次氯酸钠去蛋白化对正畸托槽粘接影响的研究进展

口腔正畸过程中的托槽脱落现象难以避免,不仅妨碍了正畸治疗的效果,而且可能延长正畸治疗的周期。次氯酸钠是口腔内科中常用的根管冲洗剂,但其较少应用于口腔正畸临床工作中。次氯酸钠对牙釉质表面的去蛋白化作用可以强化酸蚀的作用,提高牙釉质的表面粘接性能,从而减少正畸治疗过程中托槽脱落的概率。本文就次氯酸钠去蛋白化作用对牙釉质托槽粘接的影响做一综述。

EDTA对年轻恒牙釉质粘接强度的影响

目的研究乙二胺四乙酸二钠(EDTA)对年轻恒牙釉质粘接强度的影响。方法收集手术拔除的牙根未发育完全的上下颌第三磨牙,共180颗,选取全部离体牙牙冠颊面与牙长轴相对平行的釉质面(倾斜角度小于5°)作为实验粘接面,按实验方法不同随机均分为5组,每组36颗,分别进行以下处理:G0(对照组):37%磷酸常规酸蚀;G1:37%磷酸+17%EDTA;G2:37%磷酸+10%EDTA;G3:17%EDTA+37%磷酸,G4:10%EDTA+37%磷酸。以上样本均采用GLUMA通用型粘接剂粘接后,行流动树脂堆砌。使用万能材料试验机进行树脂剪切粘接强度的测试。剔除剪切测试中由于树脂块断裂而未形成树脂-釉质面分离的试件,针对剪切粘接强度采用SPSS21.0统计软件进行统计学分析。结果G0的平均剪切粘接强度(9.80±2.78 MPa)要明显低于G1(11.56±3.30 MPa)、G2(12.01±3.28 MPa)和G4(12.19±3.04 MPa)的剪切粘接强度,其差异都具有统计学意义(P<0.05)。尽管G3的平均剪切粘接强度(10.75±2.85 MPa)要高于G0,但两组之间的强度差异无统计学意义(P=0.261)。此外,G1、G2、G3和G4各组之间的平均剪切粘接强度差异无统计学意义(P>0.05)。结论:酸蚀前、后EDTA去蛋白化处理较单纯酸蚀处理更能增强树脂的剪切粘接强度,但其作用可能会受到EDTA浓度的影响。

Identification of Z-OTU protein during zebrafish oogenesis and early embryogenesis

Zebrafish（Danio rerio） Z-OTU,containing OTU and TUDOR domains,was predicted to be a member of OTU-related protease,a family of the deubiquitylating enzymes（DUBs）.A previous report from our laboratory clearly describes the expression patterns of z-otu mRNA.Here,we characterized the Z-OTU protein during zebrafish oogenesis and early embryogenesis.After prokaryotic expression,the recombinant protein of the OTU domain and GST was purified and injected into rabbits to obtain the polyclonal antibody-anti-Z-OTU,which was used for immunohistochemistry in zebrafish ovaries and embryos.Interestingly,obvious differences existed between the expression patterns of z-otu mRNA and its protein during oogenesis and early embryogenesis.In stage I oocytes,z-otu mRNA was detected in cytoplasm while its protein existed in the germinal vesicle.In addition,its protein was distributed during entire oogenesis,while mRNA was not detected in oocytes at stage IV or mature oocytes.The z-otu mRNA disappeared after midblastula transition（MBT） and its protein gradually decreased after this stage.We inferred that Z-OTU protein,like other OTU-related protease with DUB activity,was required for germinal vesicle breakdown of oocytes during meiosis,germinal vesicle migration,and embryo cleavage maintenance.

Transcriptional changes in epigenetic modifiers associated with gene silencing in the intestine of the sea cucumber,Apostichopus japonicus(Selenka),during aestivation

The sea cucumber, Apostichopusjaponicus, undergoes aestivation to improve survival during periods of high-temperature. During aestivation, the metabolic rate is depressed to reduce the consumption of reserved energy. We evaluated the role of epigenetic modification on global gene silencing during metabolic rate depression in the sea cucumber. We compared the expression of epigenetic modifiers in active and aestivating sea cucumbers. The expression of three genes involved in DNA methylation and chromatin remodeling （DNA （cytosine-5）-methyltransferase l, Methyl-CpG-binding domain protein 2）, and Chromodomain-helicase-DNA-binding protein 5） was significantly higher during aestivation （Days 20 and 40）. Similarly, we observed an increase in the expression of genes involved in histone acetylation （Histone deacetylase 3） and Histone-binding protein RBBP4） during the early （Days 5 and 10） and late phases （Days 20 and 40） of aestivation. There was no change in the expression of KAT2B, a histone acetyltransferase. However, the expression of histone methylation associated modifiers （Histone-arginine methyltransferase CARMER and Histone-lysine N-methyltransferase MLL5） was significantly higher after 5 d in the aestivating group. The results suggest that the expression of epigenetic modifiers involved in DNA methylation, chromatin remodeling, histone acetylation, and histone methylation is upregulated during aestivation. We hypothesize that these changes regulate global gene silencing during aestivation in A. japonicus.

