Understanding COVID-19 progression with longitudinal peripheral blood mononuclear cell proteomics: Changes in the cellular proteome over time.

The clinical presentation of COVID-19 is highly variable, and understanding the underlying biological processes is crucial. This study utilized a proteomic analysis to investigate dysregulated processes in the peripheral blood mononuclear cells of patients with COVID-19 compared to healthy volunteers. Samples were collected at different stages of the disease, including hospital admission, after 7 days of hospitalization, and 30 days after discharge. Metabolic pathway alterations and increased abundance of neutrophil-related proteins were observed in patients. Patients progressing to critical illness had significantly low-abundance proteins in the pentose phosphate and glycolysis pathways compared with those presenting clinical recovery. Important biological processes, such as fatty acid concentration and glucose metabolism disorder, remained altered even after 30 days of hospital discharge. Temporal proteomic changes revealed distinct pathways in critically ill and non-critically ill patients. Our study emphasizes the significance of longitudinal cellular proteomic studies in identifying disease progression-related pathways and persistent protein changes post-hospitalization.

Glucose metabolism is upregulated in the mononuclear cell proteome during sepsis and supports endotoxin-tolerant cell function.

Metabolic adaptations shape immune cell function. In the acute response, a metabolic switch towards glycolysis is necessary for mounting a proinflammatory response. During the clinical course of sepsis, both suppression and activation of immune responses take place simultaneously. Leukocytes from septic patients present inhibition of cytokine production while other functions such as phagocytosis and production of reactive oxygen species (ROS) are preserved, similarly to the in vitro endotoxin tolerance model, where a first stimulation with lipopolysaccharide (LPS) affects the response to a second stimulus. Here, we sought to investigate how cellular metabolism is related to the modulation of immune responses in sepsis and endotoxin tolerance. Proteomic analysis in peripheral blood mononuclear cells (PBMCs) from septic patients obtained at intensive care unit admission showed an upregulation of proteins related to glycolysis, the pentose phosphate pathway (PPP), production of ROS and nitric oxide, and downregulation of proteins in the tricarboxylic acid cycle and oxidative phosphorylation compared to healthy volunteers. Using the endotoxin-tolerance model in PBMCs from healthy subjects, we observed increased lactate production in control cells upon LPS stimulation, while endotoxin-tolerant cells presented inhibited tumor necrosis factor-α and lactate production along with preserved phagocytic capacity. Inhibition of glycolysis and PPP led to impairment of phagocytosis and cytokine production both in control and in endotoxin-tolerant cells. These data indicate that glucose metabolism supports leukocyte functions even in a condition of endotoxin tolerance.

Combined Transcriptome and Proteome Leukocyte's Profiling Reveals Up-Regulated Module of Genes/Proteins Related to Low Density Neutrophils and Impaired Transcription and Translation Processes in Clinical Sepsis.

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.

HIF-1α and Hypoxia Responsive Genes are Differentially Expressed in Leukocytes From Survivors and Non-Survivors Patients During Clinical Sepsis.

ABSTRACT: Hypoxia inducible factor 1 alpha (HIF-1α) is linked to the metabolic and immune alterations in septic patients. Stabilization of HIF-1α by hypoxia or inflammation promotes the expression of several genes related to glycolytic metabolism, angiogenesis, coagulation, cell proliferation, and apoptosis. Here, we analyzed public available blood transcriptome datasets from septic patients and evaluated by PCR array the expression of HIF-1α and other hypoxia responsive genes in peripheral blood mononuclear cells from patients with sepsis secondary to community acquired infections. Samples were collected at intensive care unit admission (D0, n=29) and after 7 days follow-up (D7, n = 18); healthy volunteers (n = 10) were included as controls. Hypoxia and glycolysis were among the top scored molecular signatures in the transcriptome datasets. PCR array showed that 24 out of 78 analyzed genes were modulated in septic patients compared with healthy volunteers; most of them (23/24) were downregulated at admission. This same pattern was observed in surviving patients, while non-survivors presented more upregulated genes. EGLN1, EGLN2, and HIF1AN, inhibitors of HIF-1α activation were downregulated in patients, regardless of the outcome, while HIF-1α and other target genes, such as PDK1 and HMOX1, expression were higher in non-survivors than in survivors, mainly at D7. Non-survivor patients also presented a higher SOFA score and lower PaO2/FiO2 ratio. Our results indicate a differential modulation of hypoxia pathway in leukocytes between septic patients who survived and those who did not survive with an increased intensity at D7, which is possibly influenced by disease severity and may affect the immune response in sepsis.

Utilization of increasing levels of cassava root meal in the diet of broiler / Utilização de níveis crescentes de farelo de raiz de mandioca na dieta de frangos de corte

The objective of this study was to evaluate the effect of using increasing levels of cassava root meal in the diet of broilers, estimated by the performance and yield of carcasses, cuts, and viscera over, in the period 10 to 42 days of life. A completely randomized design was carried out, with four treatments and four repetitions. The experimental unit consisted of 16 male and female Cobb 500 chickens (a total of 256 birds) that were 10 to 42 days old. The treatments consisted of four diets with 0%, 7.5%, 15%, and 22.5% of cassava root meal (CRM) in three feeding programs. At 42 days old, four birds per unit were slaughtered and the carcass yield, main cuts, and edible viscera were evaluated. The inclusion of levels of 15% and 22.5% of CRM in the diet of broiler had a negative influence on weight gain, food consumption, and feed conversion in the initial period and in the overall growth of the birds. Carcass yield, cuts, and viscera were not influenced by the diet. Based on the results, it is recommended that a maximum of 7.5% of the cassava root bran may be used in the diet of broilers of the Cobb 500 strain, over 10 to 42 days, without compromising the zootechnical performance of the birds or the yield of edible carcasses, cuts, and viscera.

