Characterization of the Effect of N-(2-Methoxyphenyl)-1-methyl-1H-benzimidazol-2-amine, Compound 8, against Leishmania mexicana and Its In Vivo Leishmanicidal Activity.

Chemotherapy currently available for leishmaniasis treatment has many adverse side effects and drug resistance. Therefore, the identification of new targets and the development of new drugs are urgently needed. Previously, we reported the synthesis of a N-(2-methoxyphenyl)-1-methyl-1H-benzimidazol-2-amine, named compound 8, with an IC50 value in the micromolar range against L. mexicana, it also inhibited 68.27% the activity of recombinant L. mexicana arginase. Herein, we report studies carried out to characterize the mechanism of action of compound 8, as well as its in vivo leishmanicidal activity. It was shown in our ultrastructural studies that compound 8 induces several changes, such as membrane blebbing, the presence of autophagosomes, membrane detachment and mitochondrial and kinetoplast disorganization, among others. Compound 8 triggers the production of ROS and parasite apoptosis. It reduced 71% of the parasite load of L. mexicana in an experimental model of cutaneous leishmaniasis in comparison with a control. Altogether, the data obtained suggest the potential use of compound 8 in the treatment of cutaneous leishmaniasis.

Rhodotorula mucilaginosa YR29 is able to accumulate Pb2+ in vacuoles: a yeast with bioremediation potential.

Microorganisms showed unique mechanisms to resist and detoxify harmful metals in response to pollution. This study shows the relationship between presence of heavy metals and plant growth regulator compounds. Additionally, the responses of Rhodotorula mucilaginosa YR29 isolated from the rhizosphere of Prosopis sp. growing in a polluted mine jal in Mexico are presented. This research carries out a phenotypic characterization of R. mucilaginosa to identify response mechanisms to metals and confirm its potential as a bioremediation agent. Firstly, Plant Growth-Promoting (PGP) compounds were assayed using the Chrome Azurol S (CAS) medium and the Salkowski method. In addition, to clarify its heavy metal tolerance mechanisms, several techniques were performed, such as optical microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) supplemented with assorted detectors. Scanning transmission electron microscopy (STEM) was used for elementary mapping of the cell. Finally, yeast viability after all treatments was confirmed by confocal laser scanning microscopy (CLSM). The results have suggested that R. mucilaginosa could be a PGP yeast capable of triggering Pb2+ biosorption (representing 22.93% of the total cell surface area, the heavy metal is encapsulated between the cell wall and the microcapsule), and Pb2+ bioaccumulation (representing 11% of the total weight located in the vacuole). Based on these results, R. mucilaginosa as a bioremediation agent and its wide range of useful mechanisms for ecological purposes are highlighted.

Detección de Chlamydia spp. en leucocitos de sangre periférica de ovinos sin aborto previo / Detection of Chlamydia spp. In Peripheral Blood Leukocytes in Ewes without any Previous Abortion

Resumen El aborto enzootico ovino es una enfermedad causada por Chlamydia abortus. Es considerada una zoonosis y una de las principales causas de pérdidas económicas en estas explotaciones. Este trabajo se enfocó en utilizar el cultivo de leucocitos de animales sin signos de abortos y la detección de anticuerpos para determinar la posible presencia de C. abortus en explotaciones de traspatio. Se obtuvieron 42 muestras de sangre periférica de ovejas de diferentes poblaciones. La detección de Chlamydia abortus se realizó mediante la tinción de Giemsa y la técnica de PCR. La detección de anticuerpos anti-C. abortus se dio mediante una técnica de ELISA comercial. Los resultados mostraron 21 muestras positivas mediante la técnica de PCR, de las cuales solo 10 fueron positivas mediante la técnica de Giemsa, mientras que 22 sueros mostraron anticuerpos anti-C. abortus. En este estudio el 38,1 % de las muestras fueron positivas a la infección por C. abortus, como se confirmó mediante PCR y serología. En conclusión, los leucocitos de sangre periférica pueden ser útiles para detectar una infección por Chlamydia spp. en explotaciones sin historial de abortos, con lo que se puede conocer la prevalencia real del aborto enzootico ovino en México.

