N-(2'-Hydroxyphenyl)-2-Propylpentanamide (HO-AAVPA) Inhibits HDAC1 and Increases the Translocation of HMGB1 Levels in Human Cervical Cancer Cells.

N-(2'-hydroxyphenyl)-2-propylpentanamide (HO-AAVPA) is a VPA derivative designed to be a histone deacetylase (HDAC) inhibitor. HO-AAVPA has better antiproliferative effect than VPA in cancer cell lines. Therefore, in this work, the inhibitory effect of HO-AAVPA on HDAC1, HDAC6, and HDAC8 was determined by in silico and in vitro enzymatic assay. Furthermore, its antiproliferative effect on the cervical cancer cell line (SiHa) and the translocation of HMGB1 and ROS production were evaluated. The results showed that HO-AAVPA inhibits HDAC1, which could be related with HMGB1 translocation from the nucleus to the cytoplasm due to HDAC1 being involved in the deacetylation of HMGB1. Furthermore, an increase in ROS production was observed after the treatment with HO-AAVPA, which also could contribute to HMGB1 translocation. Therefore, the results suggest that one of the possible antiproliferative mechanisms of HO-AAVPA is by HDAC1 inhibition which entails HMGB1 translocation and ROS increased levels that could trigger the cell apoptosis.

Sleep loss disrupts pericyte-brain endothelial cell interactions impairing blood-brain barrier function.

Sleep loss in the rat increases blood-brain barrier permeability to circulating molecules by disrupting interendothelial tight junctions. Despite the description of the ultrastructure of cerebral microvessels and the evidence of an apparent pericyte detachment from capillary wall in sleep restricted rats the effect of sleep loss on pericytes is unknown. Here we characterized the interactions between pericytes and brain endothelial cells after sleep loss using male Wistar rats. Animals were sleep-restricted 20 h daily with 4 h sleep recovery for 10 days. At the end of the sleep restriction, brain microvessels (MVs) were isolated from cerebral cortex and hippocampus and processed for Western blot and immunocytochemistry to evaluate markers of pericyte-endothelial cell interaction (connexin 43, PDGFR-ß), tight junction proteins, and proinflammatory mediator proteins (MMP9, A2A adenosine receptor, CD73, NFκB). Sleep restriction reduced PDGFR-ß and connexin 43 expression in MVs; in addition, scanning electron microscopy micrographs showed that pericytes were detached from capillary walls, but did not undergo apoptosis (as depicted by a reduced active caspase-3 expression). Sleep restriction also decreased tight junction protein expression in MVs and increased BBB permeability to low- and high-molecular weight tracers in in vivo permeability assays. Those alterations seemed to depend on a low-grade inflammatory status as reflected by the increased expression of phosphorylated NFκB and A2A adenosine receptor in brain endothelial cells from the sleep-restricted rats. Our data show that pericyte-brain endothelial cell interaction is altered by sleep restriction; this evidence is essential to understand the role of sleep in regulating blood-brain barrier function.

Identification of Immunogenic Antigens of Naegleria fowleri Adjuvanted by Cholera Toxin.

The intranasal administration of Naegleria fowleri lysates plus cholera toxin (CT) increases protection against N. fowleri meningoencephalitis in mice, suggesting that humoral immune response mediated by antibodies is crucial to induce protection against the infection. In the present study, we applied a protein analysis to detect and identify immunogenic antigens from N. fowleri, which might be responsible for such protection. A Western blot assay of N. fowleri polypeptides was performed using the serum and nasal washes from mice immunized with N. fowleri lysates, either alone or with CT after one, two, three, or four weekly immunizations and challenged with trophozoites of N. fowleri. Immunized mice with N. fowleri plus CT, after four doses, had the highest survival rate (100%). Nasal or sera IgA and IgG antibody response was progressively stronger as the number of immunizations was increased, and that response was mainly directed to 250, 100, 70, 50, 37, and 19 kDa polypeptide bands, especially in the third and fourth immunization. Peptides present in these immunogenic bands were matched by nano-LC-ESI-MSMS with different proteins, which could serve as candidates for a vaccine against N. fowleri infection.

