Showing papers by "Samuel Aparicio published in 2011"
TL;DR: It is demonstrated, using in vitro enzyme assays of reconstituted PRC2 complexes, that Y641 mutations result in a decrease in monomethylation and an increase in trimethylation activity of the enzyme relative to the wild-type enzyme.
575 citations
TL;DR: This work presents a fully integrated microfluidic device capable of performing high-precision RT-qPCR measurements of gene expression from hundreds of single cells per run, and shows that nanoliter volume processing reduced measurement noise, increased sensitivity, and provided single nucleotide specificity.
Abstract: A long-sought milestone in microfluidics research has been the development of integrated technology for scalable analysis of transcription in single cells Here we present a fully integrated microfluidic device capable of performing high-precision RT-qPCR measurements of gene expression from hundreds of single cells per run Our device executes all steps of single-cell processing, including cell capture, cell lysis, reverse transcription, and quantitative PCR In addition to higher throughput and reduced cost, we show that nanoliter volume processing reduced measurement noise, increased sensitivity, and provided single nucleotide specificity We apply this technology to 3,300 single-cell measurements of (i) miRNA expression in K562 cells, (ii) coregulation of a miRNA and one of its target transcripts during differentiation in embryonic stem cells, and (iii) single nucleotide variant detection in primary lobular breast cancer cells The core functionality established here provides the foundation from which a variety of on-chip single-cell transcription analyses will be developed
493 citations
TL;DR: Overexpression of ZNF703 in normal human breast epithelial cells enhanced the frequency of in vitro colony‐forming cells from luminal progenitors and strongly point to Z NF703 as a novel oncogene in Luminal B breast cancer.
Abstract: The telomeric amplicon at 8p12 is common in oestrogen receptor-positive (ER+) breast cancers. Array-CGH and expression analyses of 1172 primary breast tumours revealed that ZNF703 was the single gene within the minimal amplicon and was amplified predominantly in the Luminal B subtype. Amplification was shown to correlate with increased gene and protein expression and was associated with a distinct expression signature and poor clinical outcome. ZNF703 transformed NIH 3T3 fibroblasts, behaving as a classical oncogene, and regulated proliferation in human luminal breast cancer cell lines and immortalized human mammary epithelial cells. Manipulation of ZNF703 expression in the luminal MCF7 cell line modified the effects of TGFβ on proliferation. Overexpression of ZNF703 in normal human breast epithelial cells enhanced the frequency of in vitro colony-forming cells from luminal progenitors. Taken together, these data strongly point to ZNF703 as a novel oncogene in Luminal B breast cancer.
134 citations
TL;DR: A supporting role is found for miR-223* in differentiating myeloid cells in normal and leukemic cell states, which extends the complexity of miRNA-directed gene regulation of this myeloids key miRNA.
107 citations
TL;DR: A 10-protein biomarker panel that classifies breast cancer into prognostic groups that may have potential utility in the management of patients who receive anthracycline-taxane-based NST is developed.
Abstract: Purpose
To determine whether functional proteomics improves breast cancer classification and prognostication and can predict pathological complete response (pCR) in patients receiving neoadjuvant taxane and anthracycline-taxane-based systemic therapy (NST).
96 citations
TL;DR: P-cadherin positivity is associated with high-grade tumor subtypes and well-established markers of poor prognosis, and may represent a promising antibody therapeutic target.
68 citations
TL;DR: Raman spectroscopy can provide reliable estimates of the in situ glycogen content in hESCs, and this approach should also be extensible to their other biochemical constituents as well as to other cell types.
Abstract: We present a method to perform absolute quantification of glycogen in human embryonic stem cells (hESCs) in situ based on the use of Raman microspectroscopy. The proposed quantification method was validated by comparison to a commonly used commercial glycogen assay kit. With Raman microspectroscopy, we could obtain the glycogen content of hESCs faster and apparently more accurately than with the kit. In addition, glycogen distributions across a colony could be obtained. Raman spectroscopy can provide reliable estimates of the in situ glycogen content in hESCs, and this approach should also be extensible to their other biochemical constituents as well as to other cell types.
52 citations
TL;DR: Through RNA interference screening, it is discovered that knockdown of the serine/threonine phosphatase PPM1H confers trastuzumab resistance via reduction in protein levels of the tumor suppressor p27, revealing new insight into how cancer cells destabilize a well-recognized tumor suppressionor.
