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2.
Mater Sci Eng C Mater Biol Appl ; 128: 112316, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34474867

RESUMO

To develop a nanoparticle-based vaccine against necrotic enteritis, a chimeric antigen (rNA) consisting of the main antigens of Clostridium perfringens, NetB, and Alpha toxin, was prepared. Then, the rNA molecules were loaded onto the functionalized mesoporous silica nanoparticles (MSNPs) using physical adsorption or covalent conjugation methods. The characterization of synthesized nanoparticles was performed by scanning electron microscopy, dynamic light scattering, zeta potential measurement, Fourier transform infrared spectroscopy, and thermogravimetry techniques. The results revealed that the spherical nanoparticles with an average diameter of 90 ±â€¯12 nm and suitable surface chemistries are prepared. MSNPs-based formulations did not show any significant toxicity on the chicken embryo fibroblast cells. The results of the challenge experiments using subcutaneous or oral administration of the as-prepared formulations in the animal model showed that the as-prepared nanosystems, similar to those formulated with a commercial adjuvant (Montanide), present stronger humoral immune responses as compared to that of the free proteins. It was also indicated that the best protection is obtained in groups vaccinated with MSNPs-based nanovaccine, especially those who orally received covalently conjugated nanovaccine candidates. These results recommend that the MSNPs-based formulated chimeric proteinous vaccine candidates can be considered as an effective immunizing system for the oral vaccination of poultry against gastrointestinal infectious diseases.


Assuntos
Toxinas Bacterianas , Infecções por Clostridium , Enterite , Nanopartículas , Doenças das Aves Domésticas , Vacinas , Animais , Anticorpos Antibacterianos , Embrião de Galinha , Galinhas , Infecções por Clostridium/prevenção & controle , Infecções por Clostridium/veterinária , Enterite/prevenção & controle , Enterite/veterinária , Doenças das Aves Domésticas/prevenção & controle , Dióxido de Silício
3.
Int J Mol Sci ; 22(17)2021 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-34502057

RESUMO

Cereulide is one of the main food-borne toxins for vomiting synthesized by Bacillus cereus, and it widely contaminates meat, eggs, milk, and starchy foods. However, the toxicological effects and mechanisms of the long-time exposure of cereulide in vivo remain unknown. In this study, oral administration of 50 and 200 µg/kg body weight cereulide in the mice for 28 days caused oxidative stress in liver and kidney tissues and induce abnormal expression of inflammatory factors. In pathogenesis, cereulide exposure activated endoplasmic reticulum stress (ER stress) via the pathways of inositol-requiring enzyme 1α (IRE1α)/Xbox binding protein (XBP1) and PRKR-like ER kinase (PERK)/eukaryotic translation initiation factor 2α (eIF2α), and consequently led to the apoptosis and tissue damages in mouse liver and kidney. In vitro, we confirmed that the accumulation of reactive oxygen species (ROS) caused by cereulide is the main factor leading to ER stress in HepaRG and HEK293T cells. Supplementation of sodium butyrate (NaB) inhibited the activations of IRE1α/XBP1 and PERK/eIF2α pathways caused by cereulide exposure in mice, and reduced the cell apoptosis in liver and kidney. In conclusion, this study provides a new insight in understanding the toxicological mechanism and prevention of cereulide exposure.


Assuntos
Toxinas Bacterianas/toxicidade , Depsipeptídeos/toxicidade , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Animais , Apoptose , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático , Células HEK293 , Humanos , Rim/metabolismo , Fígado/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína 1 de Ligação a X-Box/metabolismo , eIF-2 Quinase/metabolismo
5.
FASEB J ; 35(10): e21875, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34533845

