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1.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360885

RESUMO

The perinuclear theca (PT) of the eutherian sperm head is a cytoskeletal-like structure that houses proteins involved in important cellular processes during spermiogenesis and fertilization. Building upon our novel discovery of non-nuclear histones in the bovine PT, we sought to investigate whether this PT localization was a conserved feature of eutherian sperm. Employing cell fractionation, immunodetection, mass spectrometry, qPCR, and intracytoplasmic sperm injections (ICSI), we examined the localization, developmental origin, and functional potential of histones from the murid PT. Immunodetection localized histones to the post-acrosomal sheath (PAS) and the perforatorium (PERF) of the PT but showed an absence in the sperm nucleus. MS/MS analysis of selectively extracted PT histones indicated that predominately core histones (i.e., H3, H3.3, H2B, H2A, H2AX, and H4) populate the murid PT. These core histones appear to be de novo-synthesized in round spermatids and assembled via the manchette during spermatid elongation. Mouse ICSI results suggest that early embryonic development is delayed in the absence of PT-derived core histones. Here, we provide evidence that core histones are de novo-synthesized prior to PT assembly and deposited in PT sub-compartments for subsequent involvement in chromatin remodeling of the male pronucleus post-fertilization.


Assuntos
Histonas/biossíntese , Cabeça do Espermatozoide/metabolismo , Espermátides/metabolismo , Espermatogênese/fisiologia , Animais , Núcleo Celular/metabolismo , Cromatografia Líquida/métodos , Feminino , Fertilização/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Injeções de Esperma Intracitoplásmicas , Espectrometria de Massas em Tandem/métodos
2.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360905

RESUMO

Some Listeria species are important human and animal pathogens that can be found in contaminated food and produce a variety of virulence factors involved in their pathogenicity. Listeria strains exhibiting multidrug resistance are known to be progressively increasing and that is why continuous monitoring is needed. Effective therapy against pathogenic Listeria requires identification of the bacterial strain involved, as well as determining its virulence factors, such as antibiotic resistance and sensitivity. The present study describes the use of liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to do a global shotgun proteomics characterization for pathogenic Listeria species. This method allowed the identification of a total of 2990 non-redundant peptides, representing 2727 proteins. Furthermore, 395 of the peptides correspond to proteins that play a direct role in Listeria pathogenicity; they were identified as virulence factors, toxins and anti-toxins, or associated with either antibiotics (involved in antibiotic-related compounds production or resistance) or resistance to toxic substances. The proteomic repository obtained here can be the base for further research into pathogenic Listeria species and facilitate the development of novel therapeutics for these pathogens.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/química , Farmacorresistência Bacteriana Múltipla , Listeria/efeitos dos fármacos , Listeria/patogenicidade , Proteoma/química , Fatores de Virulência/química , Transportadores de Cassetes de Ligação de ATP/química , Cromatografia Líquida/métodos , Genes Bacterianos , Listeria/classificação , Listeria/genética , Peptídeos/química , Proteômica/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos
3.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360914

RESUMO

Human milk is a vital biofluid containing a myriad of molecular components to ensure an infant's best start at a healthy life. One key component of human milk is ß-casein, a protein which is not only a structural constituent of casein micelles but also a source of bioactive, often antimicrobial, peptides contributing to milk's endogenous peptidome. Importantly, post-translational modifications (PTMs) like phosphorylation and glycosylation typically affect the function of proteins and peptides; however, here our understanding of ß-casein is critically limited. To uncover the scope of proteoforms and endogenous peptidoforms we utilized mass spectrometry (LC-MS/MS) to achieve in-depth longitudinal profiling of ß-casein from human milk, studying two donors across 16 weeks of lactation. We not only observed changes in ß-casein's known protein and endogenous peptide phosphorylation, but also in previously unexplored O-glycosylation. This newly discovered PTM of ß-casein may be important as it resides on known ß-casein-derived antimicrobial peptide sequences.