Abstract The Ewes Enzootic Abortion is a disease caused byChlamydia abortus. It is deemed a zoonosis and one of the leading causes of financial losses in this type of business. This article focuses on using the culture of leukocytes from animals without any abortion symptoms and antibody detection to determine the potential presence ofC. abortusin backyard exploitations. Forty-two samples of peripheral blood were obtained from ewes in different populations. The detection ofChlamydia abortuswas carried out by using the Giemsa dye and PCR technique. Anti-C. Abortusantibody detection was performed through a commercial ELISA technique. Results showed 21 positive samples using the PCR, and only ten were positive according to the Giemsa dye, while 22 serum samples showed anti-C. abortusantibody. In this study, 38.1% of the samples were positive for theC. abortusinfection, as verified with the PCR and serology. In conclusion, peripheral blood leukocytes can be helpful to detect an infection caused byChlamydiaspp. Animal exploitation without any previous abortion allows knowing the real prevalence of ewes' enzootic abortion in Mexico.

Case Report: Formation of 3D Osteoblast Spheroid Under Magnetic Levitation for Bone Tissue Engineering.

Skeletal reconstruction is necessary in cases of bone defects created by tumors, trauma, and abnormalities. Regeneration of bone defects remains a critical problem, and current approaches are based on biocompatible scaffolds. Spheroids represent a simple 3D system since no supporting material is required for cell growth. Different techniques are used to generate spheroids, such as hanging drop, low-attachment plates, and magnetic nanoparticles. The idea of using magnetic nanoparticles is to cross-link through cell membrane overnight to create complex 3D cellular spheroid by using magnets to guide the cellular response. Herein, the current study aimed to achieve 3D human fetal osteoblast (hFOB) spheroid under magnetic levitation. Formation of 3D spheroid culture under magnetic levitation was evaluated by cell viability at 3, 7, and 14 days. Morphology of the 3D hFOB spheroid was analyzed by SEM and fluorescence microscopy and the differentiation towards mineralized lineage by ALP assay, qPCR, and alizarin red staining. The cell viability indicated that the 3D hFOB spheroid still viable after 14 days of culture. ALP assay, qPCR analysis expression of Col1, ALP, and Itg-ß1 molecules, and calcium deposition with alizarin red showed a high level of bioactivity of the 3D hFOB spheroid. SEM images allowed the morphological analysis of the 3D microtissue-like spheroid with the presence of matrix deposition. These results indicate that magnetic levitation culture enables 3D stable osteoblast spheroids and could be a promising strategy for engineering application in the 3D construct in surgery regeneration of mineralized tissue.

Heterogeneity of neutrophils in arterial hypertension.

Cellular heterogeneity and diversity are recognized to contribute to the functions of neutrophils under homeostatic and pathological conditions. We previously suggested that the chronic inflammatory responses associated with hypertension (HTN) are related to the participation of different subpopulations of neutrophils. Two populations of neutrophils can be obtained by density gradient centrifugation: normal-density neutrophils (NDN) and low-density neutrophils (LDN). However, the lack of standardized functional protocols has limited phenotypic characterization and functional comparisons of LDN and NDN. Based on their capability to incorporate Na+, maturity and activation stage, we characterized NDN and LDN in blood samples from ten patients with HTN and ten healthy individuals (HI) using flow cytometry. We compared the levels of reactive oxygen species (ROS), generation of neutrophil extracellular traps (NETs) and levels of apoptosis in NDN and LDN. In general, the NDN and LDN subpopulations from patients with HTN exhibited higher levels of sodium influx and ROS, and lower levels of apoptosis than the corresponding NDN and LDN subsets from HI. Transmission electron microscopy revealed NDN and LDN from patients with HTN exhibited alterations to mitochondrial morphology and fewer cytoplasmic granules than the corresponding HI subpopulations. Our results indicate both the NDN and LDN subpopulations enhance the effects of inflammation that contribute to the pathophysiology of HTN. Further detailed studies are required to characterize the events during ontogeny of the myeloid lineage that result in the diverse phenotypic characteristics of each subpopulation of LDN and NDN.