RESUMO

Signal inhibitory receptor on leukocytes-1 (SIRL-1) is a negative regulator of myeloid cell function and dampens antimicrobial responses. We here show that different species of the genus Staphylococcus secrete SIRL-1-engaging factors. By screening a library of single-gene transposon mutants in Staphylococcus aureus, we identified these factors as phenol-soluble modulins (PSMs). PSMs are amphipathic α-helical peptides involved in multiple aspects of staphylococcal virulence and physiology. They are cytotoxic and activate the chemotactic formyl peptide receptor 2 (FPR2) on immune cells. Human cathelicidin LL-37 is also an amphipathic α-helical peptide with antimicrobial and chemotactic activities, structurally and functionally similar to α-type PSMs. We demonstrate that α-type PSMs from multiple staphylococcal species as well as human cathelicidin LL-37 activate SIRL-1, suggesting that SIRL-1 recognizes α-helical peptides with an amphipathic arrangement of hydrophobicity, although we were not able to show direct binding to SIRL-1. Upon rational peptide design, we identified artificial peptides in which the capacity to ligate SIRL-1 is segregated from cytotoxic and FPR2-activating properties, allowing specific engagement of SIRL-1. In conclusion, we propose staphylococcal PSMs and human LL-37 as a potential new class of natural ligands for SIRL-1.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Toxinas Bacterianas/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/metabolismo , Sirtuína 1/metabolismo , Staphylococcus aureus/metabolismo , Humanos , Percepção de Quorum
6.
Int J Mol Sci ; 22(18)2021 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-34576292

RESUMO

The cytotoxic necrotizing factor 1 (CNF1) toxin from uropathogenic Escherichia coli constitutively activates Rho GTPases by catalyzing the deamidation of a critical glutamine residue located in the switch II (SWII). In crystallographic structures of the CNF1 catalytic domain (CNF1CD), surface-exposed P768 and P968 peptidyl-prolyl imide bonds (X-Pro) adopt an unusual cis conformation. Here, we show that mutation of each proline residue into glycine abrogates CNF1CD in vitro deamidase activity, while mutant forms of CNF1 remain functional on RhoA in cells. Using molecular dynamics simulations coupled to protein-peptide docking, we highlight the long-distance impact of peptidyl-prolyl cis-trans isomerization on the network of interactions between the loops bordering the entrance of the catalytic cleft. The energetically favorable isomerization of P768 compared with P968, induces an enlargement of loop L1 that fosters the invasion of CNF1CD catalytic cleft by a peptide encompassing SWII of RhoA. The connection of the P968 cis isomer to the catalytic cysteine C866 via a ladder of stacking interactions is alleviated along the cis-trans isomerization. Finally, the cis-trans conversion of P768 favors a switch of the thiol side chain of C866 from a resting to an active orientation. The long-distance impact of peptidyl-prolyl cis-trans isomerizations is expected to have implications for target modification.


Assuntos
Toxinas Bacterianas/química , Domínio Catalítico , Proteínas de Escherichia coli/química , Simulação de Dinâmica Molecular , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Isomerismo , Simulação de Acoplamento Molecular , Ligação Proteica , Proteína rhoA de Ligação ao GTP/química , Proteína rhoA de Ligação ao GTP/metabolismo
7.
Clin Drug Investig ; 41(9): 829-834, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34383256

RESUMO

Moxetumomab pasudotox (Lumoxiti®), an anti-CD22 recombinant immunotoxin, is an important treatment option that is approved in adults with relapsed or refractory hairy cell leukaemia (HCL) who have received at least two prior lines of treatment with systemic therapies including purine nucleoside analogues. In a pivotal phase III trial, treatment with moxetumomab pasudotox resulted in approximately one third of patients achieving durable complete response lasting more than 6 months, as well as improvements in other haematological parameters and disease-related symptoms. Moxetumomab pasudotox had a generally manageable tolerability profile; the most common treatment-related adverse events (AEs) included nausea, peripheral oedema, headache and pyrexia. AEs of special interest (including haemolytic uraemic syndrome and capillary leak syndrome) were generally manageable and reversible with monitoring and supportive care.


Assuntos
Antineoplásicos , Toxinas Bacterianas , Leucemia de Células Pilosas , Antineoplásicos/uso terapêutico , Toxinas Bacterianas/uso terapêutico , Exotoxinas/uso terapêutico , Humanos , Leucemia de Células Pilosas/diagnóstico , Leucemia de Células Pilosas/tratamento farmacológico
8.
Nat Microbiol ; 6(9): 1199-1210, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34413503

