Oximic compounds as potential inhibitors of metacaspase-2 (TbMCA2) of Trypanosoma brucei.

Metacaspases are a distinct class of cysteine proteases predominantly found in plants, fungi, and protozoa, crucial for regulating programmed cell death (PCD). They possess unique structural features and differ markedly from caspases in their activation mechanisms and substrate specificities, with a notable preference for binding basic residues in substrates. In this study, we introduced vanillin-derived oximic compounds to explore their pharmaceutical potential. We evaluated these compounds for their inhibitory effects on TbMCA2, a metacaspase in Trypanosoma brucei, identifying AO-7, AO-12, and EO-20 as promising inhibitors. AO-12 showed significant potential as a non-competitive inhibitor with notable IC50 values. Molecular docking studies were also conducted to evaluate the binding affinity of these compounds for TbMCA2. This research is particularly relevant given the urgent need for more effective and less toxic treatments for trypanosomiasis, a parasitic disease caused by trypanosomes. The absence of available vaccines and the limitations imposed by drug toxicity underscore the importance of these findings. Our study represents a significant advancement in developing therapeutic agents targeting metacaspases in trypanosomatids and highlights the necessity of understanding metacaspase regulation across various species. It provides valuable insights into inhibitor sensitivity and potential species-specific therapeutic strategies. In conclusion, this research opens promising avenues for novel therapeutic agents targeting metacaspases in trypanosomatids, addressing a critical gap in combating neglected diseases associated with these pathogens. Further research is essential to refine the efficacy and safety profiles of these compounds, aiming to deliver more accessible and effective therapeutic solutions to populations afflicted by these debilitating diseases.

Biochemical characterization, stability, and kinetics of three substrates of the recombinant TMPRSS2 serine protease domain.

Transmembrane serine protease 2 (TMPRSS2) is a membrane-bound protease belonging to the type II transmembrane serine protease (TTSP) family. It is a multidomain protein, including a serine protease domain responsible for its self-activation. The protein has been implicated as an oncogenic transcription factor and for its ability to cleave (prime) the SARS-CoV-2 spike protein. In order to characterize the TMPRSS2 biochemical properties, we expressed the serine protease domain (rTMPRSS2_SP) in Komagataella phaffii using the pPICZαA vector and purified it using immobilized metal affinity (Ni Sepharose™ excel) and size exclusion (Superdex 75) chromatography. We explored operational fluorescence resonance energy transfer FRET peptides as substrates. We chose the peptide Abz-QARK-(Dnp)-NH2 (Abz = ortho-aminobenzoic acid, the fluorescence donor, and Dnp = 2,4-dinitrophenyl, the quencher group) as a substrate to find the optimal conditions for maximum enzymatic activity. We found that metallic ions such as Ca2+ and Na+ increased enzymatic activity, but ionic surfactants and reducing agents decreased catalytic capacity. Finally, we determined the rTMPRSS2_SP stability for long-term storage. Altogether, our results represent the first comprehensive characterization of TMPRSS2's biochemical properties, providing valuable insights into its serine protease domain.

Description of new species of Mycobacterium terrae complex isolated from sewage at the São Paulo zoological park foundation in Brazil.

Five mycobacterial isolates from sewage were classified as members of the genus Mycobacterium but presented inconclusive species assignments. Thus, the isolates (MYC017, MYC098, MYC101, MYC123 and MYC340) were analyzed by phenotypical, biochemical, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and genomic features to clarify their taxonomic position. Phenotypic analysis and biochemical tests did not distinguish these isolates from other non-pigmented mycobacteria. In contrast, MALDI-TOF MS analysis showed that isolates were not related to any previously described Mycobacterium species. Comparative genomic analysis showed values of ANI and dDDH between 81.59-85.56% and 24.4-28.8%, respectively, when compared to the genomes of species of this genus. In addition, two (MYC101 and MYC123) presented indistinguishable protein spectra from each other and values of ANI = 98.57% and dDDH = 97.3%, therefore being considered as belonging to the same species. Phylogenetic analysis grouped the five isolates within the Mycobacterium terrae complex (MTC) but in a specific subclade and separated from the species already described and supported by 100% bootstrap value, confirming that they are part of this complex but different from earlier described species. According to these data, we propose the description of four new species belonging to the Mycobacterium genus: (i) Mycobacterium defluvii sp. nov. strain MYC017T (= ATCC TSD-296T = JCM 35364T), (ii) Mycobacterium crassicus sp. nov. strain MYC098T (= ATCC TSD-297T = JCM 35365T), (iii) Mycobacterium zoologicum sp. nov. strain MYC101T (= ATCC TSD-298T = JCM 35366T) and MYC123 (= ATCC BAA-3216 = JCM 35367); and (iv) Mycobacterium nativiensis sp. nov. strain MYC340T (= ATCC TSD-299T = JCM 35368T).