Assuntos
Caseínas/metabolismo , Glicopeptídeos/química , Lactação/metabolismo , Leite Humano/química , Processamento de Proteína Pós-Traducional/fisiologia , Proteoma/química , Aleitamento Materno , Cromatografia Líquida/métodos , Feminino , Glicosilação , Voluntários Saudáveis , Humanos , Lactente , Estudos Longitudinais , Fosforilação , Espectrometria de Massas em Tandem/métodos
4.
Nat Commun ; 12(1): 4912, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389721

RESUMO

Polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) hybrid systems typically use complex protein-protein interactions to facilitate direct transfer of intermediates between these multimodular megaenzymes. In the canal-associated neurons (CANs) of Caenorhabditis elegans, PKS-1 and NRPS-1 produce the nemamides, the only known hybrid polyketide-nonribosomal peptides biosynthesized by animals, through a poorly understood mechanism. Here, we use genome editing and mass spectrometry to map the roles of individual PKS-1 and NRPS-1 enzymatic domains in nemamide biosynthesis. Furthermore, we show that nemamide biosynthesis requires at least five additional enzymes expressed in the CANs that are encoded by genes distributed across the worm genome. We identify the roles of these enzymes and discover a mechanism for trafficking intermediates between a PKS and an NRPS. Specifically, the enzyme PKAL-1 activates an advanced polyketide intermediate as an adenylate and directly loads it onto a carrier protein in NRPS-1. This trafficking mechanism provides a means by which a PKS-NRPS system can expand its biosynthetic potential and is likely important for the regulation of nemamide biosynthesis.


Assuntos
Vias Biossintéticas/genética , Proteínas de Caenorhabditis elegans/genética , Peptídeo Sintases/genética , Peptídeos/metabolismo , Policetídeo Sintases/genética , Policetídeos/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Cromatografia Líquida/métodos , Enzimas/genética , Enzimas/metabolismo , Expressão Gênica , Espectrometria de Massas/métodos , Estrutura Molecular , Mutação , Neurônios/metabolismo , Peptídeo Sintases/metabolismo , Peptídeos/química , Policetídeo Sintases/metabolismo , Policetídeos/química
5.
J Chromatogr A ; 1652: 462353, 2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34237484

RESUMO

Poloxamer 188 (P188) is formulated in proteinaceous therapeutics as an alternative surfactant to polysorbate because of its good chemical stability and surfactant properties, which enable interfacial protection, preventing visible and sub-visible particle formation. However, due to the nature of polymer heterogeneity and limited analytical approaches to resolve the superimposed components of P188, the impact of its quality variance on protein stability is still not well understood. In this study, we developed an analytical method to evaluate the components of P188 as a function of the length of polypropylene oxide (PPO), by maintaining polyethylene oxide (PEO) at the critical point of adsorption (CPA) to eliminate its chromatographic interference. The effectiveness of the separation was confirmed by nuclear magnetic resonance (NMR) spectroscopy and mass spectroscopy (MS) of the individual fractions corresponding to each peak. Additionally, a design of experiments (DoE) and method qualification were carried out to identify and optimize the key operation parameters, including column temperature and evaporative light scattering detector (ELSD) settings that need to be strictly controlled for reliable analytical results. In conclusion, this method is sensitive and reliable to compare the quality variance of commercial P188 and is suitable for routine quality control purposes. The application of this method could help in further understanding the Critical Material Attributes (CMA) that may affect the quality attributes of proteins in formulations.