Protein Phosphorylation in Serine Residues Correlates with Progression from Precancerous Lesions to Cervical Cancer in Mexican Patients.

Protein phosphorylation is a posttranslational modification that is essential for normal cellular processes; however, abnormal phosphorylation is one of the prime causes for alteration of many structural, functional, and regulatory proteins in disease conditions. In cancer, changes in the states of protein phosphorylation in tyrosine residues have been more studied than phosphorylation in threonine or serine residues, which also undergo alterations with greater predominance. In general, serine phosphorylation leads to the formation of multimolecular signaling complexes that regulate diverse biological processes, but in pathological conditions such as tumorigenesis, anomalous phosphorylation may result in the deregulation of some signaling pathways. Cervical cancer (CC), the main neoplasm associated with human papillomavirus (HPV) infection, is the fourth most frequent cancer worldwide. Persistent infection of the cervix with high-risk human papillomaviruses produces precancerous lesions starting with low-grade squamous intraepithelial lesions (LSIL), progressing to high-grade squamous intraepithelial lesions (HSIL) until CC is generated. Here, we compared the proteomic profile of phosphorylated proteins in serine residues from healthy, LSIL, HSIL, and CC samples. Our data show an increase in the number of phosphorylated proteins in serine residues as the grade of injury rises. These results provide a support for future studies focused on phosphorylated proteins and their possible correlation with the progression of cervical lesions.

Neutrophil extracellular traps and MPO in models of susceptibility and resistance against Entamoeba histolytica.

The main effector mechanisms of neutrophils are the release of neutrophil extracellular traps (NETs) and myeloperoxidase (MPO). In this work, we evaluated the role of NETs and the activity of MPO in the interactions of rodent neutrophils with amoebae and in amoebic liver abscess (ALA)-resistant and ALA-susceptible models. We showed with in vitro assays that mice produced greater amounts of NETs and MPO than did hamsters, and the elastase activity was high in both models. However, the inhibition of NETs and MPO promoted an increase in amoeba viability in the mice. The mouse ALAs showed a more profound presence of NETs and MPO than did the hamster ALAs. We concluded that both effector mechanisms were essential for the amoebic damage and could prevent the formation of ALAs in the resistant model.

Chronic sleep loss disrupts blood-testis and blood-epididymis barriers, and reduces male fertility.

Sleep loss increases blood-brain barrier permeability. As the blood-brain barrier and the blood-tissue barriers in the reproductive tract (blood-testis and blood-epididymis barriers) share common characteristics, we hypothesized that sleep restriction may also modify their barrier function. Previous reports showed that sleep loss decreased sperm viability and progressive fast mobility, which may be a consequence of altered blood-testis and blood-epididymis barrier. Therefore, we quantified changes in blood-testis and blood-epididymis barrier after sleep loss and related them to male fertility. Adult male Wistar rats were sleep restricted using the multiple-platform technique in a protocol of 20 hr daily sleep deprivation plus 4 hr of sleep recovery in the home-cage. At the 10th day, barrier permeability assays were performed with Na-fluorescein, 10 kDa Cascade blue-dextrans and Evans blue, and the expression of tight junction proteins, actin and androgen receptor was quantified. At the 10th day of sleep restriction and after sleep recovery days 1-7, males were placed with sexually receptive females, sexual behaviour was tested, and the percentage of pregnancies was calculated. Sleep restriction increased the barrier permeability to low- and high-molecular-weight tracers, and decreased the expression of tight junction proteins, actin and androgen receptor. Concomitantly, sleep restriction reduced the percentage of ejaculating males and the number of pregnancies. Sleep recovery for 2-3 days progressively re-established fertility, as indicated by a higher percentage of ejaculating males and impregnated females. In conclusion, chronic sleep loss alters fertility concomitantly with the disruption of the blood-tissue barriers at the reproductive tract, the mechanism involves androgen signalling.