RESUMO

The Type VI secretion system (T6SS) is a bacterial nanomachine that delivers toxic effectors to kill competitors or subvert some of their key functions. Here, we use transposon directed insertion-site sequencing to identify T6SS toxins associated with the H1-T6SS, one of the three T6SS machines found in Pseudomonas aeruginosa. This approach identified several putative toxin-immunity pairs, including Tse8-Tsi8. Full characterization of this protein pair demonstrated that Tse8 is delivered by the VgrG1a spike complex into prey cells where it targets the transamidosome, a multiprotein complex involved in protein synthesis in bacteria that lack either one, or both, of the asparagine and glutamine transfer RNA synthases. Biochemical characterization of the interactions between Tse8 and the transamidosome components GatA, GatB and GatC suggests that the presence of Tse8 alters the fine-tuned stoichiometry of the transamidosome complex, and in vivo assays demonstrate that Tse8 limits the ability of prey cells to synthesize proteins. These data expand the range of cellular components targeted by the T6SS by identifying a T6SS toxin affecting protein synthesis and validate the use of a transposon directed insertion site sequencing-based global genomics approach to expand the repertoire of T6SS toxins in T6SS-encoding bacteria.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Complexos Multiproteicos/metabolismo , Biossíntese de Proteínas , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Sistemas de Secreção Tipo VI/metabolismo , Proteínas de Bactérias/genética , Complexos Multiproteicos/genética , Ligação Proteica , Sistemas de Secreção Tipo VI/genética
9.
Molecules ; 26(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34361757

RESUMO

Detection of relevant contaminants using screening approaches is a key issue to ensure food safety and respect for the regulatory limits established. Electrochemical sensors present several advantages such as rapidity; ease of use; possibility of on-site analysis and low cost. The lack of selectivity for electrochemical sensors working in complex samples as food may be overcome by coupling them with molecularly imprinted polymers (MIPs). MIPs are synthetic materials that mimic biological receptors and are produced by the polymerization of functional monomers in presence of a target analyte. This paper critically reviews and discusses the recent progress in MIP-based electrochemical sensors for food safety. A brief introduction on MIPs and electrochemical sensors is given; followed by a discussion of the recent achievements for various MIPs-based electrochemical sensors for food contaminants analysis. Both electropolymerization and chemical synthesis of MIP-based electrochemical sensing are discussed as well as the relevant applications of MIPs used in sample preparation and then coupled to electrochemical analysis. Future perspectives and challenges have been eventually given.


Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Análise de Alimentos/métodos , Contaminação de Alimentos/análise , Impressão Molecular/métodos , Polímeros Molecularmente Impressos/síntese química , Animais , Toxinas Bacterianas/análise , Análise de Alimentos/instrumentação , Inocuidade dos Alimentos/métodos , Humanos , Micotoxinas/análise , Praguicidas/análise , Polimerização , Extração em Fase Sólida/métodos , Drogas Veterinárias/análise
10.
FASEB J ; 35(9): e21742, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34403506

RESUMO

Withdrawal from contact inhibition is necessary for epithelial cancer precursor cells to initiate cell growth and motility. Nevertheless, little is understood about the mechanism for the sudden initiation of cell growth under static conditions. We focused on cellular junctions as one region where breaking out of contact inhibition occurs. In well-differentiated endometrial cancer cells, Sawano, the ligand administration for tricellular tight junction protein LSR, which transiently decreased the robust junction property, caused an abrupt increase in cell motility and consequent excessive multilayered cell growth despite being under contact inhibition conditions. We observed that macropinocytosis essentially and temporarily occurred as an antecedent event for the above process at intercellular junctions without disruption of the junction apparatus but not at the apical plasma membrane. Collectively, we concluded that the formation of macropinocytosis, which is derived from tight junction-mediated signaling, was triggered for the initiation of cell growth in static precancerous epithelium.


Assuntos
Adesão Celular , Inibição de Contato , Pinocitose , Receptores de Lipoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Toxinas Bacterianas/farmacologia , Sítios de Ligação , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Humanos , Junções Intercelulares/efeitos dos fármacos , Junções Intercelulares/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fenótipo , Pinocitose/efeitos dos fármacos , Transporte Proteico , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo
11.
Front Immunol ; 12: 642373, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34413846

