TRPM4 inhibition slows neuritogenesis progression of cortical neurons.

TRPM4 is a non-selective cation channel activated by intracellular Ca2+ but only permeable to monovalent cations, its activation regulates membrane potential and intracellular calcium. This channel participates in the migration and adhesion of non-excitable cells and forms an integral part of the focal adhesion complex. In neurons, TRPM4 expression starts before birth and its function at this stage is not clear, but it may function in processes such as neurite development. Here we investigate the role of TRPM4 in neuritogenesis. We found that neurons at DIV 0 express TRPM4, the inhibition of TRPM4 using 9-Ph reduces neurite number and slows the progression of neurite development, keeping neurons in stage 1. The genetic suppression of TRPM4 using an shRNA at later stages (DIV2) reduces neurite length. Conversely, at DIV 0, TRPM4 inhibition augments the Cch-induced Ca2 + i increase, altering the calcium homeostasis. Together, these results show that TRPM4 participates in progression of neurite development and suggest a critical role of the calcium modulation during this stage of neuronal development.

Lysosomes accumulate at the perinuclear region of muscle cells during chick myogenesis.

Lysosomes are involved in a myriad of cellular functions, such as degradation of macromolecules, endocytosis and exocytosis, modulation of several signaling pathways, and regulation of cell metabolism. To fulfill these diverse functions, lysosomes can undergo several dynamic changes in their content, size, pH, and location within cells. Here, we studied some of these parameters during embryonic chick skeletal muscle cells. We used an anti-lysosome-associated membrane protein 2 (LAMP2) antibody to specifically determine the intracellular localization of lysosomes in these cells. Our data shows that lysosomes are highly enriched in the perinuclear region of chick embryonic muscle cells. We also showed that the wingless signaling pathway (Wnt)/ß-catenin signaling pathway can modulate the location of LAMP2 in chick myogenic cells. Our results highlight the role of lysosomes during muscle differentiation and particularly the presence of a subcellular population of lysosomes that are concentrated in the perinuclear region of muscle cells.

Expression of the locus of enterocyte effacement genes during the invasion process of the atypical enteropathogenic Escherichia coli 1711-4 strain of serotype O51:H40.

Atypical enteropathogenic Escherichia coli (aEPEC) is a significant cause of diarrhea in low- and middle-income countries. Certain aEPEC strains, including the Brazilian representative strain of serotype O51:H40 called aEPEC 1711-4, can use flagella to attach to, invade, and persist in T84 and Caco-2 intestinal cells. It can also translocate from the gut to extraintestinal sites in a rat model. Although various aspects of the virulence of this strain were studied and the requirement of a type III secretion system for the efficiency of the invasion process was demonstrated, the expression of the locus of enterocyte effacement (LEE) genes during the invasion and intracellular persistence remains unclear. To address this question, the expression of flagella and the different LEE operons was evaluated during kinetic experiments of the interaction of aEPEC 1711-4 with enterocytes in vitro. The genome of the strain was also sequenced. The results showed that flagella expression remained unchanged, but the expression of eae and escJ increased during the early interaction and invasion of aEPEC 1711-4 into Caco-2 cells, and there was no change 24 h post-infection during the persistence period. The number of actin accumulation foci formed on HeLa cells also increased during the 6-h analysis. No known gene related to the invasion process was identified in the genome of aEPEC 1711-4, which was shown to belong to the global EPEC lineage 10. These findings suggest that the LEE components and the intimate adherence promoted by intimin are necessary for the invasion and persistence of aEPEC 1711-4, but the detailed mechanism needs further study.IMPORTANCEAtypical enteropathogenic Escherichia coli (aEPEC) is a major cause of diarrhea, especially in low- and middle-income countries, like Brazil. However, due to the genome heterogeneity of each clonal group, it is difficult to comprehend the pathogenicity of this strain fully. Among aEPEC strains, 1711-4 can invade eukaryotic cells in vitro, cross the gut barrier, and reach extraintestinal sites in animal models. By studying how different known aEPEC virulence factors are expressed during the invasion process, we can gain insight into the commonalities of this phenotype among other aEPEC strains. This will help in developing preventive measures to control infections caused by invasive strains. No known virulence-encoding genes linked to the invasion process were found. Nevertheless, additional studies are still necessary to evaluate the role of other factors in this phenotype.