AOF1 is a histone H3K4 demethylase possessing demethylase activity-independent repression function

LSD1 （KDM1 under the new nomenclature） was the first identified lysine-specific histone demethylase belonging to the flavin-dependent amine oxidase family. Here, we report that AOF1 （KDM1B under the new nomenclature）, a mammalian protein related to LSD1, also possesses histone demethylase activity with specificity for H3K4mel and H3K4me2. Like LSD1, the highly conserved SWIRM domain is required for its enzymatic activity. However, AOF1 differs from LSD1 in several aspects. First, AOF1 does not appear to form stable protein complexes containing histone deacetylases. Second, AOF1 is found to localize to chromosomes during the mitotic phase of the cell cycle, whereas LSD1 does not. Third, AOF1 represses transcription when tethered to DNA and this repression activity is independent of its demethylase activity. Structural and functional analyses identified its unique N-terminal Zf-CW domain as essential for the demethylase activity-independent repression function. Collectively, our study identifies AOF1 as the second histone demethylase in the family of flavin-dependent amine oxidases and reveals a demethylase-independent repression function of AOF1.

Targeting histone deacetylases： perspectives for epigenetic-based therapy in cardio-cerebrovascular disease

Although the pathogenesis of cardio-cerebrovascular disease （CCVD） is multifactorial, an increasing number of experimental and clinical studies have highlighted the importance of histone deacetylase （HDAC）-mediated epigenetic processes in the development of cardio-cerebrovascular injury. HDACs are a family of enzymes to balance the acetylation activities of histone acetyltransferases on chromatin remodeling and play essential roles in regulating gene transcription. To date, 18 mammalian HDACs are identified and grouped into four classes based on similarity to yeast orthologs. The zinc-dependent HDAC family currently consists of 11 members divided into three classes （class I, II, and IV） on the basis of structure, sequence homology, and domain organization. In comparison, class III HDACs （also known as the sirtuins） are composed of a family of NAD＋-dependent protein-modifying enzymes related to the Sir2 gene. HDAC inhibitors are a group of compounds that block HDAC activities typically by binding to the zinc-containing catalytic domain of HDACs and have displayed an- ti-inflammatory and antifibrotic effects in the cardio-cerebrovascular system. In this review, we summarize the current knowledge about classifications, functions of HDACs and their roles and regulatory mechanisms in the cardio-cerebrovascular system. Pharmacological tar- geting of HDAC-mediated epigenetic processes may open new therapeutic avenues for the treatment of CCVD.

From Bench to Bedside： Targeting Epigenetics for Cancer Therapy

The initiation and progression of cancer not only involves genetic abnormalities, but also epigenetic alterations, such as DNA methylation and histone modifications. Epigenetics refers to the heritable changes that do not involve any structural changes in the target gene, i.e., DNA sequence and protein sequence. Thus, these epigenetic aberrations are potentially reversible, allowing the malignant cells to revert to a state with more normal characteristics. The use of epigenetics is emerging as an effective and promising approach to treat cancer. Epigenetic drugs, which target two well- known epigenetic pathways, namely, DNA methyltransferases and histone deacetylases, are already being applied for the cancer treatment. In the current study, an overview regarding the under-standing of epigenetic alterations in the development of cancer and the current state of epigenetic drug discovery is provided.

Combined inhibitors of angiogenesis and histone deacetylase:Efficacy in rat hepatoma