Cannabidiol Attenuates In Vivo Leukocyte Recruitment to the Spinal Cord Microvasculature at Peak Disease of Experimental Autoimmune Encephalomyelitis.

Introduction: Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system characterized by neuroinflammation leading to demyelination. The associated symptoms lead to a devastating decrease in quality of life. The cannabinoids and their derivatives have emerged as an encouraging alternative due to their management of symptom in MS. Objective: The aim of the study was to investigate the mechanism of action of cannabidiol (CBD), a nonpsychoactive cannabinoid, on molecular and cellular events associated with leukocyte recruitment induced by experimental autoimmune encephalomyelitis (EAE). Materials and Methods: C57BL/6 female mice were randomly assigned to the four experimental groups: C (control group), CBD (cannabidiol-treated group, 5 mg/kg i.p.; 14 days), EAE (experimental autoimmune encephalomyelitis-induced group), and EAE+CBD (experimental autoimmune encephalomyelitis-induced plus cannabidiol-treated group). Results: The results indicated that 5 mg/kg of CBD injected intraperitoneally between the 1st and 14th days of EAE could reduce the leukocyte rolling and adhesion into the spinal cord microvasculature as well cellular tissue infiltration. These results were supported by a decreased mRNA expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the spinal cord. Conclusion: Purified CBD reduces in vivo VCAM and ICAM-mediated leukocyte recruitment to the spinal cord microvasculature at EAE peak disease.

Activation of estrogen receptor induces differential proteomic responses mainly involving migration, invasion, and tumor development pathways in human testicular embryonal carcinoma NT2/D1 cells.

The aims of the present study were to investigate the global changes on proteome of human testicular embryonal carcinoma NT2/D1 cells treated with 17ß-estradiol (E2), and the effects of this hormone on migration, invasion, and colony formation of these cells. A quantitative proteomic analysis identified the presence of 1230 proteins in both E2-treated and control cells. The analysis revealed 75 differentially abundant proteins (DAPs), out of which 43 proteins displayed a higher abundance and, 30 proteins showed a lower abundance in E2-treated NT2/D1 cancer cells. Functional analysis using IPA highlighted some activation processes such as migration, invasion, metastasis, and tumor growth. Interestingly, the treatment with E2 and ERß-selective agonist DPN increased the migration of NT2/D1 cells. On the other hand, ERα-selective agonist PPT did not modify cell migration, indicating that ERß is the upstream receptor involved in this process. The activation of ERß increased the invasion and anchorage­independent growth of NT2/D1 cells more intensely than ERα. ERα and ERß may play overlapping roles on invasion and colony formation of these cells. Further studies are required to clarify the mechanism underlying these effects. The molecular mechanisms revealed by proteomic and functional studies might also guide the development of potential targets for a better understanding of the biology of these cells and novel treatments for non-seminoma in the future.

Diversity and emergence of multi-resistant Staphylococcus spp. isolated from subclinical mastitis in cows in of the state of Piauí, Brazil.