RESUMO

Bacterial products are able to act on nociceptive neurons during pathogenic infection. Neurogenic inflammation is an active part of pain signaling and has recently been shown to impact host-pathogen defense. Bacillus anthracis Edema Toxin (ET) produces striking edema in peripheral tissues, but the cellular mechanisms involved in tissue swelling are not completely understood. Here, we find that nociceptive neurons play a role in ET-induced edema and inflammation in mice. Subcutaneous footpad infection of B. anthracis Sterne caused ET-dependent local mechanical allodynia, paw swelling and body weight gain. Subcutaneous administration of ET induced paw swelling and vascular leakage, the early phases of which were attenuated in the absence of Trpv1+ or Nav1.8+ nociceptive neurons. Nociceptive neurons express the anthrax toxin receptor ANTXR2, but this did not mediate ET-induced edema. ET induced local cytokine expression and neutrophil recruitment, which were dependent in part on Trpv1+ nociceptive neurons. Ablation of Trpv1+ or Nav1.8+ nociceptive neurons also attenuated early increases in paw swelling and body weight gain during live B. anthracis infection. Our findings indicate that nociceptive neurons play an active role in inflammation caused by B. anthracis and Edema Toxin to potentially influence bacterial pathogenesis.


Assuntos
Antraz/complicações , Antígenos de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Inflamação/etiologia , Nociceptores/metabolismo , Animais , Antraz/fisiopatologia , Bacillus anthracis , Camundongos , Camundongos Endogâmicos C57BL , Nociceptores/efeitos dos fármacos
12.
Avian Dis ; 65(1): 77-85, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-34339126

RESUMO

Infection of poultry with Eimeria spp., the causative agent of coccidiosis, can predispose birds to necrotic enteritis (NE) caused by netB gene-positive strains of Clostridium perfringens. The detection of Eimeria spp., C. perfringens, and netB were examined in settled dust from broiler flocks under experimental and field conditions. Dust samples were collected from settle plates twice weekly from two experimental flocks inoculated with three species of pathogenic Eimeria in 9-day-old chicks, followed by netB gene-positive C. perfringens 5 days later to produce subclinical and clinical NE. A noninoculated flock was sampled weekly from day 0 and served as a control flock. An additional 227 dust samples from commercial broiler flocks were collected at the end-of-batch (6-7 wk of age; one scraped dust sample per flock). In the NE-subclinical and NE-clinical flocks, high levels of Eimeria spp. and C. perfringens were detected after inoculation followed by a gradual decline over time. In the control flock, C. perfringens and netB were detected at low levels. No significant effect of sampling location was evident on Eimeria spp., C. perfringens, and netB load within poultry houses. These results provide evidence that Eimeria spp., C. perfringens, and netB gene copies can be readily measured in poultry dust samples collected in settle plates and may provide an alternative sampling method for monitoring flock coccidiosis and NE status. Further studies are required to assess the utility for such a test in commercial flocks.


Assuntos
Toxinas Bacterianas/análise , Galinhas , Infecções por Clostridium/veterinária , Clostridium perfringens/isolamento & purificação , Coccidiose/veterinária , Eimeria/isolamento & purificação , Enterotoxinas/análise , Doenças das Aves Domésticas/epidemiologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Animais , Infecções por Clostridium/epidemiologia , Infecções por Clostridium/microbiologia , Coccidiose/epidemiologia , Coccidiose/parasitologia , Poeira , New South Wales/epidemiologia , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/parasitologia
13.
Immunity ; 54(8): 1745-1757.e7, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34348118

RESUMO

Environmental enteric dysfunction (EED) is a gastrointestinal inflammatory disease caused by malnutrition and chronic infection. EED is associated with stunting in children and reduced efficacy of oral vaccines. To study the mechanisms of oral vaccine failure during EED, we developed a microbiota- and diet-dependent mouse EED model. Analysis of E. coli-labile toxin vaccine-specific CD4+ T cells in these mice revealed impaired CD4+ T cell responses in the small intestine and but not the lymph nodes. EED mice exhibited increased frequencies of small intestine-resident RORγT+FOXP3+ regulatory T (Treg) cells. Targeted deletion of RORγT from Treg cells restored small intestinal vaccine-specific CD4 T cell responses and vaccine-mediated protection upon challenge. However, ablation of RORγT+FOXP3+ Treg cells made mice more susceptible to EED-induced stunting. Our findings provide insight into the poor efficacy of oral vaccines in EED and highlight how RORγT+FOXP3+ Treg cells can regulate intestinal immunity while leaving systemic responses intact.


