Immunogens in Balamuthia mandrillaris: a proteomic exploration.

Balamuthia mandrillaris is the causative agent of granulomatous amoebic encephalitis, a rare and often fatal infection affecting the central nervous system. The amoeba is isolated from diverse environmental sources and can cause severe infections in both immunocompromised and immunocompetent individuals. Given the limited understanding of B. mandrillaris, our research aimed to explore its protein profile, identifying potential immunogens crucial for early granulomatous amoebic encephalitis diagnosis. Cultures of B. mandrillaris and other amoebas were grown under axenic conditions, and total amoebic extracts were obtained. Proteomic analyses, including two-dimensional electrophoresis and mass spectrometry, were performed. A 50-kDa band showed a robust recognition of antibodies from immunized BALB/c mice; peptides contained in this band were matched with elongation factor-1 alpha, which emerged as a putative key immunogen. Besides, lectin blotting revealed the presence of glycoproteins in B. mandrillaris, and confocal microscopy demonstrated the focal distribution of the 50-kDa band throughout trophozoites. Cumulatively, these observations suggest the participation of the 50-kDa band in adhesion and recognition mechanisms. Thus, these collective findings demonstrate some protein characteristics of B. mandrillaris, opening avenues for understanding its pathogenicity and developing diagnostic and therapeutic strategies.

Phenolic compounds in the fruits of Enterolobium cyclocarpum and the inhibition of Haemonchus contortus eggs.

The fabacean Enterolobium cyclocarpum is a tree with multiple uses in production systems and its fruits serve as food for livestock, in addition, they contain phenolic compounds. The ovicidal effect of the secondary compounds of the Enterolobium cyclocarpum fruits extracted with hydroalcoholic solvent (HA-E) and the aqueous (Aq-F) and organic (AcOEt-F) fractions was evaluated. Additionally, a phytochemical analysis of the extract and fractions was performed by high performance liquid chromatography (HPLC). The HA-E showed an ovicidal effect close to 100% with the concentration evaluated of the 100 mg/mL. The fractionation of the extract allowed to potentiate the activity in the Aq (94.05% at 12 mg/mL) and AcOEt (99.45% at 3 mg/mL) fractions. The secondary compounds extracted from the fruits of E. cyclocarpum in the HA-E and fractions were flavonols, coumaric and ferulic acids and other derivatives of hydroxycinnamic acid and were responsible for the ovicidal activity observed against H. contortus.

Saline-Tolerant Pathogenic Acanthamoeba spp. Isolated from a Geothermal Power Plant.

Few studies have been conducted in the cooling systems of power plants; they have focused on Naegleria fowleri, leaving a gap in the knowledge of other pathogenic free-living amoebae in this environment. The objective of this study was to determine the occurrence of saline-tolerant pathogenic Acanthamoeba in a geothermal power plant. The identification of isolated amoebae at genus level was carried out, observing their morphological characteristics; the determination of genotype and species of Acanthamoeba was performed via molecular biology (PCR). Water temperature ranged from 18 to 43 °C and conductivity from 4.0 × 104 to 8.7 × 104 µS/cm; this last value was greater than the seawater value. Only five amoeba genera were found. Acanthamoeba was in all the sampling sites, showing high saline tolerance. The high temperature, but mainly high conductivity, were the environmental conditions that determined the presence of pathogenic free-living amoebae in the hot water. All the strains of Acanthamoeba culbertsoni killed the mice, having a mortality of 40 to 100%. Acanthamoeba genotypes T10 and T5 were identified, T10 is rarely isolated from the environment, while T5 is more frequent. This is the first time that genotypes T5 and T10 have been reported in the environment in Mexico.

Two MP2CL5 Antigen Vaccines from Naegleria fowleri Stimulate the Immune Response against Meningitis in the BALB/c Model.