Salmonid Rickettsial Septicemia (SRS) disease dynamics and Atlantic salmon immune response to Piscirickettsia salmonis LF-89 and EM-90 co-infection.

In Chile, Piscirickettsia salmonis contains two genetically isolated genogroups, LF-89 and EM-90. However, the impact of a potential co-infection with these two variants on Salmonid Rickettsial Septicemia (SRS) in Atlantic salmon (Salmo salar) remains largely unexplored. In our study, we evaluated the effect of P. salmonis LF-89-like and EM-90-like co-infection on post-smolt Atlantic salmon after an intraperitoneal challenge to compare changes in disease dynamics and host immune response. Co-infected fish had a significantly lower survival rate (24.1%) at 21 days post-challenge (dpc), compared with EM-90-like single-infected fish (40.3%). In contrast, all the LF-89-like single-infected fish survived. In addition, co-infected fish presented a higher presence of clinical lesions than any of the single-infected fish. The gene expression of salmon immune-related biomarkers evaluated in the head kidney, spleen, and liver showed that the EM-90-like isolate and the co-infection induced the up-regulation of cytokines (e.g., il-1ß, ifnγ, il8, il10), antimicrobial peptides (hepdicin) and pattern recognition receptors (PRRs), such as TLR5s. Furthermore, in serum samples from EM-90-like and co-infected fish, an increase in the total IgM level was observed. Interestingly, specific IgM against P. salmonis showed greater detection of EM-90-like antigens in LF-89-like infected fish serum (cross-reaction). These data provide evidence that P. salmonis LF-89-like and EM-90-like interactions can modulate SRS disease dynamics in Atlantic salmon, causing a synergistic effect that increases the severity of the disease and the mortality rate of the fish. Overall, this study contributes to achieving a better understanding of P. salmonis population dynamics.

Calcineurin activity in Fonsecaea pedrosoi: tacrolimus and cyclosporine A inhibited conidia growth, filamentation and showed synergism with itraconazole.

Fonsecaea pedrosoi is a melanized fungus that causes chromoblastomycosis (CBM), a tropical neglected disease responsible for chronic and disability-related subcutaneous mycosis. Given the challenging nature of CBM treatment, the study of new targets and novel bioactive drugs capable of improving patient life quality is urgent. In the present work, we detected a calcineurin activity in F. pedrosoi conidial form, employing primarily colorimetric, immunoblotting and flow cytometry assays. Our findings reveal that the calcineurin activity of F. pedrosoi was stimulated by Ca2+/calmodulin, inhibited by EGTA and specific inhibitors, such as tacrolimus (FK506) and cyclosporine A (CsA), and proved to be insensitive to okadaic acid. In addition, FK506 and CsA were able to affect the cellular viability and the fungal proliferation. This effect was corroborated by transmission electron microscopy that showed both calcineurin inhibitors promoted profound changes in the ultrastructure of conidia, causing mainly cytoplasm condensation and intense vacuolization that are clear indication of cell death. Our data indicated that FK506 exhibited the highest effectiveness, with a minimum inhibitory concentration (MIC) of 3.12 mg/L, whereas CsA required 15.6 mg/L to inhibit 100% of conidial growth. Interestingly, when both were combined with itraconazole, they demonstrated anti-F. pedrosoi activity, exhibiting a synergistic effect. Moreover, the fungal filamentation was affected after treatment with both calcineurin inhibitors. These data corroborate with other calcineurin studies in fungal cells and open up further discussions aiming to establish the role of this enzyme as a potential target for antifungal therapy against CBM infections.

Intracellular Streptococcus pneumoniae develops enhanced fluoroquinolone persistence during influenza A coinfection.

