SUMOylation pathway in Trypanosoma cruzi: functional characterization and proteomic analysis of target proteins.

SUMOylation is a relevant protein post-translational modification in eukaryotes. The C terminus of proteolytically activated small ubiquitin-like modifier (SUMO) is covalently linked to a lysine residue of the target protein by an isopeptide bond, through a mechanism that includes an E1-activating enzyme, an E2-conjugating enzyme, and transfer to the target, sometimes with the assistance of a ligase. The modification is reversed by a protease, also responsible for SUMO maturation. A number of proteins have been identified as SUMO targets, participating in the regulation of cell cycle progression, transcription, translation, ubiquitination, and DNA repair. In this study, we report that orthologous genes corresponding to the SUMOylation pathway are present in the etiological agent of Chagas disease, Trypanosoma cruzi. Furthermore, the SUMOylation system is functionally active in this protozoan parasite, having the requirements for SUMO maturation and conjugation. Immunofluorescence analysis showed that T. cruzi SUMO (TcSUMO) is predominantly found in the nucleus. To identify SUMOylation targets and get an insight into their physiological roles we generated transfectant T. cruzi epimastigote lines expressing a double-tagged T. cruzi SUMO, and SUMOylated proteins were enriched by tandem affinity chromatography. By two-dimensional liquid chromatography-mass spectrometry a total of 236 proteins with diverse biological functions were identified as potential T. cruzi SUMO targets. Of these, metacaspase-3 was biochemically validated as a bona fide SUMOylation substrate. Proteomic studies in other organisms have reported that orthologs of putative T. cruzi SUMOylated proteins are similarly modified, indicating conserved functions for protein SUMOylation in this early divergent eukaryote.

Proteomic analysis of papaya (Carica papaya L.) displaying typical sticky disease symptoms.

Papaya (Carica papaya L.) hosts the only described laticifer-infecting virus (Papaya meleira virus, PMeV), which is the causal agent of papaya sticky disease. To understand the systemic effects of PMeV in papaya, we conducted a comprehensive proteomic analysis of leaf samples from healthy and diseased plants grown under field conditions. First, a reference 2-DE map was established for proteins from healthy samples. A total of 486 reproducible spots were identified, and MALDI-TOF-MS/MS data identified 275 proteins accounting for 159 distinct proteins from 231 spots that were annotated. Second, the differential expression of proteins from healthy and diseased leaves was determined through parallel experiments, using 2-DE and DIGE followed by MALDI-TOF-MS/MS and LC-IonTrap-MS/MS, respectively. Conventional 2-DE analysis revealed 75 differentially expressed proteins. Of those, 48 proteins were identified, with 26 being upregulated (U) and 22 downregulated (D). In general, metabolism-related proteins were downregulated, and stress-responsive proteins were upregulated. This expression pattern was corroborated by the results of the DIGE analysis, which identified 79 differentially expressed proteins, with 23 identified (17 U and 6 D). Calreticulin and the proteasome subunits 20S and RPT5a were shown to be upregulated during infection by both 2-DE and DIGE analyses. These data may help shed light on plant responses against stresses and viral infections.

Lipidomic analysis reveals that phosphatidylglycerol and phosphatidylethanolamine are newly generated phospholipids in an early-divergent protozoan, Giardia lamblia.

The pathogenic protozoan Giardia lamblia is known to not synthesize membrane lipids de novo. Therefore, it is possible that lipids in the small intestine, where trophozoites colonize, play key roles in regulating the growth and differentiation of this important pathogen. The focus of the current study is to conduct a complete lipidomic analysis and to test the hypothesis that Giardia has some ability to generate new phospholipids (PLs). Using mass spectrometry, now we show that phosphatidylglycerols (PGs) are major PLs followed by phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) in non-encysting and encysting trophozoites, as well in cysts. The fatty acids attached to these PLs consist mostly of palmitate, palmitoleate, oleate, and linoleate. Results also indicate that PGs and PEs, unlike PCs, are not present in bovine bile and serum, the major sources of lipids of the culture medium, and that they could therefore be produced by fatty acid and headgroup remodeling reactions, circumventing the synthesis of entirely new PLs via de novo pathways. Genomic and transcriptional analyses show the presence of giardial phosphatidylglycerolphosphate synthase (gpgps) and phosphatidylserine decarboxylase (gpsd) genes, which are expressed throughout the life cycle. Bioinformatic and phylogenetic analyses further indicated that both genes are of prokaryotic origin and that they have undergone duplication in the course of evolution. Our studies suggest that the abundance of PG in Giardia is unique among eukaryotes and that its synthesis thus could serve as a potential target for developing new therapies against this waterborne parasite.

