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.

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.

Non-natural amino acids into LfcinB-derived peptides: effect in their (i) proteolytic degradation and (ii) cytotoxic activity against cancer cells.

The dimeric peptide 26[F]: (RRWQWRFKKLG)2-K-Ahx has exhibited a potent cytotoxic effect against breast cancer cell lines, with position 26 (F) being the most relevant for anti-cancer activity. In this investigation, six analogues of the 26[F] peptide were synthesized in which the 26th position was replaced by non-natural hydrophobic amino acids, finding that some modifications increased the resistance to proteolytic degradation exerted by trypsin or pepsin. Additionally, these modifications increased the cytotoxic effect against breast cancer cells and generated cell death mediated by apoptosis pathways, activating caspases 8 and 9, and did not compromise the integrity of the cytoplasmic membrane. Finally, it was found that the modified peptides have a broad spectrum of action, since they also have a cytotoxic effect against the HeLa human cervical cancer cell line. Peptide 26[F] was inoculated in mice by ip administration and the lethal dose 50 (LD50) was between 70 and 140 mg kg-1. While for the 26[1-Nal]: (RRWQWR-1-Nal-KKLG)2-K-Ahx peptide, a dose-response test was performed, and the survival rate was 100%. These results suggested that these peptides are safe in this animal model and could be considered as promissory to develop a treatment against breast cancer.

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.

The plant defensins PaDef and γ-thionin inhibit the endothelial cell response to VEGF.

Angiogenesis is involved in wound repair and tissue maintenance but is associated with diverse diseases. Pro-angiogenic factors such as vascular endothelial growth factor (VEGF) regulate this process. Therefore, searching for treatments to inhibit or promote angiogenesis is attractive. Reports from our group showed that plant antimicrobial peptides (PAPs) PaDef from avocado and γ-thionin from habanero pepper are cytotoxic on cancer cells. However, their functions as angiogenic regulators are unknown. In this work, we evaluate the effect of PaDef and γ-thionin on the angiogenic processes of two different endothelial cell lines: bovine endothelial cells (BUVEC) and the human endothelial cell line EA.hy926. The results showed that VEGF (10 ng/mL) stimulated the BUVEC (40 ± 7 %) and EA.hy926 cell proliferation (30 ± 9 %); however, peptides (5-500 ng/mL) reverted this effect. Besides, VEGF increased the migration of BUVEC (20 ± 8 %) and EA.hy926 cells (50 ± 6 %), but both PAPs (5 ng/mL) inhibited the VEGF stimulus (100 %). Furthermore, DMOG 50 µM (an inhibitor of HIF-hydroxylase) was used in BUVEC and EA.hy926 cells to determine the effect of hypoxia on VEGF and peptide activities. The DMOG reverted the inhibitory action of both peptides (100 %), indicating that peptides act through a HIF-independent pathway. Also, the PAPs do not affect the tube formation but decrease it in EA.hy926 cells stimulated with VEGF (100 %). Additionally, docking assays showed a possible interaction between PAPs and the VEGF receptor. These results suggest that plant defensins PaDef and γ-thionin are potential angiogenic modulators of the VEGF activity on endothelial cells.

Comparative virulence of the worldwide ST19 and emergent ST213 genotypes of Salmonella enterica serotype Typhimurium strains isolated from food.

Salmonella enterica Typhimurium represents one of the most frequent causal agents of food contamination associated to gastroenteritis. The sequence type ST19 is the founder and worldwide prevalent genotype within this serotype, but its replacement by emerging genotypes has been recently reported. Particularly, the ST213 genotype has replaced it as the most prevalent in clinical and contaminated food samples in Mexico and has been recently reported in several countries. In this study, the in vitro and in vivo virulence of ST213 and ST19 strains isolated from food samples in Mexico was evaluated. Three out of the five analyzed ST213 strains, showed a greater internalization capacity and increased secretion of interleukins IL-8 and IL-6 of Caco-2 cells than the ST19 strains. Microbiological counts in feces and tissues showed the ability of all strains tested to establish infection in the rat model. The ST213 strains also caused histopathological damage, characteristic of gastroenteritis in Wistar rats. In contrast to the in vitro result, one of the ST19 strains showed marked damage in the test animals. The ST213 genotype strains showed in vitro and in vivo virulence variability, but significantly higher than the observed in the ST19 genotype strains, thus such emergent genotype represents a public health concern.

Platelet Reactivity and Inflammatory Phenotype Induced by Full-Length Spike SARS-CoV-2 Protein and Its RBD Domain.

