Utilidad de la técnica de MS-MLPA en el diagnóstico de los síndromes de Beckwith-Wiedemann y Silver-Russell / Usefulness of the MS-MLPA technique in the diagnosis of Beckwith-Wiedemann syndrome and Silver-Russell syndrome

Resumen Introducción: Las alteraciones epigenéticas y genómicas de la región improntada 11p15.5 producen crecimiento excesivo o deficiente, que se manifiesta como síndrome de Beckwith-Wiedemann o síndrome de Silver-Russell, respectivamente. Objetivo: Evaluar la técnica de análisis de metilación MLPA (MS-MLPA, methylation-specific multiplex ligation-dependent probe amplification) en el diagnóstico de los síndromes de Beckwith-Wiedemann y de Silver-Russell. Métodos: Se evaluó la metilación y las variantes de 11p15.5 en pacientes con diagnóstico clínico de síndrome de Beckwith-Wiedemann y síndrome de Silver-Russell mediante la técnica MS-MLPA en ADN de sangre periférica. Resultados: Se identificó disomía uniparental paterna y pérdida de metilación del IC2 materno en dos pacientes con síndrome de Beckwith-Wiedemann, quienes presentaron onfalocele y macroglosia, respectivamente. Se registró hipometilación paterna del IC1 en dos pacientes con síndrome de Silver-Russell de fenotipo clásico. Conclusiones: Se observó adecuada correlación genotipo-fenotipo con los defectos de metilación encontrados, lo que confirma la utilidad del MLPA como estudio de primera línea en pacientes con diagnóstico de síndrome de Beckwith-Wiedemann y síndrome de Silver-Russell.

Abstract Introduction: Epigenetic and genomic imprinting alterations of the 11p15.5 region cause excessive or deficient growth, which result in Beckwith-Wiedemann syndrome (BWS) or Silver-Russell syndrome (SRS), respectively. Objective: To evaluate the methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) methylation analysis technique in the diagnosis of BWS and SRS. Methods: 11p15.5 methylation and variants were evaluated in patients with clinical diagnosis of BWS and SRS using the MS-MLPA technique in peripheral blood DNA. Results: Paternal uniparental disomy and loss of maternal IC2 methylation were identified in two patients with BWS who had omphalocele and macroglossia, respectively. Paternal IC1hypomethylation was recorded in two patients with SRS of classic phenotype. Conclusions: Adequate genotype-phenotype correlation was observed with the methylation defects that were identified, which confirms the usefulness of MLPA as a first-line study in patients diagnosed with BWS and SRS.

Genetic analysis of a family with 6q22 microdeletion and 10p15.3p13 microduplication in two consecutive pregnancies / 中华检验医学杂志

Objective:Cytogenetic and molecular genetic analysis was performed on two consecutive antenatal abnormal fetuses and their parents in a family to clarify the copy number variation(CNV) and its mechanism.Methods:The karyotypes of two fetuses and their parents were analyzed by conventional karyotyping techniques, and CNVs of two fetuses and their mother were analyzed by low-coverage whole-genome copy number variation sequencing (CNV-seq) techniques.Results:The amniotic fluid karyotype results of fetus 1 and 2 were 46, XN, der(4)t(4;10)(q35;p13). The mother′s peripheral blood karyotype result was 46, XX, t(4;10)(q35;p13), and the father′s karyotype was normal. The CNV-seq results of fetus 1 and 2 were seq[hg19]6q22.31(122740000-125440000)X1; 10p15.3p13(120000-17260000)X3, suggesting that there was a heterozygous deletion of about 2 700 000 bp in fetal 6q22.31 and a duplication of about 17 140 000 bp in fetal 10p15.3p13. The CNV-seq result of their mother was seq[hg19]6q22.31(122740000-125440000)X1, suggesting that there was a heterozygous deletion of about 2 700 000 bp in 6q22.31. The pregnant woman and her family chose to terminate the pregnancy after genetic consulting.Conclusion:The combined application of karyotyping and CNV-Seq is significantly beneficial to detecting microdeletions or microduplications of fetal chromosomes and effectively preventing the birth of defective children.