Assuntos
Toxinas Bacterianas/imunologia , Vacinas contra Escherichia coli/imunologia , Gastroenteropatias/imunologia , Intestino Delgado/imunologia , Linfócitos T Reguladores/imunologia , Administração Oral , Animais , Linhagem Celular , Modelos Animais de Doenças , Drosophila , Escherichia coli/imunologia , Feminino , Fatores de Transcrição Forkhead/metabolismo , Gastroenteropatias/microbiologia , Gastroenteropatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Vacinação
14.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445429

RESUMO

The prevalence of neurodegenerative disease (ND) is increasing, partly owing to extensions in lifespan, with a larger percentage of members living to an older age, but the ND aetiology and pathogenesis are not fully understood, and effective treatments are still lacking. Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis are generally thought to progress as a consequence of genetic susceptibility and environmental influences. Up to now, several environmental triggers have been associated with NDs, and recent studies suggest that some cyanotoxins, produced by cyanobacteria and acting through a variety of molecular mechanisms, are highly neurotoxic, although their roles in neuropathy and particularly in NDs are still controversial. In this review, we summarize the most relevant and recent evidence that points at cyanotoxins as environmental triggers in NDs development.


Assuntos
Toxinas Bacterianas/toxicidade , Cianobactérias/patogenicidade , Doenças Neurodegenerativas/etiologia , Animais , Cianobactérias/metabolismo , Predisposição Genética para Doença , Humanos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/microbiologia
15.
Biomed Res Int ; 2021: 6637617, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34395621

RESUMO

Staphylococcus aureus is a major human pathogen present on a third of the healthy population. The bacterium possesses an extensive arsenal of virulence factors. The pathogenicity is linked with S. aureus high plasticity and its exceptional ability to incorporate foreign genetic material. The aim of the present study was to perform molecular characterization of Staphylococcus aureus strains isolated from the clinical environment of the CHU-Z Abomey-Calavi/Sô-Ava. Isolation of Staphylococcus aureus bacterium was performed on Chapman agar. Toxin production by isolated S. aureus strains was investigated using the radial immunoprecipitation technique. A colorimetric assay was used to evaluate Staphylococcus aureus lipase (SA-Lipase) production. Finally, the expression of antibiotic resistance genes and genes encoding toxins production was investigated. Our data suggest that none of the isolated Staphylococcus aureus strains expressed the investigated toxin genes. Interestingly, SA-Lipase was produced by 14.28% of our isolated S. aureus strains. The mecA gene was present in 57.14% of the isolated strains, while PVL and TSST-1 genes were identified in 2.85 and 7.14% of S. aureus, respectively. Significant genetic diversity was observed along the hospital environment S. aureus strains. The present study reveals the level of virulence of S. aureus strains isolated in the different units of CHU-Z Abomey Calavi/Sô-Ava through the production of lipase, PVL, and epidermolysins. The molecular study has favored a genetic characterization within the isolated strains.


Assuntos
Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/patogenicidade , Fatores de Virulência/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Benin , Enterotoxinas/genética , Exotoxinas/genética , Hospitais Universitários , Humanos , Leucocidinas/genética , Lipase/genética , Proteínas de Ligação às Penicilinas/genética , RNA Bacteriano/genética , RNA Ribossômico/genética , RNA Ribossômico 16S/genética , Staphylococcus aureus/genética , Superantígenos/genética , Virulência
16.
Waste Manag ; 134: 32-41, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34403994