This research aimed to identify the diversity of bacterial species of the genus Staphylococcus spp. in subclinical mastitis in dairy herds in the state of Piauí, Northeastern Brazil, and to evaluate the phenotypic and genotypic resistance profile. Samples were obtained from a total of 17 dairy farms, amounting to 321 positive samples in the California Mastitis Test. Staphylococcus spp. were identified by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy. Subsequently, an antibiogram was performed, and a polymerase chain reaction was carried out to screen for resistance genes in the isolates. Among all the isolates, 59.45% (110/185) belonged to the Staphylococcus genus. Moreover, the following Staphylococcus spp. were identified Staphylococcus aureus, 68.1% (75/110); Staphylococcus chromogenes, 12.7% (14/110); Staphylococcus epidermidis, 5.4% (6/110); Staphylococcus sciuri, 4.5% (5/110); Staphylococcus warneri, 2.7% (3/110); Staphylococcus haemolyticus, 1.8% (2/110); Staphylococcus hominis, 1.8% (2/110); Staphylococcus arlettae, 0.9% (1/110); Staphylococcus capitis, 0.9% (1/110); and Staphylococcus gallinarum, 0.9% (1/110). The antibiogram showed a high frequency of resistance to penicillin and ampicillin, 70.0% (77/110) and 61.8% (68/110), respectively, and a low frequency of resistance to gentamicin and vancomycin, 10.9% (12/110) and 11.8% (13/110), respectively. In the genotypic tests for the different species of Staphylococcus spp., the occurrence of the blaZ gene was observed in 60.9% (67/110) of the isolates, followed by tetL and tetM, both with 20.0% (22/110) each, and the mecA and vanB genes were detected in 0.9% (1/110) of the samples. The identification of all Staphylococcus species isolated from subclinical mastitis cases and the phenotypic and genotypic resistance characterization in these isolates is of great importance for dairy farming in the state of Piauí, as well as for public health.

Celastrol: A lead compound that inhibits SARS-CoV-2 replication, the activity of viral and human cysteine proteases, and virus-induced IL-6 secretion.

The global emergence of coronavirus disease 2019 (COVID-19) has caused substantial human casualties. Clinical manifestations of this disease vary from asymptomatic to lethal, and the symptomatic form can be associated with cytokine storm and hyperinflammation. In face of the urgent demand for effective drugs to treat COVID-19, we have searched for candidate compounds using in silico approach followed by experimental validation. Here we identified celastrol, a pentacyclic triterpene isolated from Tripterygium wilfordii Hook F, as one of the best compounds out of 39 drug candidates. Celastrol reverted the gene expression signature from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected cells and irreversibly inhibited the recombinant forms of the viral and human cysteine proteases involved in virus invasion, such as Mpro (main protease), PLpro (papain-like protease), and recombinant human cathepsin L. Celastrol suppressed SARS-CoV-2 replication in human and monkey cell lines and decreased interleukin-6 (IL-6) secretion in the SARS-CoV-2-infected human cell line. Celastrol acted in a concentration-dependent manner, with undetectable signs of cytotoxicity, and inhibited in vitro replication of the parental and SARS-CoV-2 variant. Therefore, celastrol is a promising lead compound to develop new drug candidates to face COVID-19 due to its ability to suppress SARS-CoV-2 replication and IL-6 production in infected cells.

Activation of estrogen receptor ESR1 and ESR2 induces proliferation of the human testicular embryonal carcinoma NT2/D1 cells.

The aims of the present study were to investigate the expression of the classic estrogen receptors ESR1 and ESR2, the splicing variant ESR1-36 and GPER in human testicular embryonal carcinoma NT2/D1 cells, and the effects of the activation of the ESR1 and ESR2 on cell proliferation. Immunostaining of ESR1, ESR2, and GPER were predominantly found in the nuclei, and less abundant in the cytoplasm. ESR1-36 isoform was predominantly expressed in the perinuclear region and cytoplasm, and some weakly immunostained in the nuclei. In nonstimulated NT2/D1 cells (control), proteins of the cell cycle CCND1, CCND2, CCNE1 and CDKN1B are present. Activation of ESR1 and ESR2 increases, respectively, CCND2 and CCNE1 expression, but not CCND1. Activation of ESR2 also mediates upregulation of the cell cycle inhibitor CDKN1B. This protein co-immunoprecipitated with CCND2. Also, E2 induces an increase in the number and viability of the NT2/D1 cells. These effects are blocked by simultaneous pretreatment with ESR1-and ESR2-selective antagonists, confirming that both estrogen receptors regulate NT2/D1 cell proliferation. In addition, E2 increases SRC phosphorylation, and SRC mediates cell proliferation. Our study provides novel insights into the signatures and molecular mechanisms of estrogen receptor in NT2/D1 cells.