Assuntos
Cromatografia Líquida/métodos , Poloxâmero/química , Tensoativos/química , Adsorção , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Poloxâmero/isolamento & purificação , Polietilenoglicóis/química , Polímeros/química , Propilenoglicóis/química , Proteínas/uso terapêutico , Tensoativos/isolamento & purificação
6.
Nutrients ; 13(7)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202697

RESUMO

COVID-19 is a pandemic disease that causes severe pulmonary damage and hyperinflammation. Vitamin A is a crucial factor in the development of immune functions and is known to be reduced in cases of acute inflammation. This prospective, multicenter observational cross-sectional study analyzed vitamin A plasma levels in SARS-CoV-2 infected individuals, and 40 hospitalized patients were included. Of these, 22 developed critical disease (Acute Respiratory Distress Syndrome [ARDS]/Extracorporeal membrane oxygenation [ECMO]), 9 developed severe disease (oxygen supplementation), and 9 developed moderate disease (no oxygen supplementation). A total of 47 age-matched convalescent persons that had been earlier infected with SARS-CoV-2 were included as the control group. Vitamin A plasma levels were determined by high-performance liquid chromatography. Reduced vitamin A plasma levels correlated significantly with increased levels of inflammatory markers (CRP, ferritin) and with markers of acute SARS-CoV-2 infection (reduced lymphocyte count, LDH). Vitamin A levels were significantly lower in hospitalized patients than in convalescent persons (p < 0.01). Of the hospitalized patients, those who were critically ill showed significantly lower vitamin A levels than those who were moderately ill (p < 0.05). Vitamin A plasma levels below 0.2 mg/L were significantly associated with the development of ARDS (OR = 5.54 [1.01-30.26]; p = 0.048) and mortality (OR 5.21 [1.06-25.5], p = 0.042). Taken together, we conclude that vitamin A plasma levels in COVID-19 patients are reduced during acute inflammation and that severely reduced plasma levels of vitamin A are significantly associated with ARDS and mortality.


Assuntos
COVID-19/sangue , Vitamina A/sangue , Adulto , Idoso , Biomarcadores/sangue , Proteína C-Reativa/análise , COVID-19/mortalidade , Cromatografia Líquida/métodos , Estado Terminal , Estudos Transversais , Oxigenação por Membrana Extracorpórea/estatística & dados numéricos , Feminino , Ferritinas/sangue , Hospitalização , Humanos , Inflamação/epidemiologia , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Síndrome do Desconforto Respiratório/epidemiologia , SARS-CoV-2 , Índice de Gravidade de Doença
7.
Nutrients ; 13(7)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206219

RESUMO

(1) Background: Vitamin D, a well-established regulator of calcium and phosphate metabolism, also has immune-modulatory functions. An uncontrolled immune response and cytokine storm are tightly linked to fatal courses of COVID-19. The present retrospective study aimed to inves-tigate vitamin D status markers and vitamin D degradation products in a mixed cohort of 148 hospitalized COVID-19 patients with various clinical courses of COVID-19. (2) Methods: The serum concentrations of 25(OH)D3, 25(OH)D2, 24,25(OH)2D3, and 25,26(OH)2D3 were determined by a validated liquid-chromatography tandem mass-spectrometry method in leftover serum samples from 148 COVID-19 patients that were admitted to the University Hospital of the Medical Uni-versity of Graz between April and November 2020. Anthropometric and clinical data, as well as outcomes were obtained from the laboratory and hospital information systems. (3) Results: From the 148 patients, 34 (23%) died within 30 days after admission. The frequency of fatal outcomes did not differ between males and females. Non-survivors were significantly older than survivors, had higher peak concentrations of IL-6 and CRP, and required mechanical ventilation more frequently. The serum concentrations of all vitamin D metabolites and the vitamin D metabolite ratio (VMR) did not differ significantly between survivors and non-survivors. Additionally, the need for res-piratory support was unrelated to the serum concentrations of 25(OH)D vitamin D and the two vitamin D catabolites, as well as the VMR. (4) Conclusion: The present results do not support a relevant role of vitamin D for the course and outcome of COVID-19.


