GPER: An Estrogen Receptor Key in Metastasis and Tumoral Microenvironments.

Estrogens and their role in cancer are well-studied, and some cancer types are classified in terms of their response to them. In recent years, a G protein-coupled estrogen receptor (GPER) has been described with relevance in cancer. GPER is a pleiotropic receptor with tissue-specific activity; in normal tissues, its activation is related to correct development and homeostasis, while in cancer cells, it can be pro- or anti-tumorigenic. Also, GPER replaces estrogen responsiveness in estrogen receptor alpha (ERα)-lacking cancer cell lines. One of the most outstanding activities of GPER is its role in epithelial-mesenchymal transition (EMT), which is relevant for metastasis development. In addition, the presence of this receptor in tumor microenvironment cells contributes to the phenotypic plasticity required for the dissemination and maintenance of tumors. These characteristics suggest that GPER could be a promising therapeutic target for regulating cancer development. This review focuses on the role of GPER in EMT in tumorigenic and associated cells, highlighting its role in relation to the main hallmarks of cancer and possible therapeutic options.

The Antimicrobial Peptide γ-Thionin from Habanero Chile (Capsicum chinense) Induces Caspase-Independent Apoptosis on Human K562 Chronic Myeloid Leukemia Cells and Regulates Epigenetic Marks.

Cancer is a relevant health problem worldwide. In 2020, leukemias represented the 13th most commonly reported cancer cases worldwide but the 10th most likely to cause deaths. There has been a progressive increase in the efficacy of treatments for leukemias; however, these still generate important side effects, so it is imperative to search for new alternatives. Defensins are a group of antimicrobial peptides with activity against cancer cells. However, the cytotoxic mechanism of these peptides has been described mainly for animal defensins. This study shows that defensin γ-thionin (Capsicum chinense) is cytotoxic to the K562 leukemia cells with an IC50 = 290 µg/mL (50.26 µM) but not for human peripheral blood mononuclear cells. Results showed that γ-thionin did not affect the membrane potential; however, the peptide modified the mitochondrial membrane potential (ΔΨm) and the intracellular calcium release. In addition, γ-thionin induced apoptosis in K562 cells, but the activation of caspases 8 and 9 was not detected. Moreover, the activation of calpains was detected at one hour of treatment, suggesting that γ-thionin activates the caspase-independent apoptosis. Furthermore, the γ-thionin induced epigenetic modifications on histone 3 in K562 cells, increased global acetylation (~2-fold), and specific acetylation marks at lysine 9 (H3K9Ac) (~1.5-fold). In addition, γ-thionin increased the lysine 9 methylation (H3K9me) and dimethylation marks (H3K9me2) (~2-fold), as well as the trimethylation mark (H3K9me3) (~2-fold). To our knowledge, this is the first report of a defensin that triggers caspase-independent apoptosis in cancer cells via calpains and regulating chromatin remodelation, a novel property for a plant defensin.

Biological Activities of Organic Extracts of the Genus Aristolochia: A Review from 2005 to 2021.

Different ethnomedicinal studies have investigated the relationship between various phytochemicals as well as organic extracts and their bioactive aspects. Studies on biological effects are attributed to secondary metabolites such as alkaloids, phenolic compounds, and terpenes. Since there have been no reviews in the literature on the traditional, phytochemical, and ethnomedicinal uses of the genus Aristolochia so far, this article systematically reviews 141 published studies that analyze the associations between secondary metabolites present in organic extracts and their beneficial effects. Most studies found associations between individual secondary metabolites and beneficial effects such as anticancer activity, antibacterial, antioxidant activity, snake anti-venom and anti-inflammatory activity. The aim of this review was to analyze studies carried out in the period 2005-2021 to update the existing knowledge on different species of the genus Aristolochia for ethnomedicinal uses, as well as pharmacological aspects and therapeutic uses.

Production and characterization of a monoclonal antibody against the sialidase of Gardnerella vaginalis using a synthetic peptide in a MAP8 format.

