In this study, we are testing the hypothesis that human papillomavirus (HPV) positivity is correlated with chromatin texture in the cell. Interim analyses are important since this study involves 2000 patients and gene...In this study, we are testing the hypothesis that human papillomavirus (HPV) positivity is correlated with chromatin texture in the cell. Interim analyses are important since this study involves 2000 patients and generates 6000 biopsy specimens that will be subjected to quantitative histopathological analysis and correlated to HPV positivity as measured by the Hybrid Capture Ⅱ test (Digene; Gaithersberg, MD) and both HPV-DNA and mRNA by the polymerase chain reaction (PCR). The studies of optical technologies, from which we derive this sample, use the colposcopically directed and histopathologically classified cervical biopsy as the gold standard. In this report, we describe the results of an interim analysis of quantitative histopathology and chromatin texture as correlates of HPV infection using the cyto-savant system in cytologically and histopathologically negative specimens. Methods. A group of 1544 patients entered the optical technology trials,generating 3275 biopsies and 1544 Papanicolaou readings. Two hundred forty-eight patients were cytologically and histopathologically negative. Study pathologists reviewed histologic samples 3 times in a blinded fashion. Non-overlapping, quantitatively stained nuclei were selected from the samples by the pathologists. HPV testing was done using the PCR method and the Hybrid Capture Ⅱ test. Statistical analysis involved the creation of a classification matrix using a linear discriminant analysis. The matrix was trained on HPV-positive cells by PCR. The analysis included the random creation of both a training set and a validation set that were classified based on the discrimination score obtained by correlating nuclear texture with HPV positivity. Results. The sensitivity of the classification was 52- 54% and the specificity was 77- 78% . Overall, a 68% predicted accuracy was achieved for both the training set and the test set. The agreement of a test and training set shows that the sets created randomly are indeed similar, and that the discrimination score worked equally well in both sets of cells. Once a cell-by-cell algorithm for HPV positivity was derived, HPV positivity was recalculated on the basis of cell-by-cell texture features. HPV positivity was then recalculated on both a per-biopsy basis and a per-patient basis. For HPV 16 and 18, the positivity rate was 70% on a per-biopsy basis and 73% on a per-patient basis. Conclusions. Although these results are preliminary, they suggest that texture features reflecting chromatin condensation may correlate with HPV positivity. The current sample is histologic, the analysis suggests that in a cytologic sample, HPV positivity could be detected or confirmed by texture features computed as part of an HPV associated score. Additional biologic markers could be used as needed. While this study was performed on histologic samples, a study of cytologic samples would be more useful. Future studies will examine chromatin texture compared to HPV integration and mRNA HPV expression.展开更多
目的:探索富脯氨酸蛋白11(Proline-rich protein 11,PRR11)过表达诱发染色体凝聚异常的分子机理。方法:综合利用瞬时过表达、si RNA敲减和免疫荧光染色方法在人肺癌细胞系A549中过表达或沉默PRR11后,检测波形蛋白(Vimentin)的表达与亚...目的:探索富脯氨酸蛋白11(Proline-rich protein 11,PRR11)过表达诱发染色体凝聚异常的分子机理。方法:综合利用瞬时过表达、si RNA敲减和免疫荧光染色方法在人肺癌细胞系A549中过表达或沉默PRR11后,检测波形蛋白(Vimentin)的表达与亚细胞分布及染色质凝聚异常情况。结果:肺癌细胞中内源性PRR11在胞浆中呈现弥散型分布,而Flag-PRR11过表达后在细胞中的分布呈现弥散状和网络状两种不同形态。进一步检测发现内源性或外源过表达PRR11与Ⅲ型中间丝蛋白Vimentin共定位,并且导致Vimentin绕核分布紊乱及染色质凝聚异常。结论:在细胞周期过程中PRR11与Vimentin共定位并调节其组装过程。推测PRR11异常表达可能通过与Vimentin共定位导致核骨架核纤层不稳定并诱发染色质异常凝聚,从而促进肿瘤的发生发展。展开更多
The revolutionary induced pturipotent stem celt （iPSC） technoLogy provides a new means for celt replacement therapies and drug screening. Small molecule compounds have been found extremely useful to improve the gene...The revolutionary induced pturipotent stem celt （iPSC） technoLogy provides a new means for celt replacement therapies and drug screening. Small molecule compounds have been found extremely useful to improve the generation of iPSCs and understand the repro- gramming mechanism. Here we report the identification of a novel chemical, CYT296, which improves OSKM-mediated induction of iPSCs for 〉10 folds and enables efficient reprogramming with only Oct4 in combination with other small molecules. The derived iPSCs are genuinely pluripotent and support the development oftwo ＇All-iPSC＇ mice by tetraploid complementation. CYT296 profoundly impacts heterochromatin formation without affecting celt viability. MEFs treated with CYT296 exhibit de-condensed chromatin structure with markedly reduced loci containing heterochromatin protein 1α （HPIoL） and H3K9me3, which is very similar to the chromatin configuration in embryonic stem cells （ESCs）. Given that an open chromatin structure serves as a hallmark of pLuripotency and has to be acquired to fulfill reprogramming, we propose that CYT296 might facilitate this process by disrupting condensed chromatin, thereby creating a more favorable environment for reprogramming. In agreement of this idea, shRNA targeting HP1α also promotes the generation of iPSCs. Thus current findings not only provide a novel chemical for efficient iPSC induction, but also suggest a new approach to regulate somatic cell reprogramming by targeting chromatin de-condensation with small molecules.展开更多
