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1.
Nature ; 628(8009): 771-775, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38632399

RESUMO

Quantitative detection of various molecules at very low concentrations in complex mixtures has been the main objective in many fields of science and engineering, from the detection of cancer-causing mutagens and early disease markers to environmental pollutants and bioterror agents1-5. Moreover, technologies that can detect these analytes without external labels or modifications are extremely valuable and often preferred6. In this regard, surface-enhanced Raman spectroscopy can detect molecular species in complex mixtures on the basis only of their intrinsic and unique vibrational signatures7. However, the development of surface-enhanced Raman spectroscopy for this purpose has been challenging so far because of uncontrollable signal heterogeneity and poor reproducibility at low analyte concentrations8. Here, as a proof of concept, we show that, using digital (nano)colloid-enhanced Raman spectroscopy, reproducible quantification of a broad range of target molecules at very low concentrations can be routinely achieved with single-molecule counting, limited only by the Poisson noise of the measurement process. As metallic colloidal nanoparticles that enhance these vibrational signatures, including hydroxylamine-reduced-silver colloids, can be fabricated at large scale under routine conditions, we anticipate that digital (nano)colloid-enhanced Raman spectroscopy will become the technology of choice for the reliable and ultrasensitive detection of various analytes, including those of great importance for human health.


Assuntos
Coloides , Imagem Individual de Molécula , Análise Espectral Raman , Coloides/química , Hidroxilamina/química , Nanopartículas Metálicas/química , Distribuição de Poisson , Estudo de Prova de Conceito , Reprodutibilidade dos Testes , Prata/química , Imagem Individual de Molécula/métodos , Imagem Individual de Molécula/normas , Análise Espectral Raman/métodos , Análise Espectral Raman/normas , Vibração
2.
EMBO J ; 38(18): e100948, 2019 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-31418899

RESUMO

As a ubiquitous bacterial secondary messenger, c-di-GMP plays key regulatory roles in processes such as bacterial motility and transcription regulation. CobB is the Sir2 family protein deacetylase that controls energy metabolism, chemotaxis, and DNA supercoiling in many bacteria. Using an Escherichia coli proteome microarray, we found that c-di-GMP strongly binds to CobB. Further, protein deacetylation assays showed that c-di-GMP inhibits the activity of CobB and thereby modulates the biogenesis of acetyl-CoA. Interestingly, we also found that one of the key enzymes directly involved in c-di-GMP production, DgcZ, is a substrate of CobB. Deacetylation of DgcZ by CobB enhances its activity and thus the production of c-di-GMP. Our work establishes a novel negative feedback loop linking c-di-GMP biogenesis and CobB-mediated protein deacetylation.


Assuntos
GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Sirtuínas/metabolismo , Acetilcoenzima A/metabolismo , Acetilação , GMP Cíclico/metabolismo , Retroalimentação Fisiológica , Regulação Bacteriana da Expressão Gênica , Análise Serial de Proteínas/métodos , Proteômica/métodos , Sistemas do Segundo Mensageiro
3.
Int J Mol Sci ; 24(21)2023 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-37958853

RESUMO

Centromeric chromatin is thought to play a critical role in ensuring the faithful segregation of chromosomes during mitosis. However, our understanding of this role is presently limited by our poor understanding of the structure and composition of this unique chromatin. The nucleosomal variant, CENP-A, localizes to narrow regions within the centromere, where it plays a major role in centromeric function, effectively serving as a platform on which the kinetochore is assembled. Previous work found that, within a given cell, the number of microtubules within kinetochores is essentially unchanged between CENP-A-localized regions of different physical sizes. However, it is unknown if the amount of CENP-A is also unchanged between these regions of different sizes, which would reflect a strict structural correspondence between these two key characteristics of the centromere/kinetochore assembly. Here, we used super-resolution optical microscopy to image and quantify the amount of CENP-A and DNA within human centromere chromatin. We found that the amount of CENP-A within CENP-A domains of different physical sizes is indeed the same. Further, our measurements suggest that the ratio of CENP-A- to H3-containing nucleosomes within these domains is between 8:1 and 11:1. Thus, our results not only identify an unexpectedly strict relationship between CENP-A and microtubules stoichiometries but also that the CENP-A centromeric domain is almost exclusively composed of CENP-A nucleosomes.