Bacterial vaginosis is one of the most frequent vaginal infections. Its main etiological agent is Gardnerella vaginalis, which produces several virulence factors involved in vaginal infection and colonization, in particular, sialidase (SLD), a potential clinical biomarker that participates in immune response modulation and mucus degradation. The main objective of this work was the production and evaluation of a monoclonal antibody against G. vaginalis sialidase and its validation in immunoassays. For immunization of mice, a synthetic multiantigenic peptide was used, and hybridomas were generated. After fusion, hybridomas were evaluated for antibody production and cloned by limited dilution. One clone producing IgG1 was selected and characterized by indirect ELISA, dot blot, and Western blot, and we also tested clinical isolates and HeLa cells infected with G. vaginalis. The results showed that the anti-SLD antibody recognized a single protein of ~90 kDa that correlated with the estimated molecular weight of SLD. In addition, anti-SLD antibody recognized SLD from complete bacteria and from culture supernatants of infected Hela cells. In conclusion, our results showed that the anti-SLD antibody recognized SLD from different sources and could be considered a new tool for the diagnosis of bacterial vaginosis. KEY POINTS: • Anti-sialidase mAb was generated using a synthetic peptide • The mAb recognizes synthetic peptide and intact protein from multiple sources • The antibody was characterized by several immunological methods.

Peptides Derived from (RRWQWRMKKLG)2-K-Ahx Induce Selective Cellular Death in Breast Cancer Cell Lines through Apoptotic Pathway.

The effect on the cytotoxicity against breast cancer cell lines of the substitution of 26Met residue in the sequence of the Bovine Lactoferricin-derived dimeric peptide LfcinB (20-30)2: (20RRWQWRMKKLG30)2-K-Ahx with amino acids of different polarity was evaluated. The process of the synthesis of the LfcinB (20-30)2 analog peptides was similar to the original peptide. The cytotoxic assays showed that some analog peptides exhibited a significant cytotoxic effect against breast cancer cell lines HTB-132 and MCF-7, suggesting that the substitution of the Met with amino acids of a hydrophobic nature drastically enhances its cytotoxicity against HTB-132 and MCF-7 cells, reaching IC50 values up to 6 µM. In addition, these peptides have a selective effect, since they exhibit a lower cytotoxic effect on the non-tumorigenic cell line MCF-12. Interestingly, the cytotoxic effect is fast (90 min) and is maintained for up to 48 h. Additionally, through flow cytometry, it was found that the obtained dimeric peptides generate cell death through the apoptosis pathway and do not compromise the integrity of the cytoplasmic membrane, and there are intrinsic apoptotic events involved. These results show that the obtained peptides are extremely promising molecules for the future development of drugs for use against breast cancer.

Immunomodulatory molecules regulate adhesin gene expression in Staphylococcus aureus: Effect on bacterial internalization into bovine mammary epithelial cells.

Staphylococcus aureus is a major pathogen of subclinical bovine mastitis that usually is chronic and recurrent, which has been related to its ability to internalize into bovine mammary epithelial cells (bMECs). Previously, we reported that short and medium fatty acids and cholecalciferol reduce S. aureus internalization into pretreated-bMECs with these molecules suggesting a role as immunomodulatory agents. Hence, we assessed the role of sodium butyrate (NaB), sodium octanoate (NaO) and cholecalciferol on S. aureus adhesin expression and its internalization into bMECs. S. aureus pre-treated 2 h with 0.5 mM or 2 mM NaB showed a reduction in internalization into bMECs (∼35% and ∼55%; respectively), which coincided with a down-regulated expression of clumping factor B (ClfB). Also, the S. aureus internalization reduction by 2 mM NaB (2 h) agreed with a down-regulated expression of sdrC. Moreover, the 2 mM NaB (24 h) pre-treatment induced bacterial internalization (∼3-fold), which was related with an up-regulation of spa, clfB and sdrC genes. Also, NaO (0.25 mM and 1 mM) only reduced S. aureus internalization when bacteria were grown 2 h with this molecule but there was no relationship with adhesin expression. In addition, cholecalciferol (50 nM) reduced bacteria internalization at similar levels (∼50%) when bacteria were grown 2 and 24 h in broth supplemented with this compound, which correlated with spa and sdrC mRNA expression down-regulated at 2 h, and fnba and clfB mRNA expression decreased at 24 h. In conclusion, our data support the fact that fatty acids and cholecalciferol regulate adhesin gene expression as well as bacteria internalization in nonprofessional phagocytic cells, which may lead to development of anti-virulence agents for control of pathogens.