Streptococcus pneumoniae is a major pathogen responsible for severe complications in patients with prior influenza A virus (IAV) infection. We have previously demonstrated that S. pneumoniae exhibits increased intracellular survival within IAV-infected cells. Fluoroquinolones (FQs) are widely used to treat pneumococcal infections. However, our prior work has shown that S. pneumoniae can develop intracellular FQ persistence, a phenomenon triggered by oxidative stress within host cells. This persistence allows the bacteria to withstand high FQ concentrations. In this study, we show that IAV infection enhances pneumococcal FQ persistence during intracellular survival within pneumocytes, macrophages, and neutrophils. This enhancement is partly due to increased oxidative stress induced by the viral infection. We find that this phenotype is particularly pronounced in autophagy-proficient host cells, potentially resulting from IAV-induced blockage of autophagosome-lysosome fusion. Moreover, we identified several S. pneumoniae genes involved in oxidative stress response that contribute to FQ persistence, including sodA (superoxide dismutase), clpL (chaperone), nrdH (glutaredoxin), and psaB (Mn+2 transporter component). Our findings reveal a novel mechanism of antibiotic persistence promoted by viral infection within host cells. This underscores the importance of considering this phenomenon when using FQs to treat pneumococcal infections, especially in patients with concurrent influenza A infection.

The Trypanosoma cruzi pleiotropic protein P21 orchestrates the intracellular retention and in-vivo parasitism control of virulent Y strain parasites.

P21 is a protein secreted by all forms of Trypanosoma cruzi (T. cruzi) with recognized biological activities determined in studies using the recombinant form of the protein. In our recent study, we found that the ablation of P21 gene decreased Y strain axenic epimastigotes multiplication and increased intracellular replication of amastigotes in HeLa cells infected with metacyclic trypomastigotes. In the present study, we investigated the effect of P21 in vitro using C2C12 cell lines infected with tissue culture-derived trypomastigotes (TCT) of wild-type and P21 knockout (TcP21-/-) Y strain, and in vivo using an experimental model of T. cruzi infection in BALB/c mice. Our in-vitro results showed a significant decrease in the host cell invasion rate by TcP21-/- parasites as measured by Giemsa staining and cell count in bright light microscope. Quantitative polymerase chain reaction (qPCR) analysis showed that TcP21-/- parasites multiplied intracellularly to a higher extent than the scrambled parasites at 72h post-infection. In addition, we observed a higher egress of TcP21-/- trypomastigotes from C2C12 cells at 144h and 168h post-infection. Mice infected with Y strain TcP21-/- trypomastigotes displayed higher systemic parasitemia, heart tissue parasite burden, and several histopathological alterations in heart tissues compared to control animals infected with scrambled parasites. Therewith, we propose that P21 is important in the host-pathogen interaction during invasion, cell multiplication, and egress, and may be part of the mechanism that controls parasitism and promotes chronic infection without patent systemic parasitemia.

The SASP factor IL-6 sustains cell-autonomous senescent cells via a cGAS-STING-NFκB intracrine senescent noncanonical pathway.

Senescent cells produce a Senescence-Associated Secretory Phenotype (SASP) that involves factors with diverse and sometimes contradictory activities. One key SASP factor, interleukin-6 (IL-6), has the potential to amplify cellular senescence in the SASP-producing cells in an autocrine action, while simultaneously inducing proliferation in the neighboring cells. The underlying mechanisms for the contrasting actions remain unclear. We found that the senescence action does not involve IL-6 secretion nor the interaction with the receptor expressed in the membrane but is amplified through an intracrine mechanism. IL-6 sustains intracrine senescence interacting with the intracellular IL-6 receptor located in anterograde traffic specialized structures, with cytosolic DNA, cGAS-STING, and NFκB activation. This pathway triggered by intracellular IL-6 significantly contributes to cell-autonomous induction of senescence and impacts in tumor growth control. Inactivation of IL-6 in somatotrophic senescent cells transforms them into strongly tumorigenic in NOD/SCID mice, while re-expression of IL-6 restores senescence control of tumor growth. The intracrine senescent IL-6 pathway is further evidenced in three human cellular models of therapy-induced senescence. The compartmentalization of the intracellular signaling, in contrast to the paracrine tumorigenic action, provides a pathway for IL-6 to sustain cell-autonomous senescent cells, driving the SASP, and opens new avenues for clinical consideration to senescence-based therapies.

Presence of intracellular bacterial communities in uroepithelial cells, a potential reservoir in symptomatic and non-symptomatic people.