Objetivou-se avaliar o efeito da utilização de níveis crescentes do farelo de raiz de mandioca (FRM) na ração de frangos de corte sob o desempenho e o rendimento de carcaças, cortes e vísceras, no período 10 a 42 dias de vida. Foi realizado um delineamento inteiramente ao acaso, com quatro tratamentos e quatro repetições. A unidade experimental foi representada por 16 frangos machos e fêmeas da linhagem Cobb 500. Os tratamentos consistiram em quatro rações com 0%, 7,5%, 15% e 22,5% de farelo de raiz de mandioca em três programas de alimentação. Aos 42 dias, quatro aves por unidade foram abatidas e avaliados o rendimento de carcaça, principais cortes e vísceras comestíveis. A inclusão dos níveis de 15% e 22,5% de FRM na dieta dos frangos influenciou negativamente no ganho de peso, no consumo e na conversão alimentar no período inicial e de crescimento das aves. O rendimento de carcaça, cortes e vísceras não foram influenciados pela dieta. Com base nos resultados obtidos, recomenda-se o uso de até 7,5% do farelo de raiz de mandioca na dieta de frangos de corte da linhagem Cobb 500, no período de 10 a 42 dias, sem comprometer o desempenho zootécnico das aves e o rendimento de carcaças, dos cortes e vísceras comestíveis.

Utilização de níveis crescentes de farelo de raiz de mandioca na dieta de frangos de corte / Utilization of increasing levels of cassava root meal in the diet of broiler

Objetivou-se avaliar o efeito da utilização de níveis crescentes do farelo de raiz de mandioca (FRM) na ração de frangos de corte sob o desempenho e o rendimento de carcaças, cortes e vísceras, no período 10 a 42 dias de vida. Foi realizado um delineamento inteiramente ao acaso, com quatro tratamentos e quatro repetições. A unidade experimental foi representada por 16 frangos machos e fêmeas da linhagem Cobb 500. Os tratamentos consistiram em quatro rações com 0%, 7,5%, 15% e 22,5% de farelo de raiz de mandioca em três programas de alimentação. Aos 42 dias, quatro aves por unidade foram abatidas e avaliados o rendimento de carcaça, principais cortes e vísceras comestíveis. A inclusão dos níveis de 15% e 22,5% de FRM na dieta dos frangos influenciou negativamente no ganho de peso, no consumo e na conversão alimentar no período inicial e de crescimento das aves. O rendimento de carcaça, cortes e vísceras não foram influenciados pela dieta. Com base nos resultados obtidos, recomenda-se o uso de até 7,5% do farelo de raiz de mandioca na dieta de frangos de corte da linhagem Cobb 500, no período de 10 a 42 dias, sem comprometer o desempenho zootécnico das aves e o rendimento de carcaças, dos cortes e vísceras comestíveis.

The objective of this study was to evaluate the effect of using increasing levels of cassava root meal in the diet of broilers, estimated by the performance and yield of carcasses, cuts, and viscera over, in the period 10 to 42 days of life. A completely randomized design was carried out, with four treatments and four repetitions. The experimental unit consisted of 16 male and female Cobb 500 chickens (a total of 256 birds) that were 10 to 42 days old. The treatments consisted of four diets with 0%, 7.5%, 15%, and 22.5% of cassava root meal (CRM) in three feeding programs. At 42 days old, four birds per unit were slaughtered and the carcass yield, main cuts, and edible viscera were evaluated. The inclusion of levels of 15% and 22.5% of CRM in the diet of broiler had a negative influence on weight gain, food consumption, and feed conversion in the initial period and in the overall growth of the birds. Carcass yield, cuts, and viscera were not influenced by the diet. Based on the results, it is recommended that a maximum of 7.5% of the cassava root bran may be used in the diet of broilers of the Cobb 500 strain, over 10 to 42 days, without compromising the zootechnical performance of the birds or the yield of edible carcasses, cuts, and viscera.

Trypanosoma cruzi extracellular amastigotes selectively trigger the PI3K/Akt and Erk pathways during HeLa cell invasion.

Cell invasion by Trypanosoma cruzi extracellular amastigotes involves different signaling pathways to induce phagocytosis-like mechanisms. Previous works indicated that PI3K/Akt, Src and Erk might be involved in EA invasion; however, participation of these molecules in this process remains elusive. Here, we observed that EA activated Akt, Erk but not Src. Interference of EA invasion with specific inhibitors corroborated this observation. Our results show that EA is capable of selectively triggering complex signaling pathways. Activation of PI3K/Akt and Erk, kinases related to actin cytoskeleton rearrangement and phagocytosis, reinforces the idea that T. cruzi EA subverts the phagocytic machinery during invasion.

Trypanosoma cruzi: single cell live imaging inside infected tissues.

Although imaging the live Trypanosoma cruzi parasite is a routine technique in most laboratories, identification of the parasite in infected tissues and organs has been hindered by their intrinsic opaque nature. We describe a simple method for in vivo observation of live single-cell Trypanosoma cruzi parasites inside mammalian host tissues. BALB/c or C57BL/6 mice infected with DsRed-CL or GFP-G trypomastigotes had their organs removed and sectioned with surgical blades. Ex vivo organ sections were observed under confocal microscopy. For the first time, this procedure enabled imaging of individual amastigotes, intermediate forms and motile trypomastigotes within infected tissues of mammalian hosts.