Naegleria fowleri is an etiological agent that generates primary amoebic meningoencephalitis; unfortunately, no effective treatment or vaccine is available. The objective of this work was to determine the immunoprotective response of two vaccine antigens, as follows: (i) the polypeptide band of 19 kDa or (ii) a predicted immunogenic peptide from the membrane protein MP2CL5 (Smp145). Both antigens were administered intranasally in mice using cholera toxin (CT) as an adjuvant. The survival rate and immune response of immunized mice with both antigens and challenged with N. fowleri trophozoites were measured in the nose-associated lymphoid tissue (NALT) and nasal passages (NPs) by flow cytometry and enzyme-linked immunosorbent assay (ELISA). We also determined the immunolocalization of both antigens in N. fowleri trophozoites by confocal microscopy. Immunization with the polypeptide band of 19 kDa alone or coadministered with CT was able to confer 80% and 100% of protection, respectively. The immunization with both antigens (alone or coadministered with CT) showed an increase in T and B lymphocytes. In addition, there was an increase in the expression of integrin α4ß1 and IgA in the nasal cavity of protected mice, and the IgA, IgG, and IgM levels were increased in serum and nasal washes. The immunolocalization of both antigens in N. fowleri trophozoites was observed in the plasma membrane, specifically in pseudopod-like structures. The MP2CL5 antigens evaluated in this work were capable of conferring protection which would lead us to consider them as potential candidates for vaccines against meningitis caused by N. fowleri.

Identification of peptide epitopes of the gp120 protein of HIV-1 capable of inducing cellular and humoral immunity.

The Human Immunodeficiency Virus (HIV-1) causes Acquired Immunodeficiency Syndrome (AIDS) and a high percentage of deaths. Therefore, it is necessary to design vaccines against HIV-1 for the prevention of AIDS. Bioinformatic tools and theoretical algorisms allow us to understand the structural proteins of viruses to develop vaccines based on immunogenic peptides (epitopes). In this work, we identified the epitopes: P1, P2, P10, P27 and P30 from the gp120 protein of HIV-1. These peptides were administered intranasally alone or with cholera toxin (CT) to BALB/c mice. The population of CD4+, CD8+ T lymphocytes and B cells (CD19/CD138+, IgA+ and IgG+) from nasal-associated lymphoid tissue, nasal passages, cervical and inguinal nodes was determined by flow cytometry. In addition, anti-peptides IgG and IgA from serum, nasal and vaginal washings were measured by ELISA. The results show that peptides administered by i.n. can modulate the immune response of T and B lymphocyte populations, as well as IgA and IgG antibodies secretion in the different sites analyzed. In conclusion, bioinformatics tools help us to select peptides with physicochemical properties that allow the induction of the humoral and cellular responses that depend on the peptide sequence.

Role of FcγRIII in the nasal cavity of BALB/c mice in the primary amebic meningoencephalitis protection model.

Different mechanisms of the host immune response against the primary amebic meningoencephalitis (PAM) in the mouse protection model have been described. It has been proposed that antibodies opsonize Naegleria fowleri trophozoites; subsequently, the polymorphonuclear cells (PMNs) surround the trophozoites to avoid the infection. FcγRs activate signaling pathways of adapter proteins such as Syk and Hck on PMNs to promote different effector cell functions which are induced by the Fc portion of the antibody-antigen complexes. In this work, we analyzed the activation of PMNs, epithelial cells, and nasal passage cells via the expression of Syk and Hck genes. Our results showed an increment of the FcγRIII and IgG subclasses in the nasal cavity from immunized mice as well as Syk and Hck expression was increased, whereas in the in vitro assay, we observed that when the trophozoites of N. fowleri were opsonized with IgG anti-N. fowleri and interacted with PMN, the expression of Syk and Hck was also increased. We suggest that PMNs are activated via their FcγRIII, which leads to the elimination of the trophozoites in vitro, while in the nasal cavity, the adhesion and consequently infection are avoided.

Role of cathepsin B of Naegleria fowleri during primary amebic meningoencephalitis.