文摘In this study, we are testing the hypothesis that human papillomavirus (HPV) positivity is correlated with chromatin texture in the cell. Interim analyses are important since this study involves 2000 patients and generates 6000 biopsy specimens that will be subjected to quantitative histopathological analysis and correlated to HPV positivity as measured by the Hybrid Capture Ⅱ test (Digene; Gaithersberg, MD) and both HPV-DNA and mRNA by the polymerase chain reaction (PCR). The studies of optical technologies, from which we derive this sample, use the colposcopically directed and histopathologically classified cervical biopsy as the gold standard. In this report, we describe the results of an interim analysis of quantitative histopathology and chromatin texture as correlates of HPV infection using the cyto-savant system in cytologically and histopathologically negative specimens. Methods. A group of 1544 patients entered the optical technology trials,generating 3275 biopsies and 1544 Papanicolaou readings. Two hundred forty-eight patients were cytologically and histopathologically negative. Study pathologists reviewed histologic samples 3 times in a blinded fashion. Non-overlapping, quantitatively stained nuclei were selected from the samples by the pathologists. HPV testing was done using the PCR method and the Hybrid Capture Ⅱ test. Statistical analysis involved the creation of a classification matrix using a linear discriminant analysis. The matrix was trained on HPV-positive cells by PCR. The analysis included the random creation of both a training set and a validation set that were classified based on the discrimination score obtained by correlating nuclear texture with HPV positivity. Results. The sensitivity of the classification was 52- 54% and the specificity was 77- 78% . Overall, a 68% predicted accuracy was achieved for both the training set and the test set. The agreement of a test and training set shows that the sets created randomly are indeed similar, and that the discrimination score worked equally well in both sets of cells. Once a cell-by-cell algorithm for HPV positivity was derived, HPV positivity was recalculated on the basis of cell-by-cell texture features. HPV positivity was then recalculated on both a per-biopsy basis and a per-patient basis. For HPV 16 and 18, the positivity rate was 70% on a per-biopsy basis and 73% on a per-patient basis. Conclusions. Although these results are preliminary, they suggest that texture features reflecting chromatin condensation may correlate with HPV positivity. The current sample is histologic, the analysis suggests that in a cytologic sample, HPV positivity could be detected or confirmed by texture features computed as part of an HPV associated score. Additional biologic markers could be used as needed. While this study was performed on histologic samples, a study of cytologic samples would be more useful. Future studies will examine chromatin texture compared to HPV integration and mRNA HPV expression.
文摘目的:探索富脯氨酸蛋白11(Proline-rich protein 11,PRR11)过表达诱发染色体凝聚异常的分子机理。方法:综合利用瞬时过表达、si RNA敲减和免疫荧光染色方法在人肺癌细胞系A549中过表达或沉默PRR11后,检测波形蛋白(Vimentin)的表达与亚细胞分布及染色质凝聚异常情况。结果:肺癌细胞中内源性PRR11在胞浆中呈现弥散型分布,而Flag-PRR11过表达后在细胞中的分布呈现弥散状和网络状两种不同形态。进一步检测发现内源性或外源过表达PRR11与Ⅲ型中间丝蛋白Vimentin共定位,并且导致Vimentin绕核分布紊乱及染色质凝聚异常。结论:在细胞周期过程中PRR11与Vimentin共定位并调节其组装过程。推测PRR11异常表达可能通过与Vimentin共定位导致核骨架核纤层不稳定并诱发染色质异常凝聚,从而促进肿瘤的发生发展。
文摘The revolutionary induced pturipotent stem celt （iPSC） technoLogy provides a new means for celt replacement therapies and drug screening. Small molecule compounds have been found extremely useful to improve the generation of iPSCs and understand the repro- gramming mechanism. Here we report the identification of a novel chemical, CYT296, which improves OSKM-mediated induction of iPSCs for 〉10 folds and enables efficient reprogramming with only Oct4 in combination with other small molecules. The derived iPSCs are genuinely pluripotent and support the development oftwo ＇All-iPSC＇ mice by tetraploid complementation. CYT296 profoundly impacts heterochromatin formation without affecting celt viability. MEFs treated with CYT296 exhibit de-condensed chromatin structure with markedly reduced loci containing heterochromatin protein 1α （HPIoL） and H3K9me3, which is very similar to the chromatin configuration in embryonic stem cells （ESCs）. Given that an open chromatin structure serves as a hallmark of pLuripotency and has to be acquired to fulfill reprogramming, we propose that CYT296 might facilitate this process by disrupting condensed chromatin, thereby creating a more favorable environment for reprogramming. In agreement of this idea, shRNA targeting HP1α also promotes the generation of iPSCs. Thus current findings not only provide a novel chemical for efficient iPSC induction, but also suggest a new approach to regulate somatic cell reprogramming by targeting chromatin de-condensation with small molecules.
