Metabolic Reprogramming of Klebsiella pneumoniae Exposed to Serum and Its Potential Implications in Host Immune System Evasion and Resistance.

The aim of this study was to identify, using proteomics, the molecular alterations caused by human serum exposure to Klebsiella pneumoniae ACH2. The analysis was performed under two different conditions, native serum from healthy donors and heat-inactivated serum (to inactivate the complement system), and at two different times, after 1 and 4 h of serum exposure. More than 1,000 bacterial proteins were identified at each time point. Enterobactin, a siderophore involved in iron uptake, and proteins involved in translation were upregulated at 1 h, while the chaperone ProQ and the glyoxylate cycle were identified after 4 h. Enzymes involved in the stress response were downregulated, and the SOD activity was validated using an enzymatic assay. In addition, an intricate metabolic adaptation was observed, with pyruvate and thiamine possibly involved in survival and virulence in the first hour of serum exposure. The addition of exogenous thiamine contributes to bacterial growth in human serum, corroborating this result. During 4 h of serum exposure, the glyoxylate cycle (GC) probably plays a central role, and the addition of exogenous succinate suppresses the GC, inducing a decrease in serum resistance. Therefore, serum exposure causes important changes in iron acquisition, the expression of virulence factors, and metabolic reprogramming, which could contribute to bacterial serum resistance.

Metarhizium anisopliae E6 secretome reveals molecular players in host specificity and toxicity linked to cattle tick infection.

Although Metarhizium anisopliae is one of the most studied fungal biocontrol agents, its infection mechanism is far from being completely understood. Using multidimensional protein identification technology (MudPIT), we evaluated the differential secretome of M. anisopliae E6 induced by the host Rhipicephalus microplus cuticle. The proteomic result showed changes in the expression of 194 proteins after exposure to host cuticle, such as proteins involved in adhesion, penetration, stress and fungal defense. Further, we performed a comparative genomic distribution of differentially expressed proteins of the M. anisopliae secretome against another arthropod pathogen, using the Beauveria bassiana ARSEF2860 protein repertory. Among 47 analyzed protein families, thirty were overexpressed in the M. anisopliae E6 predicted genome compared to B. bassiana. An in vivo toxicity assay using a Galleria mellonella model confirmed that the M. anisopliae E6 secretome was more toxic in cattle tick infections compared to other secretomes, including B. bassiana with cattle ticks and M. anisopliae E6 with the insect Dysdereus peruvianus, which our proteomic results had also suggested. These results help explain molecular aspects associated with host infection specificity due to genetic differences and gene expression control at the protein level in arthropod-pathogenic fungi.

COVID-19 impacts the expression of molecular markers associated with neuropsychiatric disorders.

Coronavirus disease 2019 (COVID-19) was initially characterized due to its impacts on the respiratory system; however, many recent studies have indicated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) significantly affects the brain. COVID-19 can cause neurological complications, probably caused by the induction of a cytokine storm, since there is no evidence of neurotropism by SARS-CoV-2. In line with this, the COVID-19 outbreak could accelerate the progression or affect the clinical outcomes of neuropsychiatric conditions. Thus, we analyzed differential gene expression datasets for clinical samples of COVID-19 patients and identified 171 genes that are associated with the pathophysiology of the following neuropsychiatric disorders: alcohol dependence, autism, bipolar disorder, depression, panic disorder, schizophrenia, and sleep disorder. Several of the genes identified are associated with causing some of these conditions (classified as elite genes). Among these elite genes, 9 were found for schizophrenia, 6 for autism, 3 for depression/major depressive disorder, and 2 for alcohol dependence. The patients with the neuropsychiatric conditions associated with the genes identified may require special attention as COVID-19 can deteriorate or accelerate neurochemical dysfunctions, thereby aggravating clinical outcomes.

SARSCOVIDB-A New Platform for the Analysis of the Molecular Impact of SARS-CoV-2 Viral Infection.

