify any changes in the regulation of glycolysis and sperm motility. In contrast, there are few studies analyzing the genetic basis of hypokinesis. For this reason, we investigated the glyceraldehyde 3-phosphate dehydrogenase gene in human sperm to evaluate whether asthenozoospermia was correlated with any changes in its expression. Semen examination and glyceraldehyde 3-phosphate dehydrogenase gene expression studies were carried out on 116 semen samples divided into two groups - Group A consisted of 58 normokinetic samples and Group B of 58 hypokinetic samples. Total RNA was extracted from spermatozoa, and real-time PCR quantification of mRNA was carried out using specific primers and probes. The expression profiles for the Groups A and B were very similar. The mean delta Ct was as follows - Group A, 5.79 ＋ 1.04; Group B, 5.47 ＋ 1.27. Our study shows that in human sperm, there is no difference in glyceraldehyde 3-phosphate dehydrogenase gene expression between samples with impaired motility and samples with normal kinetics. We believe that this study could help in the understanding of the molecular mechanisms of sperm kinetics, suggesting that hypomotility may be due to a possible posttranscriptional impairment of the control mechanism, such as mRNA splicing, or to posttranslational changes.
基金the National Natural Science Foundation of China,No.30600224,30700438,30600636No.39 Grant by China Postdoctoral Science Foundation,No.20060390886
文摘BACKGROUND： Studies have shown that the Repeat superfamily, could be related to neural LRRN3, a member of the Neuron Leucine-Rich development, differentiation, information transmission, and other functions, but most studies have focused on nucleic acid levels and few have reported on LRRN3 protein levels. OBJECTIVE： To prepare rabbit anti-rat LRRN3 polyclonal antibody and to observe protein tissue expression profiles. DESIGN, TIME AND SEI-rlNG： In vitro, molecular, biological experiments were performed from October 2007 to April 2009 in Laboratory of Neurobiology at Xiangya School of Medicine, Central South University. MATERIALS： Immunization antigen, namely rat MaI-LRRN3C-His recombinant protein, was provided by the Laboratory of Neurobiology at Xiangya School of Medicine, Central South University. METHODS： Rat Mal-LRRN3C-His recombinant protein was used to immunize male, New Zealand rabbits, and rabbit anti-rat LRRN3 polyclonal antibody was prepared. MAIN OUTCOME MEASURES： Antibody purification was conducted using Protein A affinity chromatography, and the LRRN3 anti-serum titer was identified using enzyme-linked immunosorbent assay. Immunohistochemical techniques and Western blot preliminary tests were used to determine LRRN3 protein expression profiles in adult rats. RESULTS： A highly purified rabbit anti-rat LRRN3 polyclonal antibody was obtained. Western Blot results from rat brain total protein revealed a band at 79 kD, which was consistent with the size of LRRN3. Immunohistochemistry results showed that protein was mainly expressed in the central nervous system, and no significant positive signals were observed in other tissues. Positive cells included neurons of cerebral cortex and hippocampal dentate gyrus granule cell layer, and cerebellar Purkinje cells. There was no positive expression in glial cells. CONCLUSION： Rabbit anti-rat LRRN3 polyclonal antibody was successfully prepared at a high purity from the prokaryotic-expressed MaI-LRRN3C-His recombinant protein, which served as an antigen. Rat LRRN3 protein was primarily expressed in cerebral cortex neurons, hippocampal dentate gyrus granule cell layer neurons, and cerebellar Purkinje cells.
基金The Important National Science&Technology Specific Projects under contract No.2011ZX8001-003the National Natural Science Fundation of China under contract No.40706053Chinese Polar Environment Comprehensive Investigation&Assessment Programs under contract No.CHINARE2017-01-05
文摘B-3 exopolysaccharide is extracted from the Antarctic psychrophilic bacterium Psychrobacter sp. B-3. We have previously shown that it activates macrophages and affects their immunoregulatory activities. To determine what genes are affected during this process, we detected the genes differentially expressed in cells of RAW264.7 macrophages treated with B-3 exopolysaccharide by transcriptomic analysis. B-3 exopolysaccharide treatment caused differential expression of 420 genes, of which 178 were up-regulated and 242 were down-regulated. These genes were shown to be involved in many aspects of cell function, mainly metabolism and immunity. Genes were enriched in multiple immune-related pathways, and the most significantly enriched genes were involved in antigen processing and presentation pathways. The pathway in which differentially expressed genes were the most significantly enriched was the metabolic pathway; specifically, the expression of many metabolic enzyme genes was altered by B-3 exopolysaccharide treatment. Additionally, the genes involved in metabolisms of amino acids, carbohydrates, lipids and nucleotides, varied to certain degrees. B-3 exopolysaccharide, therefore, appears to directly affect the immune function of RAW264.7 macrophages as an immunostimulant, or to indirectly change intracellular metabolism. This is the first study to determine the effect of an Antarctic psychrophilic bacterial exopolysaccharide on RAW264.7 macrophages. Our findings provide an important reference for research into the regulation of macrophage immune function by different polysaccharides.
