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The incorporation of baculovirus into semi-synthetic diets has been used for the inoculation of large batches of insect larvae reared under gregarious conditions. The preparation of most artificial diets involves heating to dissolve the agar gelling agent followed by cooling to ∼55–60°C whereupon virus occlusion bodies (OBs) are incorporated into the diet. To avoid exposure of thermolabile OBs to high temperatures, a cool-textured diet using soy fiber was evaluated as a simple method to inoculate batches of larvae with the baculovirus OBs. Spodoptera exigua larvae that fed on a diet with 120, 140 or 160 g/l of soy fiber, as a substitute for agar, had a similar weight gain over a 24 h period than larvae that consumed a standard agar diet. The larval weight gain with cool-textured diets with 140 g/l of soy fiber was not improved by the addition of chlorophyll, green food dye colour, or by the removal of ascorbic acid, sodium benzoate and potassium sorbate. Preliminary tests performed to determine th...

Porous spherical aggregates of barium hexaferrite (BaFe 12 O 19 ) with 1.5 µm in diameter were synthetized by the surfactant-assisted sol-gel method. The surfactant Tween20 (C 58 H 114 O 26 ), which enables mesoporous structures, as well as polystyrene spheres (PS), as the template agent for the formation of macropores, were used. Two synthetic routes (hereafter named A and B), whose difference was the absence or presence of PS, were followed for synthesis. X-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM) in high resolution mode (HRTEM) were used for characterization. Size and morphology of the spheres were similar in both cases and they resemble a nest or ball-of-yarn type structure. Pore size and BaFe 12 O 19 crystal size produced by the two routes are different. The magnetic properties of the spheres were evaluated using a vibrating sample magnetometer (VSM) as function of the calcination temperature. The spheres present ferromagnetic behavior in both routes.

Glycogen synthase kinase 3 (GSK3) is a constitutive enzyme implicated in the regulation of cytokine expression and the inflammatory response during bacterial infections. Mammals have two GSK3 isoforms named GSK3α and GSK3β that plays different but often overlapping functions. Although the role of GSK3β in cytokine regulation during the inflammatory response caused by bacteria is well described, GSK3α has not been found to participate in this process. Therefore, we tested if GSK3α may act as a regulatory isoform in the cytokine expression by bovine endothelial cells infected with Staphylococcus aureus because this bacterium is one of the major pathogens that cause tissue damage associated with inflammatory dysfunction. Interestingly, although both isoforms were phosphorylated–inactivated, we consistently observed a higher phosphorylation of GSK3α at Ser21 than that of GSK3β at Ser9 after bacterial challenge. During a temporal course of infection, we characterized a molecular switch from pro-inflammatory cytokine expression (IL-8), promoted by nuclear factor-kappa B (NF-κB), at an early stage (2 h) to an anti-inflammatory cytokine expression (IL-10), promoted by cAMP response element binding (CREB), at a later stage (6 h). We observed an indirect effect of GSK3α activity on NF-κB activation that resulted in a low phosphorylation of CREB at Ser133, a decreased interaction between CREB and the co-activator CREB-binding protein (CBP), and a lower expression level of IL-10. Gene silencing of GSK3α and GSK3β with siRNA indicated that GSK3α knockout promoted the interaction between CREB and CBP that, in turn, increased the expression of IL-10, reduced the interaction of NF-κB with CBP, and reduced the expression of IL-8. These results indicate that GSK3α functions as the primary isoform that regulates the expression of IL-10 in endothelial cells infected with S. aureus.

Early sensing of pathogenic bacteria by the host immune system is important to develop effective mechanisms to kill the invader. Microbial recognition, activation of signaling pathways, and effector mechanisms are sequential events that must be highly controlled to successfully eliminate the pathogen. Host recognizes pathogens through pattern-recognition receptors (PRRs) that sense pathogen-associated molecular patterns (PAMPs). Some of these PRRs include Toll-like receptors (TLRs), nucleotide-binding oligomerization domain-like receptors (NLRs), retinoic acid-inducible gene-I- (RIG-I-) like receptors (RLRs), and C-type lectin receptors (CLRs). TLRs and NLRs are PRRs that play a key role in recognition of extracellular and intracellular bacteria and control the inflammatory response. The activation of TLRs and NLRs by their respective ligands activates downstream signaling pathways that converge on activation of transcription factors, such as nuclear factor-kappaB (NF-κB), activator protein-1 (AP-1) or interferon regulatory factors (IRFs), leading to expression of inflammatory cytokines and antimicrobial molecules. The goal of this review is to discuss how the TLRs and NRLs signaling pathways collaborate in a cooperative or synergistic manner to counteract the infectious agents. A deep knowledge of the biochemical events initiated by each of these receptors will undoubtedly have a high impact in the design of more effective strategies to control inflammation.