Anti-Inflammatory and Antimicrobial Effects of Estradiol in Bovine Mammary Epithelial Cells during Staphylococcus aureus Internalization.

17ß-Estradiol (E2), the predominant sexual hormone in females, is associated with the modulation of the innate immune response (IIR), and changes in its levels at parturition are related to intramammary infections, such as mastitis. In bovine mammary epithelial cells (bMECs), E2 regulates differentiation and proliferation, but its immunomodulatory functions have not been explored. Staphylococcus aureus is the predominant pathogen causing mastitis, which can persist intracellularly in bMECs. The aim of this work was to analyze whether E2 modulates the IIR of bMECs during S. aureus internalization. bMECs treated with E2 (50 pg/mL, 24 h) reduced bacteria internalization (~50%). The host receptors α5ß1 and TLR2 do not participate in this reduction. However, E2 activates ERα and modulates the IIR reducing the S. aureus induced-mRNA expression of TNF-α (~50%) and IL-1ß (90%). E2 also decreased the secretion of these cytokines as well as IL-6 production; however, in infected bMECs, E2 induced the secretion of IL-1ß. Furthermore, E2 upregulates the expression of the antimicrobial peptides DEFB1, BNBD5, and psoriasin S100A7 (~5-, 3-, and 6-fold, resp.). In addition, E2 induced the production of antimicrobial compounds in bMEC culture medium, which, together with the modulation of the IIR, could be related to the reduction of S. aureus internalization.

A new allele of γ-kafirin gene coding for a protein with high lysine content in Mexican white sorghum germplasm.

BACKGROUND: Low protein digestibility and lysine content of white sorghum grain limit its use as a foodstuff. The increase in γ-kafirin cross-linking, has an important role in the reduction of protein digestibility. The objective of this study was to characterize the γ-kafirin gene in 12 Mexican tannin-free white sorghum genotypes and its relationship with protein digestibility and lysine content. RESULTS: Two alleles of γ-kafirin gene were identified: alleles 1 and 7. The predicted amino acid sequence of allele 7 showed seven point mutations; six were silent, and one missense (C235G), causing the substitution P79A in the deduced amino acid sequence. In silico analysis showed that γ-kafirin codified by allele 1 has five α-helixes without disulfide bonds, while γ-kafirin coding by allele 7 has four α-helixes and three disulfide bonds. Genotypes with allele 7 had higher lysine content than those with allele 1, showing no differences in the kafirin electrophoretic profile, neither a correlation with the protein content nor the in vitro pepsin digestibility. CONCLUSIONS: Mexican tannin-free white sorghum genotypes showed two γ-kafirin alleles, 1 and 7. Allele 7 was associated with higher lysine content; in silico analysis showed that the substitution of P79A in this allele could modify γ-kafirin secondary structure. © 2015 Society of Chemical Industry.

A plasmid-encoded UmuD homologue regulates expression of Pseudomonas aeruginosa SOS genes.

The Pseudomonas aeruginosa plasmid pUM505 contains the umuDC operon that encodes proteins similar to error-prone repair DNA polymerase V. The umuC gene appears to be truncated and its product is probably not functional. The umuD gene, renamed umuDpR, possesses an SOS box overlapped with a Sigma factor 70 type promoter; accordingly, transcriptional fusions revealed that the umuDpR gene promoter is activated by mitomycin C. The predicted sequence of the UmuDpR protein displays 23 % identity with the Ps. aeruginosa SOS-response LexA repressor. The umuDpR gene caused increased MMC sensitivity when transferred to the Ps. aeruginosa PAO1 strain. As expected, PAO1-derived knockout lexA- mutant PW6037 showed resistance to MMC; however, when the umuDpR gene was transferred to PW6037, MMC resistance level was reduced. These data suggested that UmuDpR represses the expression of SOS genes, as LexA does. To test whether UmuDpR exerts regulatory functions, expression of PAO1 SOS genes was evaluated by reverse transcription quantitative PCR assays in the lexA- mutant with or without the pUC_umuD recombinant plasmid. Expression of lexA, imuA and recA genes increased 3.4-5.3 times in the lexA- mutant, relative to transcription of the corresponding genes in the lexA+ strain, but decreased significantly in the lexA- /umuDpR transformant. These results confirmed that the UmuDpR protein is a repressor of Ps. aeruginosa SOS genes controlled by LexA. Electrophoretic mobility shift assays, however, did not show binding of UmuDpR to 5' regions of SOS genes, suggesting an indirect mechanism of regulation.