BACKGROUND: Urinary tract infection is one of the most common infections in humans, affecting women in more proportion. The bladder was considered sterile, but it has a urinary microbiome. Moreover, intracellular bacteria (IB) were observed in uroepithelial cells from children and women with urinary tract infections (UTIs). Here, we evaluated the presence of IB in urine from healthy people and patients with UTI symptoms. METHODS: Midstream urine was self-collected from 141 donors, 77 females and 64 males; 72 belonged to the asymptomatic group and 69 were symptomatic. IB was characterized by a culture-dependent technique and visualized by confocal microscopy. Urine was also subjected to the classical uroculture and isolated bacteria were identified by MALDI-TOF. RESULTS: One-hundred and fifteen uroculture were positive. A significant association was observed between the presence of symptoms and IB (P = 0.007). Moreover, a significant association between the presence of IB, symptoms and being female was observed (P = 0.03). From the cases with IB, Escherichia coli was the most frequent microorganism identified (34.7%), followed by Stenotrophomonas maltophilia (14.2%), Staphylococcus spp (14.2%), and Enterococcus faecalis (10.7%). Intracellular E. coli was associated with the symptomatic group (P = 0.02). Most of the intracellular Staphylococcus spp. were recovered from the asymptomatic group (P = 0.006). CONCLUSIONS: Intracellular bacteria are present in patients with UTI but also in asymptomatic people. Here, we report for the first time, the presence of S. maltophilia, Staphylococcus spp., and Enterobacter cloacae as intracellular bacteria in uroepithelial cells. These findings open new insights into the comprehension of urinary tract infections, urinary microbiome and future therapies. Uroculture as the gold standard could not be enough for an accurate diagnosis in recurrent or complicated cases.

The effects of climate and soil depth on living and dead bacterial communities along a longitudinal gradient in Chile.

Soil bacterial communities play a critical role in shaping soil stability and formation, exhibiting a dynamic interaction with local climate and soil depth. We employed an innovative DNA separation method to characterize microbial assemblages in low-biomass environments such as deserts and distinguish between intracellular DNA (iDNA) and extracellular DNA (eDNA) in soils. This approach, combined with analyses of physicochemical properties and co-occurrence networks, investigated soil bacterial communities across four sites representing diverse climatic gradients (i.e., arid, semi-arid, Mediterranean, and humid) along the Chilean Coastal Cordillera. The separation method yielded a distinctive unimodal pattern in the iDNA pool alpha diversity, increasing from arid to semi-arid climates and decreasing in humid environments, highlighting the rapid feedback of the iDNA community to increasing soil moisture. In the arid region, harsh surface conditions restrict bacterial growth, leading to peak iDNA abundance and diversity occurring in slightly deeper layers than the other sites. Our findings confirmed the association between specialist bacteria and ecosystem-functional traits. We observed transitions from Halomonas and Delftia, resistant to extreme arid environments, to Class AD3 and the genus Bradyrhizobium, associated with plants and organic matter in humid environments. The distance-based redundancy analysis (dbRDA) analysis revealed that soil pH and moisture were the key parameters that influenced bacterial community variation. The eDNA community correlated slightly better with the environment than the iDNA community. Soil depth was found to influence the iDNA community significantly but not the eDNA community, which might be related to depth-related metabolic activity. Our investigation into iDNA communities uncovered deterministic community assembly and distinct co-occurrence modules correlated with unique bacterial taxa, thereby showing connections with sites and key environmental factors. The study additionally revealed the effects of climatic gradients and soil depth on living and dead bacterial communities, emphasizing the need to distinguish between iDNA and eDNA pools.

Connexin channels and hemichannels are modulated differently by charge reversal at residues forming the intracellular pocket.