This study describes four multiple nucleocapsid nucleopolyhedrovirus isolates recovered from infected larvae of beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), on crops in two different geographical regions of Mexico. Molecular and biological characterization was compared with characterized S. exigua multiple nucleopolyhedrovirus (SeMNPV) isolates from the United States (SeUS1 and SeUS2) and Spain (SeSP2). Restriction endonuclease analysis of viral DNA confirmed that all Mexican isolates were SeMNPV isolates, but molecular differences between the Mexican and the reference isolates were detected using PCR combined with restriction fragment length polymorphism (RFLP). Amplification of the variable region V01 combined with RFLP distinguished the two Mexican isolates, SeSLP6 and SeSIN6. BglII digestions showed that the majority of the isolates contained submolar bands, indicating the presence of genetic heterogeneity. Amplification of the variable regions V04 and V05 distinguished between American and the Spanish isolates. Biological characterization was performed against two laboratory colonies of S. exigua, one from Mexico, and another from Switzerland. Insects from the Mexican colony were less susceptible to infection than insects from Se-Swiss colony. In the Se-Mex colony, SeSP2 was the most pathogenic isolate followed by SeSIN6, although their virulence was similar to most of the isolates tested. In Se-Swiss colony, similar LD50 values were observed for the five isolates, although the virulence was higher for the SeSLP6 isolate, which also had the highest OB (occlusion body) yield. We conclude that the Mexican isolates SeSIN6 and SeSLP6 possess insecticidal traits of value for the development of biopesticides for the control of populations of S. exigua.

Rapid isolation and identification of pathogens is a major goal of diagnostic microbiology. In order to isolate and identify Staphylococcus aureus, a number of authors have used a variety of selective and/or differential culture media. However, to date, there are no reports comparing the efficacy of selective and differential culture media for S. aureus isolation from bovine mastitis cases using the 16S rRNA (rrs) gene sequence as a gold standard test. In the present study, we evaluated the efficacy of four selective and/or differential culture media for the isolation of S. aureus from milk samples collected from cows suffering from bovine mastitis. Four hundred and forty isolates were obtained using salt–mannitol agar (SMA, Bioxon), Staphylococcus-110 agar (S110, Bioxon), CHROMAgar Staph aureus (CSA, BD-BBL) and sheep’s blood agar (SBA, BD-BBL). All bacterial isolates were identified by their typical colony morphology in the respective media, by secondary tests (for coagulase and β-haemolysis) and by partial 16S rRNA (rrs) gene sequencing as a gold standard test. Sensitivity, positive predictive and negative predictive values were higher for SMA (86.96, 52.63 and 95.95 %, respectively) compared with S110 (70.00, 23.73 and 90.91 %, respectively), CSA (69.23, 28.13 and 95.74 %, respectively) and SBA (68.75, 37.93 and 89.58 %, respectively) while specificity values were similar for all media. Data indicated that the use of culture media for S. aureus isolation combined with determination of coagulase activity and haemolysis as secondary tests improved accuracy of the identification and was in accordance with rrs gene sequence-analysis compared with the use of the culture media alone.

El objetivo de este trabajo fue evaluar el efecto de la inoculacion del HMA Glomus intraradices en el crecimiento de las plantas de fresa variedad Camino Real, fertirrigadas con 0, 1, 3 y 10 mM de N en forma de NH4+ o NO3-. Se realizo un experimento en condiciones de invernadero, en el que se evaluo el porcentaje de colonizacion, el peso seco de las raices (PSR) y de la parte aerea (PSPA), a los 33 y 48 dias despues del establecimiento (dde). El porcentaje de colonizacion se redujo conforme al incremento de la concentracion de NH4+ o NO3- en la solucion de riego; a los 33 dde, la inoculacion de G. intraradices incremento el PSR y el PSPA en las plantas fertilizadas con NH4+; y a los 48 dde se observo el mismo efecto en las plantas que recibieron NO3-. En ambos casos, los valores mas altos de biomasa aerea y radical se registraron en las plantas fertirrigadas con bajas concentraciones de N. Estos resultados indican que la inoculacion con G. intraradices tiene el potencial de reducir la aplicacion de fertilizantes nitrogenados en el cultivo de fresa. Ademas, el exceso de N inhibe la colonizacion de HMA sin incrementar el crecimiento de la planta. Asi mismo, el efecto de la inoculacion de G. intraradices, forma y dosis de fertilizante nitrogenado depende de la fase de crecimiento de la planta.

Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium and endophyte of sugarcane, which expresses levansucrase, a fructosyltransferase exoenzyme with sucrose hydrolytic and levan biosynthetic activities. As a result of their physical properties, the levan can provide protection against stress caused by abiotic or biotic factors and participate in the formation of biofilms. In this study, we investigated the construction and function of a levansucrase-defective mutant of G. diazotrophicus. The lsdA mutant showed a decreased tolerance (65.5%) to 50-150 mM NaCl and a decrease of 89% in 876 mM (30%) sucrose, a reduction (99%) in tolerance to desiccation after 18 h, and a decrease (36.9-58.5%) in the ability to form cell aggregates on abiotic surfaces. Complementation of the mutant with the complete lsdA gene leads to a recovery of the ability to grow on sucrose-containing medium and to form slimy colonies, the ability to form the cell aggregates on abiotic surfaces and the tolerance to NaCl. This report demonstrates the importance of levansucrase in environmental adaptation of G. diazotrophicus under high osmotic stress and in biofilm formation.

Al 2 O 3 -doped TiO 2 thin films were prepared by combining electrophoretic deposition (EPD) with sputtering. A Corning* glass was used as a substrate, in which a titanium film was deposited by sputtering. Then, a precursor sol was prepared with Ti(n-OBu) 4 and Al(s-OBu) 3 and used as the medium for EPD. Next, the thin films were sintered and, finally, characterised by scanning electron microscopy (SEM), high resolution transmission electron microscopy (HRTEM) and X-ray diffraction (XRD). Several cultures of Escherichia coli , strain XL1-Blue, were prepared. Nine experiments were carried out. In three of them, an inoculum (a low amount of a product that contains bacteria) was prepared without a film; in the other six Al 2 O 3 -doped TiO 2 film-coated glass substrates were irradiated with visible light before they were introduced in the inoculum. The SEM and EDS results showed that TiO 2 –Al 2 O 3 films were obtained, covering all the glass substrate and with uniform size of particles forming them, and that the aluminium was distributed uniformly on the film. XRD results showed that rutile phase was obtained. By TEM, the structure of TiO 2 was demonstrated. Al 2 O 3 -doped TiO 2 thin films were successful at eliminating E. coli .

The ability of nanostructured TiO2 in anatase phase to eliminate Escherichia coli (E. coli) by UV light irradiation was tested using titania films supported on glass substrates. The films were obtained by electrophoretic deposition of titania sol on sputtered Ti Corning glass substrates. Experimental procedure used to obtain these films and their characterizations are discussed in this paper. Nanostructure nature of the films was analyzed using scanning electron microscopy and atomic force microscopy. Optic microscopy was used to study the photocatalytic activity of films and their interaction with E. coli bacteria, in order to measure the reduction in E. coli colonies. The structure of anatase TiO2 was determined using grazing incidence X-ray diffraction.

Microbial fructosyltransferases are polymerases that are involved in microbial fructan (levan, inulin and fructo-oligosaccharide) biosynthesis. Structurally, microbial fructosyltransferase proteins share the catalytic domain of glycoside hydrolases 68 family and are grouped in seven phylogenetically related clusters. Fructosyltransferase-encoding genes are organized in operons or in clusters associated with other genes related to carbohydrate metabolism or fructosyltransferase secretion. Fructosyltransferase gene expression is mainly regulated by two-component systems or phosphorelay mechanisms that respond to sucrose availability or other environmental signals. Microbial fructans are involved in conferring resistance to environmental stress such as water deprivation, nutrient assimilation, biofilm formation, and as virulence factors in colonization. As a result of the biological and industrial importance of fructans, fructosyltransferases have been the subject of extensive research, conducted to improve their enzymatic activity or to elucidate their biological role in nature.