Non-classical effects of prolactin on the innate immune response of bovine mammary epithelial cells: Implications during Staphylococcus aureus internalization.

Staphylococcus aureus has the ability to invade mammary epithelial cells (bMECs) causing mastitis. This event depends primarily on the α5ß1 integrin in the host cell. In addition, bMECs are a target for the hormone prolactin (PRL), which can regulate ß1 integrin-dependent actions related to differentiation and lactation. Previously, we demonstrated that bovine PRL (bPRL, 5 ng/ml) stimulates S. aureus internalization into bMECs. TLR2 is important during S. aureus infections, but its activation by PRL has not yet been established. The objective of this study was to determine the role of α5ß1 integrin and TLR2 during S. aureus internalization into bMECs stimulated with bPRL. We demonstrated that the prolactin-stimulated internalization of S. aureus decreases in response to the blockage of α5ß1 integrin (∼ 80%) and TLR2 (∼ 80%). bPRL increases the membrane abundance (MA) of α5ß1 integrin (∼ 20%) and induces TLR2 MA (∼ 2-fold). S. aureus reduces the α5ß1 integrin MA in bMECs treated with bPRL (∼ 75%) but induces TLR2 MA in bMECs (∼ 3-fold). Bacteria and bPRL did not modify TLR2 MA compared with the hormone alone. S. aureus induces the activation of the transcription factor AP-1, which was inhibited in bMECs treated with bPRL and infected. In general, bPRL induces both pro- and anti-inflammatory responses in bMECs, which are abated in response to bacterial challenge. Interestingly, the canonical Stat-5 transcription factor was not activated in the challenged bMECs and/or treated with bPRL. Taken together, these results support novel functions of prolactin as a modulator of the innate immune response that do not involve the classical prolactin pathway.

Modulation of the inflammatory response of bovine mammary epithelial cells by cholecalciferol (vitamin D) during Staphylococcus aureus internalization.

Vitamin D is an immunomodulator that exerts anti-inflammatory effects. In this work, the effects of cholecalciferol, a vitamin D precursor, on the inflammatory response of bovine mammary epithelial cells (bMECs) during the internalization of Staphylococcus aureus were analyzed. Cholecalciferol and S. aureus inhibited TLR2 mRNA expression, but cholecalciferol differentially modulated the TLR2 membrane abundance. In fact, 50 nM cholecalciferol inhibited the TLR2 membrane abundance in bMECs infected with S. aureus, and this concentration also exerted the highest inhibitory effect on internalization. Cholecalciferol down-regulated the mRNA expression of TNF-α and IL-1ß and up-regulated that of RANTES and IL-10 but did not modify IL-6 and IL-8 expression. S. aureus strongly induced the mRNA expression of TNF-α, RANTES and IL-10 and inhibited IL-8 expression. Interestingly, cholecalciferol pre-treatments inhibited the bacterial-induced expression of TNF-α, IL-1ß, RANTES and IL-10. In conclusion, cholecalciferol differentially regulates the inflammatory response of bMECs during S. aureus internalization and may be an effective innate immunity modulator in mammary gland tissues.

Evaluating the In Vitro Activity and Safety of Modified LfcinB Peptides as Potential Colon Anticancer Agents: Cell Line Studies and Insect-Based Toxicity Assessments.

