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1.
Cell ; 179(2): 527-542.e19, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31585086

RESUMO

Much of current molecular and cell biology research relies on the ability to purify cell types by fluorescence-activated cell sorting (FACS). FACS typically relies on the ability to label cell types of interest with antibodies or fluorescent transgenic constructs. However, antibody availability is often limited, and genetic manipulation is labor intensive or impossible in the case of primary human tissue. To date, no systematic method exists to enrich for cell types without a priori knowledge of cell-type markers. Here, we propose GateID, a computational method that combines single-cell transcriptomics with FACS index sorting to purify cell types of choice using only native cellular properties such as cell size, granularity, and mitochondrial content. We validate GateID by purifying various cell types from zebrafish kidney marrow and the human pancreas to high purity without resorting to specific antibodies or transgenes.


Assuntos
Separação Celular/métodos , Citometria de Fluxo/métodos , Software , Transcriptoma , Animais , Humanos , Rim/citologia , Pâncreas/citologia , Análise de Célula Única , Peixe-Zebra/anatomia & histologia
2.
Mol Cell ; 83(19): 3558-3573.e7, 2023 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-37802028

RESUMO

Cellular senescence is a stress-response mechanism implicated in various physiological processes, diseases, and aging. Current detection approaches have partially addressed the issue of senescent cell identification in clinical specimens. Effective methodologies enabling precise isolation or live tracking of senescent cells are still lacking. In-depth analysis of truly senescent cells is, therefore, an extremely challenging task. We report (1) the synthesis and validation of a fluorophore-conjugated, Sudan Black-B analog (GLF16), suitable for in vivo and in vitro analysis of senescence by fluorescence microscopy and flow cytometry and (2) the development and application of a GLF16-carrying micelle vector facilitating GLF16 uptake by living senescent cells in vivo and in vitro. The compound and the applied methodology render isolation of senescent cells an easy, rapid, and precise process. Straightforward nanocarrier-mediated GLF16 delivery in live senescent cells comprises a unique tool for characterization of senescence at an unprecedented depth.


Assuntos
Senescência Celular , Indicadores e Reagentes , Citometria de Fluxo
3.
Immunity ; 54(7): 1578-1593.e5, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34051147

RESUMO

Immune profiling of COVID-19 patients has identified numerous alterations in both innate and adaptive immunity. However, whether those changes are specific to SARS-CoV-2 or driven by a general inflammatory response shared across severely ill pneumonia patients remains unknown. Here, we compared the immune profile of severe COVID-19 with non-SARS-CoV-2 pneumonia ICU patients using longitudinal, high-dimensional single-cell spectral cytometry and algorithm-guided analysis. COVID-19 and non-SARS-CoV-2 pneumonia both showed increased emergency myelopoiesis and displayed features of adaptive immune paralysis. However, pathological immune signatures suggestive of T cell exhaustion were exclusive to COVID-19. The integration of single-cell profiling with a predicted binding capacity of SARS-CoV-2 peptides to the patients' HLA profile further linked the COVID-19 immunopathology to impaired virus recognition. Toward clinical translation, circulating NKT cell frequency was identified as a predictive biomarker for patient outcome. Our comparative immune map serves to delineate treatment strategies to interfere with the immunopathologic cascade exclusive to severe COVID-19.


Assuntos
COVID-19/imunologia , SARS-CoV-2/patogenicidade , Adulto , Enzima de Conversão de Angiotensina 2/metabolismo , Apresentação de Antígeno , Biomarcadores/sangue , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , COVID-19/patologia , Feminino , Antígenos HLA/genética , Antígenos HLA/imunologia , Humanos , Imunidade Inata , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Células T Matadoras Naturais/imunologia , Pneumonia/imunologia , Pneumonia/patologia , SARS-CoV-2/imunologia , Índice de Gravidade de Doença , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
4.
Immunity ; 51(4): 750-765.e10, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31492649