A state of immunothrombosis has been reported in COVID-19. Platelets actively participate in this process. However, little is known about the ability of SARS-CoV-2 virus proteins to induce platelet activity. Platelet-rich plasma (PRP) was incubated with spike full-length protein and the RBD domain in independent assays. We evaluated platelet activation through the expression of P-selectin and activation of glicoprotein IIbIIIa (GP IIbIIIa), determined by flow cytometry and the ability of the proteins to induce platelet aggregation. We determined concentrations of immunothrombotic biomarkers in PRP supernatant treated with the proteins. We determined that the spike full-length proteins and the RBD domain induced an increase in P-selectin expression and GP IIbIIIa activation (p < 0.0001). We observed that the proteins did not induce platelet aggregation, but favored a pro-aggregating state that, in response to minimal doses of collagen, could re-establish the process (p < 0.0001). On the other hand, the viral proteins stimulated the release of interleukin 6, interleukin 8, P-selectin and the soluble fraction of CD40 ligand (sCD40L), molecules that favor an inflammatory state p < 0.05. These results indicate that the spike full-length protein and its RBD domain can induce platelet activation favoring an inflammatory phenotype that might contribute to the development of an immunothrombotic state.

Antifungal Activity of Avocado Seed Recombinant GASA/Snakin PaSn.

The avocado fruit (Persea americana) has become a significant fruit in the human diet for its nutritional properties. However, the seed is a source of bioactive molecules and has been poorly utilized. Previously, we reported that the PaSn gene is expressed in the avocado seeds, a cysteine-rich antimicrobial peptide (GASA/Snakin), and demonstrated its antibacterial activity. In this work, we report the recombinant production of PaSn in the Escherichia coli system and evaluate its antifungal activity against plant and human pathogen fungi. The recombinant peptide showed antifungal activity at 200 µg/mL against phytopathogens Colletotrichum gloeosporioides and Fusarium oxysporum and human pathogens Candida albicans and C. glabrata. Our results demonstrate the usefulness of a prokaryotic expression system for avocado antimicrobial peptide production. In conclusion, the snakin PaSn could be helpful in the control of postharvest avocado and other fruits' fungal diseases and human fungal pathogens.

Prolactin regulates H3K9ac and H3K9me2 epigenetic marks and miRNAs expression in bovine mammary epithelial cells challenged with Staphylococcus aureus.

Epigenetic mechanisms are essential in the regulation of immune response during infections. Changes in the levels of reproductive hormones, such as prolactin, compromise the mammary gland's innate immune response (IIR); however, its effect on epigenetic marks is poorly known. This work explored the epigenetic regulation induced by bovine prolactin (bPRL) on bovine mammary epithelial cells (bMECs) challenged with Staphylococcus aureus. In this work, bMECs were treated as follows: (1) control cells without any treatment, (2) bMECs treated with bPRL (5 ng/ml) at different times (12 or 24 h), (3) bMECs challenged with S. aureus for 2 h, and (4) bMECs treated with bPRL at different times (12 or 24 h), and then challenged with S. aureus 2 h. By western blot analyses of histones, we determined that the H3K9ac mark decreased (20%) in bMECs treated with bPRL (12 h) and challenged with S. aureus, while the H3K9me2 mark was increased (50%) in the same conditions. Also, this result coincided with an increase (2.3-fold) in HDAC activity analyzed using the cellular histone deacetylase fluorescent kit FLUOR DE LYS®. ChIP-qPCRs were performed to determine if the epigenetic marks detected in the histones correlate with enriched marks in the promoter regions of inflammatory genes associated with the S. aureus challenge. The H3K9ac mark was enriched in the promoter region of IL-1ß, IL-10, and BNBD10 genes (1.5, 2.5, 7.5-fold, respectively) in bMECs treated with bPRL, but in bMECs challenged with S. aureus it was reduced. Besides, the H3K9me2 mark was enriched in the promoter region of IL-1ß and IL-10 genes (3.5 and 2.5-fold, respectively) in bMECs challenged with S. aureus but was inhibited by bPRL. Additionally, the expression of several miRNAs was analyzed by qPCR. Let-7a-5p, miR-21a, miR-30b, miR-155, and miR-7863 miRNAs were up-regulated (2, 1.5, 10, 1.5, 3.9-fold, respectively) in bMECs challenged with S. aureus; however, bPRL induced a down-regulation in the expression of these miRNAs. In conclusion, bPRL induces epigenetic regulation on specific IIR elements, allowing S. aureus to persist and evade the host immune response.

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.

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.

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.

Cytotoxicity of a Lipid-Rich Extract from Native Mexican Avocado Seed (Persea americana var. drymifolia) on Canine Osteosarcoma D-17 Cells and Synergistic Activity with Cytostatic Drugs.