Assuntos
Microscopia , Nucleossomos , Humanos , Proteína Centromérica A/genética , Proteínas Cromossômicas não Histona/metabolismo , Centrômero/metabolismo , Cromatina , Cinetocoros/metabolismo , Autoantígenos/química
4.
Int J Mol Sci ; 23(24)2022 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-36555471

RESUMO

The activation of monocytes and their trans-differentiation into macrophages are critical processes of the immune response. Prior work has characterized the differences in the expression between monocytes and macrophages, but the transitional process between these cells is poorly detailed. Here, we analyzed the temporal changes of the transcriptome during trans-differentiation of primary human monocytes into M0 macrophages. We find changes with many transcription factors throughout the process, the vast majority of which exhibit a maximally different expression at the intermediate stages. A few factors, including AP-1, were previously known to play a role in immunological transitions, but most were not. Thus, these findings indicate that this trans-differentiation requires the dynamic expression of many transcription factors not previously discussed in immunology, and provide a foundation for the delineation of the molecular mechanisms associated with healthy or pathological responses that involve this transition.


Assuntos
Monócitos , Fatores de Transcrição , Humanos , Monócitos/metabolismo , Fatores de Transcrição/metabolismo , Macrófagos/metabolismo , Diferenciação Celular/fisiologia , Transdiferenciação Celular/genética
5.
J Med Genet ; 57(6): 385-388, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-30877236

RESUMO

BACKGROUND: Spasmolytic polypeptide-expressing metaplasia (SPEM) is present in more than 90% of resected gastric cancer tissues. However, although widely regarded as a pre-cancerous tissue, its genetic characteristics have not been well studied. METHODS: Immunohistochemistry using Trefoil factor 2 (TFF2) antibodies was used to identify TFF2-positive SPEM cells within SPEM glands in the stomach of Helicobacter felis (H. felis) -infected mice and human clinical samples. Laser microdissection was used to isolate specific cells from both the infected mice and the human samples. The genetic instability in these cells was examined by measuring the lengths of microsatellite (MS) markers using capillary electrophoresis. Also, genome-wide genetic variations in the SPEM cells from the clinical sample was examined using deep whole-exome sequencing. RESULTS: SPEM cells indeed exhibit not only heightened MS instability (MSI), but also genetic instabilities that extend genome-wide. Furthermore, surprisingly, we found that morphologically normal, TFF2-negative cells also contain a comparable degree of genomic instabilities as the co-resident SPEM cells within the SPEM glands. CONCLUSION: These results, for the first time, clearly establish elevated genetic instability as a critical property of SPEM glands, which may provide a greater possibility for malignant clone selection. More importantly, these results indicate that SPEM cells may not be the sole origin of carcinogenesis in the stomach and strongly suggest the common progenitor of these cells, the stem cells, as the source of these genetic instabilities, and thus, potential key players in carcinogenesis.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/genética , Metaplasia/genética , Neoplasias Gástricas/genética , Fator Trefoil-2/genética , Animais , Feminino , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Regulação Neoplásica da Expressão Gênica/genética , Instabilidade Genômica/genética , Xenoenxertos , Humanos , Masculino , Metaplasia/patologia , Camundongos , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Neoplasias Gástricas/patologia
6.
Hereditas ; 158(1): 43, 2021 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-34740370

RESUMO

Immortalized cell lines have long been used as model systems to systematically investigate biological processes under controlled and reproducible conditions, providing insights that have greatly advanced cellular biology and medical sciences. Recently, the widely used monocytic leukemia cell line, THP-1, was comprehensively examined to understand mechanistic relationships between the 3D chromatin structure and transcription during the trans-differentiation of monocytes to macrophages. To corroborate these observations in primary cells, we analyze in situ Hi-C and RNA-seq data of human primary monocytes and their differentiated macrophages in comparison to that obtained from the monocytic/macrophagic THP-1 cells. Surprisingly, we find significant differences between the primary cells and the THP-1 cells at all levels of chromatin structure, from loops to topologically associated domains to compartments. Importantly, the compartment-level differences correlate significantly with transcription: those genes that are in A-compartments in the primary cells but are in B-compartments in the THP-1 cells exhibit a higher level of expression in the primary cells than in the THP-1 cells, and vice versa. Overall, the genes in these different compartments are enriched for a wide range of pathways, and, at least in the case of the monocytic cells, their altered expression in certain pathways in the THP-1 cells argues for a less immune cell-like phenotype, suggesting that immortalization or prolonged culturing of THP-1 caused a divergence of these cells from their primary counterparts. It is thus essential to reexamine phenotypic details observed in cell lines with their primary counterparts so as to ensure a proper understanding of functional cell states in vivo.