Assuntos
COVID-19/sangue , COVID-19/mortalidade , Hospitalização , Vitamina D/sangue , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Cromatografia Líquida/métodos , Ergocalciferóis/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Respiração Artificial/estatística & dados numéricos , Estudos Retrospectivos , SARS-CoV-2 , Espectrometria de Massas em Tandem/métodos , Vitamina D/análogos & derivados , Deficiência de Vitamina D/sangue , Vitaminas/sangue
8.
Molecules ; 26(12)2021 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-34205329

RESUMO

The content of plant secondary metabolites is not stable, and factors such as the region/location effect and seasonal variations have an impact on their chemical composition, especially in parasitic plants. Research in this area is an important step in the development of quality parameter standards of medicinal plants and their finished products. The effects of the time and place of harvest and the host tree species on the chemical composition and antioxidant activity of mistletoe extracts were investigated. Statistical tools were used to evaluate the results of the spectrophotometric and LC-ESI-MS/MS studies of the phenolic composition and antioxidant activity. The investigations indicate that the qualitative and quantitative composition, influencing the biological activity of mistletoe extracts, largely depends on the origin of the plant. The mistletoe extracts exhibited a rich phenol profile and high antioxidant activity. The chemometric analysis indicated that mistletoe collected from conifers (Viscum abietis and Viscum austriacum) had the most advantageous chemical composition and antioxidant activity. Moreover, the chemical profile and biological activity of the plant material were closely related to the climatic conditions and location of the harvested plant. Higher levels of phenolic compounds and high antioxidant activity were found in extracts obtained from plant material collected in cold weather with the presence of snow and less sunshine (autumn-winter period).


Assuntos
Antioxidantes/química , Antioxidantes/fisiologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Árvores/química , Viscum album/química , Cromatografia Líquida/métodos , Erva-de-Passarinho/química , Fenóis/química , Fenóis/farmacologia , Plantas Medicinais/química , Espectrometria de Massas em Tandem/métodos
9.
Molecules ; 26(12)2021 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-34201182

RESUMO

Most biosurfactants are obtained using costly culture media and purification processes, which limits their wider industrial use. Sustainability of their production processes can be achieved, in part, by using cheap substrates found among agricultural and food wastes or byproducts. In the present study, crude glycerol, a raw material obtained from several industrial processes, was evaluated as a potential low-cost carbon source to reduce the costs of surfactin production by Bacillus subtilis #309. The culture medium containing soap-derived waste glycerol led to the best surfactin production, reaching about 2.8 g/L. To the best of our knowledge, this is the first report describing surfactin production by B. subtilis using stearin and soap wastes as carbon sources. A complete chemical characterization of surfactin analogs produced from the different waste glycerol samples was performed by liquid chromatography-mass spectrometry (LC-MS) and Fourier transform infrared spectroscopy (FTIR). Furthermore, the surfactin produced in the study exhibited good stability in a wide range of pH, salinity and temperatures, suggesting its potential for several applications in biotechnology.


Assuntos
Bacillus subtilis/química , Glicerol/química , Tensoativos/química , Biotecnologia/métodos , Carbono/química , Cromatografia Líquida/métodos , Meios de Cultura/química , Concentração de Íons de Hidrogênio , Espectrometria de Massas/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Temperatura
10.
Molecules ; 26(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200415

RESUMO

Homovanillic acid (HVA) and vanillylmandelic acid (VMA) are end-stage metabolites of catecholamine and are clinical biomarkers for the diagnosis of neuroblastoma. For the first time in Korea, we implemented and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay to measure urinary concentrations of HVA and VMA according to Clinical and Laboratory Standards Institute guidelines. Our LC-MS/MS assay with minimal sample preparation was validated for linearity, lower limit of detection (LOD), lower limit of quantification (LLOQ), precision, accuracy, extraction recovery, carryover, matrix effect, and method comparison. A total of 1209 measurements was performed to measure HVA and VMA in spot urine between October 2019 and September 2020. The relationship between the two urinary markers, HVA and VMA, was analyzed and exhibited high agreement (89.1% agreement, kappa's k = 0.6) and a strong correlation (Pearson's r = 0.73). To our knowledge, this is the first study to utilize LC-MS/MS for simultaneous quantitation of spot urinary HVA and VMA and analyze the clinical application of both markers on a large scale for neuroblastoma patients.