Osteosarcoma is the most common malignant bone tumor in both children and dogs. It is an aggressive and metastatic cancer with a poor prognosis for long-term survival. The search for new anti-cancer drugs with fewer side effects has become an essential goal for cancer chemotherapy; in this sense, the bioactive compounds from avocado have proved their efficacy as cytotoxic molecules. The objective of this study was to determine the cytotoxic and antiproliferative effect of a lipid-rich extract (LEAS) from Mexican native avocado seed (Persea americana var. drymifolia) on canine osteosarcoma D-17 cell line. Also, the combined activity with cytostatic drugs was evaluated. LEAS was cytotoxic to D-17 cells in a concentration-dependent manner with an IC50 = 15.5 µg/mL. Besides, LEAS induced caspase-dependent cell apoptosis by the extrinsic and intrinsic pathways. Moreover, LEAS induced a significant loss of mitochondrial membrane potential and increased superoxide anion production and mitochondrial ROS. Also, LEAS induced the arrest of the cell cycle in the G0/G1 phase. Finally, LEAS improved the cytotoxic activity of cisplatin, carboplatin, and in less extension, doxorubicin against the canine osteosarcoma cell line through a synergistic effect. In conclusion, avocado could be a potential source of bioactive molecules in the searching treatments for osteosarcoma.

Defensin c-thionin from Capsicum chinense improves butyrate cytotoxicity on human colon adenocarcinoma cell line Caco-2

BACKGROUND: Butyrate is a histone deacetylase inhibitor that induces apoptosis and inhibits cell proliferation of colorectal cancer cells. To improve its anticancer activity, butyrate has been evaluated mixed with drugs and different molecules. Plant antimicrobial peptides are attractive anticancer alternative molecules because they show selective cytotoxic activity against different cancer cell lines. In this work, we explore if the plant defensin c-thionin (Capsicum chinense) can improve butyrate activity on Caco-2 cell line and we also determined the mechanism of death activated. RESULTS: The combined treatment of c-thionin (3.5 mM) and butyrate (50 mM) showed higher cytotoxicity on Caco-2 cells with respect to single treatments. Also, the combined treatment reduced cell proliferation and exhibited a higher rate of apoptosis than single treatments. Combined treatment induced caspases 8 and 9 activation to an extent comparable with that of butyrate while c-thionin did not activate caspases. Additionally, reactive oxygen species generation preceded the onset of apoptosis, and superoxide anion production was higher in cells treated with the combined treatment. CONCLUSIONS: The c-thionin from Habanero chili pepper improved the butyrate cytotoxicity on Caco-2 cells. This effect occurred through apoptosis induction associated with reactive oxygen species production. Therefore, the combination of butyrate with cytotoxic antimicrobial peptides could be an attractive strategy for cancer therapy.

Bioactive Molecules From Native Mexican Avocado Fruit (Persea americana var. drymifolia): A Review.

Avocado (Persea americana Mill.) is a tree native from central and eastern México that belongs to the Lauraceae family. Avocado has three botanical varieties known as Mexican (P. americana var. drymifolia), West Indian (P. americana var. americana), and Guatemalan (P. americana var. guatemalensis). It is an oil-rich fruit appreciated worldwide because of its nutritional value and the content of bioactive molecules. Several avocado molecules show attractive activities of interest in medicine. Avocado fatty acids have beneficial effects on cardiovascular disease risk factors. Besides, this fruit possesses a high content of carotenoids and phenolic compounds with possible antifungal, anti-cancer and antioxidant activities. Moreover, several metabolites have been reported with anti-inflammatory effects. Also, an unsaponifiable fraction of avocado in combination with soybean oil is used for the treatment of osteoarthritis. The Mexican variety is native from México and is characterized by the anise aroma in leaves and by small thin-skinned fruits of rich flavor and excellent quality. However, the study of the bioactive molecules of the fruit has not been addressed in detail. In this work, we achieved a literature review on the inflammatory, immunomodulatory and cytotoxic properties of long-chain fatty acids and derivatives from Mexican avocado seed. Also, the antioxidant and anti-inflammatory properties of the oil extracted from the avocado seed are referred. Finally, the antimicrobial, immunomodulatory, and cytotoxic activities of some antimicrobial peptides expressed in the fruit are reviewed.