RESUMO

Immunity that controls parasitemia and inflammation during Plasmodium falciparum (Pf) malaria can be acquired with repeated infections. A limited understanding of this complex immune response impedes the development of vaccines and adjunctive therapies. We conducted a prospective systems biology study of children who differed in their ability to control parasitemia and fever following Pf infection. By integrating whole-blood transcriptomics, flow-cytometric analysis, and plasma cytokine and antibody profiles, we demonstrate that a pre-infection signature of B cell enrichment, upregulation of T helper type 1 (Th1) and Th2 cell-associated pathways, including interferon responses, and p53 activation associated with control of malarial fever and coordinated with Pf-specific immunoglobulin G (IgG) and Fc receptor activation to control parasitemia. Our hypothesis-generating approach identified host molecules that may contribute to differential clinical outcomes during Pf infection. As a proof of concept, we have shown that enhanced p53 expression in monocytes attenuated Plasmodium-induced inflammation and predicted protection from fever.


Assuntos
Linfócitos B/imunologia , Proteínas Sanguíneas/metabolismo , Inflamação/metabolismo , Malária Falciparum/metabolismo , Plasmodium falciparum/fisiologia , Células Th1/imunologia , Células Th2/imunologia , Proteína Supressora de Tumor p53/metabolismo , Adolescente , Adulto , Animais , Anticorpos Antiprotozoários/metabolismo , Criança , Pré-Escolar , Resistência à Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Lactente , Interferons/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estudos Prospectivos , Receptores Fc/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Adulto Jovem
5.
J Biol Chem ; 300(7): 107482, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38897567

RESUMO

Siglecs are cell surface receptors whose functions are tied to the binding of their sialoglycan ligands. Recently, we developed an optimized liposome formulation and used it to investigate the binding of human Siglecs (hSiglec) against a panel of gangliosides. Animal models, more specifically murine models, are used to understand human biology; however, species-specific differences can complicate the interpretation of the results. Herein, we used our optimized liposome formulation to dissect the interactions between murine Siglecs (mSiglecs) and gangliosides to assess the appropriateness of mSiglecs as a proxy to better understand the biological roles of hSiglec-ganglioside interactions. Using our optimized liposome formulation, we found that ganglioside binding is generally conserved between mice and humans with mSiglec-1, -E, -F, and -15 binding multiple gangliosides like their human counterparts. However, in contrast to the hSiglecs, we observed little to no binding between the mSiglecs and ganglioside GM1a. Detailed analysis of mSiglec-1 interacting with GM1a and its structural isomer, GM1b, suggests that mSiglec-1 preferentially binds α2-3-linked sialic acids presented from the terminal galactose residue. The ability of mSiglecs to interact or not interact with gangliosides, particularly GM1a, has implications for using mice to study neurodegenerative diseases, infections, and cancer, where interactions between Siglecs and glycolipids have been proposed to modulate these human diseases.


Assuntos
Gangliosídeos , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico , Animais , Gangliosídeos/metabolismo , Camundongos , Humanos , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/genética , Lipossomos/metabolismo , Lectinas/metabolismo , Lectinas/química , Ligação Proteica , Antígenos CD/metabolismo , Antígenos CD/genética
6.
J Biol Chem ; : 107613, 2024 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-39079629