Anticancer peptides are increasingly being considered as alternative treatments for cancer due to their potency, selectivity, and low toxicity. Previously, the peptide LfcinB (21-25)Pal showed in vitro anticancer effects against the Caco-2 colon cancer cell line (half-maximal inhibitory concentration (IC50): 86 µM). In this study, we developed modifications to the peptide sequence to increase its anticancer activity. Sequence modifications were made such as the inclusion of amino hexanoic acid (Ahx), N-terminal biotinylation, acetylation, and substitutions of Orn for Arg and/or d-Arg by l-Arg. The molecules were synthesized using manual solid-phase peptide synthesis (SPPS), and their synthetic feasibility (SAScore) ranged from 6.2 to 7.6. The chromatographic purities of the synthesized peptides were greater than 89%. We found that Ahx-RWQWRWQWR and RWQWRWQW-Orn showed activity against both Caco-2 and HT-29 cell lines and decreased IC50 values by approx. 50% in Caco-2 cells (IC50: 40 µM) when compared to the parent peptide RWQWRWQWR. Moreover, the modified peptides demonstrated lower hemolytic effects, with values <10% at 200 µg/mL. Toxicity was assessed using the Galleria mellonella model and the half-maximal lethal dose (LD50) for the best peptides was >100 mg/kg, indicating that their toxicity is classified as moderately toxic or lower. In contrast, cisplatin showed an LD50 of 13 mg/Kg. The designed anticancer peptides presented good in vitro activity and low toxicity, making them promising molecules for future drug development studies.

Expression of antimicrobial peptides by bovine endothelial cells.

In this work, we explore if Gram-positive bacteria as Staphylococcus aureus or Gram-negative bacteria components as LPS, can induce the expression of seven antimicrobial peptides (AP) in an immortalized bovine umbilical vein endothelial cell line (BUVEC). By qPCR we determined the constitutive expression of all the AP evaluated. The stimulation with S. aureus or LPS induced the expression of lingual antimicrobial peptide (LAP), bovine ß-defensin 1 (DEFB1) and bovine neutrophil ß-defensin 4 (BNBD4). This expression was regulated by the autocrine production of tumor necrosis factor-α (TNF-α), indicating that bovine endothelial cells (EC) can play a more active role during infection.

PaDef (Persea americana var. drymifolia), a Plant Antimicrobial Peptide, Triggers Apoptosis, and Induces Global Epigenetic Modifications on Histone 3 in an Acute Lymphoid Leukemia Cell Line.

In recent years, it has been recognized that epigenetic alterations play an important role in the development and maintenance of cancer, including leukemias. Furthermore, it is known that these alterations are involved in the emergence of resistance to conventional chemotherapeutics. Consequently, molecules with an anticancer activity whose activity is ruled by epigenetic modifications are attractive to search for new therapies against cancer. The plant antimicrobial peptides have been widely evaluated as molecules with anticancer activity; however, the analysis of the epigenetic regulation induced by these molecules associated with this activity is scarce and still is an unexplored field. In this work, we show that the PaDef defensin, a plant antimicrobial peptide from Mexican avocado fruit (Persea americana var. drymifolia) is cytotoxic for Jurkat cell line from acute lymphoid leukemia cells, through an apoptotic process. PaDef inhibited cell viability in a concentration-dependent manner, with an IC50 = 47.3 µM. Treatment of Jurkat cells with PaDef (IC50) induced cell death by apoptosis dependent on caspases 8 and 9; besides, it was related to an increase in the production of reactive oxygen species and the loss of mitochondrial membrane potential. Interestingly, the inhibition of caspase activation by inhibitors of caspases 8 and 9 does not revert the reduction in viability, suggesting that other mechanisms, in addition to caspase activity, could be participating in the PaDef cytotoxic effect. Also, the modifications in the histone 3 tails induced by PaDef in Jurkat cells were evaluated, specifically acetylation and methylation. PaDef increased global histone 3 acetylation and lysine 9 specific marks (2-fold and up to 4-fold, respectively). These effects correlated with the reduction of the Histone Deacetylase activity (HDAC, ∼50%). Based on methylation marks, PaDef treatment increased lysine 9 di- and tri-methylation tags (2-fold in both cases). The epigenetic modulation induced by PaDef on Jurkat cells could be related to the chromatin compaction-decompaction promoting gene expression or repression; however, further studies are necessary to correlate these marks with the transcription of specific genes. Therefore, the study of new molecules that may have anticancer activity through epigenetic modulation is interesting.

Staphylococcus aureus inhibits nuclear factor kappa B activation mediated by prolactin in bovine mammary epithelial cells.

