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Post-translational methylation of proteins, which occurs in arginines and lysines, modulates several biological processes at different levels of cell signaling. Recently, methylation has been demonstrated in the regulation beyond histones, for example, in the dynamics of protein-protein and protein-nucleic acid interactions. However, the presence and role of non-histone methylation in Trypanosoma cruzi, the etiologic agent of Chagas disease, has not yet been elucidated. Here, we applied mass spectrometry-based-proteomics (LC-MS/MS) to profile the methylproteome of T. cruzi epimastigotes, describing a total of 1252 methyl sites in 824 proteins. Functional enrichment and protein-protein interaction analysis show that protein methylation impacts important biological processes of the parasite, such as translation, RNA and DNA binding, amino acid, and carbohydrate metabolism. In addition, 171 of the methylated proteins were previously reported to bear phosphorylation sites in T. cruzi, including flagellar proteins and RNA binding proteins, indicating that there may be an interplay between these different modifications in non-histone proteins. Our results show that a broad spectrum of functions is affected by methylation in T. cruzi, indicating its potential to impact important processes in the biology of the parasite and other trypanosomes.
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Histonas , Trypanosoma cruzi , Histonas/metabolismo , Trypanosoma cruzi/química , Trypanosoma cruzi/genética , Metilación , Cromatografía Liquida , Espectrometría de Masas en Tándem , Proteínas Protozoarias/genéticaRESUMEN
The study aimed to develop a multiplex qPCR to detect Leishmania infantum load in different sandfly sample settings using Leishmania kDNA and sandfly vacuolar ATPase (VATP) subunit C as internal control gene. The amplification of Lutzomyia longipalpis VATP gene was evaluated together with Leishmania infantum kDNA in a multiplex reaction. The concentration of VATP gene oligonucleotides was adjusted until no statistically significant difference was observed between all multiplex standard curves and singleplex curves, that is, only kDNA amplification. Limit of detection (LoD) was measured using a probit model and a cut-off defined by receiver operating characteristic analysis. Limit of quantification (LoQ) was assessed by a linear model using the coefficient of variation threshold of 25%. After assuring VATP gene amplification, its primer-probe concentrations were best at 100 nM/10 nM, respectively. The cut-off Cq value for L. infantum kDNA was defined as 35.46 with 100% of sensitivity and specificity. A total of 95% LoD was determined to be of 0.162 parasites while LoQ was 5.858. Our VATP/kDNA multiplex qPCR assay shows that it can be used to evaluate both DNA integrity and determine L. infantum load in L. longipalpis even for low yielded samples, that is, individual midguts.
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Leishmania infantum , Phlebotomus , Psychodidae , Animales , ADN de Cinetoplasto/genética , Leishmania infantum/genética , Psychodidae/parasitología , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinariaRESUMEN
This article describes the fabrication of a low-cost Polymerase Chain Reaction (PCR) instrument to detect diseases. In order to reduce the instrument price and simplify construction we developed an alternative fabrication process, transforming conventional printed circuit boards (PCB) in heating elements, avoiding the use of aluminum heating/cooling blocks and Peltier devices. To cool down the reaction a simple computer fan was used. The vial holder was fabricated using two double side PCB boards assembled in a sandwich-like configuration. The bottom PCB has a resistance of 0.9 Ω used to heat the reaction mix, while the top layer has a resistance of 1.1 Ω to heat the vial body, preventing vapor condensation. The top board was maintained at ~ 110 ± 1 °C during all cycles. The final device was able to heat and cool down the reaction at rates of ~ 2.0 °C/s, a rate comparable to commercial thermocyclers. An SMD NTC thermistor was used as temperature sensors, and a PID (proportional-integral-derivative) control algorithm was implemented to acquire and precisely control the temperature. We also discuss how the instrument is calibrated. The device was tested successfully for the amplification of T. pallidum (Syphilis) bacterial DNA and Zika virus RNA samples, showing similar performance to a commercial PCR instrument.
