Objective To investigate the effects of NANOG/deleted in breast cancer 1 (DBC1)pathway on biological behavior of gastric cancer cells.Methods From May 2014 to May 2015,25 patients who underwent gastric cancer resection were selected.The expression of NANOG and DBC1 was detected by real time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting in N-tera,SGC-7901,HGC-27,MKN-45,MGC803,NCI-N87,BGC823 cell lines,normal gastric epithelial cell line GES-1 and gastric cancer tissues.The proliferation,apoptosis and colony formation ability of MKN-45 cells in short hairpin (sh)NANOG-1,shNANOG-2,sh-control and shDBC1 groups were determined by MTT assay,flow cytometry and colony forming assay.The effects on the expression of the two genes in MKN-45 cells were verified by polymerase chain reaction (PCR) in shNANOG,sh-control,shDBC1 and shDBC1+NANOG groups and the effects of down regulation of DBC1 on cell biological behavior were further investigated.The differences in gene expression profile after interference which were screened by gene chips and bioinformatics were analyzed.The mechanism of NANOG regulating DBC1 was explored by Dual-luciferase assay.T test was used for two groups comparison while one-way analysis of variance was for multiple groups.Results NANOG and NANOG mRNA were highly expressed in N-tera cells,which were 1.02±0.08 and 0.95 ±0.03,respectively,and the expressions in SGC-7901,HGC-27,MKN-45 and NCI-N87 cell lines were 0.67±0.03 and 0.64±0.04,0.58±0.02 and 0.28±0.02,0.83±0.03 and 1.04 ± 0.05,and 0.61 ± 0.02 and 0.64 ± 0.08,respectively;no expression was detected in normal gastric epithelial cell line GES-1,and the expressions in MKN-45 cells were the highest in gastric cancer cells (F=21.51 and 85.53,both P＜0.01).The expression of DBC1 in HGC-27,MGC803,NCIN87,SGC-7901,BGC823 and MKN-45 cells were 0.37±0.02,0.33±0.02,0.42±0.01,0.58±0.04,0.33±0.05,and 0.87±0.02,respectively;while there was no expression of NANOG,NANOG mRNA and DBC1 in GES-1 cells.The expression of NANOG mRNA and DBC1 was detected in gastric cancer tissues of 24.0％ (6/25) patients.Compared with that of the sh control group,the apoptosis rates of MKN-45 cells in the shNANOG-1,shNANOG-2 groups were increased ((2.24±0.17)％ vs (6.03±0.24) ％ and (6.95 ± 0.38) ％),and the difference was statistically significant (F =81.18,P ＜ 0.01).Compared with that of the sh-eontrol group,the colony forming abilities of MKN-45 cells in the shNANOG-1 and shNANOG-2 groups were significantly decreased (172.03±6.35 vs 74.32±5.32 and 53.08±3.82),and the difference was statistically significant(F=171.61,P＜0.01).The results of PCR showed that compared with that of sh-eontrol group,the expression levels of NANOG mRNA and DBC1 mRNA in shNANOG group were lower (1.04±0.05 vs 0.54±0.03,1.08±0.08 vs 0.42±0.03),the level of DBC1 mRNA in shDBC1 group was lower (1.08±0.08 vs 0.50±0.04),and the differences were statistically significant (t=9.15,7.37 and 6.06,all P＜0.01).The expression level of NANOG mRNA in shDBC1 + NANOG group was higher (1.04 ± 0.05 vs 3.01 ± 0.08),while the expression level of DBC1 mRNA was lower (1.08 ± 0.08 vs 0.71 ± 0.06),and the difference was statistically significant (t=-20.22 and 3.74,both P＜0.05).The expression level of DBC1 mRNA in shDBC1±NANOG group was higher than that in shDBC1 group (0.71±0.06 vs 0.50±0.04),and the difference was statistically significant (t=4.00,P＜0.05).Bioinformatic analysis showed that DBC1 gene promoter region had the potential NANOG protein binding sites.Dual-luciferase assay indicated NANOG played the role in transcription activation in DBC1 promoter regions.Conclusion NANOG and DBC1 are highly expressed in various gastric cancer cell lines.NANOG may affect the proliferation,apoptosis and colony formation of MKN-45 cells by regulating the expression of DBC1.NANOG/DBC1 pathway may be a promising new target of gastric cancer treatment.