RESUMO

Shigella spp. are highly pathogenic members of the Enterobacteriaceae family, causing ∼269 million cases of bacillary dysentery and >200,000 deaths each year. Like many Gram-negative pathogens, Shigella rely on their type three secretion system (T3SS) to inject effector proteins into eukaryotic host cells, driving both cellular invasion and evasion of host immune responses. Exposure to the bile salt deoxycholate (DOC) significantly enhances Shigella virulence and is proposed to serve as a critical environmental signal present in the small intestine that prepares Shigella's T3SS for efficient infection of the colonic epithelium. Here, we uncover critical mechanistic details of the Shigella-specific DOC signaling process by describing the role of a π-helix secondary structure element within the T3SS tip protein IpaD. Biophysical characterization and high-resolution structures of IpaD mutants lacking the π-helix show that it is not required for global protein structure, but that it defines the native DOC binding site and prevents off target interactions. Additionally, Shigella strains expressing the π-helix deletion mutants illustrate the pathogenic importance of its role in guiding DOC interaction as flow cytometry and gentamycin protection assays show that the IpaD π-helix is essential for DOC-mediated apparatus maturation and enhanced invasion of eukaryotic cells. Together, these findings add to our understanding of the complex Shigella pathogenesis pathway and its evolution to respond to environmental bile salts by identifying the π-helix in IpaD as a critical structural element required for translating DOC exposure to virulence enhancement.

7.
J Biol Chem ; : 107637, 2024 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-39122004

RESUMO

Tissues are formed and shaped by cells of many different types and are orchestrated through countless interactions. Deciphering a tissue's biological complexity thus requires studying it at cell-level resolution, where molecular and biochemical features of different cell types can be explored and thoroughly dissected. Unfortunately, the lack of comprehensive methods to identify, isolate, and culture each cell type from many tissues has impeded progress. Here, we present a method for the breadth of cell types composing the human breast. Our goal has long been to understand the essence of each of these different breast cell types, to reveal the underlying biology explaining their intrinsic features, the consequences of interactions, and their contributions to the tissue. This biological exploration has required cell purification, deep-RNA sequencing-and a thorough dissection of the genes and pathways defining each cell type. Whereas the molecular analysis is presented in an adjoining article, we present here an exhaustive cellular dissection of the human breast and explore its cellular composition and histological organization. Moreover, we introduce a novel FACS antibody panel and rigorous gating strategy capable of isolating each of the twelve major breast cell types to purity. Finally, we describe the creation of primary cell models from nearly every breast cell type-some the first of their kind- and submit these as critical tools for studying the dynamic cellular interactions within breast tissues and tumors. Together, this body of work delivers a unique perspective of the breast, revealing insights into its cellular, molecular, and biochemical composition.

8.
Plant J ; 119(1): 525-539, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38693717

RESUMO

Regulation of gene expression in eukaryotes is controlled by cis-regulatory modules (CRMs). A major class of CRMs are enhancers which are composed of activating cis-regulatory elements (CREs) responsible for upregulating transcription. To date, most enhancers and activating CREs have been studied in angiosperms; in contrast, our knowledge about these key regulators of gene expression in green algae is limited. In this study, we aimed at characterizing putative activating CREs/CRMs from the histone genes of the unicellular model alga Chlamydomonas reinhardtii. To test the activity of four candidates, reporter constructs consisting of a tetramerized CRE, an established promoter, and a gene for the mCerulean3 fluorescent protein were incorporated into the nuclear genome of C. reinhardtii, and their activity was quantified by flow cytometry. Two tested candidates, Eupstr and Ehist cons, significantly upregulated gene expression and were characterized in detail. Eupstr, which originates from highly expressed genes of C. reinhardtii, is an orientation-independent CRE capable of activating both the RBCS2 and ß2-tubulin promoters. Ehist cons, which is a CRM from histone genes of angiosperms, upregulates the ß2-tubulin promoter in C. reinhardtii over a distance of at least 1.5 kb. The octamer motif present in Ehist cons was identified in C. reinhardtii and the related green algae Chlamydomonas incerta, Chlamydomonas schloesseri, and Edaphochlamys debaryana, demonstrating its high evolutionary conservation. The results of this investigation expand our knowledge about the regulation of gene expression in green algae. Furthermore, the characterized activating CREs/CRMs can be applied as valuable genetic tools.