RESUMO

This study examined the combined effect of hydraulic retention time (HRT), organic loading rate (OLR) and heat pretreatment of manure (70 °C, 1 h) on the fate of E. coli, enterococci, C. perfringens, C. difficile, and on chemical parameters (volatile fatty acids and ammonia) that may inactivate pathogens. Semi-continuous mesophilic anaerobic reactors were fed with pig manure and horse feed. The operating conditions were 2, 3, 4 COD.L-1.d-1 (OLR), 24, 35, 46 days (HRT) and use or not of a thermal pretreatment. The levels of the chemical parameters did not reach concentrations capable of inactivating the four bacteria. Anaerobic digestion led to a Log10 removal > 3 (E. coli), 0.9-2.1 (enterococci), 0.1-0.6 (C. perfringens) and 0-1 (C. difficile). Increasing HRT only reduced the concentration of E. coli in the digestate. Increasing OLR reduced the Log10 removal of enterococci and C. difficile. The heat pretreatment led to non-detection of E. coli in the digestate, reduced the concentration of C. perfringens by 0.8-1.3 Log10 and increased the concentration of C. difficile by 0.04-0.7 Log10. Enterococci, not detected in the heated manure, were present in the digestate. The distribution of genes encoding virulence factors of C. difficile (tcdA and tcdB) and C. perfringens (cpa, cpb2 and cpb) was not impacted by anaerobic digestion or by the heat pretreatment. Enterococci, C. perfringens, C. difficile were present in the digestate at relatively stable concentrations regardless of the operating conditions, indicating that even with heat pretreatment, the biosafety of digestate cannot be guaranteed in mesophilic conditions.


Assuntos
Toxinas Bacterianas , Clostridioides difficile , Anaerobiose , Animais , Reatores Biológicos , Clostridioides , Clostridium perfringens , Enterococcus , Escherichia coli , Cavalos , Esterco , Metano , Suínos
17.
Toxicology ; 460: 152887, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34352349

RESUMO

Microcystin-leucine-arginine (MLCR) is a cyanobacterial toxin, and has been demonstrated to cause neurotoxicity. In addition, MCLR has been identified as an inhibitor of protein phosphatase (PP)1 and PP2A, which are known to regulate the phosphorylation of various molecules related to synaptic excitability. Thus, in the present study, we examined whether MCLR exposure affects seizures induced by a low dose of kainic acid (KA; 0.05 µg, i.c.v.) administration. KA-induced seizure occurrence and seizure score significantly increased after repeated exposure to MCLR (2.5 or 5.0 µg/kg, i.p., once a day for 10 days), but not after acute MCLR exposure (2.5 or 5.0 µg/kg, i.p., 2 h and 30 min prior to KA administration), and hippocampal neuronal loss was consistently facilitated by repeated exposure to MCLR. In addition, repeated MCLR significantly elevated the membrane expression of kainate receptor GluK2 subunits, p-pan-protein kinase C (PKC), and p-extracellular signal-related kinase (ERK) at 1 h after KA. However, KA-induced membrane expression of Ca2+/calmodulin-dependent kinase II (CaMKII) was significantly reduced by repeated MCLR exposure. Consistent with the enhanced seizures and neurodegeneration, MCLR exposure significantly potentiated KA-induced oxidative stress and microglial activation, which was accompanied by increased expression of p-ERK and p-PKCδ in the hippocampus. The combined results suggest that repeated MCLR exposure potentiates KA-induced excitotoxicity in the hippocampus by increasing membrane GluK2 expression and enhancing oxidative stress and neuroinflammation through the modulation of p-CaMKII, p-PKC, and p-ERK.


Assuntos
Arginina/toxicidade , Ácido Caínico/toxicidade , Leucina/toxicidade , Microcistinas/toxicidade , Neurotoxinas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Animais , Toxinas Bacterianas/toxicidade , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Ácido Caínico/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , Neurotoxinas/administração & dosagem , Estresse Oxidativo/fisiologia , Convulsões/induzido quimicamente , Convulsões/metabolismo
18.
J Vis Exp ; (174)2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34459820

RESUMO

Mitochondrial substrate flux is a distinguishing characteristic of each cell type, and changes in its components such as transporters, channels, or enzymes are involved in the pathogenesis of several diseases. Mitochondrial substrate flux can be studied using intact cells, permeabilized cells, or isolated mitochondria. Investigating intact cells encounters several problems due to simultaneous oxidation of different substrates. Besides, several cell types contain internal stores of different substrates that complicate results interpretation. Methods such as mitochondrial isolation or using permeabilizing agents are not easily reproducible. Isolating pure mitochondria with intact membranes in sufficient amounts from small samples is problematic. Using non-selective permeabilizers causes various degrees of unavoidable mitochondrial membrane damage. Recombinant perfringolysin O (rPFO) was offered as a more appropriate permeabilizer, thanks to its ability to selectively permeabilize plasma membrane without affecting mitochondrial integrity. When used in combination with microplate respirometry, it allows testing the flux of several mitochondrial substrates with enough replicates within one experiment while using a minimal number of cells. In this work, the protocol describes a method to compare mitochondrial substrate flux of two different cellular phenotypes or genotypes and can be customized to test various mitochondrial substrates or inhibitors.