Bovine mastitis in northeastern Brazil: Occurrence of emergent bacteria and their phenotypic and genotypic profile of antimicrobial resistance.

The aim of this study was to identify emergent pathogens associated with bovine mastitis in northeastern Brazil and to characterize them for phenotypic and genotypic resistance to antimicrobials. A total of 321 milk samples from cows with subclinical mastitis were collected, and the isolates obtained in culture were identified using matrix-associated laser desorption-ionization - time of flight mass spectrometry. Phenotypic and genotypic antimicrobial resistance tests were performed. We identified 72 bacteria considered emergent in the study region: Enterococcus faecalis (26.3%; 19/72), Streptococcus agalactiae (22.2%; 16/72), Enterococcus faecium (20.0%; 15/72), Escherichia coli (6.9%; 5/72), 6.9% (5/72) Lactococcus garvieae (6.9%; 5/72), Acinetobacter baumannii (5.5%; 4/72), Bacillus subtilis (1.3%; 1/72), Kocuria marina (1.3%; 1/72), Macrococcus caseolyticus (1.3%; 1/72), Microbacterium resistens (1.3%; 1/72), Micrococcus luteus (1.3%; 1/72), Streptococcus dysgalactiae (1.3%; 1/72), Streptococcus hyovaginalis (1.3%; 1/72) and Streptococcus pluranimalium (1.3%; 1/72). The antibiogram revealed the following resistance profiles: ampicillin (77.7%; 56/72), cefoxitin (69.4%; 50/72), erythromycin (61.1%; 44/72), oxacillin (63.8%; 46/72), penicillin (79.1%; 57/72), tetracycline (63.8%; 46/72), gentamicin (25.0%; 18/72), and vancomycin (20.8%; 15/72). Of the isolates, 83.4% (60/72) showed multiple resistance to antimicrobials. The tetM gene was identified in 43.0% (31/72) of the isolates, followed by tetL (31.9%; 23/72), and blaZ (26.3%; 19/72). 83.4% (60/72) of the isolates presented a multiple antimicrobial resistance index higher than 0,2. Emergent bacteria with zoonotic and multiresistant potential occur in cows with mastitis in northeastern Brazil. It is necessary to monitor the occurrence of these and other bacteria in livestock environments and develop control strategies to prevent their spread.

Occurrence of emerging multiresistant pathogens in the production chain of artisanal goat coalho cheese in Brazil.

Sanitary-hygienic failures in cheese making can pose health risks to consumers. This study aimed to identify multiresistant pathogens in different production stages of artisanal goat coalho cheese in Brazil and characterize their phenotypic and genotypic resistance. Eleven properties in the state of Pernambuco, Brazil, participated in the study. Samples were obtained from different stages of production and the humans involved. The samples obtained were submitted to microbiological culture, then all the isolated microorganisms were submitted to the Matrix Associated Laser Desorption-Ionization - Time of Flight technique for the microbiological identification of the species. Subsequently, Staphylococcus spp., Enterococcus spp. and Macrococcus caseolyticus were subjected to polymerase chain reaction to search for resistance genes and disc diffusion technique to evaluate the resistance profile. A total of 111 isolates were obtained and 31 species were identified, with the frequency of Staphylococcus spp. (62.20%; 69/111), Enterococcus spp. (11.60%; 13/111), Macrococcus caseolyticus (10%; 11/111), Bacillus spp. (3.60%; 4/111), Enterobacter spp. (3.60%; 4/111), Aureobasidium pullulans (1.80%; 2/111), Corynebacterium camporealensis (1.80%; 2/111), Issatchenkia occidentalis (1.80%; 2/111), Kocuria kristinae (1.80%; 2/111), Aerococcus viridans (0.90%; 1/111) and Filifactor villosus (0.90%; 1/111). Phenotypic and genotypic resistance was also detected with the occurrence of 15.90% (7/44) of the mecA gene, 4% (1/25) vanA, and 4% (1/25) vanB in Staphylococcus spp. and 20% (2/10) vanB in and Enterococcus spp. Emerging multiresistant pathogens are present in the production chain of artisanal goat cheese and humans, who exert an important role in disseminating these bacteria with imminent risks to human health.