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Infección por el Virus Zika , Virus Zika , Algoritmos , Calefacción , Calor , Humanos , Reacción en Cadena de la Polimerasa , TemperaturaRESUMEN
Male breast cancer (MBC) is a rare malignancy that accounts for about 1.8% of all breast cancer cases. In contrast to the high number of the "omics" studies in breast cancer in women, only recently molecular approaches have been performed in MBC research. High-throughput proteomics based methodologies are promisor strategies to characterize the MBC proteomic signatures and their association with clinico-pathological parameters. In this study, the label-free quantification-mass spectrometry and bioinformatics approaches were applied to analyze the proteomic profiling of a MBC case using the primary breast tumor and the corresponding axillary metastatic lymph nodes and adjacent non-tumor breast tissues. The differentially expressed proteins were identified in the signaling pathways of granzyme B, sirtuins, eIF2, actin cytoskeleton, eNOS, acute phase response and calcium and were connected to the upstream regulators MYC, PI3K SMARCA4 and cancer-related chemical drugs. An additional proteomic comparative analysis was performed with a primary breast tumor of a female patient and revealed an interesting set of proteins, which were mainly involved in cancer biology. Together, our data provide a relevant data source for the MBC research that can help the therapeutic strategies for its management.
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The tissue microenvironment regulates such stem cell behaviors as self-renewal and differentiation. Attempts to mimic components of these microenvironments could provide new strategies for culturing and directing the behavior of stem cells. The aim of the present study was to mimic cardiac and umbilical cord tissue microenvironments in vitro and compare the resulting bone marrow-derived mesenchymal stem cell (BM-MSC) behaviors. We generated tissue microenvironments using conditioned medium (CM) and extracellular matrix (ECM) samples obtained from human heart and umbilical cord tissue explant cultures and by tissue decellularization. Mass spectrometry and immunostaining were used to characterize and determine the specific protein profiles of the ECMs and CMs. We demonstrated that the ECMs and CMs were not cytotoxic to BM-MSCs and could thus be tested via cell culture. The BM-MSCs showed a higher proliferation rate when cultured with umbilical cord-derived CM compared with the other analyzed conditions. Furthermore, the ECMs increased cell adhesion and migration. However, although the conditions tested in this work were able to maintain the viability and affect the proliferation, adhesion and migration of BM-MSCs in vitro, mimicking tissue microenvironments using ECM and CM was not sufficient to induce the cardiomyogenic differentiation of BM-MSCs. The present study provides a thorough characterization of the biological activity of these ECMs and CMs in human BM-MSC cultures.
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Microambiente Celular , Células Madre Mesenquimatosas/citología , Miocardio/citología , Transducción de Señal , Cordón Umbilical/citología , Muerte Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Microambiente Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Matriz Extracelular/metabolismo , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Melanogenesis-stimulated B16-F10 cells enter in a quiescent state, present inhibited mitochondrial respiration and increased reactive oxygen species levels. These alterations suggest that these cells may be under redox signaling, allowing tumor survival. The aim of this study was to evaluate redox-modified proteins in B16-F10 cells after melanogenesis stimulation and rose bengal-photodynamic therapy (RB-PDT). A redox proteomics label-free approach based on the biotin switch assay technique with biotin-HPDP and N-ethylmaleimide was used to assess the thiol-oxidized protein profile. Aconitase was oxidized at Cys-448 and Cys-451, citrate synthase was oxidized at Cys-202 and aspartate aminotransferase (Got2) was oxidized at Cys-272 and Cys-274, exclusively after melanogenesis stimulation. After RB-PDT, only guanine nucleotide-binding protein subunit beta-2-like 1 (Gnb2l1) was oxidized (Cys-168). In contrast, melanogenesis stimulation followed by RB-PDT led to the oxidation of different cysteines in Gnb2l1 (Cys-153 and Cys-249). Besides that, glyceraldehyde-3-phosphate dehydrogenase (Gapdh) presented oxidation at Cys-245, peptidyl-prolyl cis-trans isomerase A (Ppia) was oxidized at Cys-161 and 5,6-dihydroxyindole-2-carboxylic acid oxidase (Tyrp1) was oxidized at Cys-65, Cys-30, and Cys-336 after melanogenesis stimulation followed by RB-PDT. The redox alterations observed in murine melanoma cells and identification of possible target proteins are of great importance to further understand tumor resistance mechanisms.