Assuntos
Chlamydomonas reinhardtii , Histonas , Regiões Promotoras Genéticas , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Histonas/metabolismo , Histonas/genética , Regiões Promotoras Genéticas/genética , Regulação da Expressão Gênica de Plantas , Sequências Reguladoras de Ácido Nucleico/genética
9.
Mol Microbiol ; 121(3): 605-617, 2024 03.
Artigo em Inglês | MEDLINE | ID: mdl-38234267

RESUMO

Activation and function of virulence functions of bacterial pathogens are highly dynamic in time and space, and can show considerable heterogeneity between individual cells in pathogen populations. To investigate the complex events in host-pathogen interactions, single cell analyses are required. Fluorescent proteins (FPs) are excellent tools to follow the fate of individual bacterial cells during infection, and can also be deployed to use the pathogen as a sensor for its specific environment in host cells or host organisms. This Resources describes design and applications of dual fluorescence reporters (DFR) in cellular microbiology. DFR feature constitutively expressed FPs for detection of bacterial cells, and FPs expressed by an environmentally regulated promoter for interrogation of niche-specific cues or nutritional parameters. Variations of the basic design allow the generation of DFR that can be used to analyze, on single cell level, bacterial proliferation during infection, subcellular localization of intracellular bacteria, stress response, or persister state. We describe basic considerations for DFR design and review recent applications of DFR in cellular microbiology.


Assuntos
Bactérias , Proteínas de Bactérias , Bactérias/genética , Bactérias/metabolismo , Fluorescência , Regiões Promotoras Genéticas/genética , Proteínas de Bactérias/metabolismo , Virulência
10.
Eur J Immunol ; : e2451145, 2024 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-39094122

RESUMO

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection can lead to life-threatening clinical manifestations. Patients with cardiovascular disease (CVD) are at higher risk for severe courses of COVID-19. So far, however, there are hardly any strategies for predicting the course of SARS-CoV-2 infection in CVD patients at hospital admission. Thus, we investigated whether this prediction is achievable by prospectively analysing the blood immunophenotype of 94 nonvaccinated participants, including uninfected and acutely SARS-CoV-2-infected CVD patients and healthy donors, using a 36-colour spectral flow cytometry panel. Unsupervised data analysis revealed little differences between healthy donors and CVD patients, whereas the distribution of the cell populations changed dramatically in SARS-CoV-2-infected CVD patients. The latter had more mature NK cells, activated monocyte subsets, central memory CD4+ T cells, and plasmablasts but fewer dendritic cells, CD16+ monocytes, innate lymphoid cells, and CD8+ T-cell subsets. Moreover, we identified an immune signature characterised by CD161+ T cells, intermediate effector CD8+ T cells, and natural killer T (NKT) cells that is predictive for CVD patients with a severe course of COVID-19. Thus, intensified immunophenotype analyses can help identify patients at risk of severe COVID-19 at hospital admission, improving clinical outcomes through specific treatment.

11.
Development ; 149(8)2022 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-34604909

RESUMO

The adult human skin contains a vast number of T cells that are essential for skin homeostasis and pathogen defense. T cells are first observed in the skin at the early stages of gestation; however, our understanding of their contribution to early immunity has been limited by their low abundance and lack of comprehensive methodologies for their assessment. Here, we describe a new workflow for isolating and expanding significant amounts of T cells from fetal human skin. Using multiparametric flow cytometry and in situ immunofluorescence, we found a large population with a naive phenotype and small populations with a memory and regulatory phenotype. Their molecular state was characterized using single-cell transcriptomics and TCR repertoire profiling. Importantly, culture of total fetal skin biopsies facilitated T cell expansion without a substantial impact on their phenotype, a major prerequisite for subsequent functional assays. Collectively, our experimental approaches and data advance the understanding of fetal skin immunity and potential use in future therapeutic interventions.