Correction: N-(2´-Hydroxyphenyl)-2-propylpentanamide (OH-VPA), a histone deacetylase inhibitor, induces the release of nuclear HMGB1 and modifies ROS levels in HeLa cells.

[This corrects the article DOI: 10.18632/oncotarget.26077.].

Mouse neutrophils release extracellular traps in response to Naegleria fowleri.

Naegleria fowleri is a free-living amoeba, which is able to infect humans through the nasal mucosa causing a disease in the central nervous system known as primary amoebic meningoencephalitis (PAM). Polymorphonuclear cells (PMNs) play a critical role in the early phase of N fowleri infection. Recently, a new biological defence mechanism called neutrophil extracellular traps (NETs) has been attracting attention. These structures represent an important strategy to immobilize and kill invading microorganisms. In this work, we evaluate the capacity of N fowleri to induce the NETs release by PMNs cells in mice in vitro and in vivo. In vitro: Neutrophils from bone marrow were cocultured with N fowleri trophozoites. In vivo: we employed a mouse model of PAM. We evaluated DNA, histone and myeloperoxidase (MPO) and the formation of NETs by confocal microscopy. Our results showed N fowleri induce both NETs and MPO release by PMNs cells in mice after trophozoite exposure, which increased through time, in vitro and in vivo. These results demonstrate that NETs are somehow associated with the amoebas. We suggest PMNs release their traps trying to avoid N fowleri attachment at the apical side of the nasal epithelium.

N-(2'-Hydroxyphenyl)-2-propylpentanamide (OH-VPA), a histone deacetylase inhibitor, induces the release of nuclear HMGB1 and modifies ROS levels in HeLa cells.

N-(2'-Hydroxyphenyl)-2-propylpentanamide (OH-VPA) is a valproic acid (VPA) derivative with improved antiproliferative activity toward breast cancer (MCF-7, MDA-MB-231, and SKBr3) and human cervical cancer cell lines (HeLa) compared to that of VPA. However, the pharmacological mechanism of OH-VPA activity remains unknown. High-mobility group box 1 (HMGB1) is an important enzyme that is highly expressed in tumor cells and has a subcellular localization that is dependent on its acetylation or oxidative state. Therefore, in this study, we analyzed changes in HMGB1 sub-cellular localization and reactive oxygen species (ROS) as well as changes in HeLa cell viability in response to treatment with various concentrations of OH-VPA. This compound is formed by the covalent bond coupling VPA to a phenol group, which is capable of acting as a free radical scavenger due to its chemical similarities to quercetin. Our results show that OH-VPA induces nuclear to cytoplasmic translocation of HMGB1, as demonstrated by confocal microscopy observations and infrared spectra that revealed high quantities of acetylated HMGB1 in HeLa cells. Cells treated with 0.8 mM OH-VA exhibited decreased viability and increased ROS levels compared with the lower OH-VPA concentrations tested. Therefore, the antiproliferative mechanism of OH-VPA may be related to histone deacetylase (HDAC) inhibition, as is the case for VPA, which promotes high HMBG1 acetylation, which alters its subcellular localization. In addition, OH-VPA generates an imbalance in cellular ROS levels due to its biochemical activity.

Predicting peptide vaccine candidates against H1N1 influenza virus through theoretical approaches.