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Syphilis is a sexually transmitted infection (STI) caused by the spiral bacterium Treponema pallidum. Diagnosis is based on epidemiology, clinical and serology, but serodiagnosis is challenging because distinct clinical forms of the infection may influence serological performance. Several recombinant Treponema pallidum-proteins have already been tested for syphilis diagnosis and they are critical to achieve high accuracy in serological testing. A total of 647 samples were included in the study: 180 T. pallidum-positive samples, 191 T. pallidum-negative samples and 276 sera from individuals infected with unrelated diseases. The diagnostic potential was validated by analysis of ROC curves. For the indirect ELISA, TpN17 (100%) and TmpA (99%) showed excellent AUC values. Sensitivity values were 97.2% for TpN17 and 90.6% for TmpA, while specificity was 100% for both molecules. According to the clinical phase, TmpA ranged from 84% to 97%, with the highest value for secondary syphilis. TpN17 was 100% sensitive for the primary and secondary stages and 93.2% for recent latent syphilis. All clinical phases achieved 100% specificity. Accuracy values showed that TmpA (> 95%) and TpN17 (> 98%) presented high diagnostic accuracy for all clinical stages of syphilis. Cross-reactivity was only observed in one sample positive for Chagas disease (1.5%), when TpN17 was evaluated. On the other hand, TmpA showed reactivity for two samples positive for Chagas disease (3.1%), one sample positive for HBV (1.25%), two samples positive for HIV (9.5%) and one sample positive for HTLV (1.6%). The TmpA antigen's performance was evaluated in multiple studies for syphilis diagnosis, corroborating our findings. However, TpN17 sensitivity values have ranged in other studies. According to clinical stages of the infection, our findings obtained close performance values.
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COVID-19 laboratory diagnosis primarily relies on molecular tests, highly sensitive during early infection stages with high viral loads. As the disease progresses, sensitivity decreases, requiring antibody detection. Since the beginning of the pandemic, serological tests have been developed and made available in Brazil, but their diagnostic performance varies. This study evaluated the IBMP ELISA IgA/IgM/IgG COVID-19 kit performance in detecting SARS-CoV-2 antibodies. A total of 90 samples, including 64 from COVID-19 patients and 26 pre-pandemic donors, were assessed based on time post symptom onset (0-7, 8-14, and 15-21 days). The kit showed 61% sensitivity, 100% specificity, and 72% accuracy overall. Sensitivity varied with time, being 25%, 57%, and 96% for 0-7, 8-14, and 15-21 days, respectively. Similar variations were noted in other commercial tests. The Gold ELISA COVID-19 (IgG/IgM) had sensitivities of 31%, 71%, and 100%, while the Anti-SARS-CoV-2 NCP ELISA (IgG) and Anti-SARS-CoV-2 NCP ELISA (IgM) showed varying sensitivities. The IBMP ELISA kit displayed high diagnostic capability, especially as the disease progressed, complementing COVID-19 diagnosis. Reproducibility assessment revealed minimal systematic and analytical errors. In conclusion, the IBMP ELISA IgA/IgM/IgG COVID-19 kit is a robust tool for detecting anti-SARS-CoV-2 antibodies, increasing in efficacy over the disease course, and minimizing false negatives in RT-PCR COVID-19 diagnosis.
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BACKGROUND AND AIMS: The actual classification of breast tumors in subtypes represents an attempt to stratify patients into clinically cohesive groups, nevertheless, clinicians still lack reproducible and reliable protein biomarkers for breast cancer subtype discrimination. In this study, we aimed to access the differentially expressed proteins between these tumors and its biological implications, contributing to the subtype's biological and clinical characterization, and with protein panels for subtype discrimination. METHODS: In our study, we applied high-throughput mass spectrometry, bioinformatic, and machine learning approaches to investigate the proteome of different breast cancer subtypes. RESULTS: We identified that each subtype depends on different protein expression patterns to sustain its malignancy, and also alterations in pathways and processes that can be associated with each subtype and its biological and clinical behaviors. Regarding subtype biomarkers, our panels achieved performances with at least 75% of sensibility and 92% of specificity. In the validation cohort, the panels obtained acceptable to outstanding performances (AUC = 0.740 to 1.00). CONCLUSIONS: In general, our results expand the accuracy of breast cancer subtypes' proteomic landscape and improve the understanding of its biological heterogeneity. In addition, we identified potential protein biomarkers for the stratification of breast cancer patients, improving the repertoire of reliable protein biomarkers. SIGNIFICANCE: Breast cancer is the most diagnosed cancer type worldwide and the most lethal cancer in women. As a heterogeneous disease, breast cancer tumors can be classified into four major subtypes, each presenting particular molecular alterations, clinical behaviors, and treatment responses. Thus, a pivotal step in patient management and clinical decisions is accurately classifying breast tumor subtypes. Currently, this classification is made by the immunohistochemical detection of four classical markers (estrogen receptor, progesterone receptor, HER2 receptor, and the Ki-67 index); however, it is known that these markers alone do not fully discriminate the breast tumor subtypes. Also, the poor understanding of the molecular alterations of each subtype leads to a challenging decision-making process regarding treatment choice and prognostic determination. This study, through high-throughput label-free mass-spectrometry data acquisition and downstream bioinformatic analysis, advances in the proteomic discrimination of breast tumors and achieves an in-depth characterization of the subtype's proteomes. Here, we indicate how the variations in the subtype's proteome can influence the tumor's biological and clinical differences, highlighting the variation in the expression pattern of oncoproteins and tumor suppressor proteins between subtypes. Also, through our machine-learning approach, we propose multi-protein panels with the potential to discriminate the breast cancer subtypes. Our panels achieved high classification performance in our cohort and in the independent validation cohort, demonstrating their potential to improve the current tumor discrimination system as complements to the classical immunohistochemical classification.