Assuntos
Ácido Homovanílico/química , Neuroblastoma/diagnóstico , Neuroblastoma/metabolismo , Ácido Vanilmandélico/química , Bioensaio/métodos , Biomarcadores/metabolismo , Criança , Pré-Escolar , Cromatografia Líquida/métodos , Feminino , Seguimentos , Humanos , Lactente , Recém-Nascido , Limite de Detecção , Masculino , República da Coreia , Espectrometria de Massas em Tandem/métodos
11.
Molecules ; 26(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200462

RESUMO

Gastropods are among the most diverse animals. Gastropod mucus contains several glycoproteins and peptides that vary by species and habitat. Some bioactive peptides from gastropod mucus were identified only in a few species. Therefore, using biochemical, mass spectrometric, and bioinformatics approaches, this study aimed to comprehensively identify putative bioactive peptides from the mucus proteomes of seven commonly found or commercially valuable gastropods. The mucus was collected in triplicate samples, and the proteins were separated by 1D-SDS-PAGE before tryptic digestion and peptide identification by nano LC-MS/MS. The mucus peptides were subsequently compared with R scripts. A total of 2818 different peptides constituting 1634 proteins from the mucus samples were identified, and 1218 of these peptides (43%) were core peptides found in the mucus of all examined species. Clustering and correspondence analyses of 1600 variable peptides showed unique mucous peptide patterns for each species. The high-throughput k-nearest neighbor and random forest-based prediction programs were developed with more than 95% averaged accuracy and could identify 11 functional categories of putative bioactive peptides and 268 peptides (9.5%) with at least five to seven bioactive properties. Antihypertensive, drug-delivering, and antiparasitic peptides were predominant. These peptides provide an understanding of gastropod mucus, and the putative bioactive peptides are expected to be experimentally validated for further medical, pharmaceutical, and cosmetic applications.


Assuntos
Gastrópodes/metabolismo , Muco/metabolismo , Peptídeos/metabolismo , Proteoma/metabolismo , Animais , Cromatografia Líquida/métodos , Biologia Computacional/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Aprendizado de Máquina , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos
12.
Molecules ; 26(12)2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200718

RESUMO

Euterpe oleracea Mart. (açai) is a native palm from the Amazon region. There are various chemical constituents of açai with bioactive properties. This study aimed to evaluate the chemical composition and cytotoxic effects of açai seed extract on breast cancer cell line (MCF-7). Global Natural Products Social Molecular Networking (GNPS) was applied to identify chemical compounds present in açai seed extract. LC-MS/MS and molecular networking were employed to detect the phenolic compounds of açai. The antioxidant activity of açai seed extract was measured by DPPH assay. MCF-7 breast cancer cell line viability was evaluated by MTT assay. Cell death was evaluated by flow cytometry and time-lapse microscopy. Autophagy was evaluated by orange acridin immunofluorescence assay. Reactive oxygen species (ROS) production was evaluated by DAF assay. From the molecular networking, fifteen compounds were identified, mainly phenolic compounds. The açai seed extract showed cytotoxic effects against MCF-7, induced morphologic changes in the cell line by autophagy and increased the ROS production pathway. The present study suggests that açai seed extract has a high cytotoxic capacity and may induce autophagy by increasing ROS production in breast cancer. Apart from its antioxidant activity, flavonoids with high radical scavenging activity present in açai also generated NO (nitric oxide), contributing to its cytotoxic effect and autophagy induction.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Morte Celular/efeitos dos fármacos , Euterpe/química , Extratos Vegetais/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Sementes/química , Antioxidantes/química , Antioxidantes/farmacologia , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Feminino , Flavonoides/química , Flavonoides/farmacologia , Frutas/química , Humanos , Células MCF-7 , Fenóis/química , Fenóis/farmacologia , Extratos Vegetais/química , Polifenóis/química , Polifenóis/farmacologia , Espectrometria de Massas em Tandem/métodos
13.
Molecules ; 26(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203808