Immunogenicity of personalized dendritic-cell therapy in HIV-1 infected individuals under suppressive antiretroviral treatment: interim analysis from a phase II clinical trial.

BACKGROUND: We developed a personalized Monocyte-Derived Dendritic-cell Therapy (MDDCT) for HIV-infected individuals on suppressive antiretroviral treatment and evaluated HIV-specific T-cell responses. METHODS: PBMCs were obtained from 10 HIV+ individuals enrolled in trial NCT02961829. Monocytes were differentiated into DCs using IFN-α and GM-CSF. After sequencing each patient's HIV-1 Gag and determining HLA profiles, autologous Gag peptides were selected based on the predicted individual immunogenicity and used to pulse MDDCs. Three doses of the MDDCT were administered every 15 days. To assess immunogenicity, patients' cells were stimulated in vitro with autologous peptides, and intracellular IL-2, TNF, and interferon-gamma (IFN-γ) production were measured in CD4+ and CD8+ T-cells. RESULTS: The protocol of ex-vivo treatment with IFN-α and GM-CSF was able to induce maturation of MDDCs, as well as to preserve their viability for reinfusion. MDDCT administration was associated with increased expression of IL-2 in CD4+ and CD8+ T-cells at 15 and/or 30 days after the first MDDCT administration. Moreover, intracellular TNF and IFN-γ expression was significantly increased in CD4+ T-cells. The number of candidates that increased in vitro the cytokine levels in CD4+ and CD8+ T cells upon stimulation with Gag peptides from baseline to day 15 and from baseline to day 30 and day 120 after MDDCT was significant as compared to Gag unstimulated response. This was accompanied by an increasing trend in the frequency of polyfunctional T-cells over time, which was visible when considering both cells expressing two and three out of the three cytokines examined. CONCLUSIONS: MDDC had a mature profile, and this MDDCT promoted in-vitro T-cell immune responses in HIV-infected patients undergoing long-term suppressive antiretroviral treatment. Trial registration NCT02961829: (Multi Interventional Study Exploring HIV-1 Residual Replication: a Step Towards HIV-1 Eradication and Sterilizing Cure, https://www.clinicaltrials.gov/ct2/show/NCT02961829 , posted November 11th, 2016).

Semysinthetic biflavonoid Morelloflavone-7,4',7″,3‴,4‴-penta-O-butanoyl is a more potent inhibitor of Proprotein Convertases Subtilisin/Kexin PC1/3 than Kex2 and Furin.

BACKGROUND: Garcinia brasiliensis is a species native to the Amazon forest. The white mucilaginous pulp is used in folk medicine as a wound healing agent and for peptic ulcer, urinary, and tumor disease treatments. The activity of the proprotein convertases (PCs) Subtilisin/Kex is associated with the development of viral, bacterial and fungal infections, osteoporosis, hyperglycemia, atherosclerosis, cardiovascular, neurodegenerative and neoplastic diseases. METHODS: Morelloflavone (BF1) and semisynthetic biflavonoid (BF2, 3 and 4) from Garcinia brasiliensis were tested as inhibitor of PCs Kex2, PC1/3 and Furin, and determined IC50, Ki, human proinflammatory cytokines secretion in Caco-2 cells, mechanism of inhibition, and performed molecular docking studies. RESULTS: Biflavonoids were more effective in the inhibition of neuroendocrine PC1/3 than mammalian Furin and fungal Kex2. BF1 presented a mixed inhibition mechanism for Kex2 and PC1, and competitive inhibition for Furin. BF4 has no good interaction with Kex2 and Furin since carboxypropyl groups results in steric hindrance to ligand-protein interactions. Carboxypropyl groups of BF4 promote steric hindrance with Kex2 and Furin, but effective in the affinity of PC1/3. BF4 was more efficient at inhibiting PCl/3 (IC50 = 1.13 µM and Ki = 0,59 µM, simple linear competitive mechanism of inhibition) than Kex2, Furin. Also, our results strongly suggested that BF4 also inhibits the endogenous cellular PC1/3 activity in Caco-2 cells, since PC1/3 inhibition by BF4 causes a large increase in IL-8 and IL-1ß secretion in Caco-2 cells. CONCLUSIONS: BF4 is a potent and selective inhibitor of PC1/3. GENERAL SIGNIFICANCE: BF4 is the best candidate for further clinical studies on inhibition of PC1/3.