BACKGROUND: Members of the ß-subfamily of connexins contain an intracellular pocket surrounded by amino acid residues from the four transmembrane helices. The presence of this pocket has not previously been investigated in members of the α-, γ-, δ-, and ε-subfamilies. We studied connexin50 (Cx50) as a representative of the α-subfamily, because its structure has been determined and mutations of Cx50 are among the most common genetic causes of congenital cataracts. METHODS: To investigate the presence and function of the intracellular pocket in Cx50 we used molecular dynamics simulation, site-directed mutagenesis, gap junction tracer intercellular transfer, and hemichannel activity detected by electrophysiology and by permeation of charged molecules. RESULTS: Employing molecular dynamics, we determined the presence of the intracellular pocket in Cx50 hemichannels and identified the amino acids participating in its formation. We utilized site-directed mutagenesis to alter a salt-bridge interaction that supports the intracellular pocket and occurs between two residues highly conserved in the connexin family, R33 and E162. Substitution of opposite charges at either position decreased formation of gap junctional plaques and cell-cell communication and modestly reduced hemichannel currents. Simultaneous charge reversal at these positions produced plaque-forming non-functional gap junction channels with highly active hemichannels. CONCLUSIONS: These results show that interactions within the intracellular pocket influence both gap junction channel and hemichannel functions. Disruption of these interactions may be responsible for diseases associated with mutations at these positions.

Characterization, Bioaccesibility and Antioxidant Activities of Phenolic Compounds Recovered from Yellow pea (Pisum sativum) Flour and Protein Isolate.

This study focused on studying the bioaccesible phenolic compounds (PCs) from yellow pea flour (F) and protein isolate (I). Total phenolic contents (TPC), PCs composition and antioxidant activities were analysed in ethanol 60% extracts obtained by applying ultrasound assisted extraction (UAE, 15 min/40% amplitude). The preparation of I under alkaline conditions and the elimination of some soluble components at lower pH produced a change of PCs profile and antioxidant activity. After simulated gastrointestinal digestion (SGID) of both ingredients to obtain the digests FD and ID, notable changes in the PCs concentration and profiles could be demonstrated. FD presented a higher ORAC activity than ID (IC50 = 0.022 and 0.039 mg GAE/g dm, respectively), but lower ABTS•+ activity (IC50 = 0.8 and 0.3 mg GAE/g dm, respectively). After treatment with cholestyramine of extracts from FD and ID in order to eliminate bile salts and obtain the bioaccesible fractions FDb and IDb, ROS scavenging in H2O2-induced Caco2-TC7 cells was evaluated, registering a greater activity for ID respect to FD (IC50 = 0.042 and 0.017 mg GAE/mL, respectively). These activities could be attributed to the major bioaccesible PCs: OH-tyrosol, polydatin, trans-resveratrol, rutin, (-)-epicatechin and (-)-gallocatechin gallate for FD; syringic (the most concentrated) and ellagic acids, trans-resveratrol, and (-)-gallocatechin gallate for ID, but probably other compounds such as peptides or amino acids can also contribute.

Activation of Pannexin-1 channels causes cell dysfunction and damage in mesangial cells derived from angiotensin II-exposed mice.

Chronic kidney disease (CKD) is a prevalent health concern associated with various pathological conditions, including hypertensive nephropathy. Mesangial cells are crucial in maintaining glomerular function, yet their involvement in CKD pathogenesis remains poorly understood. Recent evidence indicates that overactivation of Pannexin-1 (Panx1) channels could contribute to the pathogenesis and progression of various diseases. Although Panx1 is expressed in the kidney, its contribution to the dysfunction of renal cells during pathological conditions remains to be elucidated. This study aimed to investigate the impact of Panx1 channels on mesangial cell function in the context of hypertensive nephropathy. Using an Ang II-infused mouse model and primary mesangial cell cultures, we demonstrated that in vivo exposure to Ang II sensitizes cultured mesangial cells to show increased alterations when they are subjected to subsequent in vitro exposure to Ang II. Particularly, mesangial cell cultures treated with Ang II showed elevated activity of Panx1 channels and increased release of ATP. The latter was associated with enhanced basal intracellular Ca2+ ([Ca2+]i) and increased ATP-mediated [Ca2+]i responses. These effects were accompanied by increased lipid peroxidation and reduced cell viability. Crucially, all the adverse impacts evoked by Ang II were prevented by the blockade of Panx1 channels, underscoring their critical role in mediating cellular dysfunction in mesangial cells. By elucidating the mechanisms by which Ang II negatively impacts mesangial cell function, this study provides valuable insights into the pathogenesis of renal damage in hypertensive nephropathy.