Comparative proteomic analysis of outer membrane vesicles from Brucella suis, Brucella ovis, Brucella canis and Brucella neotomae.

Gram-negative bacteria release nanovesicles, called outer membrane vesicles (OMVs), from their outer membrane. Proteomics has been used to determine their composition. OMVs contain proteins able to elicit an immune response, so they have been proposed as a model to develop acellular vaccines. In this study, OMVs of Brucella suis, B. ovis, B. canis, and B. neotomae were purified and analyzed by SDS-PAGE, transmission electron microscopy and liquid chromatography coupled to mass spectrometry to determine the pan-proteome of these vesicles. In addition, antigenic proteins were detected by western blot with anti-Brucella sera. The in silico analysis of the pan-proteome revealed many homologous proteins, such as Omp16, Omp25, Omp31, SodC, Omp2a, and BhuA. Proteins contained in the vesicles from different Brucella species were detected by anti-Brucella sera. The occurrence of previously described immunogenic proteins derived from OMVs supports the use of these vesicles as candidates to be evaluated as an acellular brucellosis vaccine.

Microscopic characterization of biofilm in mixed keratitis in a novel murine model.

PURPOSE: To report the characterization and analysis of the biofilm formation in mixed keratitis induced by the coinfection of Staphylococcus aureus and Fusarium falciforme in a novel murine model. METHODS: Clinical ocular microbial isolates and female BALB/c mice were used to develop the murine model. Immunosuppression was achieved with cyclophosphamide and methylprednisolone. A corneoscleral lesion was performed with a micro-pocket technique. Mice received an inoculum with a concentration of 1 × 105 conidia of F. falciforme and S. aureus with 1 × 105 UFC/ml. Mice were sacrificed at 72 h after induction of infection, the right eye was enucleated and preserved in 10% formaldehyde to perform the PAS staining. In addition, cuts were obtained for the labeling with the fluorophores propidium iodide and Calcofluor White, and other eye cuts were processed to transmission microscopy. RESULTS: F. falciforme and S. aureus were able to developed mono and mixed biofilm in vitro. Keratitis of F. falciforme, S. aureus and mixed, were established at immunosuppressed mice. Clinical symptoms were observed at murine cornea. Histological analysis by special stains identified bacterial, fungal and mixed biofilm structures at epithelial and stromal level. Extracellular matrix was observed surrounded clusters of bacterial, fungi and mixed by fluorescence and transmission electronic microscopy. CONCLUSION: This study provides direct evidence of the establishment and formation of mixed biofilm in vitro, as well as in vivo on the corneal surface of mice in an experimentally induced S. aureus and F. falciforme mixed keratitis infection.

The Ustilago maydis null mutant strains of the RNA-binding protein UmRrm75 accumulate hydrogen peroxide and melanin.

Ustilago maydis is a dimorphic fungus that has emerged as a model organism for the study of fungal phytopathogenicity and RNA biology. In a previous study, we isolated the U. maydis UmRrm75 gene. The deletion of the UmRrm75 gene affected morphogenesis and pathogenicity. UmRrm75 gene encodes a protein containing three RNA recognition motifs. Here we determined that UmRrm75 has chaperone activity in Escherichia coli using the transcription anti-termination assay. Subsequently, we analyzed the growth of ΔUmRrm75 mutants at 15 °C and 37 °C, observing that mutant strains had reduced growth in comparison to parental strains. UmRrm75 gene expression was induced under these non-optimal temperatures. ΔUmRrm75 mutant colonies displayed a dark-brown color at 28 °C, which was confirmed to be melanin based on spectroscopic analysis and spectrometric data. Furthermore, ΔUmRrm75 mutant strains showed the presence of peroxisomes, and increased H2O2 levels, even at 28 °C. The ΔUmRrm75 mutant strains displayed a higher expression of redox-sensor UmYap1 gene and increased catalase activity than the parental strains. Our data show that deletion of the UmRrm75 gene results in higher levels of H2O2, increased melanin content, and abiotic stress sensitivity.