AIM:To evaluate the antitumoral effect of combined inhibitors of angiogenesis and histone deacetylases in an experimental rat hepatoma model.METHODS:MH7777A hepatoma cells were injected into the liver of male Buffalo rats.After 7 d treatment with the vascular endothelial growth factor receptor antagonist PTK787/ZK222584(PTK/ZK),the histone deacetylase inhibitor MS-275,tamoxifen(TAM) and/or retinoic acid was initiated(n ≥ 8 animals/group).Natural tumor development was shown in untreated control groups(control 1 with n = 12,control 2 with n = 8).The control groups were initiated at different time points to demonstrate the stability of the hepatoma model.For documentation of possible side effects,we documented any change in body weight,loss of fur and diarrhea.After 21 d treatment,the rats were euthanized.Main target parameters were tumor size and metastasis rate.Additionally,immunohistochemistry for the proliferating cell nuclear antigen(PCNA) and TdT-mediated dUTP-biotin nick end labeling(TUNEL) assay were performed.RESULTS:The control groups developed large tumor nodules with extrahepatic tumor burden in the lung and abdominal organs(control 1:6.18 cm3 ± 4.14 cm3 and control 2:8.0 cm3 ± 4.44 cm3 28 d after tumor cell injection).The tumor volume did not differ significantly in the control groups(P = 0.13).As single agents MS-275 and PTK/ZK reduced tumor volume by 58.6% ± 2.6% and 48.7% ± 3.2% vs control group 1,which was significant only for MS-275(P = 0.025).The combination of MS-275 and PTK/ZK induced a nearly complete and highly significant tumor shrinkage by 90.3% ± 1%(P = 0.005).Addition of TAM showed no further efficacy,while quadruple therapy with retinoic acid increased antitumoral efficacy(tumor reduction by 93 ± 1%) and side effects.PCNA positive cells were not significantly reduced by the single agents,while dual therapy(MS-275 and PTK/ZK) and quadruple therapy reduced the PCNA-positive cell fraction significantly by 9.1 and 20.6% vs control 1(P < 0.05).The number of TUNEL-positive cells,markers for ongoing apoptosis,was increased significantly by the single agents(control 1:6.9%,PTK/ZK:11.4%,MS-275:12.2% with P < 0.05 vs control 1).The fraction of TUNEL-positive cells was upregulated highly significantly by dual therapy(18.4%) and quadruple therapy(24.8%,P < 0.01 vs control 1).For the proliferating(PCNA positive) and apoptotic cell fraction,quadruple therapy was significantly superior to dual therapy(P = 0.01).CONCLUSION:Combined PTK/ZK and MS-275 were highly effective in this hepatoma model.Quadruple therapy enhanced the effects microscopically,but not macroscopically.These results should be investigated further.

CONSTRUCTING ADENO-ASSOCIATED VIRUS-TGFβ_3 AND COMPARING ITS BIOLOGICAL EFFECT ON PROTEOGLYCAN SYNTHESIS IN DEDIFFERENTIATED NUCLEUS PULPOUS CELLS WITH ADENOVIRUS-TGFβ_1

Objective To construct adeno-associated virus (AAV) expression system for transforming growth factor β3 (TGFβ3) and detect its biological effect on proteoglycan synthesis of the earlier and later dedifferentiated rabbit lumbar disc nucleus pulpous (NP) cells, which was compared with that of adenovirus (AV) expression system for TGFβ1. Methods TGFβ3 gene was obtained using PCR. Its upstream contained restriction enzyme site Kpn Ⅰ, and its downstream contained restriction enzyme site SalⅠ. Using the restriction enzyme sites of PCR product of TGFβ3 and the corresponding multiple cloning site (MCS) in plasmid AAV, TGFβ3 was subcloned into AAV. The recombinant plasmid AAV-TGFβ3 was transfected into H293 cells with LipofectamineTM 2000, and the expression of TGFβ3 gene was detected using immunofluorescent analysis. After AAV-TGFβ3 virus particle with infectious activity was packaged, TGFβ3 expression in NP cells was detected by immunoblotting, and its biological effect on proteoglycan synthesis was detected by antonopulos method and compared with that of AV-TGFβ1 in the earlier and later dedifferentiated NP cells. Results For the earlier dedifferentiated NP cells, AAV-TGFβ3 slowly and stably enhanced proteoglycan synthesis, but AV-TGFβ1 rapidly and transiently enhanced its synthesis. For the later dedifferentiated NP cells, AAV-TGFβ3 stably enhanced proteoglycan synthesis, but AV-TGFβ1 inhibited its synthesis. Conclusion AAV expression system can mediate TGFβ3 gene to be expressed stably, and AAV-TGFβ3 can enhance proteoglycan synthesis of the earlier and later dedifferentiated NP cells.

The proliferation inhibition of Hela cells by histone deacetylases- 1 siRNA

Objective： To investigate the anti-proliferative effect of histone deacetylases-1 （HDAC-1） knockdown in Hela cells. Methods： The HDAC-1 protein was knockdowned using siRNA. The expression of HDAC-1 was detected by Western blotting. Apoptosis was assessed by flow cytometry. The inhibition of cell growth was assesses by MTT assay. Results： HDAC-1 siRNA knockdowned the expression of HDAC-1 protein. HDAC siRNA inhibited the proliferation of Hela cells. HDAC- 1 siRNA induced apoptosis. Conclusion： HDAC-1 siRNA may inhibit the growth of Hela cells by inducing apoptosis.