Phenotypic and Functional Characterization of Memory CD4+ and CD8+ T Cells After Antigenic Stimulation.

The encounter of T cells with the antigen through the interaction of T cell receptors with peptides and major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs) can generate effector response and memory T cells. Memory T cells developed following infections or vaccination may persist, leading to the generation of a specific immune response upon reexposure to the same pathogen through rapid clonal proliferation and activation of effector functions. T cell memory subsets can be identified based on the expression of several membrane markers such as CCR7, CD27, and CD45RA. Using fluorescent antibodies against these markers and a flow cytometer, it is possible to perform immunophenotyping via the analysis of cell surface expression of proteins by different subpopulations such as the subsets of naïve, effector, and memory T cells as well as via the analysis of functional markers that further characterize each sample. Intracellular cytokine staining allows for the evaluation of intracellular proteins expressed in T cells in response to antigenic stimulation. This chapter presents the phenotypic and functional characterization of memory T cells after antigenic stimulation, detailing the procedures for identifying intracellular and surface protein markers. Herein, we review and present a reproducible standardized protocol using antibodies for specific markers and applying flow cytometry.

Remarkable Phenotypic Virulence Factors of Microsporum canis and Their Associated Genes: A Systematic Review.

Microsporum canis is a widely distributed dermatophyte, which is among the main etiological agents of dermatophytosis in humans and domestic animals. This fungus invades, colonizes and nourishes itself on the keratinized tissues of the host through various virulence factors. This review will bring together the known information about the mechanisms, enzymes and their associated genes relevant to the pathogenesis processes of the fungus and will provide an overview of those virulence factors that should be better studied to establish effective methods of prevention and control of the disease. Public databases using the MeSH terms "Microsporum canis", "virulence factors" and each individual virulence factor were reviewed to enlist a series of articles, from where only original works in English and Spanish that included relevant information on the subject were selected. Out of the 147 articles obtained in the review, 46 were selected that reported virulence factors for M. canis in a period between 1988 and 2023. The rest of the articles were discarded because they did not contain information on the topic (67), some were written in different languages (3), and others were repeated in two or more databases (24) or were not original articles (7). The main virulence factors in M. canis are keratinases, fungilisins and subtilisins. However, less commonly reported are biofilms or dipeptidylpeptidases, among others, which have been little researched because they vary in expression or activity between strains and are not considered essential for the infection and survival of the fungus. Although it is known that they are truly involved in resistance, infection and metabolism, we recognize that their study could strengthen the knowledge of the pathogenesis of M. canis with the aim of achieving effective treatments, as well as the prevention and control of infection.

TRPV4 Activation during Guinea Pig Airway Smooth Muscle Contraction Promotes Ca2+ and Na+ Influx.

Airway smooth muscle (ASM) contraction is determined by the increase in intracellular Ca2+ concentration ([Ca2+]i) caused by its release from the sarcoplasmic reticulum (SR) or by extracellular Ca2+ influx. Major channels involved in Ca2+ influx in ASM cells are L-type voltage-dependent Ca2+ channels (L-VDCCs) and nonselective cation channels (NSCCs). Transient receptor potential vanilloid 4 (TRPV4) is an NSCC recently studied in ASM. Mechanical stimuli, such as contraction, can activate TRPV4. We investigated the possible activation of TRPV4 by histamine (His)- or carbachol (CCh)-induced contraction in guinea pig ASM. In single myocytes, the TRPV4 agonist (GSK101) evoked an increase in [Ca2+]i, characterized by a slow onset and a plateau phase. The TRPV4 antagonist (GSK219) decreased channel activity by 94%, whereas the Ca2+-free medium abolished the Ca2+ response induced by GSK101. Moreover, GSK101 caused Na+ influx in tracheal myocytes. GSK219 reduced the Ca2+ peak and the Ca2+ plateau triggered by His or CCh. TRPV4 blockade shifted the concentration-response curve relating to His and CCh to the right in tracheal rings and reduced the maximal contraction. Finally, the activation of TRPV4 in single myocytes increased the Ca2+ refilling of the SR. We conclude that contraction of ASM cells after stimulation with His or CCh promotes TRPV4 activation, the subsequent influx of Ca2+ and Na+, and the opening of L-VDCCs. The entry of Ca2+ into ASM cells via TRPV4 and L-VDCCs contributes to optimal smooth muscle contraction.