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Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/patología , Proteoma/metabolismo , Proteómica/métodos , Biomarcadores , Espectrometría de Masas , Biomarcadores de Tumor/metabolismo , Receptor ErbB-2/metabolismoRESUMEN
In the absence of validated biomarkers to control the cure of Chagas disease, PCR-based diagnosis is being used as the main tool for an early indication of therapeutic failure. However, since it is considered a technique of complex reproducibility, mainly due to difficulties in establishing accurate controls to guarantee the quality of the reaction, the use of PCR for Chagas disease diagnosis is restricted to specialized centers. In an effort to disseminate the molecular diagnosis of Chagas disease and its applications, new diagnostic kits based on qPCR have been made available in the market in recent years. Here, we show the results of the validation of the NAT Chagas kit (Nucleic Acid Test for Chagas Disease) for the detection and quantification of T. cruzi in blood samples of patients suspected of Chagas disease infection. The kit, composed of a TaqMan duplex reaction targeting the T. cruzi satellite nuclear DNA and an exogenous internal amplification control, presented a reportable range from 104 to 0.5 parasite equivalents/mL and a limit of detection (LOD) of 0.16 parasite equivalents/mL of blood. In addition, the NAT Chagas kit detected T. cruzi belonging to all six discrete typing units (DTUs-TcI to TcVI), similarly to the in-house real-time PCR performed with commercial reagents, which has been selected as the best performance assay in the international consensus for the validation of qPCR for Chagas disease. In the clinical validation presented here, the kit showed 100% sensitivity and 100% specificity when compared to the consensus in-house real-time PCR assay. Thus, the NAT Chagas kit, which is produced entirely in Brazil under the international standards of good manufacturing practices (GMP), appears as an excellent alternative to enable the molecular diagnosis of Chagas disease in public and private diagnostic centers, as well as to facilitate the monitoring of patients under etiological treatment participating in clinical trials.
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Trypanosoma cruzi is the etiologic agent of Chagas disease, which is estimated to affect over eight million people around the world. Trypanosoma cruzi has a complex life cycle, involving insect and mammalian hosts and four distinct developmental stages: epimastigotes, metacyclic trypomastigotes, amastigotes, and bloodstream trypomastigotes. Metacyclogenesis is the process by which T. cruzi epimastigotes differentiate into metacyclic trypomastigotes and acquire infectivity, and involves differential gene expression associated with acquisition of virulence. In T. cruzi, gene expression regulation is achieved mainly posttranscriptionally. Therefore, proteomics-based approaches are extremely useful for gaining a better understanding of the changes that occur in the stage-regulated gene expression program of the parasite at the molecular level. Here, we performed an in-depth quantitative MS-based proteomic study of T. cruzi metacyclogenesis and quantified almost 3000 proteins expressed during the process of differentiation. To the best of our knowledge, this work is the most comprehensive quantitative proteomics study of different cell populations of T. cruzi available so far. We identified relevant proteins and pathways involved in the parasite's differentiation and infectivity acquisition, opening new perspectives for further studies that could, ultimately, lead to the identification of new targets for chemotherapy.