The COVID-19 pandemic caused by the new coronavirus (SARS-CoV-2) has become a global emergency issue for public health. This threat has led to an acceleration in related research and, consequently, an unprecedented volume of clinical and experimental data that include changes in gene expression resulting from infection. The SARS-CoV-2 infection database (SARSCOVIDB: https://sarscovidb.org/) was created to mitigate the difficulties related to this scenario. The SARSCOVIDB is an online platform that aims to integrate all differential gene expression data, at messenger RNA and protein levels, helping to speed up analysis and research on the molecular impact of COVID-19. The database can be searched from different experimental perspectives and presents all related information from published data, such as viral strains, hosts, methodological approaches (proteomics or transcriptomics), genes/proteins, and samples (clinical or experimental). All information was taken from 24 articles related to analyses of differential gene expression out of 5,554 COVID-19/SARS-CoV-2-related articles published so far. The database features 12,535 genes whose expression has been identified as altered due to SARS-CoV-2 infection. Thus, the SARSCOVIDB is a new resource to support the health workers and the scientific community in understanding the pathogenesis and molecular impact caused by SARS-CoV-2.

Chymase inhibition: A key factor in the anti-inflammatory activity of ethanolic extracts and spilanthol isolated from Acmella oleracea.

ETHNOPHARMACOLOGICAL RELEVANCE: Acmella oleracea (L.) R. K. Jansen (Asteraceae), known as jambú in Brazil, is used in traditional medicine as analgesic and for inflammatory conditions, characterized by the presence of N-alkylamides, mainly spilanthol. This bioactive compound is responsible for the above-described pharmacological properties, including sialagogue and anesthetic. AIM OF THE STUDY: This study aimed to characterize the anti-inflammatory effects of A. oleracea leaves (AOEE-L) and flowers (AOEE-F) extracts, including an isolated alkylamide (spilanthol), using in vitro and in vivo models. The mechanism underlying this effect was also investigated. MATERIALS AND METHODS: Extracts were analyzed by HPLC-ESI-MS/MS in order to characterize the N-alkylamides content. AOEE-L, AOEE-F (25-100 µg/mL) and spilanthol (50-200 µM) were tested in vitro on VSMC after stimulation with hyperglycemic medium (25 mM glucose). Their effects over nitric oxide (NO) generation, chymase inhibition and expression, catalase (CAT), superoxide anion (SOD) radical activity were evaluated. After an acute administration of extracts (10-100 mg/mL) and spilanthol (6.2 mg/mL), the anti-inflammatory effects were evaluated by applying the formalin test in rats. Blood was collected to measure serum aminotransferases activities, NO activity, creatinine and urea. RESULTS: A number of distinct N-alkylamides were detected and quantified in AOEE-L and AOEE-F. Spilanthol was identified in both extracts and selected for experimental tests. Hyperglycemic stimulation in VSMC promoted the expression of inflammatory parameters, including chymase, NO, CAT and SOD activity and chymase expression, all of them attenuated by the presence of the extracts and spilanthol. The administration of extracts or spilanthol significantly inhibited edema formation, NO production and cell tissue infiltration in the formalin test, without causing kidney and liver toxicity. CONCLUSION: Taken together, these results provide evidence for the anti-inflammatory activity of leaves and flowers extracts of jambú associated distinctly with their chemical profile. The effects appear to be associated with the inhibition of chymase activity, suppression of the proinflammatory cytokine NO and antioxidant activities.

Ready for the journey: a comparative proteome profiling of porcine cauda epididymal fluid and spermatozoa.

In the present study, we describe the proteome of porcine cauda epididymis fluid and spermatozoa by means of Multidimensional Protein Identification Technology (MudPIT). Ten sexually mature healthy boars were surgically castrated and epididymides were dissected to obtain the cauda epididymal content. Polled protein extracts of cauda epididymal fluid (CEF) and spermatozoa (CESperm) were loaded in an Agilent 1100 quaternary HPLC and peptides eluted from the microcapillary column were electro-sprayed directly into a LTQ Orbitrap XL mass spectrometer. Using bioinformatics, identified proteins were classified by their molecular functions, involvement in biological processes and participation in relevant metabolic pathways associated with spermatozoa physiology, fertility potential and protection. A total of 645 proteins were identified in the CEF, with epididymal-specific lipocalin-5, beta-hexosaminidase subunit beta precursor and phosphatidylethanolamine-binding protein 4 being the most abundant proteins found. A total of 2886 proteins were identified in the CESperm proteome with 81 proteins being considered more abundant (spectral counts > 100). CEF and CESperm data were compared and 345 proteins were present in both proteomes. Phosphatidylethanolamine-binding protein 4 precursor was the only protein found most abundant in both CEF and CESperm proteomes. Based on Gene Ontology analysis, we identified CEF and CESperm proteins associated with sperm protection against ROS and immune mediated response, glycosaminoglycan degradation, ubiquitin-proteasome system, metabolic process and maturation, modulation of acrosome reaction and ZP binding and oocyte penetration. These results provide a better comprehension about the molecular process and biological pathways involved in sperm epididymis maturation and establishment of the cauda epididymis sperm reservoir.