Hypermethylation Analysis of p16INK4a and p15INK4b Promoters in Chronic Lymphocytic Leukaemia Patients and Normal Individuals

@#Introduction: Chronic Lymphocytic Leukaemia (CLL) is a common type of leukaemia in persons of predominantly European descent but is rare in the Asian population. Disparities in CLL incidence among people of Asian and European descent may be related to the genetic make-up of the two different populations. Hypermethylation event might be one of the silencing mechanisms that inactivate the tumour suppressor genes in CLL. The aim of this study was to determine the hypermethylation status of p16INK4a and p15INK4b among CLL patients and normal individuals. Materials & Methods: A total of 25 CLL patients and 25 normal individuals were recruited for this study and their genomic DNA were extracted from the peripheral blood. The hypermethylation status of p16INK4a and p15INK4b were determined using Methylation Specific-PCR (MS-PCR) whereas DNA sequencing method was applied to selected samples for validation of the MS-PCR results. We also evaluated the association between hypermethylation of these genes with the clinical and demographic characteristics of each group of subjects. Results: Among the CLL patients, p15INK4b partialmethylation occurred in 6 (24%) subjects while methylation occurred in 1 (4%) subject. All the remaining patients were unmethylated at p15INK4b. All the samples showed unmethylation at p16INK4a. Statistically significant associations were found between p15INK4b hypermethylation with the presence of CLL (p=0.01) and with race (p=0.02). Conclusion: Further study using a larger sample size is warranted to explore the significance of DNA methylation incidence among the CLL patients of the Malaysian population. Hence, we suggest that hypermethylation at p15INK4b has a huge influence that kick-starts CLL disease among Malaysians and MS-PCR technique is applicable to be used in methylation study.

Genetic analysis of an adult with mosaicism of uniparental disomy 11p / 中华医学遗传学杂志

Objective@#To analyze the clinical phenotype and genomic abnormality of an adult featuring congenital heart defect and multiple developmental disorders.@*Methods@#The patient was subjected to conventional G-banding chromosomal karyotyping and single nucleotide polymorphism microarray (SNP-array) analysis.@*Results@#The patient showed a normal karyotype, while SNP-array revealed a 42.7 Mb mosaic uniparental disomy (UPD) in the 11p15.5p12 region ([hg19] chr11: 491 333 - 43 189 376).@*Conclusion@#The mosaicism of UPD of 11p15.5p12 region probably underlies the congenital heart defect and developmental disorders in the patient.

Detection and clinical features of acute leukemia accompanying with 11p15/NUP98 / 实用医学杂志

Objective To observe the clinical features and therapeutic effect of 11p15/NUP98 rearrange-ments in acute leukemia. Methods A total of 598 newly diagnosed acute leukemia patients were detected by con-ventional cytogenetics analysis and fluorescence in situ hybridization(FISH)with the NUP98 double color probe, and the clinical data were analyzed retrospectively in the patients with 11p15/NUP98 abnormality. Results Six cases with 11p15/NUP98 rearrangement were found with a median age of 39 years old,one patient is male,the oth-ers are females. Three patients had acute monocytic leukemia(M5),one patient had acute monocytic leukemia (M2),one patient had acute monocytic leukemia(M4),and one patient had acute lymphoblastic leukemia. 11p15/NUP98 abnormality was detectable in all the patients. The median survival in all the patients was 9 months. Con-clusions Acute leukaemia with 11p15 abnormality frequently involves NUP98 gene and mainly occurrs in women. Patients with lower median age mainly developed in acute monocytic leukemia. The major clinical manifestations are anemia,low platelets and hyperleukocytosis. Acute leukemia with 11p15/NUP98 rearrangement is poorly re-sponsive to routine chemotherapies and to allogeneic hematopoietic stem cell transplantation,and thus has poor prognosis.