Assuntos
Feto , Citometria de Fluxo , Pele , Linfócitos T , Adulto , Feminino , Feto/citologia , Feto/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Pele/citologia , Pele/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia
12.
J Virol ; 98(7): e0068124, 2024 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-38953379

RESUMO

Serum-neutralizing antibody titers are a critical measure of vaccine immunogenicity and are used to determine flavivirus seroprevalence in study populations. An effective dengue virus (DENV) vaccine must confer simultaneous protection against viruses grouped within four antigenic serotypes. Existing flavivirus neutralization assays, including the commonly used plaque/focus reduction neutralization titer (PRNT/FRNT) assay, require an individual assay for each virus, serotype, and strain and easily become a labor-intensive and time-consuming effort for large epidemiological studies or vaccine trials. Here, we describe a multiplex reporter virus particle neutralization titer (TetraPlex RVPNT) assay for DENV that allows simultaneous quantitative measures of antibody-mediated neutralization of infection against all four DENV serotypes in a single low-volume clinical sample and analyzed by flow cytometry. Comparative studies confirm that the neutralization titers of antibodies measured by the TetraPlex RVPNT assay are similar to FRNT/PRNT assay approaches performed separately for each viral strain. The use of this high-throughput approach enables the careful serological study in DENV endemic populations and vaccine recipients required to support the development of a safe and effective tetravalent DENV vaccine. IMPORTANCE: As a mediator of protection against dengue disease and a serological indicator of prior infection, the detection and quantification of neutralizing antibodies against DENV is an important "gold standard" tool. However, execution of traditional neutralizing antibody assays is often cumbersome and requires repeated application for each virus or serotype. The optimized RVPNT assay described here is high-throughput, easily multiplexed across multiple serotypes, and targets reporter viral particles that can be robustly produced for all four DENV serotypes. The use of this transformative RVPNT assay will support the expansion of neutralizing antibody datasets to answer research and public health questions often limited by the more cumbersome neutralizing antibody assays and the need for greater quantities of test serum.


Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Vírus da Dengue , Dengue , Testes de Neutralização , Sorogrupo , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , Vírus da Dengue/imunologia , Vírus da Dengue/classificação , Humanos , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Testes de Neutralização/métodos , Dengue/imunologia , Dengue/virologia , Vacinas contra Dengue/imunologia , Vírion/imunologia , Animais
13.
Brief Bioinform ; 24(3)2023 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-37150778

RESUMO

With the aim of analyzing large-sized multidimensional single-cell datasets, we are describing a method for Cosine-based Tanimoto similarity-refined graph for community detection using Leiden's algorithm (CosTaL). As a graph-based clustering method, CosTaL transforms the cells with high-dimensional features into a weighted k-nearest-neighbor (kNN) graph. The cells are represented by the vertices of the graph, while an edge between two vertices in the graph represents the close relatedness between the two cells. Specifically, CosTaL builds an exact kNN graph using cosine similarity and uses the Tanimoto coefficient as the refining strategy to re-weight the edges in order to improve the effectiveness of clustering. We demonstrate that CosTaL generally achieves equivalent or higher effectiveness scores on seven benchmark cytometry datasets and six single-cell RNA-sequencing datasets using six different evaluation metrics, compared with other state-of-the-art graph-based clustering methods, including PhenoGraph, Scanpy and PARC. As indicated by the combined evaluation metrics, Costal has high efficiency with small datasets and acceptable scalability for large datasets, which is beneficial for large-scale analysis.


Assuntos
Algoritmos , Análise de Dados , Análise por Conglomerados
14.
EMBO Rep ; 24(4): e55789, 2023 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-36852936

RESUMO

Efficient isolation of neurons and glia from the human enteric nervous system (ENS) is challenging because of their rare and fragile nature. Here, we describe a staining panel to enrich ENS cells from the human intestine by fluorescence-activated cell sorting (FACS). We find that CD56/CD90/CD24 co-expression labels ENS cells with higher specificity and resolution than previous methods. Surprisingly, neuronal (CD24, TUBB3) and glial (SOX10) selective markers appear co-expressed by all ENS cells. We demonstrate that this contradictory staining pattern is mainly driven by neuronal fragments, either free or attached to glial cells, which are the most abundant cell types. Live neurons can be enriched by the highest CD24 and CD90 levels. By applying our protocol to isolate ENS cells for single-cell RNA sequencing, we show that these cells can be obtained with high quality, enabling interrogation of the human ENS transcriptome. Taken together, we present a selective FACS protocol that allows enrichment and discrimination of human ENS cells, opening up new avenues to study this complex system in health and disease.