Assuntos
Monócitos , Transcriptoma , Diferenciação Celular , Humanos , Macrófagos , Células THP-1
7.
Angew Chem Int Ed Engl ; 60(6): 3055-3061, 2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33084179

RESUMO

Owing to the challenges to acquire detailed spatial information of gut bacteria in situ, three-dimensional (3D) microbiota distributions in the gut remain largely uncharted. Here, we propose a tissue clearing-based and D-amino acid labeling-facilitated (TiDaL) strategy that combines a novel microbiota in vivo labeling protocol, CUBIC-based tissue clearing and whole-mount tissue imaging, to achieve 3D imaging of indigenous gut microbiota. We demonstrate high-resolution 3D acquisition of their biogeography in different gut sections, and present quantitative spatial details in relation to the host epithelium. We unexpectedly observe microbiota in the small intestine crypts, which were thought to be bacteria-free. Significant bacterial overgrowth in the first two-thirds of the small intestine is detected in an enteritis model. We expect that this quantitative 3D imaging strategy for native gut microbiota will provide insightful information into the host-microbiota interactions.


Assuntos
Microbioma Gastrointestinal , Imageamento Tridimensional/métodos , Aminoácidos/química , Aminoácidos/metabolismo , Animais , Bactérias/crescimento & desenvolvimento , Bactérias/metabolismo , Carbocianinas/química , Sulfato de Dextrana/química , Sulfato de Dextrana/metabolismo , Corantes Fluorescentes/química , Intestinos/microbiologia , Camundongos , Imagem Óptica
8.
Mol Cell Proteomics ; 17(9): 1720-1736, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29871872

RESUMO

Proteins, as the major executer for cell progresses and functions, its abundance and the level of post-translational modifications, are tightly monitored by regulators. Genetic perturbation could help us to understand the relationships between genes and protein functions. Herein, to explore the impact of the genome-wide interruption on certain protein, we developed a cell lysate microarray on kilo-conditions (CLICK) with 4837 knockout (YKO) and 322 temperature-sensitive (ts) mutant strains of yeast (Saccharomyces cerevisiae). Taking histone marks as examples, a general workflow was established for the global identification of upstream regulators. Through a single CLICK array test, we obtained a series of regulators for H3K4me3, which covers most of the known regulators in S. cerevisiae We also noted that several group of proteins are involved in negatively regulation of H3K4me3. Further, we discovered that Cab4p and Cab5p, two key enzymes of CoA biosynthesis, play central roles in histone acylation. Because of its general applicability, CLICK array could be easily adopted to rapid and global identification of upstream protein/enzyme(s) that regulate/modify the level of a protein or the posttranslational modification of a non-histone protein.


Assuntos
Redes Reguladoras de Genes , Código das Histonas/genética , Saccharomyces cerevisiae/genética , Acil Coenzima A/metabolismo , Acilação , Química Click , Histonas/metabolismo , Lisina/metabolismo , Metilação , Modelos Biológicos , Mutação/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico
9.
Acta Biochim Biophys Sin (Shanghai) ; 52(5): 546-553, 2020 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-32324846

RESUMO

Monocyte-to-macrophage trans-differentiation has long been studied to better understand this immunological response and aspects of developmental processes more generally. A key question is the nature of the corresponding changes in chromatin conformation and its relationship to the transcriptome during this process. This question is especially intriguing since this trans-differentiation is not associated with progression through mitosis, often considered a necessary step for gross changes in chromosomal structure. Here, we characterized the transcriptional and genomic structural changes during macrophage development of primary human monocytes using RNA-seq and in situ Hi-C. We found that, during this transition, the genome architecture undergoes a massive remodeling to a degree not observed before between structured genomes, with changes in ~90% of the topologically associating domains (TADs). These changes in the TADs are associated with changed expression of immunological genes. These structural changes, however, differ extensively from those described recently in a study of the leukemia cell line, THP-1. Furthermore, up-regulation of the AP-1 family of genes that effected functionally important changes in the genomic structure during the differentiation of the THP-1 cells was not corroborated with the primary cells. Taken together, our results provide a comprehensive characterization of the changes in genomic structure during the monocyte-to-macrophage transition, establish a framework for the elucidation of processes underlying differentiation without proliferation, and demonstrate the importance of verifying with primary cells the mechanisms discovered with cultured cells.