RESUMO

This work represents a comparative metabolomic study of extracts of wild grapes obtained from six different places in the Primorsky and Khabarovsk territories (Far East Russia) and extracts of grapes obtained from the collection of N.I. Vavilov All-Russian Institute of Plant Genetic Resources (St. Petersburg). The metabolome analysis was performed by liquid chromatography in combination with ion trap mass spectrometry. The results showed the presence of 118 compounds in ethanolic extracts of V. amurensis grapes. In addition, several metabolites were newly annotated in V. amurensis. The highest diversity of phenolic compounds was identified in the samples of the V. amurensis grape collected in the vicinity of Vyazemsky (Khabarovsk Territory) and the floodplain of the Arsenyevka River (Primorsky Territory), compared to the other wild samples and cultural grapes obtained in the collection of N.I. Vavilov All-Russian Institute of Plant Genetic Resources.


Assuntos
Fenóis/análise , Vitis/química , Vitis/metabolismo , Cromatografia Líquida/métodos , Frutas/química , Metabolômica/métodos , Fenóis/química , Federação Russa , Espectrometria de Massas em Tandem/métodos
14.
Molecules ; 26(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208735

RESUMO

Saccharides are the most common carbon source for Streptococcus thermophilus, which is a widely used bacterium in the production of fermented dairy products. The performance of the strain is influenced by the consumption of different saccharides during fermentation. Therefore, a precise measurement of the concentrations of saccharides in the fermentation media is essential. An 18-min long method with limits of quantitation in the range of 0.159-0.704 mg/L and with 13C labelled internal standards employing hydrophilic interaction chromatography coupled to mass spectrometric detection-(HILIC-LC-MS) allowed for simultaneous quantification of five saccharides: fructose, glucose, galactose, sucrose, and lactose in the fermentation samples. The method included a four-step sample preparation protocol, which could be easily applied to high-throughput analysis. The developed method was validated and applied to the fermentation samples produced by Streptococcus thermophilus.


Assuntos
Cromatografia Líquida/métodos , Fermentação/fisiologia , Espectrometria de Massas em Tandem/métodos , Dissacarídeos/química , Dissacarídeos/metabolismo , Frutose/metabolismo , Galactose/metabolismo , Glucose/metabolismo , Lactose/metabolismo , Monossacarídeos/química , Monossacarídeos/metabolismo , Streptococcus thermophilus/metabolismo , Sacarose/metabolismo
15.
Methods Mol Biol ; 2320: 261-281, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302664

RESUMO

Identifying causative genes in a given phenotype or disease model is important for biological discovery and drug development. The recent development of the CRISPR/Cas9 system has enabled unbiased and large-scale genetic perturbation screens to identify causative genes by knocking out many genes in parallel and selecting cells with desired phenotype of interest. However, compared to cancer cell lines, human somatic cells including cardiomyocytes (CMs), neuron cells, and endothelial cells are not easy targets of CRISPR screens because CRISPR screens require a large number of isogenic cells to be cultured and thus primary cells from patients are not ideal. The combination of CRISPR screens with induced pluripotent stem cell (iPSC) technology would be a powerful tool to identify causative genes and pathways because iPSCs can be expanded easily and differentiated to any cell type in principle. Here we describe a robust protocol for CRISPR screening using human iPSCs. Because each screening is different and needs to be customized depending on the cell types and phenotypes of interest, we show an example of CRISPR knockdown screening using CRISPRi system to identify essential genes to differentiate iPSCs to CMs.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Sequência de Bases , Causalidade , Células Cultivadas , Cromatografia Líquida/métodos , DNA/isolamento & purificação , Doxiciclina/farmacologia , Citometria de Fluxo , Estudos de Associação Genética , Vetores Genéticos/genética , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Lentivirus/genética , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , RNA Guia/genética , Transfecção
16.
Commun Biol ; 4(1): 825, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34211107