Label-free quantitative proteomics reveals differentially regulated proteins in the latex of sticky diseased Carica papaya L. plants.

Papaya meleira virus (PMeV) is so far the only described laticifer-infecting virus, the causal agent of papaya (Carica papaya L.) sticky disease. The effects of PMeV on the laticifers' regulatory network were addressed here through the proteomic analysis of papaya latex. Using both 1-DE- and 1D-LC-ESI-MS/MS, 160 unique papaya latex proteins were identified, representing 122 new proteins in the latex of this plant. Quantitative analysis by normalized spectral counting revealed 10 down-regulated proteins in the latex of diseased plants, 9 cysteine proteases (chymopapain) and 1 latex serine proteinase inhibitor. A repression of papaya latex proteolytic activity during PMeV infection was hypothesized. This was further confirmed by enzymatic assays that showed a reduction of cysteine-protease-associated proteolytic activity in the diseased papaya latex. These findings are discussed in the context of plant responses against pathogens and may greatly contribute to understand the roles of laticifers in plant stress responses.

Características epidemiológicas de los pacientes con enfermedad crítica crónica. Un estudio observacional ambispectivo / Epidemiological characteristics of patients with chronic critical illness. An ambispective observational study / Características epidemiolológicas dos pacientes con doença crítica crónica. Um estudo observacional ambispectivo

Resumen: Introducción: La población de pacientes que requiere de cuidados intensivos por tiempo prolongado se ha incrementado en las últimas décadas, lo que ha dado lugar a una nueva población de individuos con enfermedad crítica crónica (ECC). El objetivo de este trabajo fue describir las características epidemiológicas de los sujetos con enfermedad crítica crónica en la Unidad de Cuidados Intensivos (UCI) del Hospital Regional de Alta Especialidad de Oaxaca. Material y métodos: Se realizó un estudio ambispectivo, observacional y descriptivo que incluyó a las personas ingresadas entre el 01 de enero de 2012 y el 31 de diciembre de 2015. Se definió a los pacientes con ECC como aquéllos con ventilación mecánica prolongada (más de 21 días de ventilación mecánica por más de seis horas al día). Los individuos fueron seguidos hasta su egreso hospitalario o defunción. Se excluyeron los sujetos con expedientes clínicos incompletos, edad menor de 16 años, enfermedades neuromusculares (síndrome de Guillain-Barre y miastenia gravis) y aquéllos sin ventilación mecánica o requerimiento menor a 48 horas. Resultados: Se incluyeron 284 pacientes; la incidencia de ECC fue de 9.8%. En los individuos con ECC, la escala de APACHE II fue 19.4 ± 9.7, y en aquéllos sin ECC, fue 15.94 ± 8.6 (p = 0.044). La escala SOFA en los sujetos con ECC fue de 8.7 ± 2.6, y aquéllos sin ECC, de 7.01 ± 4.4 (p = 0.007). Los días de estancia en la UCI fueron 17.1 ± 9.2 en las personas con ECC y 8 ± 4.8 días en aquéllas sin ECC (p = 0.0000). Los días de estancia hospitalaria fueron 45.9 ± 19.7 en los pacientes con ECC y 18.3 ± 12.4 en los individuos sin ECC (p = 0.0000). La mortalidad en la UCI fue de 17.8% en los sujetos con ECC y de 27.7% en las personas sin ECC (p = 0.0000). La mortalidad hospitalaria fue de 50% en los pacientes con ECC y de 16% en aquéllos sin ECC (p = 0.0000). Fueron egresados del hospital 32.2% de los individuos con ECC y 56.3% de los sujetos sin ECC (p = 0.0000). Conclusiones: La incidencia de ECC en nuestro estudio fue similar a la reportada en la literatura. La severidad de la enfermedad aguda al ingreso (de acuerdo con las escalas de SOFA y APACHE) estuvo relacionada con el desarrollo de ECC. Las personas con enfermedad crítica crónica tuvieron mayor estancia en la UCI, más días de sedación, así como una estancia hospitalaria más prolongada y una mortalidad hospitalaria aumentada.