OBJECTIVETo understand the characteristics of embB gene mutation of Mycobacterium tuberculosis (MTB) isolates from tuberculosis patients in Chongqing, and the value of embB306 as a molecular marker used to diagnose ethambutol (EMB)-resistant MTB strains.

METHODSDirect sequencing was used to analyze the polymorphism of embB mutation in 51 EMB-resistant MTB strains and 50 EMB-sensitive MTB strains. And diagnostic testing was used to evaluate the value of embB306 as a molecular marker of EMB -resistant MTB strains as compared with the traditional sensitivity test.

RESULTSAll 34 of 51 EMB-resistant strains (66.7%) and 3 of 51 EMB-sensitive strains (6%) had had embB306 mutation. The embB306 mutation rate in EMB-resistant strains coming from previously treated case was 87.5%, showing significantly higher than that from new cases (48.1%, P < 0.01); embB306 mutation rate was increased with the number of the resistant drugs; embB306 mutation serving as a marker to diagnose EMB-resistant MTB strains comparing with the traditional sensitivity test, had the rate of sensitivity = 66.7%, specificity = 94.0%, accuracy = 80.2% and Youden index = 60.7%.

CONCLUSIONembB306 mutation should be the main mechanism of MTB resistance to EMB in Chongqing, showing an association with the history of the treated and numbers of the resistant drugs. embB306 mutation should be a good marker to diagnose EMB-resistant MTB strains.

China ; DNA Mutational Analysis ; DNA, Bacterial ; genetics ; Genes, Bacterial ; Humans ; Mutation ; Mycobacterium tuberculosis ; drug effects ; genetics ; isolation & purification ; Pentosyltransferases ; genetics ; Tuberculosis, Multidrug-Resistant ; microbiology

Objective To evaluate the safety and efficacy of cross-linked hyaluronic acid Revanesse Ultra for treatment of nasolabial folds in Chinese population .Methods A total of 120 participants were enrolled in this prospective , randomized , positive controlled , non-inferiority clinical trial in accordance to inclusion and ex-clusion criteria , and signed informed consents were obtained .The participants were numbered in the order of en-rollment and randomized into Restylane group and Revanesse group , receiving hyaluronic acid injection to correct bilateral nasolabial folds .Each person received 1 or 2 times of injection ( a touch-up injection could be adminis-tered 4 weeks post the first injection ) .According to the original depth of the wrinkles , no more than 2 ml hyalu-ronic acid was injected into each side .All the participants were followed up at 1, 3, 6, and 12 months after the last injection and standardized photographs were taken at each visit .All the participants were asked to fill the form of local adverse events within the first 15 days after injection.Investigators and the participants both evalu-ated wrinkles based on Wrinkle Severity Rating Scale ( WSRS) .The WSRS score according to the pictures of 6 months post-injection were compared with the pictures which were taken before the injection ( baseline) by in-dependent staff at the end of the trial .One or more grades of WSRS improvement compared with the baseline was considered as effective .Laboratory tests including blood and urine routine , liver and renal function tests were carried out at screening visit and 6 months after injection.Results The baseline features between the two groups were comparable (all P>0.05).There was no significant difference in the WSRS 6 months after injec-tion between the two groups ( P>0.05 ) .There was no significant difference in the WSRS improvement com-pared with baseline between the two groups ( P=0.105 ) .There was no significant difference in the rate of ef-fectiveness between the two groups ( 93.0% vs.96.7%, P=0.431 ) .Two participants reported minor ad-verse events, although both of which might not be associated with the product or procedure .No laboratory test change was found during the trial .Conclusions No severe adverse event associated with the injection material was observed during this clinical trial .According to the result , Revanesse Ultra may have good histocompati-bility and tolerance .It could provide obvious improvement in the nasolabial folds , with effectiveness comparable to that of Restylane .