Assuntos
Diferenciação Celular , Genoma Humano , Macrófagos/metabolismo , Monócitos/metabolismo , Feminino , Humanos , Macrófagos/citologia , Masculino , Monócitos/citologia
10.
Small ; 15(42): e1902637, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31468738

RESUMO

The atomic structure of free-standing graphene comprises flat hexagonal rings with a 2.5 Å period, which is conventionally considered the only atomic period and determines the unique properties of graphene. Here, an unexpected highly ordered orthorhombic structure of graphene is directly observed with a lattice constant of ≈5 Å, spontaneously formed on various substrates. First-principles computations show that this unconventional structure can be attributed to the dipole between the graphene surface and substrates, which produces an interfacial electric field and induces atomic rearrangement on the graphene surface. Further, the formation of the orthorhombic structure can be controlled by an artificially generated interfacial electric field. Importantly, the 5 Å crystal can be manipulated and transformed in a continuous and reversible manner. Notably, the orthorhombic lattice can control the epitaxial self-assembly of amyloids. The findings reveal new insights about the atomic structure of graphene, and open up new avenues to manipulate graphene lattices.

11.
Mol Cell Proteomics ; 16(8): 1491-1506, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28572091

RESUMO

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, the leading cause of death among all infectious diseases. There are 11 eukaryotic-like serine/threonine protein kinases (STPKs) in Mtb, which are thought to play pivotal roles in cell growth, signal transduction and pathogenesis. However, their underlying mechanisms of action remain largely uncharacterized. In this study, using a Mtb proteome microarray, we have globally identified the binding proteins in Mtb for all of the STPKs, and constructed the first STPK protein interaction (KPI) map that includes 492 binding proteins and 1,027 interactions. Bioinformatics analysis showed that the interacting proteins reflect diverse functions, including roles in two-component system, transcription, protein degradation, and cell wall integrity. Functional investigations confirmed that PknG regulates cell wall integrity through key components of peptidoglycan (PG) biosynthesis, e.g. MurC. The global STPK-KPIs network constructed here is expected to serve as a rich resource for understanding the key signaling pathways in Mtb, thus facilitating drug development and effective control of Mtb.


Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/metabolismo , Mapas de Interação de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Proteoma/metabolismo , Proteínas de Bactérias/genética , Parede Celular , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteoma/genética , Proteômica , Transdução de Sinais
12.
Nucleic Acids Res ; 44(10): 4934-46, 2016 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-27016738

RESUMO

Mitotic chromosomes are one of the most commonly recognized sub-cellular structures in eukaryotic cells. Yet basic information necessary to understand their structure and assembly, such as their composition, is still lacking. Recent proteomic studies have begun to fill this void, identifying hundreds of RNA-binding proteins bound to mitotic chromosomes. However, by contrast, there are only two RNA species (U3 snRNA and rRNA) that are known to be associated with the mitotic chromosome, suggesting that there are many mitotic chromosome-associated RNAs (mCARs) not yet identified. Here, using a targeted protocol based on 5'-tag sequencing to profile the mammalian mCAR population, we report the identification of 1279 mCARs, the majority of which are ncRNAs, including lncRNAs that exhibit greater conservation across 60 vertebrate species than the entire population of lncRNAs. There is also a significant enrichment of snoRNAs and specific SINE RNAs. Finally, ∼40% of the mCARs are presently unannotated, many of which are as abundant as the annotated mCARs, suggesting that there are also many novel ncRNAs in the mCARs. Overall, the mCARs identified here, together with the previous proteomic and genomic data, constitute the first comprehensive catalogue of the molecular composition of the eukaryotic mitotic chromosomes.


Assuntos
Cromossomos de Mamíferos/química , Mitose/genética , RNA não Traduzido/análise , Células 3T3 , Animais , Sequenciamento de Nucleotídeos em Larga Escala , Metáfase/genética , Camundongos , RNA não Traduzido/química , RNA não Traduzido/isolamento & purificação , Análise de Sequência de RNA , Sitios de Sequências Rotuladas
13.
Proc Natl Acad Sci U S A ; 112(49): 15084-9, 2015 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-26598702

RESUMO

Arsenic is highly effective for treating acute promyelocytic leukemia (APL) and has shown significant promise against many other tumors. However, although its mechanistic effects in APL are established, its broader anticancer mode of action is not understood. In this study, using a human proteome microarray, we identified 360 proteins that specifically bind arsenic. Among the most highly enriched proteins in this set are those in the glycolysis pathway, including the rate-limiting enzyme in glycolysis, hexokinase-1. Detailed biochemical and metabolomics analyses of the highly homologous hexokinase-2 (HK2), which is overexpressed in many cancers, revealed significant inhibition by arsenic. Furthermore, overexpression of HK2 rescued cells from arsenic-induced apoptosis. Our results thus strongly implicate glycolysis, and HK2 in particular, as a key target of arsenic. Moreover, the arsenic-binding proteins identified in this work are expected to serve as a valuable resource for the development of synergistic antitumor therapeutic strategies.