Naegleria fowleri causes primary amoebic meningoencephalitis in humans and experimental animals. It has been suggested that cysteine proteases of parasites play key roles in metabolism, nutrient uptake, host tissue invasion, and immune evasion. The aim of this work was to evaluate the presence, expression, and role of cathepsin B from N. fowleri in vitro and during PAM. Rabbit-specific polyclonal antibodies against cathepsin B were obtained from rabbit immunization with a synthetic peptide obtained by bioinformatic design. In addition, a probe was designed from mRNA for N. fowleri cathepsin B. Both protein and messenger were detected in fixed trophozoites, trophozoites interacted with polymorphonuclear and histological sections of infected mice. The main cathepsin B distribution was observed in cytoplasm or membrane mainly pseudopods and food-cups while messenger was in nucleus and cytoplasm. Surprisingly, both the messenger and enzyme were observed in extracellular medium. To determine cathepsin B release, we used trophozoites supernatant recovered from nasal passages or brain of infected mice. We observed the highest release in supernatant from recovered brain amoebae, and when we analyzed molecular weight of secreted proteins by immunoblot, we found 30 and 37 kDa bands which were highly immunogenic. Finally, role of cathepsin B during N. fowleri infection was determined; we preincubated trophozoites with E-64, pHMB or antibodies with which we obtained 60%, 100%, and 60% of survival, respectively, in infected mice. These results suggest that cathepsin B plays a role during pathogenesis caused by N. fowleri mainly in adhesion and contributes to nervous tissue damage.

Effect of soybean grain (Glycine max L.) supplementation on the production and fatty acid profile in milk of grazing cows in the dry tropics of Mexico.

Improving the lipid profile in milk of cows with the use of soybean grain (Glycine max L.) can be favored in the grazing systems in the dry tropics of Mexico. The objective was to evaluate the milk production, the chemical composition, and the fatty acids profile (FAs) of the milk of cows in continuous grazing and supplemented with and without ground roasted soybean in the dry tropics of Mexico. Ten cows randomly distributed in two equal groups were used. Daily during confinement for milking, the cows individually received the treatments on dry basis T0: supplement with 4.6 kg commercial concentrate® without soybean, T1: supplement with 3.7 kg commercial concentrate® with 380 g of soybean. During the 78 days of the experiment, milk production was measured in all cows, and samples were collected to determine the chemical composition and FAs profile. Milk production, protein, milk total fat, lactose, and non-fat solids did not vary with treatment (p >0.05). Linoleic acid content (C18: 2, cis, cis-∆9, ∆12) increased by 22% in milk fat of cows of the T1 (p ˂0.05). The sum of the mono- and polyunsaturated FAs 29.1%, the ratio of saturated-unsaturated FAs (1.65), and the atherogenicity index (1.71) also improved in the milk of cows supplemented with T1 (p ˂0.05). It was concluded that ground roasted soybean supplement in the diet of grazing dairy cows did not affect production and did improve the lipid profile in milk fat with favorable index to promote human health.

A 250-kDa glycoprotein of Naegleria fowleri induces protection and modifies the expression of α4ß1 and LFA-1 on T and B lymphocytes in mouse meningitis model.

The aims of this work were to evaluate the protective role of the 250-kDa polypeptide band of Naegleria fowleri. We designed an immunization strategy in Balb/c mice which were inoculated by i.n. route with an electrocuted 250-kDa polypeptide band of N. fowleri. We observed that the 250-kDa band induced 80% of protection, whereas the coadministration with Cholera Toxin induced 100% of protection. Moreover, high levels of IgA- and IgG-specific antibodies were detected by ELISA assay. We also analysed migration molecules (α4ß1 and LFA-1) on T and B lymphocytes in nose-associated lymphoid tissue (NALT), cervical lymph nodes (CN) and nasal passages (NP) by flow cytometry. We observed that the percentage of B cells (B220/α4ß1) and T cells (CD4/α4ß1) in NP were higher in all immunized groups compared with the other compartments analysed. Finally, we detected by immunohistochemistry ICAM-1 and V-CAM-1 in the nasal cavity. The immunization with the 250-kDa polypeptide band, protect mice against N. fowleri challenge and modifies migration molecules and their ligands.

Isolation and Identification of Naegleria Species in Irrigation Channels for Recreational Use in Mexicali Valley, Mexico.

Members of the genus Naegleria are free-living amoebae that are widely distributed in water and soil environments. Moreover, Naegleria fowleri is a pathogenic amoeba species that causes a fatal disease in the central nervous system known as primary amoebic meningoencephalitis (PAM) in humans. Since most reported infections due to N. fowleri are reported in recreational waters worldwide, this study was aimed to describe the presence of these amoebic genus in Mexicali Valley irrigation channels of recreational use. A total of nine water samples were collected and processed by triplicate, in nine different sites of the Valley. After filtering and culturing the samples, plates were examined, and the observed amoebae were morphologically identified at the genus level. In addition, the pathogenicity of these amoebic isolates was checked, and molecular characterization was performed by PCR/sequencing. The results revealed the presence of Naegleria spp. in all the channels sampled. Finally, molecular identification confirmed the presence of five different species of Naegleria: N. fowleri, N. australiensis, N. gruberi, N. clarki and N. pagei. The presence of these protists, particularly N. fowleri, should be considered as a potential human health risk in the region.