RESUMO

Extracellular vesicles can modulate diverse processes ranging from proliferation and tissue repair, to chemo-resistance and cellular differentiation. With the advent of tissue and immunological targeting, extracellular vesicles are also increasingly viewed as promising vectors to deliver peptide-based cancer antigens to the human immune system. Despite the clinical relevance and therapeutic potential of such 'cell-free' approaches, the natural antigen presentation landscape exported in extracellular vesicles is still largely uncharted, due to the challenging nature of such preparations and analyses. In the context of therapeutic vesicle production, a critical evaluation of the similarity in vesicular antigen presentation is also urgently needed. In this work, we compared the HLA-I peptide ligandomes of extracellular vesicles against that of whole-cells of the same cell line. We found that extracellular vesicles not only over-represent HLA-B complexes and peptide ligands, but also cysteinylated peptides that may modulate immune responses. Collectively, these findings describe the pre-existing provision of vesicular HLA complexes that may be utilized to carry peptide vaccines, as well as the propensity for different peptide and post-translationally modified ligands to be presented, and will outline critical considerations in devising novel EV vaccination strategies.


Assuntos
Apresentação do Antígeno/imunologia , Cisteína/imunologia , Vesículas Extracelulares/imunologia , Antígenos HLA-B/imunologia , Linhagem Celular , Cromatografia Líquida/métodos , Cisteína/metabolismo , Vesículas Extracelulares/metabolismo , Antígenos HLA-B/metabolismo , Humanos , Ligantes , Peptídeos/imunologia , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional/imunologia , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos
17.
Commun Biol ; 4(1): 856, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244602

RESUMO

Mosquito vectors transmit various diseases through blood feeding, required for their egg development. Hence, blood feeding is a major physiological event in their life cycle, during which hundreds of genes are tightly regulated. Blood is a rich source of proteins for mosquitoes, but also contains many other molecules including microRNAs (miRNAs). Here, we found that human blood miRNAs are transported abundantly into the fat body tissue of Aedes aegypti, a key metabolic center in post-blood feeding reproductive events, where they target and regulate mosquito genes. Using an artificial diet spiked with the mimic of an abundant and stable human blood miRNA, hsa-miR-21-5p, and proteomics analysis, we found over 40 proteins showing differential expression in female Ae. aegypti mosquitoes after feeding. Of interest, we found that the miRNA positively regulates the vitellogenin gene, coding for a yolk protein produced in the mosquito fat body and then transported to the ovaries as a protein source for egg production. Inhibition of hsa-miR-21-5p followed by human blood feeding led to a statistically insignificant reduction in progeny production. The results provide another example of the involvement of small regulatory molecules in the interaction of taxonomically vastly different taxa.


Assuntos
Aedes/metabolismo , MicroRNAs/sangue , Mosquitos Vetores/metabolismo , Vitelogeninas/metabolismo , Aedes/citologia , Aedes/genética , Animais , Linhagem Celular , Cromatografia Líquida/métodos , Corpo Adiposo/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Proteínas de Insetos/metabolismo , MicroRNAs/genética , Mosquitos Vetores/genética , Proteômica/métodos , RNA-Seq/métodos , Espectrometria de Massas em Tandem/métodos , Vitelogeninas/genética
18.
Commun Biol ; 4(1): 860, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34253841

RESUMO

Schistosomes require both molluscan and mammalian hosts for development. The larval cercaria exits the snail host and swims to identify and invade the mammalian host. The cercaria has two macrostructures, the head and the tail. The head invades the host, where it matures into an adult worm. The tail is lost after host invasion. Translation in the cercaria differs in each macrostructure, with higher levels of translation in the cercarial tail and little to no translational activity in the cercarial head. We compared the transcriptome and proteome of the cercarial head and tail and observed stark differences between the two macrostructures. We identified unique and differentially expressed transcripts and proteins, including ribosomal components expressed in higher levels in tails than in heads, which may explain the differences in translation levels between heads and tails. We also characterized the weak correlation between transcription and translation in infectious cercarial heads and tails.


