Thermal Proteome Profiling for Drug Target Identification and Probing of Protein States.

Proteins are central drivers of physiological and pathological processes in the cell. Methods evaluating protein functional states are therefore vital to fundamental research as well as drug discovery. Thermal proteome profiling (TPP) to this date constitutes the only approach that permits examining protein states in live cells, under native conditions and at a proteome-wide scale. TPP harnesses ligand/perturbation-induced changes in protein thermal stability, which are monitored by multiplexed quantitative mass spectrometry. In this chapter, we describe a modular experimental workflow for TPP experiments using live cells or crude cell extracts. We provide the tools to perform different TPP formats, i.e., temperature range experiments, TPP-TR; isothermal compound titrations, TPP-CCR; and a combination thereof, 2D-TPP.

Fatores de risco para o desenvolvimento de doença cardiovascular em pacientes com depressão em um hospital do sul do Brasil / l Risk factors for the development of cardiovascular disease in patients with depression in a hospital in Southern Brazil

Introdução: A associação entre depressão e fatores de risco cardiovascular é recorrente. O aumento de risco em deprimidos está relacionado à fatores como obesidade, sedentarismo, dislipidemia, alcoolismo e tabagismo. O objetivo deste trabalho foi identificar a presença de fatores de risco para o desenvolvimento de doenças cardiovasculares em pacientes com quadro de depressão internados em um hospital do sul do Brasil. Métodos: Estudo transversal que envolveu adultos de ambos os sexos internados por episódio depressivo. O estado nutricional foi avaliado pelas medidas antropométricas de peso, altura e circunferência da cintura. Um questionário foi aplicado englobando perfil sociodemográfico, histórico familiar de doenças, consumo de produtos de tabaco e de álcool, atividade física, além do questionário autoaplicável para medida da severidade da depressão. Para avaliação do risco cardiovascular global, foi calculado o escore de Framingham. Os testes qui-quadrado de Pearson (χ2 ) ou exato de Fisher foram utilizados para testar a associação entre as variáveis categóricas, considerando o nível de significância quando p ≤ 0,05 e IC95%. Resultados: Foram avaliados 54 indivíduos, predominantemente mulheres (n = 32), com idade média de 40,2 ± 10,8 anos. A depressão foi classificada como grave na maioria dos pacientes (n = 29). Fatores de risco relacionados ao nível de atividade física (sedentarismo), dislipidemia e estado nutricional (sobrepeso e obesidade) estiveram presentes em 81,5%, 73,1% e 66,7% da amostra, respectivamente. Percentual de risco obtido por meio do escore de Framingham foi encontrado acima do normal em 42,9% dos indivíduos. Depressão leve associou-se positivamente aos pacientes com magreza/eutrofia e, ao serem estratificados como severos e não-severos, o primeiro grupo teve associação positiva com histórico familiar de excesso de peso e hipertensão. Conclusões: Diversos fatores de risco cardiovascular foram encontrados, alertando para a importância do cuidado integral da saúde do paciente e avaliação destes indicadores. (AU)

Introduction: The association between depression and cardiovascular risk factors is recurrent. Increased risk of depression is related to factors such as obesity, sedentary lifestyle, dyslipidemia, alcoholism, and smoking. The aim of this study was to identify the presence of risk factors for the development of cardiovascular disease in patients with depression admitted to a hospital in southern Brazil. Methods: A cross-sectional study involving adults of both sexes hospitalized for a depressive episode. Nutritional status was assessed by the anthropometric measurements of weight, height and waist circumference. A questionnaire was applied covering sociodemographic data, family history of diseases, consumption of tobacco and alcohol products, and physical activity, in addition to a self-administered questionnaire to measure the severity of depression. The Framingham risk score was calculated for global cardiovascular risk evaluation. Pearson's chi-square test (χ2 ) or Fisher's exact Test were used to test the association between categorical variables, considering the level of significance at p ≤ 0.05 and 95% CI. Results: Fifty-four individuals were evaluated, most were women (n = 32), with a mean age of 40.2 ± 10.8 years were evaluated. Depression was classified as severe in most patients (n = 29). Risk factors related to the physical activity level (sedentary lifestyle), dyslipidemia and nutritional status (overweight and obesity) were present in 81.5%, 73.1% and 66.7% of the sample, respectively. Percentage of risk obtained by the Framingham risk score was found above normal in 42.9% of the individuals. Mild depression was positively associated with thin/eutrophic patients and, when stratified as severe and non-severe, the first group had a positive association with family history of overweight and hypertension. Conclusions: Several cardiovascular risk factors were found, alerting to the importance of integral health care for patients and evaluation of these indicators. (AU)