Analysis of genetic defects in the 11p15.5 region in Russell-Silver syndrome / 临床儿科杂志

Objective To explore the pathogenesis of Russell-Silver syndrome (RSS). Methods Two milliliter peripheral blood samples were collected from 6 male patients aged 6 to 8 years with suspected RSS phenotype, the parents of 2 patients and 5 healthy boys. Mononuclear cells were isolated and genomic DNA was extracted. The methylation level of the H19 imprinting control region(ICR)1 on chromosome 11p15.5 was detected by pyrosequencing.The methylation status and the copy number variation in the corresponding region of one RSS patient with positive results by pyrosequencing were analysed by methylation-specific multiplex-ligation-dependent probe amplification assay (MS-MLPA). Results Pyrosequencing analysis revealed that the methylation rates on the 6 CpG targeting sites in H19 differentially methylated region(DMR)in the 6 RSS patients were about 11%~29%, which were significantly lower than those in their parents and normal controls (44%~59%). The MS-MLPA results of one patient with positive pyrosequencing showed that the methylation rates of 4 sites in H19-DMR were about 10%,which was obviously lower than the normal level.The methylation rates of the 4 sites in KCNQ1OT1 gene were about 50%, which was in the normal range. The copy number variations from all samples detected were in the normal range. Conclusion There is methylation aberration of H19-DMR in ICR1 in children with RSS.

Optimizing a methylation-specific polymerase chain reaction technology / 中国职业医学

OBJECTIVE: To establish a highly specific methylation-specific polymerase chain reaction( MSP) method.METHODS: Bisulphited conversion hypermethylated human genomic DNA and unmethylated human genomic DNA were used as template and p15 gene as the target gene. The Cp G island of p15 gene promoter was predicted by Methprimer software.The methylated and unmethylated primers for polymerase chain reaction( PCR) of methylated and unmethylated DNA templates were designed. The PCR stage of MSP was divided into normal temperature group( methylation primer and unmethylated primer annealing temperature of 58 and 55 ℃,respectively) and elevated temperature group( methylation primer and unmethylated primer annealing temperature were 60 and 57 ℃ respectively). The PCR reaction system was optimized based on the normal temperature group by increasing the concentration of magnesium ions( Mg2 +) and adding dimethyl sulfoxide( DMSO) in the PCR reaction system. RESULTS: Nonspecific amplification products appeared in normal temperature group in the PCR reaction stage of MSP,indicating false positive and false negative reactions. The non-specific amplification was weakened and the specific amplification was equally reduced in the elevated temperature group. The nonspecific amplification disappeared in the optimized PCR system and normal temperature group,and the false positive and false negative reactions were eliminated completely. CONCLUSION: Setting the normal annealing temperature and the concentration of Mg2 +and DMSO is beneficial to ensure the specificity of MSP results.

p15(Ink4b) Loss of Expression by Promoter Hypermethylation Adds to Leukemogenesis and Confers a Poor Prognosis in Acute Promyelocytic Leukemia Patients / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The p15(Ink4b) gene exerts its influence as an inhibitor of cyclin-dependent kinases and is frequently associated with hematological malignancies. Inactivation of this gene through DNA methylation has been found to be the most prevalent epigenetic alteration reported, with a high frequency in all French-American-British subtypes of acute myeloid leukemias, including acute promyelocytic leukemia (APL). In this study,we investigated the prognostic significance of p15 gene promoter hypermethylation and its expression in APL patients of Kashmir (North India). MATERIALS AND METHODS: p15 gene promoter hypermethylation was conducted by methylation-specific polymerase chain reaction, while its subsequent expression analysis was carried out by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Of the 37 patients, 16 (43.2%) were found to have methylated p15 genes. Of these 16 cases, seven (43.8%) were methylated partially and nine (56.2%) were found to have complete methylation. Moreover, nine of the 37 patients (24.3%) who presented with leukocytosis at their baseline had complete p15 gene methylation as well (p < 0.05). Semiquantitative RT-PCR showed a complete loss of p15 expression in nine patients with complete methylation coupled with leukocytosis (p=0.031), while seven patients with partial methylation showed decreased p15 expression. Six patients relapsed during the maintenance phase of treatment and were found to have a completely methylated p15 gene and no p15 mRNA. CONCLUSION: Complete methylation and loss of p15 gene expression causes susceptibility to relapse and decreased survival in APL patients. Thus, p15 promoter hypermethylation is a prospective prognostic indicator and a reliable clinical aid in assessment of patients with APL.