基金supported by the National Natural Science Foundation of China (31360543 and 31760655)the earmarked fund for the China Agriculture Research System (CARS-39)+1 种基金the Open Project of Key Laboratory of Genetics Breeding and Reproduction of Xinjiang Cashmere and Wool Sheep, China (2016D03017)the Postdoctoral Science Foundation, China (2017M623287)
文摘As a member of the Frizzled family, Frizzled3 (FZD3) is a receptor of the canonical Wnt signaling pathway and plays a vital role in mammalian hair follicle developmental processes. However, its effects on wool traits are not clear. The objectives of this study were to identify the single nucleotide polymorphisms (SNPs) and the expression patterns of FZD3 gene, and then to determine whether it affected wool traits of Chinese Merino sheep (Xinjiang Type) or not. PCR-single stranded conformational polymorphism (PCR-SSCP) and sequencing were used to identify mutation loci, and general linear model (GLM) with SAS 9.1 was used for the association analysis between wool traits and SNPs. Quantitative real-time PCR (qRT-PCR) was used to investigate FZD3 gene expression levels. The results showed that six exons of FZD3 gene were amplified and two mutation loci were identified in exon 1 (NC_019459.2: g.101771685 T>C (SNP1)) and exon 3 (NC_019459.2: g.101810848, A>C (SNP2)), respectively. Association analysis showed that SNP1 was significantly associated with mean fiber diameter (MFD)(P=0.04) and live weight (LW)(P=0.0004), SNP2 was significantly associated with greasy fleece weight (GFW)(P=0.04). The expression level of FZD3 gene in skin tissues of the superfine wool (SF) group was significantly lower (P<0.05) than that of the fine wool (F) group. Moreover, it had a higher expression level (P<0.01) in skin tissues than in other tissues of Chinese Merino ewes. While, its expression level had a fluctuant expression in skin tissues at different developmental stages of embryos and born lambs, with the highest expression levels (P<0.01) at the 65th day of embryos. Our study revealed the genetic relationship between FZD3 variants and wool traits and two identified SNPs might serve as potential and valuable genetic markers for sheep breeding and lay a molecular genetic foundation for sheep marker-assisted selection (MAS).
文摘AIM To obtain greater antigenicity of HCV NS3 protein. METHODS The HCV NS3 cDNA fragment was amplified by reverse transcription polymerase chain reaction from the sera of the HCV infected patients. The DNA sequence was determined by dideoxy mediated chain termination method using T7 polymerase. HCV NS3 protein was expressed in E. coli . RESULTS Sequence analysis indicated that the HCV isolate of this study belongs to HCV Ⅱ; SDS PAGE demonstrated an M r 23800 and an M r 22000 recombinant protein band which amount to 14% and 11% of the total bacterial proteins separately. Western blotting and ELISA showed NS3 protein possessed greater antigenicity. CONCLUSION Recombinant HCV NS3 protein was expressed successfully, which provided the basis for developing HCV diagnostic reagents.
文摘To find optimal conditions for expressing the soluble form of sFv-2F3 and to study the purification and property of its derivative Se-sFv-2F3,the preferred expression conditions were investigated by means of orthogonal design. These culture conditions included incubation temperature,inducer concentration, induction time and cell concentration. The evaluation of expression was accomplished by the analysis of whole cell lysates and the yield of soluble sFv-2F3 was calculated according to the analysis of Profinder(FTI-500,Pharmacia). The purification procedure was carried out via a two-step purification procedure consisting of ion-exchange chromatography,followed by immobilized metal affinity chromatography(IMAC). The antioxidant efficacy of Se-sFv-2F3 was demonstrated by the determination of the content of the main product of lipid peroxidation,MDA,the viability of cells and the activity of LDH. We obtained the preferred culture conditions to grow the engineered bacteria and the procedure for preparing soluble sFv-2F3 and confirmed the antioxidant efficacy of Se-sFv-2F3.
基金Supported by the National Natural Science Foundation of China(No.31402305)the Sichuan Science and Technology Program(Nos.2017JY0161,2018NZ0119)。
文摘Circadian clock genes are crucial for generating and sustaining most rhythmic daily functions in the animal kingdom,which entrain the rhythms of biochemical,physiological,and behavioural processes.To better understand the molecular oscillations of the circadian rhythms in darkbarbel catfish(Pelteobagrus vachellii),we isolated and characterized two circadian clock genes in P.vachellii,period 1(per1),and period 3(per3).The circadian clock gene per1 was found to encode a 1428-amino acid polypeptide,including PER-ARNT-SIM(PAS)dimerisation domains,a PAS-associated C-terminal motif(PAC),a short mutable domain(S/M),and a nuclear export signal(NES).The 4902-bp per3 cDNA includes an open reading frame encoding a 1292-amino acid residue polypeptide with a PER-ARNT-SIM(PAS)domain,cytoplasmic localisation domain(CLD),interaction site(TIS),and a nuclear localisation signal(NLS).The per1 and per3 gene was constitutively expressed in all examined tissues.Moreover,per1 expression within a light/dark cycles showed rhythmic expression in the diencephalon,brain,liver and intestine,with the acrophase at 15:15,12:52,7:51 and 12:55,respectively.Daily expression of per3 was rhythmic in the diencephalonbrain,liver and intestine,with the acrophase at 8:15,9:54,10:39,and 10:25 h,respectively.These findings expand our understanding of circadian mechanism at the molecular level in this species.