Single-Fluorescent Protein Reporters Allow Parallel Quantification of Natural Killer Cell-Mediated Granzyme and Caspase Activities in Single Target Cells.

Natural killer (NK) cells eliminate infected and tumorigenic cells through delivery of granzymes via perforin pores or by activation of caspases via death receptors. In order to understand how NK cells combine different cell death mechanisms, it is important to quantify target cell responses on a single cell level. However, currently existing reporters do not allow the measurement of several protease activities inside the same cell. Here, we present a strategy for the comparison of two different proteases at a time inside individual target cells upon engagement by NK cells. We developed single-fluorescent protein reporters containing the RIEAD or the VGPD cleavage site for the measurement of granzyme B activity. We show that these two granzyme B reporters can be applied in combination with caspase-8 or caspase-3 reporters. While we did not find that caspase-8 was activated by granzyme B, our method revealed that caspase-3 activity follows granzyme B activity with a delay of about 6 min. Finally, we illustrate the comparison of several different reporters for granzyme A, M, K, and H. The approach presented here is a valuable means for the investigation of the temporal evolution of cell death mediated by cytotoxic lymphocytes.

The quorum-sensing regulator ComA from Bacillus subtilis activates transcription using topologically distinct DNA motifs.

ComA-like transcription factors regulate the quorum response in numerous Gram-positive bacteria. ComA proteins belong to the tetrahelical helix-turn-helix superfamily of transcriptional activators, which bind as homodimers to inverted sequence repeats in the DNA. Here, we report that ComA from Bacillus subtilis recognizes a topologically distinct motif, in which the binding elements form a direct repeat. We provide in vitro and in vivo evidence that the canonical and non-canonical site play an important role in facilitating type I and type II promoter activation, respectively, by interacting with different subunits of RNA polymerase. We furthermore show that there is a variety of contexts in which the non-canonical site can occur and identify new direct target genes that are located within the integrative and conjugative element ICEBs1. We therefore suggest that ComA acts as a multifunctional transcriptional activator and provides a striking example for complexity in protein-DNA interactions that evolved in the context of quorum sensing.

Association between LAP Index (Lipid Accumulation Product) and metabolic profile in hospitalized patients.

INTRODUCTION: Lipid Accumulation Product (LAP) Index correlates to cardiovascular risk factors in general population but it has not been tested in hospitalized patients. OBJECTIVES: We aimed to evaluate associations between LAP Index and metabolic profile in a tertiary hospital. METHODS: Cross-sectional study with 90 inpatients. Lipid profile, fasting glucose, systolic and diastolic blood pressure measurements were obtained from electronic medical records. Weight, height and waist circumferences (WC) were assessed; Body Mass Index (BMI) and LAP Index were calculated. Data were expressed as mean ± standard deviation or percentage. Pearson's correlation and Multiple Linear Regression were used to assess the objectives. RESULTS: Mean age of participants was 55.03 ± 14.86 years and 47.8% (n = 43) were men. After adjustment for sex, age and physic activity LAP Index (log-transformed) was significantly associated with HDL-cholesterol (p < 0.001), fasting glucose (p = 0.02), systolic blood pressure (p = 0.03) and a trend toward total cholesterol (p = 0.07). CONCLUSION: There are independent association between LAP Index (log-transformed) and metabolic profile in hospitalized patients.