p15(Ink4b) Loss of Expression by Promoter Hypermethylation Adds to Leukemogenesis and Confers a Poor Prognosis in Acute Promyelocytic Leukemia Patients / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The p15(Ink4b) gene exerts its influence as an inhibitor of cyclin-dependent kinases and is frequently associated with hematological malignancies. Inactivation of this gene through DNA methylation has been found to be the most prevalent epigenetic alteration reported, with a high frequency in all French-American-British subtypes of acute myeloid leukemias, including acute promyelocytic leukemia (APL). In this study,we investigated the prognostic significance of p15 gene promoter hypermethylation and its expression in APL patients of Kashmir (North India). MATERIALS AND METHODS: p15 gene promoter hypermethylation was conducted by methylation-specific polymerase chain reaction, while its subsequent expression analysis was carried out by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Of the 37 patients, 16 (43.2%) were found to have methylated p15 genes. Of these 16 cases, seven (43.8%) were methylated partially and nine (56.2%) were found to have complete methylation. Moreover, nine of the 37 patients (24.3%) who presented with leukocytosis at their baseline had complete p15 gene methylation as well (p < 0.05). Semiquantitative RT-PCR showed a complete loss of p15 expression in nine patients with complete methylation coupled with leukocytosis (p=0.031), while seven patients with partial methylation showed decreased p15 expression. Six patients relapsed during the maintenance phase of treatment and were found to have a completely methylated p15 gene and no p15 mRNA. CONCLUSION: Complete methylation and loss of p15 gene expression causes susceptibility to relapse and decreased survival in APL patients. Thus, p15 promoter hypermethylation is a prospective prognostic indicator and a reliable clinical aid in assessment of patients with APL.

Digoxin Inhibits Retinoblastoma through Suppressing a Non-canonical TGFβ Signaling Pathway.

Aims: Retinoblastoma is a childhood ocular tumor rapidly developing from the immature cells of the retina due to loss of functional retinoblastoma protein. Digoxin, a cardiac glycoside, has been reported to be effective in inducing apoptosis, cell cycle arrest, and cytotoxic effects on human cancers. In this regard, the present study aims to investigate whether digoxin could suppress retinoblastoma cancer through the regulation of transforming growth factor-β (TGF-β) signaling pathway. Methodology: The effects of digoxin on Y-79 cells, retinoblastoma cancer cell line, were investigated using MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazoli-umbromide) and BrdU (bromodeoxyuridine) assays to measure cellular cytotoxicity effects and cell apoptosis, respectively. Also, a qPCR assay was employed to analyze the mRNA expression levels of TGFβ signaling pathway including C-MYC, P21, P15, TGFβRI, TGFβRII, and SMAD2, 3, and 4 genes. Results: The results of the cell function assays revealed that digoxin inhibited the cell viability and proliferation of Y-79 cells. In addition, it was found that digoxin significantly suppressed C-MYC expression and enhanced the expression of P21, P15, SMAD2 and SMAD4 genes in a dose-and time-dependent manner. However, the obtained results could not detect any significant effect of digoxin on TGFβRI, TGFβRII and SMAD3 genes. Conclusion: Taken together, the findings of the present study suggest that digoxin could be a potential therapeutic agent in the treatment of retinoblastoma by regulating the cell cycle genes via a non-canonical TGF-β signaling pathway.