Progress in clinical trial of histone deacetylase(HDAC) inhibitors for non-small cell lung cancers

Histone deacetylase(HDAC) inhibitors, which represent a structurally diverse group of molecules, have emerged as a novel therapeutic class of molecules with significant anticancer potential. Vorinostat and romidepsin, known as the first generation of HDAC inhibitors, were approved in the United States for the treatment of T-cell lymphomas. Preliminary activity of HDAC inhibitors has also been observed in non-small cell lung cancer(NSCLC) in combination with the existing treatment regimens, of which is the focus of the current review.

The relationship between cell apoptosis and dephosphorylated RB protein and proliferating cell nuclear antigen in human breast cancer

Objective:The aim of our study was to investigate the relationship between cell apoptosis and dephosphorylated RB protein and proliferating cell nuclear antigen(PCNA) in human breast cancer.Methods:MTT colorimetric assay was applied to examine the growth inhibition,and the apoptosis was determined by flow cytometry(FCM).The expressing quantity of dephosphorylated RB protein and PCNA pre-and post the action of ADR were detected with immunocytochemistry.Results:MTT assay revealed that ADR inhibited proliferation of MCF-7/S cells in a dose dependent manner,the 50% inhibition concentration(IC50) value was 0.128 mg/L.Tumor cell apoptotic rate(AR) in ADR group(χ= 0.259) was significantly higher than that in the control group(χ = 0.045)(P < 0.01).The expressive levels of dephosphorylated RB protein in ADR group(MOD × area = 986.8 ± 207.4) was significantly higher than that in control group(MOD × area =131.7 ± 31.9)(P < 0.01).PCNA positive expression rate in ADR group(χ = 0.3371) was significantly lower than that in the control group(χ = 0.5152)(P < 0.01).Conclusion:In ADR group,there was significant positive correlation between AR and the expressing quantity of dephosphorylated RB protein,but there was significant negative correlation between AR and PCNA.

Expression Grade Sirtuin-1 （SIRT-1） in Tumor Tissue in Women with Breast Cancer： A New Biomarker Prognosis？

Breast cancer is a complex and heterogeneous disease with different clinical outcomes. The investigations of new biomolecular markers are essential to know the prognosis and improve the clinical management of patients. The SIRT-1 （sirtuin- 1） is a histone deacetylase implicated in various epigenetic critical functions for the cells and the maintenance of genomic stability. The objective of this study is to investigate the grade of expression of the SIRT-I （sirtuin-1） and the prognostic value in overall survival of women with breast cancer. Retrospective cohort of 457 women with breast cancer has been researched, undergoing treatment in Erechim-RS from 2003 to 2013 and followed until July 2015. The degree of SIRT-1 expression was investigated by immunohistochemistry in 123 patients （26.9%） of the cohort. The OS （overall survival） from specific disease and risk of death from breast cancer were estimated by Kaplan-Meier and Cox＇s proportional risks. The median age of 57.4 years cohort with OS of 79.6% in 5 years and 69.1% at 10 years, with follow-up time of 61.9 months are revealed in this work. The SIRT-1 overexpression was found in 6.5% of cases and characterized a subgroup of women with shorter survival and increased risk of death from breast cancer （HR = 2.66; 95% CI 1.03 to 6.86; p = 0.043） and adjusted by age （HR = 2.86; 95% CI 1.11 to 7.38; p = 0.030）, histology （HR = 2.79; 95% CI 1.07 to 7.28; p = 0.036）, lymph nodes （HR = 2.73; 95% CI 1.06 to 7.04; p = 0.037）, Her-2 （HR = 2.82; 95% CI 1.07 to 7.44; p = 0.036）; chemotherapy （HR = 2.90; 95% CI 1.11 to 7.60; p = 0.030） and radiotherapy （HR = 2.71; 95% CI 1.05 to 7.01; p = 0.040）. In regressive multivariate models adjusted for age, status of axillary lymph nodes, Her-2 expression and proliferation index （Ki-67）, the grade of expression of the SIRT-1 maintained association with poor prognosis. From the study, it can be concluded that the assessment of the SIRT-1 expression is an independent prognostic biomarker in breast cancer.