MicroRNAs(miRNAs)are small non-coding RNAs that are closely associated with the occur-rence and progression of tumors and other diseases.However,miRNAs require high-sensitivity and high-specificity detection due to their low abundance,high sequence homology,and rapid degradation.The RNA-cleaving deoxyribozyme(RCD)is a functional single-stranded DNA molecule that enables specific cleavage of substrates to release miRNAs that can be recycled with the assistance of metal ions,prompting cyclic signal amplification.Recently,developing new methods based on RCD catalytic amplification for miRNA high-sensitivity detection has become the focus of researchers.Based on combing with the various new technologies and materials in miRNA biosensors,this study classifies and reviews the new methods for detecting miRNAs based on deoxyribozyme catalytic amplification developed in recent years.We sepa-rate these miRNA detection strategies into three categories:RCDs combined with DNA self-assembly,i-sothermal amplification,and nanomaterials.We explore the basic principles of each approach,the latest research advancements,and application scenarios in biomedical sensors and medical detection.This re-view provides a foundation and reference for further research of highly sensitive and accurate miRNA de-tection strategies.

This study was aimed to investigate the transfection efficacy of recombinant adeno-associated virus 2/1 (rAAV2/1) on bone marrow mesenchymal stem cells (BMMSCs) at different multiplicities of infection (MOI) and time, and effect of transfection on growth of rat BMMSCs. The rat BMMSCs cultured in vitro were transfected by using rAAV2/1 with enhanced green fluorescent protein (rAAV2/1-EGFP) at MOI of 1 x 10(4), 1 x 10(5) and 1 x 10(6); the EGFP expression was observed by fluorescent microscopy at 3, 7 and 14 days. The viability, proliferation multiple, differentiation ability of daughter cells were detected for evaluating the effect of rAAV2/1 on survival, proliferation and differentiation of BMMSCs and the fluorescence index (FI) were determined by flow cytometry. The results indicated that after transfection with rAAV2/1 for 24 hours the green fluorescence in BMMSCs were observed, but also the fluorescence gradually was enhanced along with prolonging of time, and reached to steady level after 7 days; the viability, proliferation multiple, differentiation ability of BMMSCs transfected by rAAV2/1-EGFP at different MOI showed no significant changes at 3,7 and 14 days (p > 0.05), meanwhile at same MOI the proliferation multiple obviously increased in comparison between 7 day vs 3 day and 14 days vs 7 days (p < 0.01). The flow cytometric detection showed that the transfection efficacy of rAAV2/1-EGFP on BMMSCs and FI increased significantly as the multiplicity of infection and culture time increased (p < 0.05). It is concluded that rAAV2/1-EGFP is able to transfect into BMMSCs effectively, but the transfection efficiency and fluorescence index increase significantly along with increase of multiplicity of infection and culture time. rAAV2/1-EGFP do not affect viability, proliferation multiple and differentiation ability of BMMSCs. rAAV2/1 is a kind of active vector for gene transfer to reform BMMSCs.
Animals ; Bone Marrow Cells ; cytology ; Dependovirus ; genetics ; Genetic Vectors ; Male ; Mesenchymal Stromal Cells ; cytology ; Rats ; Rats, Sprague-Dawley ; Transfection

OBJECTIVETo investigate the clinical features and molecular diagnostic methods of three patients with DiGeorge anomaly.

METHODThe clinical manifestations and immunological features of the three cases with DiGeorge anomaly were analyzed. We detected the chromosome 22q11.2 gene deletion by fluorescence in situ hybridization (FISH).

RESULT(1) CLINICAL MANIFESTATIONS: All three cases had varying degrees of infection, congenital heart disease and small thymus by imaging; two cases had significant hypocalcemia (1.11 mmol/L and 1.22 mmol/L, respectively), accompanied by convulsions; only 1 case had cleft palate and all had no significant facial deformity. (2) Immunological characteristics: All three cases had varying degrees of T-cell immune function defects (percentage of T lymphocytes was 24% - 43%, absolute count was 309 - 803/µl), and levels of immunoglobulin G, A, M, and percent of B lymphocytes and absolute count were normal. (3) Detection of the chromosome 22q11.2 gene deletion: 400 cells of each case were detected. All cells showed two green and one red hybridization signal, indicating the presence of gene deletions in chromosome 22q11.2. (4) OUTCOME: All three cases were treated with thymosin, and appropriate clinical intervention for cardiac malformations, hypocalcemia, and were followed-up for 4 - 18 months, the prognosis was good.