Expressions of antisense non-coding RNA in INK4 locus and tumor suppressors in peripheral blood lymphocytes of patient with cirrhosis and hepatocellular carcinoma / 中国中西医结合急救杂志

Objective To investigate the clinical diagnostic and differential diagnostic values of antisense non-coding RNA in the INK4 locus (ANRIL) and tumor suppressors (p14ARF, p15INK4b and p16INK4a) mRNA expression levels in peripheral blood lymphocytes of patients with cirrhosis and hepatocellular carcinoma. Methods The patients with hepatocellular carcinoma and cirrhosis admitted in Shantou Central Hospital from October 2013 to April 2014 were selected. The real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect ANRIL, p14ARF, p15INK4b and p16INK4a mRNA expression levels of peripheral blood lymphocytes. The subjects having taken physical health examinations in outpatient clinics were assigned in the healthy control group. Results During the study period, 19 cases of hepatocellular carcinoma, 24 cases of cirrhosis, and 31 healthy controls were finally enrolled. In the hepatocellular carcinoma group, the mRNA expression level of ANRIL was significantly higher than that of the healthy control group (?Ct:13.07±0.62 vs. 12.45±0.84, P0.05). There were also no statistically significant differences in p14ARF and p16INK4a mRNA expressions among the three groups (all P>0.05). Conclusion The elevation of ANRIL and descent of p15INK4b mRNA expression levels in peripheral blood lymphocytes in patients with liver lesion can be used as the reference indicators for the early diagnosis of hepatocellular carcinoma and to predict their prognoses.

A combination of STI571 and BCR-ABL1 siRNA with overexpressed p15INK4B induced enhanced proliferation inhibition and apoptosis in chronic myeloid leukemia

p15INK4B, a cyclin-dependent kinase inhibitor, has been recognized as a tumor suppressor. Loss of or methylation of the p15INK4B gene in chronic myeloid leukemia (CML) cells enhances myeloid progenitor formation from common myeloid progenitors. Therefore, we examined the effects of overexpressed p15INK4B on proliferation and apoptosis of CML cells. Overexpression of p15INK4B inhibited the growth of K562 cells by downregulation of cyclin-dependent kinase 4 (CDK4) and cyclin D1 expression. Overexpression of p15INK4B also induced apoptosis of K562 cells by upregulating Bax expression and downregulating Bcl-2 expression. Overexpression of p15INK4B together with STI571 (imatinib) or BCR-ABL1 small interfering RNA (siRNA) also enhanced growth inhibition and apoptosis induction of K562 cells. The enhanced effect was also mediated by reduction of cyclin D1 and CDK4 and regulation of Bax and Bcl-2. In conclusion, our study may provide new insights into the role of p15INK4B in CML and a potential therapeutic target for overcoming tyrosine kinase inhibitor resistance in CML.

Diagnosis of one case with 7p15.3p22.1 microdeletion by applying array-based comparative genomic hybridization / 临床儿科杂志

Objective To investigate the diagnosis of a case with 7p15.3p22.1 microdeletion by applying array-based comparative genomic hybridization (array-CGH) and to analyze the relationship between the clinical manifestations and 7p15.3p22.1 microdeletion. Method Array-CGH technique was used to detect genomic copy number variations (CNVs) in an infant with normal karyotype after conventional chromosomal karyotyping. Results Array-CGH detected 7p15.3p22.1 deletion (chr7: 6777262-23981753), which was confirmed as pathogenic CNV after comparative analysis with database. Conclusion Array-CGH could serve as a useful complement for G-banding to be used in the clinical cytogenetic diagnosis.

Study on aberrant methylations of SHP-1, p15 and SOCS-1 genes in diffuse large B-cell lymphoma / 白血病·淋巴瘤

Objective To detect the significance of the SHP-1,p15 and SOCS-1 genes methylation status in the genesis and development of diffuse large B-cell lymphoma (DLBCL).Methods The methylation state of SHP-1,p15 and SOCS-1 genes CpG islands were measured by methylation-specific polymerase chain (MSP) reaction.Results The positive rates of aberrant methylation for SHP-1,p15 and SOCS-1 genes in 50 specimens of DLBCL were 96 ％ (48/50),52 ％ (26/50) and 56 ％ (28/50) respectively.In control group,however,15 specimens of benign lymph node proliferation showed no methylation in CpG island of SHP-1,p15 and SOCS-1 genes promoter.Conclusion Aberrant methylations of the SHP-1,p15 and SOCS-1 genes exist in the patients with DLBCL.The methylations of SHP-1,p15 and SOCS-1 genes may be associated with the occurrence and development of DLBCL.This study provides a theoretical basis of treatment methylation for DLBCL.