Staphylococcus aureus is a pathogenic bacterium causing clinical and subclinical bovine mastitis. Infections of the udder by S. aureus are frequently associated with the presence of Staphylococcus epidermidis, an opportunistic pathogen. We reported previously that the capacity of bovine endothelial cells (BEC) to endocytize S. aureus is associated with the activation of NF-kappaB and modulated by the proinflammatory cytokines TNF-alpha and IL-1beta. In this work, we explore the ability of BEC to eliminate intracellular S. aureus and S. epidermidis and their response to these cytokines. Time-kinetics survival experiments indicated that BEC eliminate intracellular S. epidermidis more efficiently. Replication of S. aureus, but not S. epidermidis, inside BEC was evident by an increase in intracellular bacteria recovered at 2 h postinfection. Afterwards, the intracellular number of staphylococci decreased gradually, reaching the lowest value at 24 h. Treatment of BEC with TNF-alpha or IL-1beta potentiated the capacity of BEC to eliminate both Staphylococcus species at the times tested. These results indicate that activation of the intrinsic antistaphylococcal response in BEC, enhanced by TNF-alpha and IL-1beta, is effective to eliminate S. aureus and S. epidermidis and suggest that endothelial cells may play a prominent role in the defense against infections caused by these bacteria.

Bacterial internalization is an important process in the pathogenesis of infectious diseases in which nuclear factor kappaB (NF-kappaB) plays a prominent role. We present pharmacological evidence indicating that in bovine endothelial cells (BEC) the internalization of Staphylococcus aureus, a pathogenic bacterium that causes mastitis in bovine cattle, was associated with the activation of NF-kappaB. The internalization of S. aureus increased when BEC were stimulated with alpha-tumour necrosis factor (TNF-alpha) or beta-interleukin 1 (IL-1beta) which are known activators of NF-kappaB. SN50 (an inhibitor peptide of NF-kappaB nuclear translocation) and BAY 11-7083 (a chemical that inhibits the IkappaBalpha phosphorylation) caused significant reduction in S. aureus intracellular number, indicating that its internalization was associated with the NF-kappaB activity. Furthermore, specific inhibition of c-Jun N-terminal kinase with SP600125 (SP) or p-38 with SB203580 (SB) did not cause any change in the S. aureus intracellular number compared with the untreated control. Finally, TNF-alpha treatment of BEC after the addition of both SP and SB, induced a significant increase in S. aureus internalization above the control value. These data indicate that NF-kappaB activity is associated with S. aureus internalization and suggest that this transcription factor may play a role in the pathophysiology of bovine mastitis caused by this bacterium.

Internalization of Staphylococcus aureus in bovine endothelial cells (BEC) is increased by tumor necrosis factor alpha stimulation and NF-κB activation. Because the phosphoinositide-3-kinase (PI3K)–Akt signaling pathway also modulates NF-κB activity, we considered whether the internalization of S. aureus by BEC is associated with the activity of PI3K and Akt. We found a time- and multiplicity of infection-dependent phosphorylation of Akt on Ser473 in BEC infected with S. aureus . This phosphorylation was inhibited by LY294002 (LY), indicating the participation of PI3K. Inhibition of either PI3K with LY or wortmannin, or Akt with SH-5, strongly reduced the internalization of S. aureus . Transfection of BEC with a dominant-negative form of the Akt gene significantly decreased S. aureus internalization, whereas transfection with the constitutively active mutant increased the number of internalized bacterium. Inhibition of PDK1 activity with OSU-03012 did not affect the level of S. aureus internalization, demonstrating that phosphorylation of Akt on Thr308 is not important for this process. Compared to the untreated control, the adherence of S. aureus to the surface of BEC was unaltered when cells were transfected or incubated with the pharmacological inhibitors. Furthermore, Akt activation by internalized S. aureus triggered a time-dependent phosphorylation of glycogen synthase kinase-3α (GSK-3α) on Ser21 and GSK-3β on Ser9 that was partially inhibited with SH-5. Finally, treatment of BEC with LY prior to S. aureus infection inhibited the NF-κB p65 subunit phosphorylation on Ser536, indicating the involvement of PI3K. These results suggest that PI3K-Akt activity is important for the internalization of S. aureus and phosphorylation of GSK-3α, GSK-3β, and NF-κB.