Assuntos
Sistema Nervoso Entérico , Humanos , Citometria de Fluxo , Sistema Nervoso Entérico/metabolismo , Intestinos , Neurônios/metabolismo , Neuroglia
15.
Mol Cell ; 68(3): 626-640.e5, 2017 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-29107535

RESUMO

Eukaryotic cells spend most of their life in interphase of the cell cycle. Understanding the rich diversity of metabolic and genomic regulation that occurs in interphase requires the demarcation of precise phase boundaries in situ. Here, we report the properties of two genetically encoded fluorescence sensors, Fucci(CA) and Fucci(SCA), which enable real-time monitoring of interphase and cell-cycle biology. We re-engineered the Cdt1-based sensor from the original Fucci system to respond to S phase-specific CUL4Ddb1-mediated ubiquitylation alone or in combination with SCFSkp2-mediated ubiquitylation. In cultured cells, Fucci(CA) produced a sharp triple color-distinct separation of G1, S, and G2, while Fucci(SCA) permitted a two-color readout of G1 and S/G2. Fucci(CA) applications included tracking the transient G1 phase of rapidly dividing mouse embryonic stem cells and identifying a window for UV-irradiation damage in S phase. These results show that Fucci(CA) is an essential tool for quantitative studies of interphase cell-cycle regulation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiologia , Proteínas Culina/metabolismo , Células-Tronco Embrionárias/fisiologia , Corantes Fluorescentes/metabolismo , Proteínas Luminescentes/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteínas Culina/genética , Células-Tronco Embrionárias/citologia , Genes Reporter , Células HeLa , Humanos , Proteínas Luminescentes/genética , Camundongos
16.
Mol Cell Proteomics ; 22(6): 100556, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37087050

RESUMO

Non-obstructive azoospermia (NOA), the most severe form of male infertility, could be treated with intracytoplasmic sperm injection, providing spermatozoa were retrieved with the microdissection testicular sperm extraction (mTESE). We hypothesized that testis-specific and germ cell-specific proteins would facilitate flow cytometry-assisted identification of rare spermatozoa in semen cell pellets of NOA patients, thus enabling non-invasive diagnostics prior to mTESE. Data mining, targeted proteomics, and immunofluorescent microscopy identified and verified a panel of highly testis-specific proteins expressed at the continuum of germ cell differentiation. Late germ cell-specific proteins AKAP4_HUMAN and ASPX_HUMAN (ACRV1 gene) revealed exclusive localization in spermatozoa tails and acrosomes, respectively. A multiplex imaging flow cytometry assay facilitated fast and unambiguous identification of rare but morphologically intact AKAP4+/ASPX+/Hoechst+ spermatozoa within debris-laden semen pellets of NOA patients. While the previously suggested markers for spermatozoa retrieval suffered from low diagnostic specificity, the multistep gating strategy and visualization of AKAP4+/ASPX+/Hoechst+ cells with elongated tails and acrosome-capped nuclei facilitated fast and unambiguous identification of the mature intact spermatozoa. AKAP4+/ASPX+/Hoechst+ assay may emerge as a noninvasive test to predict retrieval of morphologically intact spermatozoa by mTESE, thus improving diagnostics and treatment of severe forms of male infertility.