Introducción: el índice PAL (Producto de Acumulación Lipídica) se correlaciona con factores de riesgo cardiovasculares en la población general, pero no ha sido probado en pacientes hospitalizados. Objetivos: evaluar las asociaciones entre el índice PAL y el perfil metabólico en un hospital de tercer nivel. Métodos: estudio transversal con 90 pacientes hospitalizados. Se obtuvieron de los registros médicos electrónicos el perfil lipídico, la glucosa en ayunas y las mediciones de la presión arterial sistólica y diastólica. Fueron evaluados el peso, la talla y la circunferencia de la cintura (CC); se calcularon el Índice de Masa Corporal (IMC) y el Índice de PAL. Los datos se expresan como media ± desviación estándar o porcentaje. Para evaluar los objetivos se utilizaron la correlación de Pearson y la regresión lineal múltiple. Resultados: la edad promedio de los participantes fue de 55,03 ± 14,86 años y el 47,8% (n = 43) eran hombres. Después de ajustar por sexo, edad y actividad física, el índice PAL (transformado-log) se asoció significativamente con el HDL-colesterol (p < 0,001), la glucosa en ayunas (p=0,02), la presión arterial sistólica (p = 0,03) y una tendencia hacia el colesterol total (p = 0,07). Conclusión: en los pacientes hospitalizados existe una asociación independiente entre el índice PAL (transformado- log) y el perfil metabólico.

Association between LAP Index (lipid accumulation product) and metabolic profile in hospitalized patients / Asociación entre el índice de PAL (el Producto de Acumulación Lipídica) y el perfil metabólico en pacientes hospitalizados

Introduction: lipid Accumulation Product (LAP) Index correlates to cardiovascular risk factors in general population but it has not been tested in hospitalized patients. Objectives: we aimed to evaluate associations between LAP Index and metabolic profile in a tertiary hospital. Methods: Cross-sectional study with 90 inpatients. Lipid profile, fasting glucose, systolic and diastolic blood pressure measurements were obtained from electronic medical records. Weight, height and waist circumferences (WC) were assessed; Body Mass Index (BMI) and LAP Index were calculated. Data were expressed as mean ± standard deviation or percentage. Pearson’s correlation and Multiple Linear Regression were used to assess the objectives. Results: mean age of participants was 55.03 ± 14.86 years and 47.8% (n = 43) were men. After adjustment for sex, age and physic activity LAP Index (log-transformed) was significantly associated with HDL-cholesterol (p< 0.001), fasting glucose (p = 0.02), systolic blood pressure (p = 0.03) and a trend toward total cholesterol (p = 0.07). Conclusion: There are independent association between LAP Index (log-transformed) and metabolic profile in hospitalized patients (AU)

Introducción: el índice PAL (Producto de Acumulación Lipídica) se correlaciona con factores de riesgo cardiovasculares en la población general, pero no ha sido probado en pacientes hospitalizados. Objetivos: evaluar las asociaciones entre el índice PAL y el perfil metabólico en un hospital de tercer nivel. Métodos: estudio transversal con 90 pacientes hospitalizados. Se obtuvieron de los registros médicos electrónicos el perfil lipídico, la glucosa en ayunas y las mediciones de la presión arterial sistólica y diastólica. Fueron evaluados el peso, la talla y la circunferencia de la cintura (CC); se calcularon el Índice de Masa Corporal (IMC) y el Índice de PAL. Los datos se expresan como media ± desviación estándar o porcentaje. Para evaluar los objetivos se utilizaron la correlación de Pearson y la regresión lineal múltiple. Resultados: la edad promedio de los participantes fue de 55,03 ± 14,86 años y el 47,8% (n = 43) eran hombres. Después de ajustar por sexo, edad y actividad física, el índice PAL (transformado-log) se asoció significativamente con el HDL-colesterol (p < 0,001), la glucosa en ayunas (p=0,02), la presión arterial sistólica (p = 0,03) y una tendencia hacia el colesterol total (p = 0,07). Conclusión: en los pacientes hospitalizados existe una asociación independiente entre el índice PAL (transformado-log) y el perfil metabólico (AU)