Staphylococcus epidermidis is an environmental opportunistic pathogen associated with bovine intramammary infections. In bacterial infections, the endothelial tissue plays an important role during inflammation and it is the target of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha). Therefore, this work was designed to explore the effect of TNF-alpha on the interaction of S. epidermidis with bovine endothelial cells (BEC). We show that cell signaling activated by TNF-alpha caused a marked reduction in the number of intracellular S. epidermidis, suggesting that molecules participating in this pathway were involved in the internalization of this bacterium. We also found that S. epidermidis internalization was not associated with basal levels of nuclear factor kappa B (NF-kappaB) activity because the intracellular number of bacteria recovered after treating BEC with the NF-kappaB inhibitors, SN50 or BAY 11-7083, was similar to that of the untreated control. Interestingly, inhibition of the basal activity of JNK with SP600125 and p38 with SB203580 caused a decrease in the number of intracellular S. epidermidis. These results suggest that activation of the signaling pathway initiated by TNF-alpha could play an important role in the phagocytosis of this bacterium. However, the basal activity of NF-kappaB was shown not to be important for the internalization process of S. epidermidis.

Glycogen synthase kinase 3 (GSK3) is a constitutively active regulatory enzyme that is important in cancer, diabetes, and cardiovascular, neurodegenerative, and psychiatric diseases. While GSK3α is usually important in neurodegenerative and psychiatric diseases GSK3β is fundamental in the inflammatory response caused by bacterial components. Peptidoglycan (PGN), one of the most abundant cell-wall structures of Gram-positive bacteria, is an important inducer of inflammation. To evaluate whether inhibition of GSK3α and GSK3β activity in bovine endothelial cells (BEC) regulates the expression of the pro-inflammatory cytokine IL-12p40, we treated BEC with SDS-purified PGN from Staphylococcus aureus. We found that PGN triggered a TLR2/PI3K/Akt-dependent phosphorylation of GSK3α at Ser21, GSK3β at Ser9, and NF-κB p65 subunit (p65) at Ser536, and the phosphorylation of GSK3α was consistently higher than that of GSK3β. The expression of IL-12p40 was inhibited in BEC stimulated with PGN and pre-treated with a specific neutralizing anti-TLR2 antibody that targets the extracellular domain of TLR2 or by the addition of Akt-i IV (an Akt inhibitor). Inhibition of GSK3α and GSK3β with LiCl or SB216763 induced an increase in IL-12p40 mRNA and protein. The effect of each isoform on IL-12p40 expression was evaluated by siRNA-gene expression silencing of GSK3α and GSK3β. GSK3α gene silencing resulted in a marked increase in IL-12p40 mRNA and protein while GSK3β gene silencing had the opposite effect on IL-12p40 expression. These results indicate that the TLR2/PI3K/Akt-dependent inhibition of GSK3α activity also plays an important role in the inflammatory response caused by stimulation of BEC with PGN from S. aureus.

Mastitis (mammary gland inflammation) is one of the most important bovine diseases causing economic losses to dairy producers. Mammary gland inflammation is a consequence of the activity of a number of cell and soluble factors that function together to eliminate invading microorganisms. The factors involved in this inflammatory response differ depending on the infectious agent. This review analyzes the factors involved in the immunologic mechanisms against the main pathogenic bacteria causing mastitis, and emphasizes the innate immune response of the mammary gland. Knowledge, at the molecular level, of the mammary gland immune response during infection by pathogenic bacteria is fundamental to the design of effective therapies to control and eradicate bovine mastitis.

La mastitis es una enfermedad que afecta al ganado bovino lechero a nivel mundial, provocando una disminución en la calidad y cantidad de la leche producida, lo cual genera pérdidas económicas considerables. Esta enfermedad es causada por una variedad de microorganismos, entre los cuales Staphyloccocus aureus es uno de los más importantes. El objetivo del trabajo fue caracterizar 20 aislamientos de Staphylococcus spp. obtenidos de la glándula mamaria de vacas lactantes con mastitis en un sistema de producción de traspatio, mediante el uso de polimorfismos derivados de la amplificación al azar de ADN (RAPD’s) y su sensibilidad hacia trece antibióticos y 1 detergente. Las pruebas de sensibilidad a los 13 antibióticos demostraron que todos los aislamientos fueron resistentes a penicilina y sensibles a cefalotina, eritromicina y trimetoprim. Todos los aislamientos fueron susceptibles a una concentración menor de 6 µg/ml de bromuro de cetil trimetil amonio (CTAB). La caracterización molecular permitió determinar que los aislamientos SA7 y SA8, SA30 y SA32, presentaron una similitud superior al 90 % en el patrón de bandas obtenida por RAPD, mientras que los aislamientos SA35 y SA36 fueron idénticos, sin embargo, el resto de los aislamientos presentaron perfiles de amplificación distintos. Los aislamientos SA7 y SA8, SA30 y SA32, SA35 y SA36, tuvieron patrones de resistencia a antibióticos y de RAPD similares, por lo que probablemente se trató de reaislamientos. Los aislamientos de Staphylococcus de Tarímbaro, Michoacán presentaron una diversidad de perfiles de amplificación.