Assuntos
Arsênio/farmacologia , Proteínas de Transporte/análise , Hexoquinase/antagonistas & inibidores , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Arsênio/metabolismo , Trióxido de Arsênio , Arsenicais/farmacologia , Proteínas de Transporte/metabolismo , Biologia Computacional , Glicólise , Humanos , Metabolômica , Dados de Sequência Molecular , Óxidos/farmacologia , Proteoma
14.
J Med Genet ; 53(9): 643-6, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27287394

RESUMO

BACKGROUND: The transformation of healthy gastric tissue into intestinal metaplasia (IM) is thought to be a critical premalignant step in the development of intestinal-type gastric adenocarcinoma (GA). How such premalignancies contribute to the development of GA is, however, poorly understood. METHODS: In this study, the extent and clonal complexity in IM tissue from patients without gastric cancer were analysed by measuring variations of multiple microsatellite (MS) markers. RESULTS: Even though these tissues are generally regarded as clinically benign, we found extensive MS length heterogeneity between and within individual IM glands, indicating that complex genome diversity is already pervasive in these tissues. Based on a clonal relationship analysis, we found that there exist multiple clones within individual IM glands and that MS alterations can accumulate in these clones. Moreover, we found spatially distant IM glands with the same MS phenotype, suggesting that these MS alterations were progressed by gland fission. CONCLUSIONS: These results provide evidence that genetic instability is an early event, present within metaplastic tissues of otherwise non-cancer patients, and such frequent genetic alterations can be part of the pathophysiological rationale for the requirement of this phase during gastric carcinogenesis.


Assuntos
Metaplasia/genética , Mutação/genética , Neoplasias Gástricas/genética , Adenocarcinoma/genética , Adulto , Feminino , Humanos , Masculino , Repetições de Microssatélites/genética , Pessoa de Meia-Idade , Mosaicismo , Lesões Pré-Cancerosas/genética
17.
Breast Cancer Res ; 17: 36, 2015 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-25848723

RESUMO

INTRODUCTION: Triple-negative breast cancer (TNBC) patients often face a high risk of early relapse characterized by extensive metastasis. Previous works have shown that aberrant cell surface glycosylation is associated with cancer metastasis, suggesting that altered glycosylations might serve as diagnostic signatures of metastatic potential. To address this question, we took TNBC as an example and analyzed six TNBC cell lines, derived from a common progenitor, that differ in metastatic potential. METHODS: We used a microarray with 91 lectins to screen for altered lectin bindings to the six TNBC cell lines. Candidate lectins were then verified by lectin-based flow cytometry and immunofluorescent staining assays using both TNBC/non-TNBC cancer cells. Patient-derived tissue microarrays were then employed to analyze whether the staining of Ricinus communis agglutinin I (RCA-I), correlated with TNBC severity. We also carried out real-time cell motility assays in the presence of RCA-I. Finally, liquid chromatography-mass spectrometry/tandem spectrometry (LC-MS/MS) was employed to identify the membrane glycoproteins recognized by RCA-I. RESULTS: Using the lectin microarray, we found that the bindings of RCA-I to TNBC cells are proportional to their metastatic capacity. Tissue microarray experiments showed that the intensity of RCA-I staining is positively correlated with the TNM grades. The real-time cell motility assays clearly demonstrated RCA-I inhibition of adhesion, migration, and invasion of TNBC cells of high metastatic capacity. Additionally, a membrane glycoprotein, POTE ankyrin domain family member F (POTEF), with different galactosylation extents in high/low metastatic TNBC cells was identified by LC-MS/MS as a binder of RCA-I. CONCLUSIONS: We discovered RCA-I, which bound to TNBC cells to a degree that is proportional to their metastatic capacities, and found that this binding inhibits the cell invasion, migration, and adhesion, and identified a membrane protein, POTEF, which may play a key role in mediating these effects. These results thus indicate that RCA-I-specific cell surface glycoproteins may play a critical role in TNBC metastasis and that the extent of RCA-I cell binding could be used in diagnosis to predict the likelihood of developing metastases in TNBC patients.


