Dados do Trabalho

Título

DIFFERENCES IN THE PROTEOMIC LANDSCAPE OF BOTHROPS ATROX ENVENOMING VICTIM’S URINE ACCORDING TO ACCIDENT SEVERITY

Introdução

Snakebite envenoming is a neglected public health issue, with high morbidity and mortality rates, which is preventable by antivenom's effective administration. Brazilian guidelines define antivenom posology according to the severity of clinical manifestations. However, delayed local and systemic symptoms may occur, leading to additional antivenom administration. This scenario might result in antivenom overdosage, or even initial under-dosage, allowing clinical complications.

Objetivo (s)

This work aims to compare the urinary proteomic profile of Bothrops atrox envenomations in different severity grades to explore potential severity biomarkers.

Material e Métodos

We collected the urine of 12 bothropic envenomed patients before antivenom treatment, 4 of each accident classification grade - mild, moderate, and severe. For protein extraction, we applied a protocol of methanol precipitation followed by Urea/Thiourea (7M/2M) on 2 mL of snakebite patients’ urine and 10 mL of healthy controls urine; 100 µg of each sample was reduced with dithiothreitol (10 mM), alkylated with iodoacetamide (30 mM), and then incubated with dithiothreitol (5 mM). The urea concentration was diluted to 1M with ammonium bicarbonate (50 mM) and the samples were digested with trypsin. Later, the peptide mixture was desalted, concentrated, resuspended in acid formic 0.1%, and analyzed using an UltiMate 3000 Basic online with a Fusion Lumos Orbitrap mass spectrometer set in data-dependent acquisition (DDA) mode. For data analysis, we used the software PatternLab for Proteomics V (PLV).

Resultados e Conclusão

We identified a total of 1765 proteins (with redundancy) and 1002 were quantified. Regarding the proteins differentially abundant, we highlight Tetranectin (CLEC3B) and Maltase-glucoamylase (MGAM) in the mild group, Fibulin-3 (EFEMP1) and Hemoglobin subunit alpha (HBA1) in moderate cases, and Tenascin (TNC) in severe conditions. Additionally, protein YIPF3 is shown to be more abundant in mild/moderate conditions when compared to severe cases. In conclusion, our results suggest a panel of biomarkers candidates for early diagnosis of bothropic envenoming severity. Although further studies are necessary to endorse our findings; we believe our work enlightens interesting pathophysiology differences in snakebite envenoming accidents.

Palavras-chave

Snakebite, envenoming, Bothrops atrox, proteomic shotgun, urine analysis.

Agradecimentos

We would like to thank the Dr. Heitor Vieira Dourado Tropical Medicine Foundation and the Leônidas & Maria Deane Institute, Fiocruz Amazônia.