Glycogen synthase kinase 3β (GSK3β) plays a fundamental role during the inflammatory response induced by bacteria. Depending on the pathogen and its virulence factors, the type of cell and probably the context in which the interaction between host cells and bacteria takes place, GSK3β may promote or inhibit inflammation. The goal of this review is to discuss recent findings on the role of the inhibition or activation of GSK3β and its modulation of the inflammatory signaling in monocytes/macrophages and epithelial cells at the transcriptional level, mainly through the regulation of nuclear factor-kappaB (NF-κB) activity. Also included is a brief overview on the importance of GSK3 in non-inflammatory processes during bacterial infection.

The Notch signaling pathway is a reiteratively used cell to cell communication pathway that triggers pleiotropic effects. The correct regulation of the pathway permits the efficient regulation of genes involved in cell fate decision throughout development. This activity relies notably on the CSL proteins, (an acronym for CBF-1/RBPJ-κ in Homo sapiens/Mus musculus respectively, Suppressor of Hairless in Drosophila melanogaster, Lag-1 in Caenorhabditis elegans) which is the unique transcription factor and DNA binding protein involved in this pathway. The CSL proteins have the capacity to recruit activation or repression complexes according to the cellular context. The aim of this review is to describe the different co-repressor proteins that interact directly with CSL proteins to form repression complexes thereby regulating the Notch signaling pathway in animal cells to give insights into the paralogous evolution of these co-repressors in higher eumetazoans and their subsequent effects at developmental processes.

Instituto de Investigaciones Agropecuarias y Forestales. Instituto de Investigaciones sobre los Recursos Naturales. Instituto de Investigaciones Químico Biológicas. Facultad de Biología. Facultad de Medicina Veterinaria y Zootecnia. Facultad de Químico Farmacobiología. Programa Institucional de Doctorado en Ciencias Biológicas The Notch signaling pathway (NSP) is an ancestral cell-cell communication circuit that controls key processes to maintain metazoan homeostasis such as differentiation and organogenesis in early stages of embryonic development, cell identity and specialization in adult organisms. NSP regulates critical processes in the transition of the cell cycle in the G1 / S phase, participates in cross-communication with other cell signaling pathways such as Wingless (WNT) and Hedgehog (Hh) and participates in the regulation of apoptotic events. In mammals the NSP is mediated by the interaction of 4 receptors (Notch1-4) and 4 ligands (Delta1, 4; Jagged1, 2) and several regulatory proteins that may be species-specific. Among these proteins is Hairless (H), a negative regulator of NSP present in insects and mollusks. Hairless protein has been shown to be capable of downregulating the Notch/CSL activator complex in mammals by protein-protein binding and affinity experiments. The deregulation or constitutive overexpression of the NSP has been related to the appearance and maintenance of various types of human cancers. In breast cancer, constitutive activation of the NSP plays a major role in aberrant cell cycle progression, promotes poor and incomplete cell differentiation, and inhibits apoptosis mechanisms. Given the great evolutionary conservation among NSP proteins in metazoans; a plasmid expressing a peptide derived from the Drosophila melanogaster Hairless protein domain was designed and used to bind to the CSL protein and downregulate La vía de señalización Notch (vN) es un circuito molecular de comunicación ancestral entre célula-célula que controla procesos clave para mantener la homeostasis de los metazoarios; como la diferenciación y organogénesis en etapas tempranas del desarrollo embrionario, la especialización e identidad celular en organismos adultos, regula procesos críticos en la transición del ciclo celular en la fase G1/S, participa en la comunicación cruzada con otras vías de señalización celular como Wingless (WNT) y Hedgehog (Hh) y participa en la regulación de eventos apoptóticos. En mamíferos la vía Notch es mediada por la interacción de 4 receptores (Notch1-4) y 4 ligandos (Delta1, 4; Jagged1, 2) y varias proteínas reguladoras que pueden ser específicas en cada especie. Dentro de estas proteínas se encuentra Hairless (H), un regulador negativo de la vN presente en insectos y moluscos. Por experimentos de afinidad y unión proteína-proteína se ha comprobado que la proteína H es capaz de regular negativamente el complejo activador Notch/CSL (CBF-1, Su[H], Lag1) en mamíferos. La desregularización o sobrexpresión constitutiva de la vN se ha relacionado con la aparición y mantenimiento de varios tipos de cánceres humanos. En el cáncer de mama la activación constitutiva de la vN juega un rol principal en la progresión aberrante del ciclo celular, promueve una deficiente e incompleta diferenciación celular e inhibe los mecanismos de apoptosis. Dada la gran conservación evolutiva entre las proteínas de la vN en metazoarios; se diseñó un plásmido que expresa un péptido derivado del dominio que usa la proteína H de Drosophila melanogaster para unirse a la proteína CSL y regular negativamente la vN.