Abstract: The HER2 oncogene is overexpressed or amplified in 20% of breast cancers. HER2-positive cancer historically portends a poor prognosis, but the HER2-targeted therapy trastuzumab mitigates this otherwise ominous distinction. Nevertheless, some patients suffer disease recurrence despite trastuzumab, and metastatic disease remains largely incurable due to innate and acquired resistance. Thus, understanding trastuzumab resistance remains an unmet medical need. Through RNA interference screening, we discovered that knockdown of the serine/threonine phosphatase PPM1H confers trastuzumab resistance via reduction in protein levels of the tumor suppressor p27. PPM1H dephosphorylates p27 at threonine 187, thus removing a signal for proteasomal degradation. We further determined that patients whose tumors express low levels of PPM1H trend towards worse clinical outcome on trastuzumab. Identifying PPM1H as a novel p27 phosphatase reveals new insight into how cancer cells destabilize a well-recognized tumor suppressor. Furthermore, low PPM1H expression may identify a subset of HER2-positive tumors that are harder to treat.
Significance: PPM1H is identified as a phosphatase impacting p27 stability. Low expression of PPM1H may be associated with poor outcome in breast cancer. Cancer Discovery; 1(4) ; 326–337. ©2011 AACR .
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49 citations
TL;DR: A clear role of Mll5 in mammalian spermatogenesis at the level of terminal differentiation is demonstrated providing further support for its classification in the SET3 branch of proteins.
Abstract: Background
Mll5 is currently a member of the Mll family of SET domain histone methyltransferase proteins but studies have also showed that it could be part of the SET3 branch of proteins. Recently, constitutive knock out animal studies have shown that Mll5 is required for proper haematopoietic stem cell differentiation, and loss of Mll5 results in synthetic lethality for genome de-methylation. Mll5 deficient male mice are infertile and here we analyse the consequences of Mll5 deficiency for spermatogenesis.
Methodology/Principal Findings
Mll5 deficient male mice, but not female mice, are infertile. Here we show using RNA in-situ hybridization that Mll5 is expressed in the germ cells of the testes of wild type mice. Consistent with the expression of Mll5, we demonstrate by electron microscopy, video microscopy and in vitro fertilisation techniques that Mll5 deficient mice have defects in terminal maturation and packaging of sperm. The defects seen include detachment of the acrosomal cap and impaired excess cytoplasm removal. Functional tests of sperm motility show a lack of progressive motility of spermatozoa from Mll5 deficient animals. None of these defects could be rescued by in vitro fertilization. Using microarray analysis we show that transcripts implicated in spermatogenesis are dysregulated.
Conclusions/Significance
Our data demonstrate a clear role of Mll5 in mammalian spermatogenesis at the level of terminal differentiation providing further support for its classification in the SET3 branch of proteins. Moreover, this study identifies Tlk2, Utx, Gpr64, Sult4a1, Rap2ip, Vstm2 and HoxA10 as possible Mll5 targets that together may account for the observed spermatozoa maturation defects.
45 citations
TL;DR: New candidate positive regulators of humanPS cells are revealed, providing evidence of their ability to regulate expression of pluripotency, cell cycle, and differentiation genes in human PS cells.
32 citations
TL;DR: Raman mapping is used to contrast supra-cellular variations in cytochemical composition across pluripotent and partly differentiated hESC colonies to gain a better understanding of the early-stage effects of the differentiation process on the nature and evolution of these patterns.
Abstract: Measuring spatial and temporal patterns of cytochemical variation in human embryonic stem cell (hESC) colonies is necessary for understanding the role of cellular communication in spontaneous differentiation, the mechanisms of biological niche creation, and structure-generating developmental processes. Such insights will ultimately facilitate directed differentiation and therewith promote advances in tissue engineering and regenerative medicine. However, the patterns of cytochemical inhomogeneities of hESC colonies are not well studied and their causes are not fully understood. We used Raman spectroscopic mapping to contrast supracellular variations in cytochemical composition across pluripotent and partly differentiated hESC colonies to gain a better understanding of the early-stage (i.e., 5 days) effects of the differentiation process on the nature and evolution of these patterns. Higher protein-to-nucleic acid ratios, a differentiation status indicator observed previously using Raman spectroscopy, confirmed reported results that spontaneous differentiation is more pronounced on the edges of a colony than elsewhere. In addition, pluripotent and partly differentiated colonies also showed higher lipid concentrations relative to nucleic acids at colony edges in contrast to relative glycogen concentrations, which were up to 400% more pronounced in the colony centers compared to their edges. Pluripotent and partly differentiated colonies differed, with the latter having higher average protein-to-nucleic acid and lipid-to-nucleic acid ratios but a lower glycogen-to-nucleic acid ratio. In both cases, cell density, pluripotency, and high glycogen appeared to vary in tandem. Spatial variations in glycogen- and protein-to-nucleic acid ratios have features on the order of 100 l m and larger. These dimensions are consistent with those reported for stem cell niches and suggest that cytochemical inhomogeneities may provide colony-level information about niches and niche formation. These results demonstrate Raman mapping to be a potentially useful technique for revealing the complexities in the spatial organization of hESC cultures and thus for monitoring the evolution of engineered hESC niches.