Identification of potential epitopes that might activate the immune system has been facilitated by the employment of algorithms that use experimental data as templates. However, in order to prove the affinity and the map of interactions between the receptor (major histocompatibility complex, MHC, or T-cell receptor) and the potential epitope, further computational studies are required. Docking and molecular dynamics (MDs) simulations have been an effective source of generating structural information at molecular level in immunology. Herein, in order to provide a detailed understanding of the origins of epitope recognition and to select the best peptide candidate to develop an epitope-based vaccine, docking and MDs simulations in combination with MMGBSA free energy calculations and per-residue free energy decomposition were performed, taking as starting complexes those formed between four designed epitopes (P1-P4) from hemagglutinin (HA) of the H1N1 influenza virus and MHC-II anchored in POPC membrane. Our results revealed that the energetic contributions of individual amino acids within the pMHC-II complexes are mainly dictated by van der Waals interactions and the nonpolar part of solvation energy, whereas the electrostatic interactions corresponding to hydrogen bonds and salt bridges determine the binding specificity, being the most favorable interactions formed between p4 and MHC-II. Then, P1-P4 epitopes were synthesized and tested experimentally to compare theoretical and experimental results. Experimental results show that P4 elicited the highest strong humoral immune response to HA of the H1N1 and may induce antibodies that are cross-reactive to other influenza subtypes, suggesting that it could be a good candidate for the development of a peptide-based vaccine.

Cytotoxic effect of the immunotoxin constructed of the ribosome-inactivating protein curcin and the monoclonal antibody against Her2 receptor on tumor cells.

The toxicity of the curcin on cancer cells allows to consider this protein as the toxic component of an immunotoxin directed to Her2, which is associated with cancer. Reductive amination was proposed to conjugate curcin and an anti-Her2; the binding was tested using Polyacrylamide gel electrophoresis, western blot, and immunocytochemistry. The in vitro cytotoxicity of curcin and the immunotoxin was assessed on breast cancer cell lines SK-BR-3 (Her2(+)) and MDA-MB-231 (Her2(-)). IC50 values for curcin were 15.5 ± 8.3 and 18.6 ± 2.4 µg/mL, respectively, statistically equivalent (p < 0.05). While to the immunotoxin was 2.2 ± 0.08 for SK-BR-3 and 147.6 ± 2.5 µg/mL for MDA-MB-231. These values showed that the immunotoxin was seven times more toxic to the SK-BR-3 than curcin and eight times less toxic to the MDA-MB-231. The immunotoxin composed of curcin and an antibody against Her2 and constructed by reductive amination could be a therapeutic candidate against Her2(+) cancer.

Intranasal coadministration of Cholera toxin with amoeba lysates modulates the secretion of IgA and IgG antibodies, production of cytokines and expression of pIgR in the nasal cavity of mice in the model of Naegleria fowleri meningoencephalitis.

The nasal mucosa is the first contact with antigens to induce IgA response. The role of this site has rarely been studied. We have shown than intranasal administration with Naegleria fowleri lysates plus Cholera toxin (CT) increased the protection (survival up to 100%) against N. fowleri infection in mice and apparently antibodies IgA and IgG together with polymorphonuclear (PMN) cells avoid the attachment of N. fowleri to apical side of the nasal epithelium. We also observed that nasal immunization resulted in the induction of antigen-specific IgG subclasses (IgG1 and IgG2a) in nasal washes at days 3 and 9 after the challenge and IgA and IgG in the nasal cavity, compared to healthy and infected mice. We found that immunization with both treatments, N. fowleri lysates plus CT or CT alone, increased the expression of the genes for alpha chain, its receptor (pIgR), and it also increased the expression of the corresponding proteins evidenced by the ∼65 and ∼74kDa bands, respectively. Since the production of pIgR, IgA and IgG antibodies, is up-regulated by some factors, we analyzed the expression of genes for IL-10, IL-6, IFN-γ, TNF-α and IL-1ß by using RT-PCR of nasal passages. Immunization resulted in an increased expression of IL-10, IL-6, and IFN-γ cytokines. We also aimed to examine the possible influences of immunization and challenge on the production of inflammatory cytokines (TNF-α and IL-1ß). We observed that the stimulus of immunization inhibits the production of TNF-α compared to the infected group where the infection without immunization causes an increase in it. Thus, it is possible that the coexistence of selected cytokines produced by our immunization model may provide a highly effective immunological environment for the production of IgA, IgG and pIgR as well as a strong activation of the PMN in mucosal effector tissue such as nasal passages.