Assuntos
Azoospermia , Infertilidade Masculina , Masculino , Humanos , Azoospermia/genética , Azoospermia/metabolismo , Azoospermia/terapia , Sêmen/metabolismo , Espermatozoides/metabolismo , Testículo/metabolismo , Infertilidade Masculina/metabolismo , Estudos Retrospectivos , Proteínas de Ancoragem à Quinase A/metabolismo
17.
Proc Natl Acad Sci U S A ; 119(29): e2200553119, 2022 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-35858317

RESUMO

Loss of activity of the lysosomal glycosidase ß-glucocerebrosidase (GCase) causes the lysosomal storage disease Gaucher disease (GD) and has emerged as the greatest genetic risk factor for the development of both Parkinson disease (PD) and dementia with Lewy bodies. There is significant interest into how GCase dysfunction contributes to these diseases, however, progress toward a full understanding is complicated by presence of endogenous cellular factors that influence lysosomal GCase activity. Indeed, such factors are thought to contribute to the high degree of variable penetrance of GBA mutations among patients. Robust methods to quantitatively measure GCase activity within lysosomes are therefore needed to advance research in this area, as well as to develop clinical assays to monitor disease progression and assess GCase-directed therapeutics. Here, we report a selective fluorescence-quenched substrate, LysoFQ-GBA, which enables measuring endogenous levels of lysosomal GCase activity within living cells. LysoFQ-GBA is a sensitive tool for studying chemical or genetic perturbations of GCase activity using either fluorescence microscopy or flow cytometry. We validate the quantitative nature of measurements made with LysoFQ-GBA using various cell types and demonstrate that it accurately reports on both target engagement by GCase inhibitors and the GBA allele status of cells. Furthermore, through comparisons of GD, PD, and control patient-derived tissues, we show there is a close correlation in the lysosomal GCase activity within monocytes, neuronal progenitor cells, and neurons. Accordingly, analysis of clinical blood samples using LysoFQ-GBA may provide a surrogate marker of lysosomal GCase activity in neuronal tissue.


Assuntos
Doença de Gaucher , Glucosilceramidase , Doença de Parkinson , Doença de Gaucher/enzimologia , Doença de Gaucher/genética , Glucosilceramidase/análise , Glucosilceramidase/genética , Humanos , Corpos de Lewy/enzimologia , Doença por Corpos de Lewy/enzimologia , Lisossomos/enzimologia , Mutação , Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Especificidade por Substrato , alfa-Sinucleína/metabolismo
18.
BMC Biol ; 22(1): 181, 2024 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-39183273

RESUMO

BACKGROUND: Pathologists commonly employ the Ki67 immunohistochemistry labelling index (LI) when deciding appropriate therapeutic strategies for patients with breast cancer. However, despite several attempts at standardizing the Ki67 LI, inter-observer and inter-laboratory bias remain problematic. We developed a flow cytometric assay that employed tissue dissociation, enzymatic treatment and a gating process to analyse Ki67 in formalin-fixed paraffin-embedded (FFPE) breast cancer tissue. RESULTS: We demonstrated that mechanical homogenizations combined with thrombin treatment can be used to recover efficiently intact single-cell nuclei from FFPE breast cancer tissue. Ki67 in the recovered cell nuclei retained reactivity against the MIB-1 antibody, which has been widely used in clinical settings. Additionally, since the method did not alter the nucleoskeletal structure of tissues, the nuclei of cancer cells can be enriched in data analysis based on differences in size and complexity of nuclei of lymphocytes and normal mammary cells. In a clinical study using the developed protocol, Ki67 positivity was correlated with the Ki67 LI obtained by hot spot analysis by a pathologist in Japan (rho = 0.756, P < 0.0001). The number of cancer cell nuclei subjected to the analysis in our assay was more than twice the number routinely checked by pathologists in clinical settings. CONCLUSIONS: The findings of this study showed the application of this new flow cytometry method could potentially be used to standardize Ki67 assessments in breast cancer.