Assuntos
Membrana Celular/metabolismo , Lectinas de Plantas/metabolismo , Polissacarídeos/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Feminino , Humanos , Glicoproteínas de Membrana/metabolismo , Metástase Neoplásica , Ligação Proteica , Mapeamento de Interação de Proteínas , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem
18.
Anal Chem ; 87(19): 9761-8, 2015 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-26378744

RESUMO

The high glucose uptake and activation of oncogenic signaling pathways in cancer cells has long made these features, together with the mutational spectrum, prime diagnostic targets of circulating tumor cells (CTCs). Further, an ability to characterize these properties at a single cell resolution is widely believed to be essential, as the known extensive heterogeneity in CTCs can obscure important correlations in data obtained from cell population-based methods. However, to date, it has not been possible to quantitatively measure metabolic, proteomic, and genetic data from a single CTC. Here we report a microchip-based approach that allows for the codetection of glucose uptake, intracellular functional proteins, and genetic mutations at the single-cell level from rare tumor cells. The microchip contains thousands of nanoliter grooves (nanowells) that isolate individual CTCs and allow for the assessment of their glucose uptake via imaging of a fluorescent glucose analog, quantification of a panel of intracellular signaling proteins using a miniaturized antibody barcode microarray, and retrieval of the individual cell nuclei for subsequent off-chip genome amplification and sequencing. This approach integrates molecular-scale information on the metabolic, proteomic, and genetic status of single cells and permits the inference of associations between genetic signatures, energy consumption, and phosphoproteins oncogenic signaling activities in CTCs isolated from blood samples of patients. Importantly, this microchip chip-based approach achieves this multidimensional molecular analysis with minimal cell loss (<20%), which is the bottleneck of the rare cell analysis.


Assuntos
Análise Mutacional de DNA/instrumentação , Glucose/metabolismo , Dispositivos Lab-On-A-Chip , Células Neoplásicas Circulantes/metabolismo , Fosfoproteínas/metabolismo , Análise de Célula Única/instrumentação , Desenho de Equipamento , Genômica/instrumentação , Glucose/análise , Humanos , Mutação , Células Neoplásicas Circulantes/patologia , Imagem Óptica/instrumentação , Fosfoproteínas/análise , Análise Serial de Proteínas/instrumentação
19.
Mol Genet Genomics ; 290(1): 217-24, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25213602

RESUMO

The identification of structural and functional elements encoded in a genome is a challenging task. Although the transcriptome of budding yeast has been extensively analyzed, the boundaries and untranslated regions of yeast genes remain elusive. To address this least-explored field of yeast genomics, we performed a transcript profiling analysis through paired-end ditag (PET) approach coupled with deep sequencing. With 562,133 PET sequences we accurately defined the boundaries and untranslated regions of 3,409 ORFs, suggesting many yeast genes have multiple transcription start sites (TSSs). We also identified 85 previously uncharacterized transcripts either in intergenic regions or from the opposite strand of reported genomic features. Furthermore, our data revealed the extensive 3' end heterogeneity of yeast genes and identified a novel putative motif for polyadenylation. Our results indicate the yeast transcriptome is more complex than expected. This study would serve as an invaluable resource for elucidating the regulation and evolution of yeast genes.


Assuntos
Perfilação da Expressão Gênica , Genoma Fúngico/genética , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA/métodos , Transcriptoma/genética , Regiões não Traduzidas/genética , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Regulação Fúngica da Expressão Gênica , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sítio de Iniciação de Transcrição
20.
Nanomedicine ; 11(1): 119-25, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25220907

RESUMO

Invented in the 1990s, near-field optical microscopy (NSOM) was the first optical microscopy method to hold the promise of finally breaking the diffraction barrier in studies of biological samples. This promise, though, failed to materialize at that time, largely owing to the inability to image soft samples, such as cell surfaces, without damage. However, steady technical improvements have now produced NSOM devices that can routinely achieve images of cell surfaces with sub-100nm resolution in aqueous solution. Further, beyond just optical information, these instruments can also provide simultaneous topographic, mechanical, and/or chemical details of the sample, an ability not yet matched by any other optics-based methodology. With the long recognized important roles of many biological processes at cell surfaces in human health and disease, near-field probing of cell surfaces is indeed now well poised to directly illume in biomedicine what has, until recently, been unknowable with classic light microscopy.


Assuntos
Membrana Celular/metabolismo , Microscopia/métodos , Nanotecnologia/métodos , Íons , Luz , Neurônios/metabolismo , Óptica e Fotônica , Estrutura Terciária de Proteína , Processos Estocásticos
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