Molecular modeling of alkaloids bouchardatine and orirenierine binding to sirtuin-1(SIRT1)

Objective Bouchardatine(1)is a β-indoloquinazoline alkaloid isolated from the plant Bouchardatia neurococca,acting as a modulator of adipogenesis and lipogenesis,and as an anticancer agent.The natural product functions as an activator of proteins adenosine 5’-monophosphate(AMP)-activated protein kinase(AMPK)and sirtuin 1(SIRT1).We used molecular modeling to investigate the SIRT1-binding capacity of compound 1 and various structural analogues,such as orirenierine A(2)and orirenierine B(3)isolated from the medicinal plant Oricia renieri.Methods We investigated the binding to human SIRT1(hSIRT1)of 25 natural products including theβ-indoloquinazoline alkaloids 1−3 and analogues,in comparison with the reference product sirtinol(R and S isomers).A sirtinol binding model was elaborated starting from the closed and open state conformations of the catalytic domain of hSIRT1(PDB structures 4KXQ and 4IG9).For each compound bound to SIRT1,the empirical energy of interaction(ΔE)was calculated and compared to that of sirtinol.Results In our model,compound 1 was found to bind modestly to the sirtinol site of SIRT1.In contrast,the presence of a phenolic OH group at position 7 on the quinazolinone moiety conferred a much higher binding capacity.Compound 2 provided SIRT1 protein complexes as stable as those observed with sirtinol.The replacement of the hydroxy substituent(2)with a methoxy group(3)reduced the SIRT1 binding capacity.Other SIRT1-binding natural products were identified,such as the alkaloids orisuaveolines A and B.Structure-binding relationships were discussed.Conclusion The study underlines the capacity of β-indoloquinazoline alkaloids to interact with SIRT1.This deacetylase enzyme could represent a molecular target for the alkaloid 2.This compound merits further attention for the design of drugs active against SIRT1-dependent pathologies.

Chemical Composition of Meat in Castrated Male Brahman Cattle in Venezuela

Chemical composition of Brahman cattle was studied and determined in this study. Fifty males had been castrated at birth, farmed semi-intensively on the Venezuelan plain and then slaughtered at the age of 31 months with an approximate weight of 500 kilograms. The average pH value of beef was 5,84 after obtained for 24 hours after staughtering, which matches the ~atue found in similar studies made on the same breed in Venezuela （5.8）. The moisture, crude protein and ash contents correspond to the expected values of cattle, while the intramuscular fat values are slightly higher than those of the different breeds ofBos indicus. The total collagen values found in this study are either similar or lower than those found by other researches from the different breeds ofBos indicus and their crossbreeds, while the collagen solubility value （39%） was higher than the one found in other studies on the Brahman breed.

Design, synthesis and biological evaluation of novel HDAC inhibitors: sulphur-containing zinc binding groups

Zinc binding group(ZBG)is the crucial moiety in the chemical structure of any HDAC inhibitor.In the present study,a series of sulphur-containing ZBG were designed and synthesized in the novel HDAC inhibitors to replace the classical ZBGs of SAHA and BML-210,hydroxamic acids and benzamides,respectively.The HDAC inhibitory activity and the structure-activity relationships of these molecules were analyzed.A sulphur-rich group,diethylcarbamo(dithioperoxo)thioate,was finally identified as a novel potent ZBG.Among all the synthesized compounds,4 d was much more potent compared with BML-210,and it showed similar inhibitory effect of SAHA against HDAC isoforms 1 and 2.Therefore,it was chosen as a lead compound.

Retinoic acid inhibits white adipogenesis by disrupting GADD45A-mediated Zfp423 DNA demethylation

Retinoic acid （RA）, a bioactive metaboUte of vitamin A, is a critical mediator of cell differentiation. RA blocks adipogenesis, but mechanisms remain to be established. ZFP423 is a key transcription factor maintaining white adipose identity. We found that RA inhibits Zfp423 expression and adipogenesis via blocking DNA demethylation in the promoter of Zfp423, a process mediated by growth arrest and DNA-damage-inducible protein alpha （GADD45A）. RA induces the partnering between retinoic acid receptor （PAR） and tumor suppressor inhibitor of growth protein 1 （ING1）, which prevents the formation of GADD45A and ING1 complex necessary for locus-specific Zfp423 DNA demethylation. In vivo, vitamin A supplementation prevents obesity, downregulates Godd45a expression, and reduces GADD45A binding and DNA demethylation in the Zfp423 promoter. Inhibition of Zfp423 expres- sion due to PA contributes to the enhanced brown adipogenesis. In summary, PA inhibits white adipogenesis by inducing PAR and ING1 interaction and inhibiting Godd45a expression, which prevents GADD45A-mediated DNA demethylation.