Ultraestructura del ovocito maduro y en reabsorción de Spondylus limbatus G. B. Sowerby II, 1847 (= S. calcifer ) (Bivalvia: Spondylidae) / Ultrastructure of the mature and in resorption oocytes of Spondylus limbatus G. B. Sowerby II, 1847 (= S. calcifer ) (Bivalvia: Spondylidae)

Spondylus limbatus es una especie bajo protección especial en México, de la que existe poca información biológica y nada sobre estudios histológicos o de ultraestructura del ovario. El objetivo de esta investigación fue caracterizar la morfología ultraestructural de los gametos femeninos maduros y en degeneración. La gónada femenina de S. limbatus en estado de madurez presentó ovocitos postvitelogénicos de 60-70 µm de diámetro, que presentan el aspecto característico de células metabólicamente activas y altamente sintetizadoras. La membrana citoplasmática posee especializaciones destinadas a aumentar la superficie de absorción de la célula, las microvellosidades; el citoplasma presenta numerosos sistemas membranosos relacionados con la síntesis de material de reserva y secreción; y el patrón de organización nuclear altamente lobulado, y por consiguiente con una gran superficie que asegura el intercambio núcleo-citoplasma, se incorpora de forma estructural al proceso de vitelogénesis. Finalmente, se describen los cambios ultraestructurales resultantes de la lisis de los ovocitos: colapso de las membranas nuclear y citoplásmica, y presencia de células hemocíticas macrófagas.

Spondylus limbatus is a species under special protection in Mexico, of which there is little or no information in the literature of biological, histological or ultrastructural studies of the ovary. The objective of this research was to characterize the ultrastructural morphology of mature and degenerating female gametes. The female gonad of S. limbatus in mature state presented post-vitellogenic oocytes 60-70 µm in diameter, which have characteristics of metabolically active and highly synthesizing cells. The cytoplasmic membrane has specializations designed to increase the absorption surface of the cell, the microvilli; the cytoplasm presents numerous membranous systems related to synthesis of reserve and secretion material as well as the highly lobed nuclear organization pattern; a large surface that ensures core-cytoplasm exchange, is structurally incorporated into the vitellogenesis process. Finally, ultrastructural changes resulting from the lysis of the oocytes are described: collapse of nuclear and cytoplasmic membranes, and the presence of macrophage hemocytic cells.

Enhanced expression of the Epithelial Sodium Channel in neutrophils from hypertensive patients.

Hypertension (HTN), i.e. abnormally high blood pressure, is a major risk factor for heart attack, stroke, and kidney failure. The Epithelial Sodium Channel (ENaC), one of the main transporters regulates blood pressure by tightly controlling the sodium reabsorption along the nephron. Recently, we have shown an α-ENaC overexpression in platelets from hypertensive patients compared to platelets from normotensive subjects, suggesting it makes a contribution to the activation state of platelets and the physiopathology of hypertension. However, the involvement of the α-ENaC localized in neutrophils to this disease remains unknown. Neutrophils are the first leukocytes to be recruited to an inflammatory site and are equipped with a strong ability to eliminate intra- or extracellular pathogens using reactive oxygen species or antibacterial proteins contained in their granules. Using the Western blotting (Wb), flow cytometry, and qRT-PCR approaches; we determined α-ENaC neutrophil overexpression at the protein and messenger RNA (mRNA) levels. By confocal and cytometry analysis, we determined the α-ENaC distribution and the heterogeneity of HTN neutrophils population, respectively. Immunoprecipitation and Wb assays demonstrated the presence of both α-ENaC and caveolin-1 phosphorylated forms, compared with neutrophils from healthy individuals. Although neutrophils from hypertensive subjects circulating in an activated state were exhibiting important oxidative stress and modifications registered by confocal, atomic force, and scanning electron microscope, they conserved their defense capabilities. The features described above for neutrophils from hypertensive patients could be attributed to α-ENaC overexpression, as its drug inhibition diminished their activation state modulating the actin cytoskeleton reorganization triggered during the activation process.

First Report of Carrot Cyst Nematode Heterodera carotae in Mexico: Morphological, Molecular Characterization, and Host Range Study.