Assuntos
Neoplasias da Mama , Citometria de Fluxo , Antígeno Ki-67 , Inclusão em Parafina , Antígeno Ki-67/metabolismo , Antígeno Ki-67/análise , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Humanos , Citometria de Fluxo/métodos , Feminino , Inclusão em Parafina/métodos , Formaldeído , Fixação de Tecidos/métodos
19.
Genes Chromosomes Cancer ; 63(2): e23228, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-38380728

RESUMO

An emerging group of spindle cell neoplasms harboring fusions involving NTRK or non-NTRK kinase genes often share characteristic S100 and/or CD34 expression; however, the diagnostic utility of immunohistochemical stains is not well established in this family owing to their lack of specificity. Recently, CD30 expression in spindle cell neoplasms with kinase gene fusions, such as NTRK, BRAF, RAF1, and RET, has been increasingly identified. We herein report a 10-year-old girl with high-grade spindle cell sarcoma of the neck. Prior to histopathological evaluation, flow cytometry (FCM) analysis and touch smear cytology of the tumor tissue revealed CD34+ and dimCD30+ spindle cell populations. Histopathologically, the case was characterized by monomorphic spindle-shaped cytomorphology with CD30, S100, and CD34 positivity and harbored close similarities with spindle cell neoplasms with NTRK or non-NTRK gene fusions. Subsequently, a comprehensive next-generation sequencing sarcoma panel identified a rare PLEKHH2::ALK fusion, and a diagnosis of ALK-rearranged spindle cell neoplasm was made. The patient showed significant tumor response to single-agent treatment with alectinib, an ALK-tyrosine kinase inhibitor. This case supports that CD30 is expressed in an ALK-rearranged mesenchymal neoplasm. The benefit of the early detection of CD30 expression by FCM for a prompt diagnosis and treatment is highlighted in the context of an aggressive clinical course. This case represents a learning experience regarding the need to the check the status of CD30 expression in these tumors and suggests the potential clinical benefits of CD30-targeted therapy.


Assuntos
Sarcoma , Neoplasias de Tecidos Moles , Feminino , Humanos , Criança , Imuno-Histoquímica , Citometria de Fluxo , Sarcoma/patologia , Neoplasias de Tecidos Moles/genética , Neoplasias de Tecidos Moles/patologia , Fusão Gênica , Receptores Proteína Tirosina Quinases/genética , Biomarcadores Tumorais/genética
20.
Traffic ; 23(11): 538-553, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36117140

RESUMO

Those who study macrophage biology struggle with the decision whether to utilize primary macrophages derived directly from mice or opt for the convenience and genetic tractability of immortalized macrophage-like cell lines in in vitro studies. Particularly when it comes to studying phagocytosis and phagosomal maturation-a signature cellular process of the macrophage-many commonly used cell lines are not representative of what occurs in primary macrophages. A system developed by Mark Kamps' group, that utilizes conditionally constitutive activity of Hox transcription factors (Hoxb8 and Hoxa9) to immortalize differentiation-competent myeloid cell progenitors of mice, offers an alternative to the macrophage/macrophage-like dichotomy. In this resource, we will review the use of Hoxb8 and Hoxa9 as hematopoietic regulators to conditionally immortalize murine hematopoietic progenitor cells which retain their ability to differentiate into many functional immune cell types including macrophages, neutrophils, basophils, osteoclasts, eosinophils, dendritic cells, as well as limited potential for the generation of lymphocytes. We further demonstrate that the use of macrophages derived from Hoxb8/Hoxa9 immortalized progenitors and their similarities to bone marrow-derived macrophages. To supplement the existing data, mass spectrometry-based proteomics, flow cytometry, cytology, and in vitro phagosomal assays were conducted on macrophages derived from Hoxb8 immortalized progenitors and compared to bone marrow-derived macrophages and the macrophage-like cell line J774. We additionally propose the use of a standardized nomenclature to describe cells derived from the Hoxb8/Hoxa9 system in anticipation of their expanded use in the study of leukocyte cell biology.


Assuntos
Células-Tronco Hematopoéticas , Macrófagos , Animais , Diferenciação Celular , Macrófagos/metabolismo , Camundongos , Fatores de Transcrição/metabolismo
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