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Enfermedad de Chagas/parasitología , Regulación del Desarrollo de la Expresión Génica , Proteómica/métodos , Proteínas Protozoarias/genética , Trypanosoma cruzi/crecimiento & desarrollo , Humanos , Proteínas Protozoarias/metabolismo , Espectrometría de Masas en Tándem/métodos , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismoRESUMEN
INTRODUCTION: Yellow fever (YF) is a public health threat with frequent outbreaks in tropical and subtropical areas, despite the existence of a safe and effective vaccine. The diagnosis of acute infection of the etiologic agent relies mainly on real-time reverse transcription-polymerase chain reaction (RT-qPCR)-based assays. OBJECTIVES: The aim of this study was to evaluate and compare this novel protocol for yellow fever virus (YFV) diagnosis against assays developed in-house by reference laboratories for arboviruses. METHODS: We developed a novel molecular protocol for the detection of YFV that includes an Internal Control to validate the reaction and an External Control to monitor the RNA extraction efficiency. RESULTS AND DISCUSSION: Our assay detects one viral genome per reaction and displays no cross-reactions with dengue (1-4), Zika, or Chikungunya viruses. This novel assay yielded 95% of agreement with the reference method recommended by the Pan American Health Organization when analyzing 204 clinical samples and cultured viruses, these samples were analyzed in 3 different diagnosis centers for arboviruses in Brazil. The data suggest the use of the proposed multiplex assay protocol to do routine tests in a clinical laboratory. This product adds higher specificity and sensitivity in addition to reduced cost per test due to hands-on time and reagent spending.
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Arbovirus , Fiebre Chikungunya , Virus del Dengue , Dengue , Fiebre Amarilla , Infección por el Virus Zika , Virus Zika , Fiebre Chikungunya/diagnóstico , Virus del Dengue/genética , Humanos , Fiebre Amarilla/prevención & control , Virus de la Fiebre Amarilla/genética , Virus Zika/genética , Infección por el Virus Zika/diagnósticoRESUMEN
Several evidence has demonstrated the involvement of the ribosomal proteins (RPs) in many malignancies, however, the function and clinical relevance of the RPs in breast cancer remains unclear. The present study aims to contribute to the understanding of the role of the RPs in breast tumorigenesis and its clinical implications in the field of biomarker discovery and outcome prediction. We investigated the proteomic and transcriptomic expression of the RPs in non-tumor and tumor tissues of different breast cancer subtypes, and integrated bioinformatics approaches and online databases to comprehensively evaluate the potential functions, regulatory networks, mutational landscape, and prognostic values of the ribosomal proteins in breast cancer. Our results show that 33 RPs have deregulated expression in breast cancer and its subtypes and that 26 RPs have potential as prognostic markers in a subtype-dependent way, with mutations in RP genes being frequent in breast tumors and related to overall survival and relapse-free status. Our RP gene regulatory network indicates the transcription factors MYC, ETS1, and SPI1, and the miRNAs has-let-7c-5p, has-mir-20b-5p, and has-mir-4668-3p as regulators of the RPs expression in breast cancer. The RPs were associated with several clinicopathological parameters of breast cancer and predicted to be involved in ribosomal-independent mechanisms such as regulation of the SLITS-ROBO pathway. This study comprehensively investigated the ribosomal proteins in breast cancer, suggesting that the RPs have clinical potential as biomarkers of diagnostic and prognostic, also providing an in-depth view of the RPs significance in breast cancer.
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Neoplasias de la Mama , MicroARNs , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/genética , Mutación , Pronóstico , Proteómica , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , TranscriptomaRESUMEN
COVID-19 disease is spread worldwide and diagnostic techniques have been studied in order to contain the pandemic. Immunochromatographic (IC) assays are feasible and a low-cost alternative especially in low and middle-income countries, which lack structure to perform certain diagnostic techniques. Here we evaluate the sensitivity and specificity of eleven different IC tests in 145 serum samples from confirmed cases of COVID-19 using RT-PCR and 100 negative serum samples from blood donors collected in February 2019. We also evaluated the cross-reactivity with dengue using 20 serum samples from patients with confirmed diagnosis for dengue collected in early 2019 through four different tests. We found high sensitivity (92%), specificity (100%) and an almost perfect agreement (Kappa 0.92) of IC assay, especially when we evaluated IgG and IgM combined after 10 days from the onset of symptoms with RT-PCR. However, we detected cross-reactivity between dengue and COVID-19 mainly with IgM antibodies (5 to 20% of cross-reaction) and demonstrated the need for better studies about diagnostic techniques for these diseases.