During 2008 to 2016 in several nematological surveys in the Tepeaca Valley, Puebla, Mexico, carrot cyst nematode, Heterodera carotae was found parasitizing carrots, Daucus carota . The nematode was present in 61% of the sampled fields with high population densities, causing severe carrot yield losses in the Tepeaca Valley. The aim of this work was to study morphology, morphometrics, host range, and molecular characterization of the nematode. The morphological and morphometric characterization was made using light and scanning electron microscopy of the second stage juveniles, females, males and cysts, and the host range study, was performed with nine different plants from five families. The molecular identification was made by sequencing and analysing the ITS rRNA and partial COI genes. It was shown that using presently available molecular tools it is not possible to make an accurate differentiation of H. carotae from H. cruciferae . The host range test allowed to distinguish these species from each other. Our study showed that male stylet length is longer for H. carotae compared with that for H. cruciferae . This is an example of importance of combination of several methods for the correct identification of cyst nematodes. To our knowledge, this is the first report of H. carotae in Mexico.

Monosodium urate crystals induce oxidative stress in human synoviocytes.

BACKGROUND: Gout is the most common inflammatory arthropathy of metabolic origin and it is characterized by intense inflammation, the underlying mechanisms of which are unknown. The aim of this study was to evaluate the oxidative stress in human fibroblast-like synoviocytes (FLS) exposed to monosodium urate (MSU) crystals, which trigger an inflammatory process. METHODS: Human FLS isolated from synovial tissue explants were stimulated with MSU crystals (75 µg/mL) for 24 h. Cellular viability was evaluated by crystal violet staining, apoptosis was assessed using Annexin V, and the cellular content of reactive oxygen species (ROS) and nitrogen species (RNS) (O2 (-), H2O2, NO) was assessed with image-based cytometry and fluorometric methods. In order to determine protein oxidation levels, protein carbonyls were detected through oxyblot analysis, and cell ultrastructural changes were assessed by transmission electron microscopy. RESULTS: The viability of FLS exposed to MSU crystals decreased by 30 % (P < 0.05), while apoptosis increased by 42 % (P = 0.01). FLS stimulated with MSU crystals exhibited a 2.1-fold increase in H2O2 content and a 1.5-fold increase in O2 (-) and NO levels. Oxyblots revealed that the spots obtained from FLS protein lysates exposed to MSU crystals exhibited protein carbonyl immunoreactivity, which reflects the presence of oxidatively modified proteins. Concomitantly, MSU crystals triggered the induction of changes in the morphostructure of FLS, such as the thickening and discontinuity of the endoplasmic reticulum, and the formation of vacuoles and misfolded glycoproteins. CONCLUSIONS: Our results prove that MSU crystals induce the release of ROS and RNS in FLS, subsequently oxidizing proteins and altering the cellular oxidative state of the endoplasmic reticulum, which results in FLS apoptosis.

Roles of bacterial membrane vesicles.

Outer membrane vesicles (OMVs) are released from the outer membrane of Gram-negative bacteria. Moreover, Gram-positive bacteria also produce membrane-derived vesicles. As OMVs transport several bacterial components, especially from the cell envelope, their interaction with the host cell, with other bacteria or as immunogens, have been studied intensely. Several functions have been ascribed to OMVs, especially those related to the transport of virulence factors, antigenic protein composition, and development as acellular vaccines. In this work, we review some of the recent findings about OMVs produced by specific pathogenic bacterial species.

Metabolic requirements for neutrophil extracellular traps formation.