Multiresistant zoonotic pathogens isolated from goat milk in Northeastern Brazil.

Dairy goats play a significant role in socio-economic, cultural, and nutritional development in many countries. This study aimed to identify multiresistant zoonotic pathogens causing mastitis in goats, in addition to characterizing them for the presence of resistance genes and phenotypic resistance. A total of 714 milk samples from 357 lactating goats in 12 farms in the Northeast region of Brazil were analyzed. The isolates were submitted to Matrix Associated Laser Desorption-Ionization - Time of Flight to identify bacterial species, Polymerase Chain Reaction (PCR) to search for resistance genes, and an antibiogram to evaluate the phenotypic profile of antimicrobial resistance. A total of 214 pathogens were identified and bacterial prevalence was 83.29 % (178/214) Staphylococcus spp.; 6.50 % (14/214) Micrococcus luteus; 3.73 % (8/214) Corynebacterium spp.; 2.80 % (6/214) Bacillus spp.; 1.38 % (3/214) Escherichia coli; 0.92 % (2/214) Enterobacter cloacae; 0.46 % (1/214) Aerococcus viridans; 0.46 % (1/214) Morganella morganii; and 0.46 % (1/214) Turicella otitidis. As for gene frequency, 64.60 % (115/178) of the isolates carried the blaZ gene; 37.07 % (66/178) norA; 22.47 % (40/178) tet(L); 16.85 % (30/178) tet(M); 14.04 % (25/178) norB; 8.42 % (15/178) vanA; 7.30 % (13/178) msrA; 6.41 % (5/178) tet-38; 4.49 % (8/178) norC; 2.25 % (4/178) mecA; and 0.56 % (1/178) vanB. Emerging multiresistant zoonotic pathogens are present in the goat milk production chain, especially the coagulase-negative Staphylococcus species that pose a risk to human and animal health.

Interfering with cholesterol metabolism impairs tick embryo development and turns eggs susceptible to bacterial colonization.

Cholesterol is a known precursor of arthropod molecules such as the hormone 20-hydroxyecdysone and the antimicrobial boophiline, a component of tick egg wax coat. Because the cholesterol biosynthetic pathway is absent in ticks, it is necessarily obtained from the blood meal, in a still poorly understood process. In contrast, dietary cholesterol absorption is better studied in insects, and many proteins are involved in its metabolism, including Niemann-Pick C (NPC) transporter and acyl-CoA:cholesterol acyltransferase (ACAT), as well as enzymes to convert between free cholesterol and esterified cholesterol. The present work addresses the hypothesis that tick viability can be impaired by interfering with cholesterol metabolism, proposing this route as a target for novel tick control methods. Two drugs, ezetimibe (NPC inhibitor) and avasimibe (ACAT inhibitor) were added to calf blood and used to artificially feed Rhipicephalus microplus females. Results show that, after ingesting avasimibe, tick reproductive ability and egg development are impaired. Also, eggs laid by females fed with avasimibe did not hatch and were susceptible to Pseudomonas aeruginosa adhesion and biofilm formation in their surfaces. The immunoprotective potential of ACAT against ticks was also accessed using two selected ACAT peptides. Antibodies against these peptides were used to artificially feed female ticks, but no deleterious effects were observed. Taken together, data presented here support the hypothesis that enzymes and other proteins involved in cholesterol metabolism are suitable as targets for tick control methods.

Copper mining bacteria: Converting toxic copper ions into a stable single-atom copper.

The chemical synthesis of monoatomic metallic copper is unfavorable and requires inert or reductive conditions and the use of toxic reagents. Here, we report the environmental extraction and conversion of CuSO4 ions into single-atom zero-valent copper (Cu0) by a copper-resistant bacterium isolated from a copper mine in Brazil. Furthermore, the biosynthetic mechanism of Cu0 production is proposed via proteomics analysis. This microbial conversion is carried out naturally under aerobic conditions eliminating toxic solvents. One of the most advanced commercially available transmission electron microscopy systems on the market (NeoArm) was used to demonstrate the abundant intracellular synthesis of single-atom zero-valent copper by this bacterium. This finding shows that microbes in acid mine drainages can naturally extract metal ions, such as copper, and transform them into a valuable commodity.