Assuntos
Toxinas Bacterianas , Respiração Celular , Toxinas Bacterianas/metabolismo , Proteínas Hemolisinas/metabolismo , Mitocôndrias/metabolismo , Consumo de Oxigênio
19.
Toxins (Basel) ; 13(7)2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34357943

RESUMO

Cyanobacteria are ubiquitous photosynthetic microorganisms considered as important contributors to the formation of Earth's atmosphere and to the process of nitrogen fixation. However, they are also frequently associated with toxic blooms, named cyanobacterial harmful algal blooms (cyanoHABs). This paper reports on an unusual out-of-season cyanoHAB and its dynamics during the COVID-19 pandemic, in Lake Avernus, South Italy. Fast detection strategy (FDS) was used to assess this phenomenon, through the integration of satellite imagery and biomolecular investigation of the environmental samples. Data obtained unveiled a widespread Microcystis sp. bloom in February 2020 (i.e., winter season in Italy), which completely disappeared at the end of the following COVID-19 lockdown, when almost all urban activities were suspended. Due to potential harmfulness of cyanoHABs, crude extracts from the "winter bloom" were evaluated for their cytotoxicity in two different human cell lines, namely normal dermal fibroblasts (NHDF) and breast adenocarcinoma cells (MCF-7). The chloroform extract was shown to exert the highest cytotoxic activity, which has been correlated to the presence of cyanotoxins, i.e., microcystins, micropeptins, anabaenopeptins, and aeruginopeptins, detected by molecular networking analysis of liquid chromatography tandem mass spectrometry (LC-MS/MS) data.


Assuntos
Cianobactérias , Proliferação Nociva de Algas , Lagos/microbiologia , Toxinas Bacterianas/análise , Toxinas Bacterianas/toxicidade , COVID-19/epidemiologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cianobactérias/genética , DNA Bacteriano/análise , Monitoramento Ambiental , Atividades Humanas , Humanos , Itália/epidemiologia , Microcystis , Pandemias , SARS-CoV-2 , Imagens de Satélites
20.
Toxicon ; 200: 38-47, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34237340

RESUMO

Induction of CD8+ T cell responses against tumor cells and intracellular pathogens is an important goal of modern vaccinology. One approach of translational interest is the use of liposomes encapsulating pore-forming proteins (PFPs), such as Listeriolysin O (LLO), which has shown efficacy at priming strong and sustained CD8+ T cell responses. Recently, we have demonstrated that Sticholysin II (StII), a PFP from the sea anemone Stichodactyla helianthus, co-encapsulated into liposomes with ovalbumin (OVA) was able to stimulate, antigen presenting cells, antigen-specific CD8+ T cells and anti-tumor activity in mice. In the present study, we aimed to compare StII and LLO in terms of their abilities to stimulate dendritic cells and to induce major histocompatibility complex (MHC) class I restricted T cell responses against OVA. Interestingly, StII exhibited similar abilities to LLO in vitro of inducing dendritic cells maturation, as measured by increased expression of CD40, CD80, CD86 and MHC-class II molecules, and of stimulating OVA cross-presentation to a CD8+ T cell line. Remarkably, using an ex vivo Enzyme-Linked ImmunoSpot Assay (ELISPOT) to monitor gamma interferon (INF-γ) producing effector memory CD8+ T cells, liposomal formulations containing either StII or LLO induced comparable frequencies of OVA-specific INF-γ producing CD8+ T cells in mice that were sustained in time. However, StII-containing liposomes stimulated antigen-specific memory CD8+ T cells with a higher potential to secrete IFN-γ than liposomes encapsulating LLO. This StII immunostimulatory property further supports its use for the rational design of T cell vaccines against cancers and intracellular pathogens. In summary, this study indicates that StII has immunostimulatory properties similar to LLO, despite being evolutionarily distant PFPs.


Assuntos
Linfócitos T CD8-Positivos , Linfócitos T Citotóxicos , Animais , Toxinas Bacterianas , Venenos de Cnidários , Células Dendríticas , Proteínas de Choque Térmico , Proteínas Hemolisinas , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina
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