As part of the innate immune response, neutrophils are at the forefront of defence against infection, resolution of inflammation and wound healing. They are the most abundant leucocytes in the peripheral blood, have a short lifespan and an estimated turnover of 10(10) to 10(11) cells per day. Neutrophils efficiently clear microbial infections by phagocytosis and by oxygen-dependent and oxygen-independent mechanisms. In 2004, a new neutrophil anti-microbial mechanism was described, the release of neutrophil extracellular traps (NETs) composed of DNA, histones and anti-microbial peptides. Several microorganisms, bacterial products, as well as pharmacological stimuli such as PMA, were shown to induce NETs. Neutrophils contain relatively few mitochondria, and derive most of their energy from glycolysis. In this scenario we aimed to analyse some of the metabolic requirements for NET formation. Here it is shown that NETs formation is strictly dependent on glucose and to a lesser extent on glutamine, that Glut-1, glucose uptake, and glycolysis rate increase upon PMA stimulation, and that NET formation is inhibited by the glycolysis inhibitor, 2-deoxy-glucose, and to a lesser extent by the ATP synthase inhibitor oligomycin. Moreover, when neutrophils were exposed to PMA in glucose-free medium for 3 hr, they lost their characteristic polymorphic nuclei but did not release NETs. However, if glucose (but not pyruvate) was added at this time, NET release took place within minutes, suggesting that NET formation could be metabolically divided into two phases; the first, independent from exogenous glucose (chromatin decondensation) and, the second (NET release), strictly dependent on exogenous glucose and glycolysis.

Pathogenic determinants of clinical Klebsiella pneumoniae strains associated with their persistence in the hospital environment

Background: Klebsiella pneumoniae is considered an opportunistic pathogen associated with nosocomial infections occurring mainly in pediatric patients, such as premature infants placed in intensive care units. The aim of this study was to characterize K. pneumoniae strains isolated from different clinical sources based on their resistance to antibiotics and the presence of virulence factors associated with their persistence in the hospital environment. Methods: Fifty clinical strains of K. pneumoniae isolated from urine, blood, catheters, and cerebrospinal fluid sources were characterized. Susceptibility testing of antibiotics was performed by the Kirby-Bauer method (Clinical Laboratory Standards Institute, 2010). The ability to form a biofilm was determined by the 96-well microplate method. Capsule and fimbrial structures were visualized by transmission electron microscopy (TEM). Adherence was evaluated on A549 and HT29 cells. Assessment for the presence and expression of the ecpA, fimH, and mrkA genes was performed by PCR and RT-PCR. Results: Clinical strains of K. pneumoniae were isolated from 48% of urine, 24% of blood, 18% of catheters, and 10% of cerebrospinal fluid. Ninety-two percent of the strains showed resistance to cefpodoxime, whereas few strains showed resistance to imipenem and meropenem (4 and 2%, respectively). The extended spectrum-type beta-lactamase (ESBL) phenotype was identified in 97% of the strains positive for resistance to third-generation cephalosporins. In addition, 88% of the strains were multidrug resistant. All strains were able to form biofilms. Capsule and fimbirial structures were visualized by TEM. Based on our adhesion assays, the A549 cell line was more permissive to K. pneumoniae strains than the HT-29 cell line. K. pneumoniae strains amplified and expressed ecpA (100/70%), fimH (98/2%), and mrkA (84/48%) genes, respectively. Conclusion: The K. pneumoniae strains exhibited features that allowed them to survive in the hospital environment (formation of biofilm) and resist antimicrobial therapy (multidrug resistant MDR strains). These strains also possessed a capsule, adhesive properties, and expression of genes encoding colonization factors that favor the selection and persistence of these strains in hospitals.

Análisis in silico de proteínas potencialmente involucradas en la biogénesis de fimbrias en Helicobacter pylori / In silico analysis of proteins potentially involved in fimbrial biogenesis in Helicobacter pylori