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COVID-19 , Dengue , Anticuerpos Antivirales , COVID-19/diagnóstico , Dengue/diagnóstico , Humanos , Inmunoensayo/métodos , Inmunoglobulina G , Inmunoglobulina M , SARS-CoV-2 , Sensibilidad y EspecificidadRESUMEN
Human immunodeficiency virus (HIV) is still considered a pandemic, and the detection of p24-HIV protein has an important role in the early diagnosis of HIV in adults and newborns. The accessibility of these trials depends on the price and execution difficulty of the method, which can be reduced using electrochemical methods by using enzymeless approaches, disposable and accurate devices. In this work, graphene quantum dots were acquired by a simple synthesis and employed as an electrochemical signal amplifier and support for the aptamer immobilization through a feasible and stable modification of disposable screen-printed electrodes. The device has been easily assembled and used to detect p24-HIV protein without the interference of similar proteins or sample matrix. Using the best set of experimental conditions, a linear correlation between analytical signal and log of p24-HIV concentration from 0.93 ng mL-1 to 93 µg mL-1 and a limit of detection of 51.7 pg mL-1 were observed. The developed device was applied to p24 determination in spiked human serum and provided distinct levels of signal for positive and negative samples, successfully identifying real samples with the target protein. This sensor is a step towards the development of point-of-care devices and the popularization of electrochemical methods for trials and diagnostics of relevant diseases.
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Aptámeros de Nucleótidos , Técnicas Biosensibles , Grafito , Infecciones por VIH , Puntos Cuánticos , Adulto , Técnicas Electroquímicas , Electrodos , Infecciones por VIH/diagnóstico , Proteínas del Virus de la Inmunodeficiencia Humana , Humanos , Recién Nacido , Límite de DetecciónRESUMEN
The use of RT-LAMP (reverse transcriptase-loop mediated isothermal amplification) has been considered as a promising point-of-care method to diagnose COVID-19. In this manuscript we show that the RT-LAMP reaction has a sensitivity of only 200 RNA virus copies, with a color change from pink to yellow occurring in 100% of the 62 clinical samples tested positive by RT-qPCR. We also demonstrated that this reaction is 100% specific for SARS-CoV-2 after testing 57 clinical samples infected with dozens of different respiratory viruses and 74 individuals without any viral infection. Although the majority of manuscripts recently published using this technique describe only the presence of two-color states (pink = negative and yellow = positive), we verified by naked-eye and absorbance measurements that there is an evident third color cluster (orange), in general related to positive samples with low viral loads, but which cannot be defined as positive or negative by the naked eye. Orange colors should be repeated or tested by RT-qPCR to avoid a false diagnostic. RT-LAMP is therefore very reliable for samples with a RT-qPCR Ct < 30 being as sensitive and specific as a RT-qPCR test. All reactions were performed in 30 min at 65 °C. The use of reaction time longer than 30 min is also not recommended since nonspecific amplifications may cause false positives.
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COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Viral/metabolismo , SARS-CoV-2/genética , COVID-19/virología , Prueba de Ácido Nucleico para COVID-19 , Colorimetría , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , SARS-CoV-2/aislamiento & purificación , Carga ViralRESUMEN
Trypanosoma cruzi is a pathogenic protozoan that still has an impact on public health, despite the decrease in the number of infection cases along the years. T. cruzi possesses an heteroxenic life cycle in which it differentiates in at least four forms. Among the differentiation processes, metacyclogenesis has been exploited in different views by researchers. An intriguing question that rises is how metacyclogenesis is triggered and controlled by cell signaling and which are the differentially expressed proteins and posttranslational modifications involved in this process. An important cell signaling pathway is the protein phosphorylation, and it is reinforced in T. cruzi in which the gene expression control occurs almost exclusively posttranscriptionally. Additionally, the number of protein kinases in T. cruzi is relatively high compared to other organisms. A way to approach these questions is evaluating the cells through phosphoproteomics and proteomics. In this chapter, we will describe the steps from the cell protein extraction, digestion and fractionation, phosphopeptide enrichment, to LC-MS/MS analysis as well as a brief overview on peptide identification. In addition, a published method for in vitro metacyclogenesis will be detailed.