Methods for Intracellular Peptidomic Analysis.

Peptides have broad biological significance among different species. Intracellular peptides are considered a particular class of bioactive peptides, whose generation is initiated by proteasomal degradation of cytosolic, nuclear, or mitochondrial proteins. To extract and purify intracellular peptides, which may apply for biological peptides in general, it is important to consider the initial source: tissue, cell, or fluid. First, it is important to proceed fast with inactivation of proteases and/or peptidases commonly present in the biological source of peptides, which might rapidly degrade peptides during the initial process of extraction. The incubation of biological tissues, cells, and fluids at 80 °C for up to 20 min have been sufficient to fully inactivate proteases or peptidases activities. It is particularly important not to acidify the samples at high temperature, because it can lead to nonspecific hydrolysis reactions; particularly, the Asp-Pro peptide bond can be cleaved at acidic environments and elevated temperatures. Unfortunately, not every sample can have proteinases and peptidases denatured by heating the biological source of intracellular peptides. Plasma, for example, when heated at temperatures higher than 55 °C can clot and trap peptides within the fibrin net. Therefore, alternative conditions for inactivating proteinases and peptidases must apply for plasma samples. In this chapter, the most successful methods used in our laboratory to extract intracellular peptides are described.

Peptidomics of Zebrafish Brain in a 6-OHDA-Induced Neurodegeneration Model.

Bioactive peptides such as neuropeptides and peptide hormones are largely understood in their involvement in a variety of physiologic systems. In addition to the neuropeptides produced and processed by the classic secretory pathway, intracellular peptides (InPeps) have shown biological activity in studies involving different organisms. A model that has become attractive in many research fields is the zebrafish (Danio rerio), which has allowed correlating behavioral responses or physiological processes with underlying molecular pathways or signaling cascades, improving the understanding of homeostasis mechanisms of the central nervous system, as well as pathological processes such as neurodegenerative diseases. Here, we provide a detailed description of the protocol of treatment with 6-OHDA, which mimics some features of Parkinson's Disease, as well as the validation of the treatment by evaluation of the locomotor activity and the protocol of peptide extraction followed by isotopic labeling to peptide relative quantitation by mass spectrometry.

Corrigendum: The role of cholesterol and mitochondrial bioenergetics in activation of the inflammasome in IBD.

[This corrects the article DOI: 10.3389/fimmu.2022.1028953.].

Expression of the locus of enterocyte effacement genes during the invasion process of the atypical enteropathogenic Escherichia coli 1711-4 strain of serotype O51:H40.

Atypical enteropathogenic Escherichia coli (aEPEC) is a significant cause of diarrhea in developing countries. Some aEPEC strains, including the Brazilian representative strain of serotype O51:H40 called aEPEC 1711-4, can use flagella to attach to, invade, and persist in T84 and Caco-2 intestinal cells. They can even translocate from the gut to extraintestinal sites in a rat model. Although various aspects of the virulence of this strain were studied and the requirement of the T3SS for the efficiency of the invasion process was demonstrated, the expression of the LEE genes during the invasion and intracellular persistence remains unclear. To address this, the expression of flagella and the different LEE operons was evaluated during kinetic experiments of the interaction of aEPEC 1711-4 with enterocytes in vitro. The genome of the strain was also sequenced. The results showed that flagella expression remained unchanged, but the expression of eae and escJ increased during the early interaction and invasion of aEPEC 1711-4 into Caco-2 cells, and there was no change 24 hours post-infection during the persistence period. The number of pedestal-like structures formed on HeLa cells also increased during the 24-hour analysis. No known gene related to the invasion process was identified in the genome of aEPEC 1711-4, which was shown to belong to the global EPEC lineage 10. These findings suggest that LEE components and the intimate adherence promoted by intimin are necessary for the invasion and persistence of aEPEC 1711-4, but the detailed mechanism needs further study.