Molecular characterization of bacterial microbiota associated with infectious bovine keratoconjunctivitis in Michoacán, Mexico / Caracterización molecular de la microbiota bacteriana asociada con queratoconjuntivitis infecciosa bovina en Michoacán, México / Caracterização molecular da microbiota bacteriana associada à ceratoconjuntivite bovina infecciosa em Michoacán, México

Abstract Background: The most common ocular disease affecting cattle worldwide is infectious bovine keratoconjunctivitis (IBK), which has been associated with Moraxella bovis bacterium. Objective: To report the molecular characterization of the ocular bacterial microbiota and its relation to IBK in cattle in two dairy regions in Michoacán, Mexico. Methods: A total population of 761 bovines were evaluated, of which 17 (2.23%) showed symptoms of IBK. Thirty-eight bacterial isolates from ocular samples of bovines with IBK were characterized by Gram-staining and antimicrobial sensitivity. In addition, isolates were identified by sequence comparisons of the 16S ribosomal gene. Results: The genus Moraxella was one of the most abundant bacteria and M. bovoculi was the most predominant species. Conclusion: The bacterial isolates identified in eye lesions of cattle and associated to IBK are diverse. To the author´s knowledge, this is the first study on the subject in Mexico; therefore, more research is needed to estimate the incidence of IBK and determine its associated microbiota.

Resumen Antecedentes: la enfermedad ocular más común que afecta al ganado en todo el mundo es la queratoconjuntivitis infecciosa bovina (IBK), que se ha asociado con la bacteria Moraxella bovis. Objetivo: reportar la caracterización molecular de la microbiota bacteriana ocular y su relación con IBK en ganado de dos regiones lecheras en Michoacán, México. Métodos: se evaluó una población total de 761 bovinos de los cuales 17 (2,23%) mostraron síntomas de IBK. Se obtuvieron treinta y ocho aislamientos bacterianos de muestras oculares de bovinos con IBK, los cuales se caracterizaron por tinción de Gram y sensibilidad antimicrobiana. Además, los aislamientos se identificaron mediante comparaciones de secuencias del gen ribosomal 16S. Resultados: el género Moraxella fue una de las bacterias más abundantes y M. bovoculi fue la especie más predominante. Conclusión: los aislamientos bacterianos identificados en lesiones oculares de bovinos y asociados a IBK son diversos. Hasta donde sabemos, este es el primer estudio sobre el tema realizado en México; por lo tanto, es necesario ampliar esta investigación para estimar la incidencia de IBK y determinar la microbiota asociada con la misma.

Resumo Antecedentes: a doença ocular mais comum que afeta o gado no mundo é a ceratoconjuntivite bovina (IBK), que tem sido associada à bactéria Moraxella bovis. Objetivo: relatar a caracterização molecular da microbiota bacteriana ocular e sua relação com a IBK em bovinos de duas regiões leiteiras de Michoacán, México. Métodos: foi avaliada uma população total de 761 bovinos, más apenas 17 (2,23%) apresentaram sintomas de IBK. Trinta e oito isolados bacterianos de amostras de olho bovino com IBK foram caracterizados por coloração de Gram e sensibilidade antimicrobiana. Além disso, os isolados foram identificados por comparação de sequências do gene ribossômico 16S. Resultados: a microbiota bacteriana associada à IBK foi diversa, sendo o gênero Moraxella uma das mais abundantes e M. bovoculi a espécie predominante. Conclusão: de acordo com o conhecimento dos autores, este é o primeiro estudo sobre o tema no México até o momento, portanto é necessário expandir essa pesquisa para estimar a incidência de IBK e determinar a microbiota associada à mesma.

Long-term activation of hippocampal glial cells and altered emotional behavior in male and female adult rats after different neonatal stressors.

Early life stress increases the risk of developing psychiatric diseases in adulthood. Severe neonatal infections can also contribute to the development of affective illnesses. Stress and infections both trigger the immediate activation of the neuroimmune system. We compared the long-term effects of neonatal single or combined stress-immune challenges on emotional behavior and glial cell responses in the hippocampus. Male and female Sprague Dawley rats were randomly allocated across four conditions: (1) control + vehicle; (2) maternal separation (MS, 3 h/day on postnatal days [PN] 1-14) + vehicle; (3) control + lipopolysaccharide (LPS, 0.5. mg/kg, PN14); (4) MS + LPS. The rats' behaviors were analyzed from PN120 in males and from PN150 in diestrous females. LPS, but not MS, increased anxiety-like behavior in male rats; however, in females, it increased with both challenges. Depressive-like behavior increased after MS-but not LPS-in males and females. Combined stressors increased depressive-like behavior in both sexes. All stressors promoted microglial activation in CA3 and hilus in males and females. MS and LPS increased the astrocytic density within the male hilus, but LPS only increased it in CA3. MS prevented the rise in astrocytic density with LPS. In females, MS reduced the astrocytic population of the hilus and CA3 areas. Taken together, the behavioral and glial cell responses to early life challenges are sex-dependent and cell-type specific. This suggests a sexual dimorphism in the nature of the adverse event faced. These results have implications for understanding the emergence of psychiatric illnesses.

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.

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.

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.