Assuntos
Proteínas de Helminto/metabolismo , Proteoma/metabolismo , Schistosoma mansoni/genética , Schistosoma mansoni/metabolismo , Transcriptoma/genética , Animais , Cercárias/genética , Cercárias/crescimento & desenvolvimento , Cercárias/metabolismo , Cromatografia Líquida/métodos , Perfilação da Expressão Gênica/métodos , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Proteômica/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Schistosoma mansoni/crescimento & desenvolvimento , Espectrometria de Massas em Tandem/métodos
19.
Artigo em Inglês | MEDLINE | ID: mdl-34224962

RESUMO

Monitoring estrogen levels, especially estradiol (E2), is amongst others important for determining menopausal status and guidance of breast cancer treatment. We validated a serum E2 and estrone (E1) liquid chromatography tandem-mass spectrometry assay (LC-MS/MS) suitable for quantitation in human subjects. In addition, we compared our method with an E2 immunoassay (IA) and established preliminary reference values. Validation parameters were within the predetermined acceptance criteria. Assay linearity ranges were 4-1500 pmol/L for E1 and 4-2500 pmol/L for E2. Imprecision ranged from 7.4 to 9.6%. The lower limit of quantitation for E2 (8.0 pmol/L) was 11.4 times lower than the IA. The method comparison revealed differences in E2 quantitation up to 155% between both methods. The method allowed quantitation of E1 in all healthy volunteers, while E2 could not be detected in 95% versus 40% of the post-menopausal women using IA and LC-MS/MS, respectively. Male, pre-, peri- and postmenopausal female reference values were estimated. An LC-MS/MS based method combining E1 and E2 analysis was validated with superior E2 analytical sensitivity when compared to the IA.


Assuntos
Cromatografia Líquida/métodos , Estradiol/sangue , Estrona/sangue , Imunoensaio/métodos , Espectrometria de Massas em Tandem/métodos , Adolescente , Adulto , Feminino , Humanos , Limite de Detecção , Modelos Lineares , Masculino , Menopausa , Pessoa de Meia-Idade , Valores de Referência , Reprodutibilidade dos Testes , Adulto Jovem
20.
Artigo em Inglês | MEDLINE | ID: mdl-34224963

RESUMO

Anti-doping analysis requires an exceptional level of accuracy and precision given the stakes that are at play. Current methods rely on the application of chromatographic techniques linked with mass spectrometry to provide this. However, despite the effectiveness of these techniques in achieving good selectivity and specificity, some issues still exist. In order to reach the minimum required performance level as set by WADA, labs commonly use selective monitoring by quadrupole mass spectrometry. This can be potentially fooled through the use of masking agents or by moving the peaks, as often only a small portion of the spectrum is used for analysis. Further issues exist in the inability to detect new or modified compounds, or to reanalyse samples/spectra. One technique that could overcome these problems is that of comprehensive 2D chromatography. Here a second separation column is employed to generate greater separative power. Compared to conventional separation, GCxGC allows for a greater peak capacity (i.e., number of peaks that can be resolved within a given time) and greater separation of coeluting compounds, which makes the technique promising for the complex task required in anti-doping. When combined with Time of Flight Mass Spectrometry this technique demonstrates vast potential allowing for full mass range datasets to be obtained for retroactive analysis. Similarly, LCxLC provides improvements in resolving power compared to its 1D counterpart and can be used both online as part of the analysis or offline solely as a purification step. In this review we summarise the work in this field so far, how comprehensive chromatography has been applied to anti-doping studies, and discuss the future application for this technique.


Assuntos
Anabolizantes/análise , Cromatografia Gasosa/métodos , Cromatografia Líquida/métodos , Doping nos Esportes , Drogas Ilícitas/análise , Animais , Cavalos , Humanos
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