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Fosfoproteínas/análisis , Proteómica/métodos , Proteínas Protozoarias/análisis , Trypanosoma cruzi/fisiología , Cromatografía Liquida/métodos , Regulación del Desarrollo de la Expresión Génica , Estadios del Ciclo de Vida/genética , Parasitología/métodos , Fosfoproteínas/metabolismo , Fosforilación/fisiología , Proteínas Protozoarias/metabolismo , Espectrometría de Masas en Tándem/métodosRESUMEN
Phosphorylation is an important event in cell signaling that is modulated by kinases and phosphatases. In Trypanosoma cruzi, the etiological agent of Chagas disease, approximately 2% of the protein-coding genes encode for protein kinases. This parasite has a heteroxenic life cycle with four different development stages. In the midgut of invertebrate vector, epimastigotes differentiate into metacyclic trypomastigotes in a process known as metacyclogenesis. This process can be reproduced in vitro by submitting parasites to nutritional stress (NS). Aiming to contribute to the elucidation of mechanisms that trigger metacyclogenesis, we applied super-SILAC (super-stable isotope labeling by amino acids in cell culture) and LC-MS/MS to analyze different points during NS. This analysis resulted in the identification of 4205 protein groups and 3643 phosphopeptides with the location of 4846 phosphorylation sites. Several phosphosites were considered modulated along NS and are present in proteins associated with various functions, such as fatty acid synthesis and the regulation of protein expression, reinforcing the importance of phosphorylation and signaling events to the parasite. These modulated sites may be triggers of metacyclogenesis.
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Enfermedad de Chagas/parasitología , Estadios del Ciclo de Vida/fisiología , Proteoma/metabolismo , Proteómica/métodos , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/metabolismo , Animales , FosforilaciónRESUMEN
Data present here describe a comparative proteomic analysis among the malignant [primary breast tumor (PT) and axillary metastatic lymph nodes (LN)], and the non-tumor [contralateral (NCT) and adjacent (ANT)] breast tissues. Protein identification and quantification were performed through label-free mass spectrometry using a nano-liquid chromatography coupled to an electrospray ionization-mass spectrometry (nLC-ESI-MS/MS). The mass spectrometry proteomic data have been deposited to the ProteomeXchange Consortium via PRIDE partner repository with the dataset identifier PXD012431. A total of 462 differentially expressed proteins was identified among these tissues and was analyzed in six groups' comparisons (named NCTxANT, PTxNCT, PTxANT, LNxNCT, LNxANT and PTxLN). Proteins at 1.5 log2 fold change were submitted to the Ingenuity® Pathway Analysis (IPA) software version 2.3 (QIAGEN Inc.) to identify biological pathways, disease and function annotation, and interaction networks related to cancer biology. The detailed data present here provides information about the proteome alterations and their role on breast tumorigenesis. This information can lead to novel biological insights on cancer research. For further interpretation of these data, please see our research article 'Quantitative label-free mass spectrometry using contralateral and adjacent breast tissues reveal differentially expressed proteins and their predicted impacts on pathways and cellular functions in breast cancer' [2].
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Proteins play an essential role in the biological processes associated with cancer. Their altered expression levels can deregulate critical cellular pathways and interactive networks. In this study, the mass spectrometry-based label-free quantification followed by functional annotation was performed to investigate the most significant deregulated proteins among tissues of primary breast tumor (PT) and axillary metastatic lymph node (LN) and corresponding non-tumor tissues contralateral (NCT) and adjacent (ANT) from patients diagnosed with invasive ductal carcinoma. A total of 462 proteins was observed as differentially expressed (DEPs) among the groups analyzed. A high level of similarity was observed in the proteome profile of both non-tumor breast tissues and DEPs (nâ¯=â¯12) were mainly predicted in the RNA metabolism. The DEPs among the malignant and non-tumor breast tissues [nâ¯=â¯396 (PTxNCT) and nâ¯=â¯410 (LNxNCT)] were related to pathways of the LXR/RXR, NO, eNOS, eIF2 and sirtuins, tumor-related functions, fatty acid metabolism and oxidative stress. Remarkable similarity was observed between both malignant tissues, which the DEPs were related to metastatic capabilities. Altogether, our findings revealed differential proteomic profiles that affected cancer associated and interconnected signaling processes. Validation studies are recommended to demonstrate the potential of individual proteins and/or pathways as biological markers in breast cancer. SIGNIFICANCE: The proteomic analysis of this study revealed high similarity in the proteomic profile of the contralateral and adjacent non-tumor breast tissues. Significant differences were identified among the proteome of the malignant and non-tumor tissue groups of the same patients, providing relevant insights into the hallmarks, signaling pathways, biological functions, and interactive protein networks that act during tumorigenesis and breast cancer progression. These proteins are suggested as targets of relevant interest to be explored as potential biological markers related to tumor development and metastatic progression in the breast cancer disease.