CONCLUSIONDiGeorge anomaly showed diverse clinical manifestations. We should consider the disease if patients had congenital heart disease, thymic hypoplasia, hypocalcemia and/or impaired immune function. FISH for detecting chromosome 22q11.2 gene deletion can be used as accurate and rapid diagnostic method. Thymosin treatment and other clinical intervention may help to improve the prognosis of patients with partial DiGeorge anomaly.

Cells, Cultured ; Chromosome Deletion ; Chromosomes, Human, Pair 22 ; genetics ; DiGeorge Syndrome ; diagnosis ; genetics ; immunology ; Female ; Heart Defects, Congenital ; diagnosis ; genetics ; Humans ; Hypocalcemia ; diagnosis ; genetics ; In Situ Hybridization, Fluorescence ; Infant, Newborn ; Male ; T-Lymphocytes ; immunology ; Thymus Gland ; immunology ; pathology

OBJECTIVETo evaluate the application value of magnetic resonance diffusion-weighted imaging (DWI) combined with routine T2WI sequence in the determination of pathological complete response (pCR) after neoadjuvant therapy for rectal cancer.

METHODSClinical data of 51 cases with locally advanced mid-low rectal cancer undergoing neoadjuvant therapy plus radical resection in the Rectal Cancer Center at The Sixth Affiliated Hospital of Sun Yat-sen University from June 2012 to April 2013 were analyzed retrospectively. Magnetic resonance DWI and T2WI sequences scanning were performed within 1 week before neoadjuvant therapy and within 1 week before operation. Routine single T2WI sequence and DWI combined with T2WI sequence were used separately to predict the residual tumor and to compare with postoperative pathological examination. The prediction values of two methods were compared.

RESULTSOf 51 patients, 12 cases had pathological complete response (pCR). Prediction of DWI combined T2WI sequence was correct in 8 cases of pCR, whose sensitivity and specificity were higher than those of routine single T2WI sequence (66.7%, 94.9% vs. 33.3%, 84.6%). Prediction value of DWI combined T2WI sequence for pCR was significantly higher as compared to routine single T2WI sequence (AUC, 0.808 vs. 0.590, P=0.001).

CONCLUSIONCompared with the routine single T2WI sequence, DWI combined with T2WI sequence can improve the prediction accuracy of pathological complete response.

Adult ; Aged ; Aged, 80 and over ; Diffusion Magnetic Resonance Imaging ; Female ; Humans ; Male ; Middle Aged ; Neoadjuvant Therapy ; Predictive Value of Tests ; Rectal Neoplasms ; pathology ; therapy ; Retrospective Studies ; Sensitivity and Specificity

