RESUMO
This chapter describes the application of genomic, transcriptomic, proteomic, and metabolomic methods in the study of SARS-CoV-2 variants of concern. We also describe the important role of machine learning tools to identify the most significant biomarker signatures and discuss the latest point-of-care devices that can be used to translate these findings to the physician's office or to bedside care. The main emphasis is placed on increasing our diagnostic capacity and predictability of disease outcomes to guide the most appropriate treatment strategies.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Proteômica , COVID-19/diagnóstico , COVID-19/genética , GenômicaRESUMO
Blood serum or plasma proteins are potentially useful in COVID-19 research as biomarkers for risk prediction, diagnosis, stratification, and treatment monitoring. However, serum protein-based biomarker identification and validation is complicated due to the wide concentration range of these proteins, which spans more than ten orders of magnitude. Here we present a combined affinity purification-liquid chromatography mass spectrometry approach which allows identification and quantitation of the most abundant serum proteins along with the nonspecifically bound and interaction proteins. This led to the reproducible identification of more than 100 proteins that were not specifically targeted by the affinity column. Many of these have already been implicated in COVID-19 disease.
Assuntos
COVID-19 , Soro , Biomarcadores , Proteínas Sanguíneas/química , COVID-19/diagnóstico , Cromatografia de Afinidade/métodos , Cromatografia Líquida/métodos , Humanos , Soro/química , Espectrometria de Massas em Tandem/métodosRESUMO
COVID-19 disease is caused by infection with the SARS-CoV-2 virus and is associated with a cytokine storm effect in some patients. This can lead to decreased ability of the host to cope with the infection and result in severe disease outcomes. Here, we present a protocol for isolation of peripheral blood mononuclear cells (PBMCs) from COVID-19 patients followed by liquid chromatography-mass spectrometry (LC-MS) profiling to identify the affected molecules and molecular pathways. It is hoped that this will lead to the identification of potential biomarkers for monitoring the disease as well as treatment responses. This approach could also be used in the study of other respiratory viruses.
Assuntos
COVID-19 , SARS-CoV-2 , Cromatografia Líquida , Humanos , Leucócitos Mononucleares , Espectrometria de MassasRESUMO
Approximately one in three people infected with the SARS-CoV-2 virus have mild symptoms or are asymptomatic. However, these individuals can still spread the virus. Regular self-testing can help to detect these individuals and thereby slow the spread and protect the more vulnerable members of society. Here, we present a protocol for use of the COVID-19 rapid antigen test which was made freely available to residents of the United Kingdom in April of this year. This using the lateral flow technique for detection of antigens and is amenable to multiplexing.
Assuntos
COVID-19 , COVID-19/diagnóstico , Teste para COVID-19 , Humanos , Imunoensaio/métodos , SARS-CoV-2 , Sensibilidade e Especificidade , Medicina EstatalRESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent driving the current COVID-19 pandemic. The acute respiratory distress that occurs in some severe COVID-19 cases has been linked with hypercoagulation or thrombotic events as well as a worse prognosis and increased risk of death. Thus, point-of-care devices that can be used for early detection of coagulation abnormalities would assist in COVID-19 management. This chapter describes the use of the Roche Diagnostics CoaguChek® XS test kit for potential use in COVID-19 personalized medicine approaches.
Assuntos
COVID-19 , Coagulação Sanguínea , COVID-19/diagnóstico , Humanos , Pandemias , Sistemas Automatizados de Assistência Junto ao Leito , SARS-CoV-2RESUMO
This chapter describes a protocol for proteomic profiling of the rat hippocampal proteome using a combination of liquid chromatography tandem mass spectrometry (LC-MS/MS) and data analysis to determine the cellular location of the identified proteins. In the example given, many of these proteins were localized in the plasma membrane and nucleus. These could be of interest in further studies of neurological and neurodegenerative disorders linked with hippocampal dysfunction, such as aging, major depression, and Alzheimer's disease.
Assuntos
Envelhecimento/metabolismo , Cognição/fisiologia , Hipocampo/metabolismo , Transtornos da Memória/fisiopatologia , Proteoma/metabolismo , Proteômica/métodos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Animais , Cromatografia Líquida/métodos , Hipocampo/fisiopatologia , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem/métodosRESUMO
This chapter presents characterization of the db/db mouse model of type 2 diabetes. The protocol describes generation of the mice along with measurements of physical characteristics such as body weight and body composition, and behaviours including food and water consumption. Such a procedure can also be used for omics-based analyses of brain tissues to investigate the potential use of db/db mouse model in neuropsychiatric research. In addition, we present measurement of blood glucose and plasma insulin levels which tend to be elevated in this model and can modulated post treatment with anti-diabetic agents. All of these characteristics can be used for studying the pathophysiology and for monitoring response to potential therapeutics.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Resistência à Insulina/genética , Biologia Molecular/métodos , Animais , Glicemia/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/fisiopatologia , Modelos Animais de Doenças , Humanos , Insulina/sangue , CamundongosRESUMO
A major challenge in proteomic biomarker discovery and validation for psychiatric diseases is the inherent biological complexity underlying these conditions. There are also many technical issues which hinder this process such as the lack of standardization in sampling, processing and storage of bio-samples in preclinical and clinical settings. This chapter describes a reproducible procedure for sampling blood serum and plasma that is specifically designed for maximizing data quality output in two-dimensional gel electrophoresis, multiplex immunoassay and mass spectrometry profiling studies.
Assuntos
Biomarcadores/sangue , Coleta de Amostras Sanguíneas/métodos , Transtornos Mentais/sangue , Proteômica/métodos , Coagulação Sanguínea , Preservação de Sangue/métodos , Coleta de Amostras Sanguíneas/instrumentação , Centrifugação , Criopreservação/métodos , Humanos , Prontuários Médicos , Flebotomia/métodos , Plasma , Soro , Manejo de Espécimes/instrumentação , Manejo de Espécimes/métodosRESUMO
This chapter describes a protocol for measuring prolyl oligopeptidase (POP) activity using a biotinylated peptide substrate coupled to magnetic microspheres. The complex is incubated in the presence of a pituitary extract and activity can be detected by loss of the biotin label. The assay can be multiplexed for measuring multiple proprotein-cleaving enzymes simultaneously and can be used in analyses of neuropsychiatric diseases in which proteolytic cleavage of biologically active peptides is known to play a role.
Assuntos
Separação Imunomagnética/métodos , Proteínas do Tecido Nervoso/análise , Hipófise/enzimologia , Serina Endopeptidases/análise , Biotinilação , Humanos , Microesferas , Fragmentos de Peptídeos/química , Prolil Oligopeptidases , Esquizofrenia/metabolismo , EstreptavidinaRESUMO
A major challenge in single or panel of biomarker discovery and validation is the inherent biological complexity underlying disease heterogeneity and inconsistent responses to treatment. Moreover, the lack of standardization in the sampling, processing, and storage of biological fluids such as plasma and serum disrupts the discovery and validation of blood-based biomarker tests in preclinical and clinical settings. This chapter presents a reproducible sample collection and handling procedure that aims to enhance analyte stability and ensure compatibility with the corresponding multiplex biomarker profiling platforms. The importance of defining bio-sample acquisition and processing, study design, and profiling platform guidelines for blood-based biomarker measurements is paramount for the success of personalized healthcare strategy and development of companion diagnostics.