Interaction of Trypanosoma cruzi Gp82 With Host Cell LAMP2 Induces Protein Kinase C Activation and Promotes Invasion.

The surface molecule gp82 of metacyclic trypomastigote (MT) forms of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, mediates the host cell invasion, a process critical for the establishment of infection. Gp82 is known to bind to the target cell in a receptor-dependent manner, triggering Ca2+ signal, actin cytoskeleton rearrangement and lysosome spreading. The host cell receptor for gp82 was recently identified as LAMP2, the major lysosome membrane-associated protein. To further clarify the mechanisms of MT invasion, we aimed in this study at identifying the LAMP2 domain that interacts with gp82 and investigated whether target cell PKC and ERK1/2, previously suggested to be implicated in MT invasion, are activated by gp82. Interaction of MT, or the recombinant gp82 (r-gp82), with human epithelial HeLa cells induced the activation of Ca2+-dependent PKC and ERK1/2. The LAMP2 sequence predicted to bind gp82 was mapped and the synthetic peptide based on that sequence inhibited MT invasion, impaired the binding of r-gp82 to HeLa cells, and blocked the PKC and ERK1/2 activation induced by r-gp82. Treatment of HeLa cells with specific inhibitor of focal adhesion kinase resulted in inhibition of r-gp82-induced PKC and ERK1/2 activation, as well as in alteration of the actin cytoskeleton architecture. PKC activation by r-gp82 was also impaired by treatment of HeLa cells with inhibitor of phospholipase C, which mediates the production of diacylglycerol, which activates PKC, and inositol 1,4,5-triphosphate that releases Ca2+ from intracellular stores. Taken together, our results indicate that recognition of MT gp82 by LAMP2 induces in the host cell the activation of phosholipase C, with generation of products that contribute for PKC activation and the downstream ERK1/2. This chain of events leads to the actin cytoskeleton disruption and lysosome spreading, promoting MT internalization.

Contact System Activation in Plasma from Dengue Patients Might Harness Endothelial Virus Replication through the Signaling of Bradykinin Receptors.

Since exacerbated inflammation and microvascular leakage are hallmarks of dengue virus (DENV) infection, here we interrogated whether systemic activation of the contact/kallikrein-kinin system (KKS) might hamper endothelial function. In vitro assays showed that dextran sulfate, a potent contact activator, failed to generate appreciable levels of activated plasma kallikrein (PKa) in the large majority of samples from a dengue cohort (n = 70), irrespective of severity of clinical symptoms. Impaired formation of PKa in dengue-plasmas correlated with the presence of cleaved Factor XII and high molecular weight kininogen (HK), suggesting that the prothrombogenic contact system is frequently triggered during the course of infection. Using two pathogenic arboviruses, DENV or Zika virus (ZIKV), we then asked whether exogenous BK could influence the outcome of infection of human brain microvascular endothelial cells (HBMECs). Unlike the unresponsive phenotype of Zika-infected HBMECs, we found that BK, acting via B2R, vigorously stimulated DENV-2 replication by reverting nitric oxide-driven apoptosis of endothelial cells. Using the mouse model of cerebral dengue infection, we next demonstrated that B2R targeting by icatibant decreased viral load in brain tissues. In summary, our study suggests that contact/KKS activation followed by BK-induced enhancement of DENV replication in the endothelium may underlie microvascular pathology in dengue.

Prediction, mapping and validation of tick glutathione S-transferase B-cell epitopes.