The hormone prolactin (PRL) regulates differentiation and lactation in the bovine mammary epithelium. This tissue is especially prone to contracting mastitis, a disease characterized by an inflammatory response in the mammary gland. Staphylococcus aureus is the infectious agent primarily responsible for mastitis. In a previous study, we have shown that bovine PRL (bPRL) stimulates S. aureus internalization in bovine mammary epithelial cells (bMECs) by regulating several host innate immune elements, which are often modulated by nuclear factor kappa B (NF-κB). However, it is unknown whether the activation of the NF-κB transcription factor is regulated by bPRL during S. aureus internalization. The objective of this study was to determine the role of NF-κB in bPRL-stimulated bMECs during S. aureus internalization. Our results showed that bPRL (5 ng/ml) induced NF-κB activation in bMECs; however, it was inhibited by S. aureus in presence of the hormone. When we blocked NF-κB activation with acetylsalicylic acid, we detected an inhibition in S. aureus internalization (48%) in bPRL-stimulated bMECs. The infection-induced inhibition of NF-κB activation in the presence of bPRL correlates with the downregulation in bPRL-mediated tumor necrosis factor (TNF)-α (27%) and tracheal antimicrobial peptide (TAP, 70%) mRNA expression and nitric oxide (NO) production in bMECs. We also detected an inhibition in the expression of the bPRL target gene κ-casein (50%) under these conditions. Interestingly, these effects are not achieved through increased PRL receptor expression (PRLR), as it was inhibited (48%) compared to control cells. In conclusion, NF-κB activation in bMECs is inhibited by S. aureus in the presence of bPRL, suggesting a mechanism by which the host innate immune response may be compromised during subclinical mastitis.

In vitro cytotoxic potential of extracts from Aristolochia foetida Kunth against MCF-7 and bMECs cell lines.

The aim of this study was to evaluate the cytotoxic potential of Aristolochia foetida Kunth. Stems and leaves of A. foetida Kunth (Aristolochiaceae) have never been investigated pharmacologically. Recent studies of species of the Aristolochiaceae family found significant cytotoxic activities. Hexane, dichloromethane, ethyl acetate and methanol extracts were analyzed by 1H NMR and GC-MS to know the metabolites in each extract. In GC-MS analysis, the main compounds were methyl hexadecanoate (3); hexadecanoic acid (4); 2-butoxyethyl dodecanoate (9); ethyl hexadecanoate (20); methyl octadeca-9,12,15-trienoate (28) and (9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid (40). The results showed a significant reduction in cell viability of the MCF-7 (breast cancer) cell line caused by organic extracts in a dose-dependent manner. The cytotoxicity activity of the dichloromethane extract from the stems (DSE) showed IC50 values of 45.9 µg/mL and the dichloromethane extract of the leaves (DLE) showed IC50 values of 47.3 µg/mL. DSE and DLE had the highest cytotoxic potential in an in vitro study against the MCF-7 cell line and non-tumor cells obtained from the bovine mammary epithelial (bMECs). DSE and DLE induced a loss in mitochondrial membrane potential (ΔΨm) and can cause cell death by apoptosis through the intrinsic pathway in the MCF-7 cell line. DSE and DLE are cytotoxic in cancer cells and cause late apoptosis. Higher concentrations of DSE and DLE are required to induce a cytotoxic effect in healthy mammary epithelial cells. This is the first report of the dichloromethane extract of A. foetida Kunth that induces late apoptosis in MCF-7 cancer cells and may be a candidate for pharmacological study against breast cancer.

Plant Defensin γ-Thionin Induces MAPKs and Activates Histone Deacetylases in Bovine Mammary Epithelial Cells Infected With Staphylococcus aureus.