Molecular mechanism of apoptosis induced by valproate in primary cells from acute lymphoblastic leukemia in vitro / 白血病·淋巴瘤

Objective To investigate the relation between demethylation effect and apoptosis of valproate (VPA) in primary acute lymphoblastic leukemia (ALL) cells in vitro.Methods 10 cases of ALL patients was choosed to acquire leukemia cells.Cell growth curve were assessed by the MTT assay,the apoptosist of primary ALL was analyzed with DNA Ladder and Annexin-V-FITC/PI by flow cytometry.The expression methylation level of p15 was detected by hn-MSPCR,and p15mRNA were detected by RT-PCR.All dates was analyzed by SPSS16.0.Results The 50 ％ inhibition rate of VPA were 1.898 mmol/L to primary ALL cells assayed by MTT respectively.DNA ladder showed the apoptosis of primary ALL cells increased by adding VPA dose.Annexin-V-FITC/PI tests showed that the apoptosis percentage of primary ALL cells were (0.44±0.04) ％ in control group,(5.80±0.65) ％ in 1.0 mmol/L VPA group,(48.46±2.49) ％ in 2.0 mmol/L VPA group,(76.45±2.98) ％ in 4.0 mmol/L VPA group,the apoptosis percentage increased significantly (P ＜ 0.05).The demethylation of p15 INK4B gene decreased by adding VPA dose,the expression of p15 mRNA expression increased significantly compared with control group by RT-PCR (P ＜ 0.05).Conclusion It is found that VPA could induce demethylation of p15 INK4B gene,which could upregulate the p15 mRNA expression,due to the apoptosis of primary ALL cells.

Detection of p15 methylation and deletion status in acute lymphoblastic leukemia using hn-MSP / 白血病·淋巴瘤

Objective To explore the characteristics of hemi-nested methylation specific polymerase chain reaction (hn-MSP) and to find out the possible relationship between patterns of methylation or deletion and the developmet of adult acute lymphoblastic leukemia(ALL).Methods hn-MSP and bisulfit-sequencing PCR (BSP) were designed and adopted to analyze p15 gene methylation or deletion patterns in 25 adult ALL patients,malignant hematopathy cell lines and normal lymphocytes. hn-MSP and BSP products were cloned and sequenced.The sensitivity and specificity of hn-MSP were also analized.Results The sequencing results of hn-MSP and BSP products were consistent, and the sensitivity of detection of p15 methylation was up to 1.0×10-5.17 adult ALL patients (68 ％) were p15 gene hypermethylation and 3 patients were with deletion of p15 gene exon 1.There were no hypermethylation or deletion in the 10 controls.Conclusions The detection rate of p15 methylation in many tumors,especially in adult ALL,is frequent high.hn-MSP is highly sensitive and specific in analyzing p15 methylation.

Estudo clínico da aplicação de matriz inorgânica de osso associado a peptídeo sintético de adesão celular (MIO/P-15), PepGen P-15®, em lesões periodontais avançadas de cães / Clinical study of effectiveness of an anorganic bone matrix and cell-binding peptide (ABM/P-15), PepGen-P15®: application in advanced periodontal lesions of dogs