Introducción. La colonización e infección crónica por Helicobacter pylori es el factor de mayor contribución al desarrollo de cáncer gástrico. Se ha descrito un gran repertorio de adhesinas que contribuyen a la adaptación específica de la bacteria al nicho gástrico y, para H. pylori , al igual que en otras bacterias patógenas, la formación de biopelícula es fundamental en la supervivencia a ambientes no favorables. Las fimbrias o pili tipo IV son responsables de la adhesión de diversas bacterias patógenas ( Escherichia coli , Pseudomonas aeruginosa y Vibrio cholerae) a distintas superficies. El objetivo de este trabajo fue identificar y analizar genes que pudieran codificar para proteínas involucradas en la biogénesis de fimbrias en H. pylori y caracterizar su expresión durante la formación de biopelícula. Métodos. Se emplearon herramientas bioinformáticas y moleculares, tales como la base de datos del NCBI para la búsqueda de secuencias de proteínas relacionadas con la biogénesis de fimbrias, así como la herramienta de PSI BLAST. Los alineamientos múltiples se realizaron con el programa T-COFFEE y HMMER. La predicción de las estructuras secundarias se realizó con ANTHEPROT y las estructuras terciarias se predijeron con el programa I-TASSER. Resultados. Se identificaron dos homólogos, jhp0257 y HP0272, de la proteína PilN de Campylobacter rectus y Xilella fastidiosa , la cual es parte de la maquinaria del ensamble de la fimbria tipo IV. Asimismo, las proteínas jhp0887 y HP0953 presentaron homología a nivel del péptido señal de PilA de P. aeruginosa , y la proteína HP0953 se sobreexpresó durante la formación de la biopelícula. Conclusiones. H. pylori posee proteínas homólogas a las proteínas de familias fimbriales, específicamente PilN y PilA, que ensamblan fimbria tipo IV en otras bacterias. Esta última tiene un nivel de expresión mayor durante la etapa inicial del proceso de formación de biopelícula.

Background. Colonization and chronic infection with Helicobacter pylori is the major contributing factor to the development of gastric cancer. A large repertoire of adhesins has been described that contribute to the adaptation of bacteria to a specific gastric niche. As in other pathogenic bacteria, H. pylori biofilm formation is central to survival on unfavorable environments. Type IV pili or fimbriae are responsible for the adhesion of many pathogenic bacteria (e.g., Escherichia coli, Pseudomonas aeruginosa and Vibrio cholerae ) to various surfaces. The aim of this study was to identify and analyze genes that might encode proteins involved in the biogenesis of fimbriae on H. pylori and characterize their expression during biofilm formation. Methods. PSI BLAST, bioinformatics and molecular tools were used as well as the NCBI database search for sequences related to protein biogenesis of fimbriae. Multiple alignments were performed using the HMMer and T-COFFEE programs. The secondary structure prediction was performed with ANTHEPROT and the tertiary structures were predicted with the I-Tasser. Results. We identified two counterparts-jhp0257 and HP0272-from protein of Campylobacter rectus and PilN Xilella fastidiosa , which is part of the machinery of assembly type IV fimbria. Similarly, proteins jhp0887 and HP0953 show homology from peptide PilA level of P. aeruginosa , and the HP0953 protein is overexpressed during the formation of the biofilm. Conclusions. H. pylori possesses proteins homologous to fimbrial protein families, specifically PilN and PilA, which join type IV fimbriae in other bacteria. The latter has a higher expression level during the initial stage of the formation of biofilm.

Cell vacuolation induced by Haemophilus influenzae supernatants in HEp-2 cells.

Haemophilus influenzae belongs to respiratory tract microbiota. We observed vacuoles formation in previous studies with H. influenzae culture supernatants, so in this work we characterised that cytotoxic effect. We observed an abundant production of acidic cytoplasmic vacuoles due to the presence of a "vacuolating factor" in H. influenzae supernatants which was characterised as thermolabile. Greatest vacuolating activity was observed when utilizing the fraction > 50 kDa. The presence of a large number of vacuoles in HEp-2 cells was verified by transmission electron microscopy and some vacuoles were identified with a double membrane and/or being surrounded by ribosomes. These results suggest similar behaviour to that of vacuolating effects described by autotransporter proteins an undescribed cytotoxic effect induced by H. influenzae.

Characterization of outer membrane vesicles from Brucella melitensis and protection induced in mice.

