Sepsis is a global health emergency, which is caused by various sources of infection that lead to changes in gene expression, protein-coding, and metabolism. Advancements in “omics” technologies have provided valuable tools to unravel the mechanisms involved in the pathogenesis of this disease. In this study, we performed shotgun mass spectrometry in peripheral blood mononuclear cells (PBMC) from septic patients (N=24) and healthy controls (N=9) and combined these results with two public microarray leukocytes datasets. Through combination of transcriptome and proteome profiling, we identified 170 co‐differentially expressed genes/proteins. Among these, 122 genes/proteins displayed the same expression trend. Ingenuity Pathway Analysis revealed pathways related to lymphocyte functions with decreased status, and defense processes that were predicted to be strongly increased. Protein-protein interaction network analyses revealed two densely connected regions, which mainly included down‐regulated genes/proteins that were related to the transcription of RNA, translation of proteins, and mitochondrial translation. Additionally, we identified one module comprising of up‐regulated genes/proteins, which were mainly related to low-density neutrophils (LDNs). LDNs were reported in sepsis and in COVID-19. Changes in gene expression level were validated using quantitative real-time PCR in PBMCs from patients with sepsis. To further support that the source of the upregulated module of genes/proteins found in our results were derived from LDNs, we identified an increase of this population by flow cytometry in PBMC samples obtained from the same cohort of septic patients included in the proteomic analysis. This study provides new insights into a reprioritization of biological functions in response to sepsis that involved a transcriptional and translational shutdown of genes/proteins, with exception of a set of genes/proteins related to LDNs and host‐defense system.

Cryptococcosis is a fungal disease caused by C . neoformans . To adapt and survive in diverse ecological niches, including the animal host, this opportunistic pathogen relies on its ability to uptake nutrients, such as carbon, nitrogen, iron, phosphate, sulfur, and amino acids. Genetic circuits play a role in the response to environmental changes, modulating gene expression and adjusting the microbial metabolism to the nutrients available for the best energy usage and survival. We studied the sulfur amino acid biosynthesis and its implications on C . neoformans biology and virulence. CNAG_04798 encodes a BZip protein and was annotated as CYS3 , which has been considered an essential gene. However, we demonstrated that CYS3 is not essential, in fact, its knockout led to sulfur amino acids auxotroph. Western blots and fluorescence microscopy indicated that GFP-Cys3, which is expressed from a constitutive promoter, localizes to the nucleus in rich medium (YEPD); the addition of methionine and cysteine as sole nitrogen source (SD–N + Met/Cys) led to reduced nuclear localization and protein degradation. By proteomics, we identified and confirmed physical interaction among Gpp2, Cna1, Cnb1 and GFP-Cys3. Deletion of the calcineurin and GPP2 genes in a GFP-Cys3 background demonstrated that calcineurin is required to maintain Cys3 high protein levels in YEPD and that deletion of GPP2 causes GFP-Cys3 to persist in the presence of sulfur amino acids. Global transcriptional profile of mutant and wild type by RNAseq revealed that Cys3 controls all branches of the sulfur amino acid biosynthesis, and sulfur starvation leads to induction of several amino acid biosynthetic routes. In addition, we found that Cys3 is required for virulence in Galleria mellonella animal model.

Background: Crotalus durissus is considered one of the most important species of venomous snakes in Brazil, due to the high mortality of its snakebites. The venom of Crotalus durissus contains four main toxins: crotoxin, convulxin, gyroxin and crotamine. Venoms can vary in their crotamine content, being crotamine-negative or-positive. This heterogeneity is of great importance for producing antivenom, due to their different mechanisms of action. The possibility that antivenom produced by Butantan Institute might have a different immunorecognition capacity between crotamine-negative and crotamine-positive C. durissus venoms instigated us to investigate the differences between these two venom groups. Methods: The presence of crotamine was analyzed by SDS-PAGE, western blotting and ELISA, whereas comparison between the two types of venoms was carried out through HPLC, mass spectrometry analysis as well as assessment of antivenom lethality and efficacy. Results: The results showed a variation in the presence of crotamine among the subspecies and the geographic origin of snakes from nature, but not in captive snakes. Regarding differences between crotamine-positive and-negative venoms, some exclusive proteins are found in each pool and the crotamine-negative pool presented more phospholipase A 2 than crotamine-positive pool. This variation could affect the time to death, but the lethal and effective dose were not affected. Conclusion: These differences between venom pools indicate the importance of using both, crotamine-positive and crotamine-negative venoms, to produce the antivenom.

Snake venoms are complex protein mixtures with different biological activities that can act in both their preys and human victims. Many of these proteins play a role in prey capture and in the digestive process of these animals. It is known that some snakes are resistant to the toxicity of their own venom by mechanisms not yet fully elucidated. However, it was observed in the Laboratory of Herpetology of Instituto Butantan that some Bothrops moojeni individuals injured by the same snake species showed mortalities caused by envenoming effects. This study analyzed the biochemical composition of 13 venom and plasma samples from Bothrops moojeni specimens to assess differences in their protein composition. Application of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed distinct venom protein profiles, but very homogeneous plasma profiles. Western Blotting (WB) was performed with plasma samples, which were submitted to incubation with the respective venom. Some individuals showed an immunorecognized band zone around 25 kDa, indicating interaction between the same individual plasma and venom proteins. Crossed-WB assay using non-self-plasma and venom showed that this variability is due to venom protein composition instead of plasma composition. These venoms presented higher caseinolytic, collagenolytic and coagulant activities than the venoms without these regions recognized by WB. Mass spectrometry analyses performed on two individuals revealed that these individuals present, in addition to higher protein concentrations, other exclusive proteins in their composition. When these same two samples were tested in vivo, the results also showed higher lethality in these venoms, but lower hemorrhagic activity than in the venoms without these regions recognized by WB. In conclusion, some Bothrops moojeni specimens differ in venom composition, which may have implications in envenomation. Moreover, the high individual venom variability found in this species demonstrates the importance to work with individual analyses in studies involving intraspecific venom variability and venom evolution.

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