Abstract: Introduction: The population of patients requiring intensive care for a prolonged time has increased in recent decades, resulting in a new population of individuals with chronic critical illness (CCI). The aim of this study was to describe the epidemiological characteristics of subjects with CCI in the ICU of the High Specialty Regional Hospital of Oaxaca. Material and methods: It was a retrospective and prospective, observational and descriptive study that included people admitted between January 1, 2012 and December 31, 2015. Patients with CCI were defined as those with prolonged mechanical ventilation (more than 21 days of ventilation for more than six hours/day). The individuals were followed until their hospital discharge or death. Subjects with incomplete medical records, under 16 years of age, neuromuscular diseases (Guillain-Barre syndrome and Myasthenia gravis) and those without mechanical ventilation or who required it for less than 48 hours were excluded. Results: Two-hundred eighty-four people were included; the incidence of CCI was 9.8%. In patients with CCI, the APACHE II score was 19.4 ± 9.7, and in those without CCI, it was 15.94 ± 8.6 (p = 0.044). The SOFA scale in individuals with CCI was 8.7 ± 2.6, and in those without CCI, 7.01 ± 4.4 (p = 0.007). The days of ICU stay were 17.1 ± 9.2 for subjects with CCI and 8 ± 4.8 for those without CCI (p = 0.0000). The days of hospital stay were 45.9 ± 19.7 in people with CCI and 18.3 ± 12.4 in patients without CCI (p = 0.0000). ICU mortality was 17.8% in individuals with CCI and 27.7% in subjects without CCI (p = 0.0000). The hospital mortality was 50% in people with CCI and 16% in those without CCI (p = 0.0000). Of those patients with CCI, 32.2% were discharged from hospital, compared to 56.3% of individuals without CCI (p = 0.0000). Conclusions: The incidence of CCI in our study was similar to that reported in the literature. The severity of acute disease upon admission, according to the APACHE and SOFA scales, was related to the development of CCI. Subjects with chronic critical illness had a longer ICU stay, more days on sedation and of hospital stay, and increased hospital mortality.

Resumo: Introdução: A população de pacientes que necessitam de cuidados intensivos por tempo prolongado tem aumentado nas últimas décadas, resultando em uma nova população de pacientes com doença crítica crônica (DCC). O objetivo deste estudo foi descrever as características epidemiológicas dos pacientes com a doença crônica crítica na UTI do Hospital Regional de Alta Especialidade de Oaxaca. Material e métodos: Estudo ambispectivo, observacional e descritivo que incluiu pacientes admitidos entre 1 de janeiro de 2012 a 31 de dezembro de 2015. Definiu-se os pacientes com DCC como aqueles com ventilação mecânica prolongada > 21 dias ventilação mecânica por > 6 horas por dia. Os pacientes foram acompanhados até a alta hospitalar ou morte. Foram excluídos pacientes com prontuários incompletos, idade inferior a 16 anos, as doenças neuromusculares (síndrome de Guillain Barré e Miastenia Gravis) e pacientes sem ventilação mecânica ou exigência ventilatória menor à 48 horas. Resultados: 284 pacientes foram incluídos no estudo, a incidência de DCC foi de 9.8%. Em pacientes com DCC a pontuação APACHE II foi de 19.4 + 9.7 e sem DCC foi 15.94 + 8.6 (p = 0.044), a escala SOFA em paciente com DCC foi de 8.7 + 2.6, e sem DCC 7.01 + 4.4 (p = 0.007). Os dias de permanência na UTI foram de 17.1 + 9.2 dias para os pacientes com DCC e 8 + 4.8 dias para os pacientes sem DCC (p = 0.0000). Os dias de permanência hospitalar foram de 45.9 + 19.7 dias para os pacientes com DCC e para os pacientes sem DCC 18.3 + 12.4 dias (p = 0.0000). A mortalidade na UTI foi de 17.8% nos pacientes com DCC e 27.7% nos pacientes sem DCC (p = 0.0000). A mortalidade hospitalar foi de 50% nos pacientes com DCC e 16% (p = 0.0000) nos pacientes sem DCC. 32.3% dos pacientes com DCC tiveram alta e 56.3% dos pacientes sem DCC (p = 0.0000). Conclusões: A incidência de DCC em nosso estudo foi semelhante ao relatado na literatura. A gravidade da doença aguda ao ingresso do paciente, de acordo com a pontuação APACHE e SOFA está relacionada com o desenvolvimento da DCC. Os pacientes com doença crítica crônica tiveram um maior tempo de permanência na UTI, mais dias de sedação, maior tempo de internação e maior mortalidade hospitalar.