Defensins are an important group of host defense peptides. They have immunomodulatory properties, which have been mainly described for mammal defensins, but similar effects for plant defensins remain unknown. Previously, we showed that the defensin γ-thionin (Capsicum chinense) reduces Staphylococcus aureus internalization into bovine mammary epithelial cells (bMECs) while inducing Toll-like receptor 2 (TLR2), modulating the inflammatory response. Here, we analyze the effect of γ-thionin on the TLR2 pathway in bMECs infected with S. aureus and determine if it modulates epigenetic marks. Pre-treated bMECs with γ-thionin (100 ng/ml) reduced the basal activation of p38 and ERK1/2 (~3-fold), but JNK was increased (~1.5-fold). Also, infected bMECs induced p38, but this effect was reversed by γ-thionin, whereas ERK1/2 was reduced by infection but stimulated by γ-thionin. Likewise, γ-thionin reduced the activation of Akt kinase ~50%. Furthermore, γ-thionin induced the activation of transcriptional factors of inflammatory response, highlighting EGR, E2F-1, AP-1, and MEF, which were turned off by bacteria. Also, γ-thionin induced the activation of histone deacetylases (HDACs, ~4-fold) at 24 h in infected bMECs and reduced LSD1 demethylase (HDMs, ~30%) activity. Altogether, these results demonstrated the first time that a plant defensin interferes with inflammatory signaling pathways in mammalian cells.

Analysis of virulence traits of Staphylococcus aureus isolated from bovine mastitis in semi-intensive and family dairy farms.

BACKGROUND: Staphylococcus aureus is one of the main microorganisms that causes bovine mastitis, and its well-known virulence characteristics and interactions with the environment are used to aid the design of more efficient therapies. OBJECTIVES: To determine whether the virulence traits, such as antibiotic resistance and biofilm-forming and internalization abilities, of S. aureus isolated from bovine mastitis are related to dairy production system types. METHODS: The study was performed in the Mexican states of Guanajuato and Michoacan. Semi-intensive dairy farms (SIDFs) and family dairy farms (FDFs) (454 and 363 cows, respectively) were included. The 194 milk samples from mastitis affected quarters were collected and 92 strains of S. aureus were isolated and identified by biochemical and molecular tests. Antibiotic resistance, biofilm and internalization assays were performed on 30 randomly selected isolated strains to determine virulence traits, and these strains were equally allocated to the 2 dairy production systems. RESULTS: All 30 selected strains displayed a high degree of resistance (50%-91.7%) to the antibiotics tested, but no significant difference was found between SIDF and FDF isolates. S. aureus strains from SIDFs had an average biofilm forming capacity of up to 36% (18.9%-53.1%), while S. aureus strains from FDFs registered an average of up to 53% (31.5%-77.8%) (p > 0.05). Internalization assays revealed a higher frequency of internalization capacity for strains isolated from FDFs (33.3%) than for those isolated from SIDFs (6.7%) (p > 0.05). fnbpA gen was detected in 46.6% of FDF strains and 33.3% of SIDF strains, and this difference was significant (p < 0.05). CONCLUSIONS: Our findings show that the virulence traits of S. aureus isolates analyzed in this study, depend significantly on several factors, such as phenotype, genotype, and environmental conditions, which are significantly related to dairy production system type and daily management practices.

Sodium butyrate inhibits Staphylococcus aureus internalization in bovine mammary epithelial cells and induces the expression of antimicrobial peptide genes.

A distinctive feature of bovine milk fat is the presence of butyrate, molecule with recognized antimicrobial and antiinflammatory properties. Bovine mastitis is a pathology characterized by inflammatory and infectious processes; however, the role of sodium butyrate on Staphylococcus aureus infection in mammary epithelium has not been studied. In this work we assess the role of sodium butyrate on the invasion of bovine mammary epithelial cells (bMEC) by S. aureus responsible of mastitis and on the expression of antimicrobial peptide genes. Our data show that sodium butyrate (0.25-0.5mM) reduces approximately 50% the internalization of S. aureus (ATCC 27543) into bMEC. By RT-PCR analysis, we showed that sodium butyrate is able to up-regulate the expression of tracheal antimicrobial peptide (TAP), beta-defensin and inducible nitric oxide synthase (iNOS) mRNAs, as well as nitric oxide production. Also, sodium butyrate and infection increased acetylation of histone H3 in bMEC. These results indicate that sodium butyrate could be effective to modulate innate immune gene expression in mammary gland that leads to a better defense against bacterial infection. To our knowledge, this is the first report that shows a role of sodium butyrate during the internalization of S. aureus into bMEC.