Renal and vascular effects of kallikrein inhibition in a model of Lonomia obliqua venom-induced acute kidney injury.

BACKGROUND: Lonomia obliqua venom is nephrotoxic and acute kidney injury (AKI) is the main cause of death among envenomed victims. Mechanism underlying L. obliqua-induced AKI involves renal hypoperfusion, inflammation, tubular necrosis and loss of glomerular filtration and tubular reabsorption capacities. In the present study, we aimed to investigate the contribution of kallikrein to the hemodynamic instability, inflammation and consequent renal and vascular impairment. METHODOLOGY/PRINCIPAL FINDINGS: Addition of L. obliqua venom to purified prekallikrein and human plasma in vitro or to vascular smooth muscle cells (VSMC) in culture, was able to generate kallikrein in a dose-dependent manner. Injected in rats, the venom induced AKI and increased kallikrein levels in plasma and kidney. Kallikrein inhibition by aprotinin prevented glomerular injury and the decrease in glomerular filtration rate, restoring fluid and electrolyte homeostasis. The mechanism underlying these effects was associated to lowering renal inflammation, with decrease in pro-inflammatory cytokines and matrix metalloproteinase expression, reduced tubular degeneration, and protection against oxidative stress. Supporting the key role of kallikrein, we demonstrated that aprotinin inhibited effects directly associated with vascular injury, such as the generation of intracellular reactive oxygen species (ROS) and migration of VSMC induced by L. obliqua venom or by diluted plasma obtained from envenomed rats. In addition, kallikrein inhibition also ameliorated venom-induced blood incoagulability and decreased kidney tissue factor expression. CONCLUSIONS/SIGNIFICANCE: These data indicated that kallikrein and consequently kinin release have a key role in kidney injury and vascular remodeling. Thus, blocking kallikrein may be a therapeutic alternative to control the progression of venom-induced AKI and vascular disturbances.

Comparative proteomic analysis of foodborne Salmonella Enteritidis SE86 subjected to cold plasma treatment.

The increasing demand for high quality and safe food led to important technological innovations in food processing. Cold plasma appears as an emerging technology that has demonstrated efficiency in the removal of microbial contamination from fresh and minimally processed food. In this study, the proteomic profile of Salmonella Enteritidis SE86 subjected to cold plasma treatment was investigated. The number of viable S. Enteritidis SE86 cells was analyzed at different time intervals upon exposure to cold plasma and approximately 100 µg of S. Enteritidis SE86 protein extracts were analyzed by Multidimensional Protein Identification Technology (MudPIT). The results demonstrated that no significant changes in cell counts were detected for up to 20 min exposure to cold plasma, and 2 log reduction was achieved after 60 min. Overall, 1096 proteins were identified, with 249 detected only in plasma-treated samples, and 9 exclusive in non-treated control samples. The proteins uniquely detected in cold plasma-treated cells that showed higher abundance were glyoxalase I, ABC transporter substrate-binding protein and transcriptional activator OsmE, followed by some oxidoreductases. Proteins related with carbohydrate and nucleotide metabolism were mostly overexpressed in cold plasma treated cells, suggesting energy metabolism was increased.

A potential source for cellulolytic enzyme discovery and environmental aspects revealed through metagenomics of Brazilian mangroves.