Instituto de Investigaciones Agropecuarias y Forestales. Facultad de Biología. Facultad de Medicina Veterinaria y Zootecnia. Facultad de Agrobiología. Facultad de Químico Farmacobiología. Programa Institucional de Maestría en Ciencias Biológicas The PRRSV virus is a reemergent pathogen that causes enormous economic losses over the world. PRRSV is endemic and generates repetitive outbreaks that become more aggressive than the original outbreaks. GP5 protein is the structural protein from PRRSV virus envelope, it participates in the virus recognition of the target cell and it has been associated to the neutralizing antibodies induction. In addition, GP5 protein participates in the evasion of the host's immune response. GP5 is an insoluble protein, this characteristic complicates the study of the entire protein. Nowadays, little is known about the role of epitopes from GP5 protein in the protective immune response mediated by antibodies and cells. Therefore, to know the role of the epitopes from GP5 protein and to explore their possible immunomodulatory function, we selected two peptides from GP5 protein through in silico prediction, one of them has a T lymphocyte recognition epitope (GP5-T) and the other has a B lymphocyte recognition epitope (GP5-B). The goal of this study was to investigate the epitopes participation in humoral and cellular immune response in vivo. The specific and total antibodies against peptides were analyzed, at 15 days of reimmunization scheme intervals, for 45 days. The results showed that both peptides increased total antibodies production for each reimmunization. However, only GP5-B group showed a strong and sustained specific antibodies response, which suggested that GP5-B peptide was capable to induce memory B lymphocytes. El virus del PRRS (PRRSV) es un patógeno reemergente que causa cuantiosas pérdidas económicas en todo el mundo. PRRSV se encuentra de forma endémica y genera brotes reincidentes que llegan a ser más agresivos que los brotes originales. La proteína GP5 es una proteína estructural presente en la envoltura del PRRSV, la cual participa en el reconocimiento del virus a la célula blanco y se ha asociado a la inducción de anticuerpos neutralizantes. GP5 es una proteína insoluble, esta característica dificulta el estudio de la proteína completa. Además, la proteína GP5 participa en la evasión de la respuesta inmune del hospedero. A la fecha se conoce poco sobre el papel que desempeñan los epítopos de la proteína GP5 en la inducción de una repuesta inmune protectora mediada por anticuerpos y células. Por lo tanto, para conocer el papel de los epítopos de la proteína GP5 y además explorar su posible función inmunomoduladora, nosotros seleccionamos dos péptidos a partir de la proteína GP5 a través de predicción in silico, uno de ellos posee un epítopo de reconocimiento a Linfocitos T (GP5-T) y otro con un epítopo de reconocimiento a linfocitos B (GP5-B). El objetivo de este estudio fue determinar la participación de dichos epítopos en la respuesta inmune celular y humoral en un modelo in vivo. Se analizó la respuesta inmune mediada por anticuerpos totales y específicos contra dichos péptidos en un esquema de reinmunización a intervalos de 15 días, durante un periodo de 45 días. Los resultados indicaron que ambos péptidos aumentaron la producción de anticuerpos totales en cada reinmunización. Sin embargo, solo el grupo del péptido GP5-B mostró una respuesta fuerte y sostenida de anticuerpos específicos, lo que sugirió que el péptido GP5-B fue capaz de inducir linfocitos B de memoria.