TL;DR: Global transcriptional profiling shows that loss of GPR54 and kisspeptin are not fully equivalent in the mouse hypothalamus.
Abstract: Humans and mice with loss of function mutations in GPR54 (KISS1R) or kisspeptin do not progress through puberty, caused by a failure to release GnRH. The transcriptional networks regulated by these proteins in the hypothalamus have yet to be explored by genome-wide methods. We show here, using 1 million exon mouse arrays (Exon 1.0 Affymetrix) and quantitative polymerase chain reaction (QPCR) validation to analyse microdissected hypothalamic tissue from Gpr54 and Kiss1 knockout mice, the extent of transcriptional regulation in the hypothalamus. The sensitivity to detect important transcript differences in microdissected RNA was confirmed by the observation of counter-regulation of Kiss1 expression in Gpr54 knockouts and confirmed by immunohistochemistry (IHC). Since Gpr54 and Kiss1 knockout animals are effectively pre-pubertal with low testosterone (T) levels, we also determined which of the validated transcripts were T-responsive and which varied according to genotype alone. We observed four types of transcriptional regulation (i) genotype only dependent regulation, (ii) T only dependent regulation, (iii) genotype and T-dependent regulation with interaction between these variables, (iv) genotype and T-dependent regulation with no interaction between these variables. The results implicate for the first time several transcription factors (e.g. Npas4, Esr2), proteases (Klk1b22), and the orphan 10-transmembrane transporter TMEM144 in the biology of GPR54/kisspeptin function in the hypothalamus. We show for the neuronal activity regulated transcription factor NPAS4, that distinct protein over-expression is seen in the hypothalamus and hippocampus in Gpr54 knockout mice. This links for the first time the hypothalamic-gonadal axis with this important regulator of inhibitory synapse formation. Similarly we confirm TMEM144 up-regulation in the hypothalamus by RNA in situ hybridization and western blot. Taken together, global transcriptional profiling shows that loss of GPR54 and kisspeptin are not fully equivalent in the mouse hypothalamus.
TL;DR: It is demonstrated that heat and cold stress had quite different effects on the characteristic Raman signatures of hESCs, demonstrating that Raman spectroscopy can be used to detect deviation from optimal culturing temperatures and therefore it could be of considerable value in the routine and noninvasive determination of h ESC culture quality.
Abstract: Human embryonic stem cells (hESCs) have large nucleus-to-cytoplasm ratios and nucleic acid spectral bands are prominent in their characteristic Raman signatures. Under normal conditions, the major variations in these signatures are due to changes in glycogen content, but how these signatures vary in response to different external conditions is largely unknown. In this study we investigated the influences of temperature variations on hESC Raman signatures. At 32 °C, compared to the 37 °C control condition, cell proliferation rates were markedly reduced and glycogen Raman band intensities were elevated. In addition, at both temperatures, an inverse relationship between cell proliferation rates (i.e., onset of exponential growth phase vs. end of exponential phase) and glycogen Raman band intensities was observed. This relationship suggested a role for glycogen in the energy metabolism of hESC self-renewal. Protein and lipid spectral variations were small and co-varied with those of nucleic acids, suggesting that they were related to changes in cellular dimensions occurring during the cell cycle. When the temperature was elevated to 39 °C, increased glycogen band intensities, compared to controls, were also observed. In addition, spectral evidence of differentiation emerged that was supported by reduced SSEA-3 expression. Taken together, these results demonstrated that heat and cold stress had quite different effects on the characteristic Raman signatures of hESCs. Thus, Raman spectroscopy can be used to detect deviation from optimal culturing temperatures and therefore it could be of considerable value in the routine and noninvasive determination of hESC culture quality.
TL;DR: A mouse line transgenic for EZH2 Y641F can collaborate with Emu-myc in high efficiency lymphoma induction and provide a powerful tool to study global changes in the epigenome caused by this frequent mutation.
TL;DR: A dual, inhibitor of EGFR and HER2/neu tyrosine kinase activity improves overall survival in patients with metastatic BC when added to paclitaxel (SABCS 2010:P3-1424) and further research using standardized methodology is required to define prognostic and predictive molecular markers.
Abstract: TPS108 Background: The EGFR pathways are important therapeutic targets in HER2/neu+ BC. Lapatinib (L) a dual, inhibitor of EGFR and HER2/neu tyrosine kinase activity improves overall survival (OS) ...