The mangroves are among the most productive and biologically important environments. The possible presence of cellulolytic enzymes and microorganisms useful for biomass degradation as well as taxonomic and functional aspects of two Brazilian mangroves were evaluated using cultivation and metagenomic approaches. From a total of 296 microorganisms with visual differences in colony morphology and growth (including bacteria, yeast and filamentous fungus), 179 (60.5%) and 117 (39.5%) were isolated from the Rio de Janeiro (RJ) and Bahia (BA) samples, respectively. RJ metagenome showed the higher number of microbial isolates, which is consistent with its most conserved state and higher diversity. The metagenomic sequencing data showed similar predominant bacterial phyla in the BA and RJ mangroves with an abundance of Proteobacteria (57.8% and 44.6%), Firmicutes (11% and 12.3%) and Actinobacteria (8.4% and 7.5%). A higher number of enzymes involved in the degradation of polycyclic aromatic compounds were found in the BA mangrove. Specific sequences involved in the cellulolytic degradation, belonging to cellulases, hemicellulases, carbohydrate binding domains, dockerins and cohesins were identified, and it was possible to isolate cultivable fungi and bacteria related to biomass decomposition and with potential applications for the production of biofuels. These results showed that the mangroves possess all fundamental molecular tools required for building the cellulosome, which is required for the efficient degradation of cellulose material and sugar release.

Acute Lonomia obliqua caterpillar envenomation-induced physiopathological alterations in rats: evidence of new toxic venom activities and the efficacy of serum therapy to counteract systemic tissue damage.

The clinical manifestations of Lonomia obliqua caterpillar envenomation are systemic hemorrhage and acute kidney injury. In an effort to better understand the physiopathological mechanisms of envenomation, a rat model was established to study systemic tissue damage during L. obliqua envenomation. An array of acute venom effects was characterized, including biochemical, hematological, histopathological, myotoxic and genotoxic alterations. Rapid increases in serum alanine and aspartate transaminases, γ-glutamyl transferase, lactate dehydrogenase, hemoglobin, bilirubin, creatinine, urea and uric acid were observed, indicating that intravascular hemolysis and liver and kidney damage had occurred. Treatment with a specific antivenom (antilonomic serum) for up to 2 h post-venom injection neutralized the biochemical alterations. However, treatment after 6 h post-venom injection failed to normalize all biochemical parameters, despite its efficacy in reversing coagulation dysfunction. The hematological findings were consistent with hemolytic anemia and neutrophilic leukocytosis. The histopathological alterations were mainly related to hemorrhage and inflammation in the subcutaneous tissue, lung, heart and kidneys. Signs of congestion and hemosiderosis were evident in the spleen, and hemoglobin and/or myoglobin casts were also detected in the renal tubules. Increased levels of creatine kinase and creatine kinase-MB were correlated with the myocardial necrosis observed in vivo and confirmed the myotoxicity detected in vitro in isolated extensor digitorum longus muscles. Significant DNA damage was observed in the kidneys, heart, lung, liver and lymphocytes. The majority of the DNA lesions in the kidney were due to oxidative damage. The results presented here will aid in understanding the pathology underlying Lonomia's envenomation.

Properties of a non collagen-degrading Bacillus subtilis keratinase.

Bacillus subtilis S14 produces a keratinase (KerS14) with non collagen-degrading activity. Indeed, this is the first keratinase described so far that does not have any detectable effect on collagen, which is a crucial property for an enzyme intended to be used in skin dehairing. Because of its importance as an industrial tanning enzyme, we report the biochemical characterization of KerS14. This protein exhibited an apparent molecular mass of 27 kDa, a pI of 6.5, and an optimum pH in the range of 8.0-9.0. The enzyme's activity was stimulated by Mn2+ (7.7-fold), Ca2+ (6.1-fold), Mg2+ (4.9-fold), and Co2+ (4.0-fold) but was inhibited by Cu2+ and Zn2+. Using p-nitroanilide and methylcoumarine derivatized peptides, we observed that KerS14 prefered Arg at subsite P1, small amino acid residues at subsite P2, and Gln or Glu at subsite P3. KerS14 presented higher keratin degradation specificity than other commercial proteases. Its high keratinolytic activity and the absence of virtually any activity against collagen remark the biotechnological potential of this enzyme to be used at larger scales in tannery dehairing processes.