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.

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.

Identification of differential protein recognition pattern between Naegleria fowleri and Naegleria lovaniensis.

Many pathogenicity factors are involved in the development of primary amoebic meningoencephalitis (PAM) caused by N fowleri. However, most of them are not exclusive for N fowleri and they have not even been described in other nonpathogenic Naegleria species. Therefore, the objective of this work was to identify differential proteins and protein pattern recognition between Naegleria fowleri and Naegleria lovaniensis using antibodies anti-N fowleri as strategy to find vaccine candidates against meningoencephalitis. Electrophoresis and Western blots conventional and 2-DE were performed for the identification of antigenic proteins, and these were analysed by the mass spectrometry technique. The results obtained in 2-DE gels and Western blot showed very notable differences in spot intensity between these two species, specifically those with relative molecular weight of 100, 75, 50 and 19 kDa. Some spots corresponding to these molecular weights were identified as actin fragment, myosin II, heat shock protein, membrane protein Mp2CL5 among others, with differences in theoretical post-translational modifications. In this work, we found differences in antigenic proteins between both species, proteins that could be used for a further development of vaccines against N fowleri infection.

Bioinformatics design and experimental validation of influenza A virus multi-epitopes that induce neutralizing antibodies.

Pandemics caused by influenza A virus (IAV) are responsible for the deaths of millions of humans around the world. One of these pandemics occurred in Mexico in 2009. Despite the impact of IAV on human health, there is no effective vaccine. Gene mutations and translocation of genome segments of different IAV subtypes infecting a single host cell make the development of a universal vaccine difficult. The design of immunogenic peptides using bioinformatics tools could be an interesting strategy to increase the success of vaccines. In this work, we used the predicted amino acid sequences of the neuraminidase (NA) and hemagglutinin (HA) proteins of different IAV subtypes to perform multiple alignments, epitope predictions, molecular dynamics simulations, and experimental validation. Peptide selection was based on the following criteria: promiscuity, protein surface exposure, and the degree of conservation among different medically relevant IAV strains. These peptides were tested using immunological assays to test their ability to induce production of antibodies against IAV. We immunized rabbits and mice and measured the levels of IgG and IgA antibodies in serum samples and nasal washes. Rabbit antibodies against the peptides P11 and P14 (both of which are hybrids of NA and HA) recognized HA from both group 1 (H1, H2, and H5) and group 2 (H3 and H7) IAV and also recognized the purified NA protein from the viral stock (influenza A Puerto Rico/916/34). IgG antibodies from rabbits immunized with P11 and P14 were capable of recognizing viral particles and inhibited virus hemagglutination. Additionally, intranasal immunization of mice with P11 and P14 induced specific IgG and IgA antibodies in serum and nasal mucosa, respectively. Interestingly, the IgG antibodies were found to have neutralizing capability. In conclusion, the peptides designed through in silico studies were validated in experimental assays.

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.].

In silico search, chemical characterization and immunogenic evaluation of amino-terminated G4-PAMAM-HIV peptide complexes using three-dimensional models of the HIV-1 gp120 protein.