Os mecanismos biológicos desenvolvidos para aumentar a qualidade da regeneração óssea e da reparação tecidual de sítios periodontais específicos continuam a ser um desafio e têm sido complementado pela capacidade de adesão celular do colágeno do tipo I, promovida por um peptídeo sintético de adesão celular (P-15), associado a uma matriz inorgânica de osso (MIO) para formar MIO/P-15. O objetivo deste estudo foi avaliar a perda do nível clínico de inserção e a resposta da bolsa periodontal em dentes após 3 e 6 meses da aplicação de enxerto com MIO/P-15. Vinte e um cães do Hospital Veterinário da Universidade de São Paulo foram anestesiados para realização de tratamento periodontal e 132 faces dentais com perda de nível clínico de inserção foram tratadas, sendo que 36,4 por cento (48 faces) receberam o peptídeo de adesão celular e 63,6 por cento (84 faces) compuseram o grupo controle que recebeu tratamento convencional (retalho muco-gengival e aplainamento radicular). O procedimento foi documentado através de radiografia intra-oral e todas as sondagens de bolsas periodontais foram fotografadas. Depois de 3 e de 6 meses, os animais foram re-anestesiados a fim de se obter novas avaliações, radiografias, fotografias e sondagens periodontais. As 48 faces com perda de nível clínico de inserção que receberam material de enxertia apresentaram taxa de 40 por cento de recuperação do nível clínico de inserção após 6 meses. O grupo controle de faces dentais não apresentou alteração do nível clínico de inserção. A face palatina foi a que apresentou melhor taxa de regeneração (40 por cento) e os dentes caninos e molares mostraram as melhores respostas (57,14 por cento e 65 por cento, respectivamente). Não houve sinais de infecção pós-cirúrgica relacionadas à falta de higienização oral dos animais. Pode-se concluir que o MIO/P-15 auxilia na regeneração e re-aderência das estruturas periodontais, incluindo osso alveolar. Sua aplicação mostrou-se fácil...

The development of biologic modalities designed to enhance bone regeneration and wound healing of specific periodontal sites continues to be a challenge and has been accomplished through the cell binding activity of Type-I collagen. These have been provided by a synthetic cell biding peptide (P-15), associated to a anorganic bone matrix (ABM) to form ABM/P-15. The aim of this study was to evaluate the attachment loss and periodontal pocket response in teeth after 3 and 6 months with ABM/P-15 graft application. Twenty one dogs from the Veterinary Hospital, University of São Paulo, were anesthetized in order to accomplish periodontal treatment and 132 teeth faces with attachment loss were treated. From these, 36.4 percent (48 faces) received cell binding peptide and 63.6 percent (84 faces) compounded the control group that received conventional treatment (muco-gingival flap and root planning). The procedure was documented by intra-oral radiography and all periodontal probings were photographed. After 3 and 6 months, the animals were re-anesthetized in order to accomplish new photography, radiography and periodontal probing exams. The 48 attachment loss faces that received graft material exhibited 40 percent of regeneration rate after 6 months. The control faces did not change their attachment level. The palatal face presented the better regeneration rates (40 percent) and the canines and molars teeth showed the better responses (57.14 percent and 65 percent, respectively). There was no post-surgical infection related to absence of oral home care. It can be concluded that ABM/P-15 helps a more rapidly periodontal structure re-attachment and regeneration, including alveolar bone. Its application was easy and practical, and the post-surgical complications incidence was low. Nevertheless, more work is necessary to evaluate the amount and the quality of formed bone and periodontal ligament.

Inhibitory effect of p15~(INK4B) gene on proliferation of human pancreatic cancer cell line BxPC / 中华胰腺病杂志