Cardiovascular and cerebrovascular diseases, usually result from atherosclerosis, has the highest mortality rate globally. Lipid metabolism disorder is the main cause of atherosclerotic cardiovascular and cerebrovascular diseases, which not only lead to acute diseases such as myocardial infarction, stroke, acute pancreatitis, but also chronic kidney disease. In recent years, the advancement of gene therapy technologies has provided novel means for lipid metabolism study, and has also made it possible to cure patients with congenital lipid metabolism abnormalities. Adeno-associatd virus has a wide host range, high safety, low immunogenicity, and especially the ability of long-term stable expression in vivo, making it the preferred delivery tool for gene therapy of monogenic genetic diseases. Alipogene triprivec, also known as Glybera, was approved by the European Medicines Agency in 2012. It is the first gene therapy drug that uses recombinant AAV1 vector to directly deliver a highly active LPL protein S447X mutant to muscle cells for the treatment of patients with hereditary LPL deficiency. To enhance the targeted transduction efficiency of AAV carriers, recombinantAAV8.TBG.hLDLR utilizes the tissue tropsim of AAV8 to liver, meanwhile utilizes a liver specific thyroxine binding globulin promoter to control gene transcription, thereby achieving liver cell specific high expressionof human low-density lipoprotein receptors (LDLR). In patients with familial hypercholesterolemia,AAV8.TBG.hLDLR treatment effectively lower the level of plasma LDL for a long time, thus preventing the occurrence of atherosclerosis.Proprotein convert subunit kexin 9 (PCSK9) is secreted by liver cells. PCSK9 binds and transports LDLR to lysosomes for degradation, preventing the circulation and regeneration of LDLR, leading to accelerated degradation of LDLR and finally resulting in the accumulation of low-density lipoprotein cholesterol in plasma. Using AAV to deliver Cas9 of Staphylococcus aureus and gRNA targeting the Pcsk9 gene can knock out Pcsk9 in mouse liver, leading to a long-term significant decrease in plasma cholesterol levels in mice. Hepatocyte specific angiopoietin related protein 3 (Angptl3) is an endogenous inhibitor of LPL. Using the AAV9 mediated AncBE4max system and the dCas9 mediated single base gene editing system to introduce early termination codons, the knockout of Angptal3 in liver cells was achieved with an average knockout efficiency of 63.3%. After 2-4 weeks of administration in mice, the Angptl3 protein was completely undetectable in the peripheral blood, and serum triglycerides and total cholesterol decreased by 58% and 61%, respectively. Ring finger containing protein 130 (RNF130) is an E3 ubiquitin ligase. Research has shown that overexpression of RNF130 using AAV2/8 leads to ubiquitination degradation and redistribution of LDLR on the cell membrane, significantly reducing LDLR expression on liver cells and increasing plasma LDLC levels, while knocking out Rnf130 gene using the AAV-CRISPR system results in the opposite effect. This AAV mediated RNF130 function study proves that RNF130 is a posttranslational regulatory protein of LDLR and plays an important role in the regulation of serum LDLC. As mentioned above, recently, various lipid-lowering gene therapy drugs carried by different serotypes of adeno-associated virus have been applied in clinic or are undergoing clinical trials, and adeno-associated virus has emerging to be an important tool for lipid metabolism research.This article reviews the new progress of adeno-associated virus vectors in lipid metabolism study and lipid-lowering gene therapy.

Jumonji domain-containing protein D3(JMJD3)is a 2-oxoglutarate-dependent dioxygenase that specif-ically removes transcriptional repression marks di-and tri-methylated groups from lysine 27 on histone 3(H3K27me2/3).The erasure of these marks leads to the activation of some associated genes,thereby influencing various biological processes,such as development,differentiation,and immune response.However,comprehensive descriptions regarding the relationship between JMJD3 and inflammation are lacking.Here,we provide a comprehensive overview of JMJD3,including its structure,functions,and involvement in inflammatory pathways.In addition,we summarize the evidence supporting JMJD3's role in several inflammatory diseases,as well as the potential therapeutic applications of JMJD3 inhibitors.Additionally,we also discuss the challenges and opportunities associated with investigating the functions of JMJD3 and developing targeted inhibitors and propose feasible solutions to provide valuable insights into the functional exploration and discovery of potential drugs targeting JMJD3 for inflammatory diseases.

The current study aims to rapidly and comprehensively profile the chemical composition of Cistanche salsa using direct infusion coupled with MS/MS~(ALL)(DI-MS/MS~(ALL)). The C. salsa extract was directly imported into electrospray ionization(ESI) source of quadrupole time-of-flight(Q-TOF) mass spectrometer with an infusion pump at a flow rate of 10 μL·min~(-1). Acquisition program was applied under negative ionization polarity to collect one MS~1 spectrum(m/z 50-1 200), followed by 1 150 MS~2 spectra with precursor isolation window(m/z 1) amongst mass range m/z 50-1 200. After each MS~2 spectrum was matched to its precursor ion, putative identification was conducted through matching mass spectral data with literature and database. A total of 31 components were identified from C. salsa, including 9 phenylethanoid glycosides, 2 iridoids, 4 saccharides, 9 organic acids, and 7 other compounds, similar to those from C. tubulosa and C. deserticola. In conclusion, DI-MS/MS~(ALL), a facile and reliable analytical tool, can be employed for qualitative analysis of chemical constituents in C. salsa. The research offers a promising strategy to achieve rapid chemome profiling of herbal medicine and provides an alternative source of Cistanches Herba.
Chromatography, High Pressure Liquid ; Cistanche ; Drugs, Chinese Herbal ; Glycosides ; Plants, Medicinal ; Tandem Mass Spectrometry