In search of ways to address the increasing incidence of global acaricide resistance, tick control through vaccination is regarded as a sustainable alternative approach. Recently, a novel cocktail antigen tick-vaccine was developed based on the recombinant glutathione S-transferase (rGST) anti-sera cross-reaction to glutathione S-transferases of Rhipicephalus appendiculatus (GST-Ra), Amblyomma variegatum (GST-Av), Haemaphysalis longicornis (GST-Hl), Rhipicephalus decoloratus (GST-Rd) and Rhipicephalus microplus (GST-Rm). Therefore, the current study aimed to predict the shared B-cell epitopes within the GST sequences of these tick species. Prediction of B-cell epitopes and proteasomal cleavage sites were performed using immunoinformatics algorithms. The conserved epitopes predicted within the sequences were mapped on the homodimers of the respective tick GSTs, and the corresponding peptides were independently used for rabbit immunization experiments. Based on the dot blot assay, the immunogenicity of the peptides and their potential to be recognized by corresponding rGST anti-sera raised by rabbit immunization in a previous work were investigated. This study revealed that the predicted conserved B-cell epitopes within the five tick GST sequences were localized on the surface of the respective GST homodimers. The epitopes of GST-Ra, GST-Rd, GST-Av, and GST-Hl were also shown to contain a seven residue-long peptide sequence with no proteasomal cleavage sites, whereas proteasomal digestion of GST-Rm was predicted to yield a 4-residue fragment. Given that a few proteasomal cleavage sites were found within the conserved epitope sequences of the four GSTs, the sequences could also contain a T-cell epitope. Finally, the peptide and rGST anti-sera reacted against the corresponding peptide, confirming their immunogenicity. These data support the claim that the rGSTs, used in the previous study, contain conserved B-cell epitopes, which elucidates why the rGST anti-sera cross-reacted to non-homologous tick GSTs. Taken together, the data suggest that the B-cell epitopes predicted in this study could be useful for constituting epitope-based GST tick vaccines.

Evaluation of the milk clotting properties of an aspartic peptidase secreted by Rhizopus microsporus.

Traditionally, chymosin has been used for milk-clotting, but this naturally occurring enzyme is in short supply and its use has raised religious and ethical concerns. Because milk-clotting peptidases are a promising substitute for chymosin in cheese preparation, there is a need to find and test the specificity of these enzymes. Here, we evaluated the milk-clotting properties of an aspartic peptidase secreted by Rhizopus microsporus. The molecular mass of this enzyme was estimated at 36 kDa and Pepstatin A was determined to be an inhibitor. Optimal activity occurred at a pH of 5.5 and a temperature range of 50-60 °C, but the peptidase was stable in the pH range of 4-7 and a temperature as low as 45 °C. Proteolytic activity was significantly reduced in the presence of Cu2+ and Al3+. When enzyme substrates based on FRET were used, this peptidase exhibited the highest catalytic efficiency for Abz-KNRSSKQ-EDDnp (4,644 ± 155 mM-1.s-1), Abz-KLRSSNQ-EDDnp (3,514 ± 130 mM-1.s-1), and Abz-KLRQSKQ-EDDnp (3,068 ± 386 mM-1.s-1). This study presents a promising peptidase for use in cheese making, due to its high stability in the presence of Ca2+ and broad pH range of 4-7, in addition to its ability to efficiently clot milk.

Identification of Angiotensin I-Converting Enzyme-Inhibitory and Anticoagulant Peptides from Enzymatic Hydrolysates of Chicken Combs and Wattles.

Peptides from protein hydrolysate of a mixture of chicken combs and wattles (CCWs) were obtained through enzymatic hydrolysis, and their anticoagulant and inhibitory effects on angiotensin I-converting enzyme (ACE) were investigated. The protein hydrolysate exhibited anticoagulant capacity by the intrinsic pathway (activated partial thromboplastin time) and potent ACE-inhibitory activity. The peptides were sequenced by LC-MS to identify those with higher inhibitory potential. From the pool of sequenced peptides, the following three peptides were selected and synthesized based on their low molecular weight and the presence of amino acids with ACE-inhibitory potential at the C-terminus: peptide I (APGLPGPR), peptide II (Piro-GPPGPT), and peptide III (FPGPPGP). Peptide III (FPGPPGP) showed the highest ACE-inhibitory capacity among the peptides selected. In conclusion, a peptide (FPGPPGP) of unknown sequence was identified as having potent ACE-inhibitory capacity. This peptide originated from unconventional hydrolysates from poultry slaughter waste, including combs and wattles.