Peptide epitopes have been widely used to develop synthetic vaccines and immunotherapies. However, peptide epitopes may exhibit poor absorption or immunogenicity due to their low molecular weights. Conversely, fourth-generation polyamidoamine (G4-PAMAM) dendrimers are nonimmunogenic and relatively nontoxic synthetic nanoparticles that have been used as adjuvants and nanocarriers of small peptides and to improve nasal absorption. Based on this information, we hypothesized that the combination of intranasal immunization and G4-PAMAM dendrimers would be useful for enhancing the antibody responses of HIV-1 gp120 peptide epitopes. Therefore, we first used structural data, peptide epitope predictors and docking and MD simulations on MHC-II to identify two peptide epitopes on the CD4 binding site of HIV-1 gp120. The formation of G4-PAMAM-peptide complexes was evaluated in silico (molecular docking studies using different G4-PAMAM conformations retrieved from MD simulations as well as the MMGBSA approach) and validated experimentally (electrophoresis, 1H NMR and cryo-TEM). Next, the G4-PAMAM dendrimer-peptide complexes were administered intranasally to groups of female BALB/cJ mice. The results showed that both peptides were immunogenic at the systemic and mucosal levels (nasal and vaginal), and G4-PAMAM dendrimer-peptide complexes improved IgG and IgA responses in serum and nasal washes. Thus, G4-PAMAM dendrimers have potential for use as adjuvants and nanocarriers of peptides.

Anti-pathogenic, antibiofilm, and technological properties of fermented food associated Staphylococcus succinus strain AAS2.

The present investigation was aimed to determine the anti-pathogenic, antibiofilm, and technological properties of fermented food associated Staphylococcus succinus strain AAS2. The anti-pathogenic attribute of cell-free neutralized supernatant (CFNS) of strain AAS2 was assessed against food-borne and enteric pathogens that revealed promising activity against Staphylococcus aureus and Enterobacter aerogenes with high arbitrary unit of 220.25 ± 3.3 and 170.2 ± 4.6 AU/mL, respectively. Furthermore, the antibiofilm and time-kill assay of CFNS of strain AAS2 depicted remarkable reduction in biofilm formation of indicator pathogens in a dose-dependent manner. Moreover, time-kill assay data revealed the drastic reduction in the viability (log cfu/mL) of S. aureus and E. aerogenes in the presence of varied minimum inhibitory concentration ranges of CFNS. The distinct technological properties of strain AAS2 were demonstrated using standard methodologies. Reported results estimated moderate level of exopolysaccharide (41.3 ± 0.6 mg/L) and lipase production (8.3 ± 0.3 mm), followed by remarkable autolytic (30.1 ± 1.2-43.1 ± 1.3%), catalase (13.82 ± 0.3 AU), and nitrate reductase (10.25 ± 0.3 mM nitrite/mg dry weight) activities under standard conditions. Most importantly, the strain cleared the specific in vitro safety assessment tests. The described anti-pathogenic and technological traits of strain AAS2 paved the way to utilize it in pharmaceutical as well as food processing industries as starter/adjunct culture.

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.

Posterior Subdiaphragmatic Vagotomy Downmodulates the IgA Levels in the Small Intestine of BALB/c Mice.

OBJECTIVE: The posterior vagus nerve trunk innervates the entire small intestine, and elucidating its modulatory role in the IgA response was the aim of this study. METHODS: Two groups of six male BALB/c mice underwent sham or posterior subdiaphragmatic vagotomy and were euthanized on the 14th postoperative day; then, the small intestines were dissected. The intestinal fluid was harvested for antibody analysis by ELISA, and cell suspensions from Peyer's patches and lamina propria were prepared for cytofluorometric analysis of plasma cells and T lymphocytes. The CD4+ T cells were labeled for the intracellular IgA-producing interleukins (ILs)-4, -5, -6, and -10; transforming growth factor (TGF)-ß; and the inflammatory cytokines tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and IL-12. In the intestinal tissue samples, myeloperoxidase (MPO) visualization and the enzymatic activity were assessed by immunohistochemistry and ELISA, respectively. The data were analyzed by Student's t test, and the differences were considered significant at p < 0.05. RESULTS: In the vagotomy group, the IgA levels and the CD4+ T cells labeled with mediators that promote IgA secretion, including IL-4 (only at lamina propria), TNF-α, and IFN-γ, were decreased, whereas the lamina propria IgA+ plasma cells and MPO presence/activity were increased; changes in the IgM levels, IgM+ plasma cells, and CD4+ T cells labeled with TGF-ß, which have a role in class switch recombination, were not observed. CONCLUSION: The downmodulating impact of vagotomy on IgA levels may result from defective IgA secretion without affecting class switch recombination, whereas vagotomy evoked a proinflammatory response regarding MPO. These findings may reflect the role of the vagus nerve on the control of the IgA response in the small intestine.

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.