Objective To investigate the effect of p15~(INK4B)(p15)gene transfection on the proliferation of pancreatic cancer cell line BxPC3.Methods In p15 transfection group.pCDNA3.1(+)p15 was transfected into BxPC3 cell by the vector of Lipofectamine2000.In empty plasmid transfection group, pCDNA3.1(+)neo was transfected into BxPC3 cell with the sa/ne method as a blank control group.In non-transfection group.the BxPC3 cell was not transfected as a negative control group.The p15 mRNA expressions were assayed by RT-PCR,and p15 protein expressions were assayed by Western blot.The proliferation was determined by MTY assay,ultra-structure changes were measured by transmission electron microscope.Cell cycle and apoptosis were measured by flow cytometry.Results In the pCDNA3.1(+)p15 transfeetion group,the expression of p15 mRNA and protein were resumed.Since the 2nd day of culture,the growth of pCDNA3.1(+)p15 transfeetion group was inhibited,till the 7th day,the inhibitory rate was 47.9%,G_0/G_1 Dhase cell accounted for(61.56±3.96)% of all the cells,which was significantly higher than(47.44±6.35)%ofthe black control groups and(49.22±7.23)%0f tlle negative control group(P<0.05).G_1apoptosis peak occurred and the apoptosis rate was(5.27±1.04)%in pCDNA3.1(+)p15 transfection group,which was significantly higher than(0.11±0.06)% of the black control groups and(0.09±0.07)% of the negative control group(P<0.05).Apoptosis was also observed by transmission electron microscope in the pCDNA3.1(+)-p15 transfection group cells.Conclusions After p15 gene transfection,BLPC3 cell proliferation could be significantly inhibited and apoptosis could be induced.

Mutational analysis of genes p14ARF, p15INK4b, p16INK4a, and PTEN in human nervous system tumors

The cancer is one of the most common and severe problems in clinical medicine, and nervous system tumors represent about 2% of the types of cancer. The central role of the nervous system in the maintenance of vital activities and the functional consequences of the loss of neurons can explain how severe brain cancers are. The cell cycle is a highly complex process, with a wide number of regulatory proteins involved, and such proteins can suffer alterations that transform normal cells into malignant ones. The INK4 family members (CDK inhibitors) are the cell cycle regulators that block the progression of the cycle through the R point, causing an arrest in G1 stage. The p14ARF (alternative reading frame) gene is a tumor suppressor that inhibits p53 degradation during the progression of the cell cycle. The PTEN gene is related to the induction of growth suppression through cell cycle arrest, to apoptosis and to the inhibition of cell adhesion and migration. The purpose of the present study was to assess the mutational state of the genes p14ARF, p15INK4b, p16INK4a, and PTEN in 64 human nervous system tumor samples. Homozygous deletions were found in exon 2 of the p15INK4b gene and exon 3 of the p16INK4a gene in two schwannomas. Three samples showed a guanine deletion (63 codon) which led to a loss of heterozygosity in the p15 gene, and no alterations could be seen in the PTEN gene. Although the group of patients was heterogeneous, our results are in accordance with other different studies that indicate that homozygous deletion and loss of heterozygosity in the INK4 family members are frequently observed in nervous system tumors.

High Frequency of Genetic Alterations in Non-small Cell Lung Cancer Detected by Multi-target Fluorescence In Situ Hybridization

Detection of genetic alterations could provide a tool as an adjuvant for the diagnosis of non-small cell lung cancer (NSCLC) and to define patients at risk for early relapse. In this study, a multi-target fluorescence in situ hybridization (FISH) assay was conducted to investigate the correlation between the alterations of chromosomes, including 5p15.2, 6p11.1-q11, 7p12, and 8q24.12-q24.13 (LaVysion Test), and clinicopathological variables, and to clarify the potential of the multi-target FISH assay in 37 NSCLC. The most notable finding was the higher frequency of a gain in chromosome 5p15.2 in early-stage (I+IIa) lung cancers. The frequency of the gain was 81.3% (16/22) in stage I tumors. The frequencies of gains in 6p11.1-q11 and 8q24.12-q24.13 were 61.5% (8/13) and 84.6% (11/13) in stage IIIa cancers, as compared with lower frequencies in stage I tumors at 25.0% and 31.3%, respectively. There was also a significant difference in the histological type. Our results suggest that a gain in 6p11.1-q11 and 8q24.12-q24.13 plays an important role in tumor progression and is associated with histological differentiation. On the other hand, gene amplification in the 5p region was one of the most consistent alterations in early-stage lung cancer, and thus a series of genes in the critical 5p15.2 region might potentially associated with the development of lung cancer.