The outer membrane vesicles (OMVs) from smooth B. melitensis 16 M and a derived rough mutant, VTRM1 strain, were purified and characterized with respect to protein content and induction of immune responses in mice. Proteomic analysis showed 29 proteins present in OMVs from B. melitensis 16 M; some of them are well-known Brucella immunogens such as SOD, GroES, Omp31, Omp25, Omp19, bp26, and Omp16. OMVs from a rough VTRM1 induced significantly higher expression of IL-12, TNFα, and IFNγ genes in bone marrow dendritic cells than OMVs from smooth strain 16 M. Relative to saline control group, mice immunized intramuscularly with rough and smooth OMVs were protected from challenge with virulent strain B. melitensis 16 M just as well as the group immunized with live strain B. melitensis Rev1 (P < 0.005). Additionally, the levels of serum IgG2a increased in mice vaccinated with OMVs from rough strain VTRM1 consistent with the induction of cell-mediated immunity.

Descripción histológica de la cámara incubatriz de moina hutchinsoni (Brem, 1937) / Histological description of the brood-chamber of moina hutchinsoni (Brem, 1937)

Los cladóceros son partenogenéticos por lo que la mayor parte del año, las poblaciones consisten enteramente de hembras que se reproducen asexualmente, en ellas el ovario se comunica por medio de un oviducto, con la cámara incubatriz, la cual se localiza en el margen interno posterior del caparazón, cerca del corazón y antes del intestino. Los huevos provenientes de los oviductos se depositan en la cámara y se incuban hasta terminar el desarrollo embrionario. Se considera que Moina presenta un desarrollo postembrionario directo, porque los organismos juveniles o neonatos, salen completamente formados e independientes durante la muda. En la reproducción asexual la cámara contiene a las diferentes etapas del desarrollo embrionario llamadas; ovocito, huevo y embrión. En la etapa sexual o gamogenética la cámara contiene un efipio con dos huevos. La cámara incubatriz histológicamente esta conformada por un epitelio plano simple, que descansa sobre una membrana basal evidente, la cual se continúa con el tejido conjuntivo laxo, después del cual se encuentra el caparazón. En el interior de la cámara se identificaron los ovocitos, huevos y algunas etapas del desarrollo embrionario, en cortes semifinos y finos por microscopia óptica y de transmisión, respectivamente.

Cladocerans are parthenogenetic during the greater part of the year. Populations consist entirely of females that reproduce asexually; in these females the ovary communicates through an oviduct with the brood chamber which is located at the rear inner shell border, near the heart and before the intestine. The eggs originating from the oviducts are deposited in the brood chamber and are incubated until the end of embryonic development. Moina is considered post embryonic development because juvenile or neonate organisms emerge fully formed and independent during moulting. In asexual reproduction the brood chamber contains the various stages of embryonic development denominated, oocyte, egg and embryo. At this stage the sexual gamogenetic contains an ephippium with two eggs. The brood chamber is formed histologically by a simple, plane epithelium that rests on a clear basal membrane continuous with the loose connective tissue, after which the shell can be located. Inside the brood chamber oocytes, eggs and some stages of embryonic development are identified, as well as fine semi optical and transmission microscopy cuts respectively.

Ultrastructural changes on clinical isolates of Trichophyton rubrum, Trichophyton mentagrophytes, and Microsporum gypseum caused by Solanum chrysotrichum saponin SC-2.

Worldwide, dermatophytoses represent a high percentage of all superficial mycoses. The most frequently isolated dermatophyte is Trichophyton rubrum. Solanum chrysotrichum is a vegetal species widely used in Mexican traditional medicine to treat skin infections; its extract has been used to formulate an herbal medicinal product that is used successfully to treat Tinea pedis. Spirostanic saponin SC-2 from S. Chrysotrichum possesses high activity against dermatophytes. The present study reports the ultrastructural changes observed by means of transmission electron microscopy (TEM) in clinical isolates of T. rubrum, T. mentagrophytes, and Microsporum gypseum induced by saponin SC-2. Strains were grown in RPMI 1640 containing SC-2 (1600 microg/mL). Fungi were harvested at 6, 12, 24, and 48 h; controls without SC-2 were included. T. mentagrophytes was the most susceptible to the SC-2 saponin, followed by M. gypseum, while T. rubrum was the most resistant. The main alterations caused by the SC-2 saponin were as follows: i) loss of cytoplasmic membrane continuity; ii) organelle degradation; iii) to a lesser extent, irreversible damage to the fungal wall; and iv) cellular death.