Instituto de Investigaciones Agropecuarias y Forestales. Facultad de Biología. Facultad de Medicina Veterinaria y Zootecnia. Facultad de Agrobiología. Facultad de Químico Farmacobiología. Programa Institucional de Maestría en Ciencias Biológicas The Notch signaling pathway (NSP) is a highly conserved cell communication mechanism in most animals and its deregulation is involved with several diseases such as cancer. Several proteins responsible for the negative regulation of the NSP target genes have been reported and all these co-repressor proteins interact with the DNA-binding transcription factor CSL to, potentially, repress the transcription of these genes. Recently, in our laboratory, the transcriptional repressor protein NAC-2 was identified as a protein possibly involved in the regulation of NSP in mammals. Therefore, we decided to investigate the possible participation of NAC-2 in the regulation of the NSP target genes. Using a vector that contains a reporter gene of the luciferase enzyme sensitive to the activity of the NSP, that contains has some consensus regulatory elements of binding to CSL, we evaluate the effect of NAC-2 on the activity of the luciferase enzyme, by transfection of MCF-7 cells. The data obtained showed that NAC-2 decreased the activity of luciferase by 50%, which suggests that NAC-2 functions as an inhibitor of the expression of the target genes of the NSP. It is likely that this NAC-2 inhibitory activity requires the interaction with the transcriptional factor CSL. La vía de señalización Notch (VSN) es un mecanismo de comunicación celular altamente conservado en casi todos los animales, y su desregulación está involucrada con diversas enfermedades como el cáncer. Varias proteínas responsables de la regulación negativa de los genes blanco de la VSN, han sido reportadas, y todas estas proteínas co-represoras interaccionan con el factor transcripcional de unión a ADN, CSL, para eventualmente, reprimir la transcripción de estos genes. Recientemente, en nuestro laboratorio se identificó a la proteína represora transcripcional NAC-2, como una proteína probablemente involucrada en la regulación de la VSN en mamíferos. Por lo anterior, decidimos investigar la posible participación de NAC-2, en la regulación de los genes blanco de la VSN. Empleando un vector que contiene un gen reportero de la enzima luciferasa sensible a la actividad de la VSN, debido a que tiene algunos elementos regulatorios consenso de unión a CSL, evaluamos el efecto de NAC-2 sobre la actividad de la enzima luciferasa, por transfección de las células MCF-7. Los datos obtenidos demostraron que NAC-2 disminuyó en un 50% la actividad de la luciferasa, lo cual sugiere que NAC-2 funciona como un inhibidor de la expresión de los genes blanco de la VSN. Es probable que esta actividad inhibidora de NAC-2 requiera de la interacción con el factor transcripcional CSL.

Instituto de Investigación en Metalurgia y Materiales. Maestría en Metalurgia y Ciencias de los Materiales In the present investigation, nanoparticles and gold assemblies were synthesized using the plant extract of Taraxacum officinale (T. officinale) and the precursor salt tetrachloroauric acid (HAuCl4), which were characterized by Visible Ultraviolet Spectroscopy, Infrared Spectroscopy, Electron Microscopy of Scanning, Electron Microscopy of Transmission, X-ray Diffraction, Fluorescence Spectroscopy, Atomic Absorption Spectroscopy and Nuclear Magnetic Resonance. An important application of nanoparticles and gold assemblies is the use of these materials for treatments against breast cancer, which is primarily tested in the cell lines from which this cancer originates (MCF-12F regular cell line, MCF- 7 carcinoma, MDA-MB-231 metastatic carcinoma). In some studies drugs linked to the nanomaterial are additionally used to combat cancer in two ways, that is, by the release of the drug that is bound to its surface and by the generation of heat of gold nanomaterials, due to radiation incident by chemotherapy to treat breast cancer. Now, an effective alternative against this type of disease is to use cisplatin as a treatment drug, for its rapid elimination from the body and its high effectiveness against various types of cancer. The problem is that when administered to the patient, this drug destroys both healthy and cancerous cells, causing secondary damage such as DNA damage, kidney diseases, ototoxicity, etc. The solution to this problem is to bind using organic substances from the extract of the plant T. officinale which could be done in future studies to the present investigation. En la presente investigación se sintetizaron nanopartículas y ensambles de oro por medio del extracto de la planta Taraxacum officinale (T. officinale) y del precursor ácido tetracloroáurico (HAuCl4), las cuales se caracterizaron por medio de Espectroscopía Ultravioleta Visible, Espectroscopía de Infrarrojo, Microscopía Electrónica de Barrido, Microscopía Electrónica de Trasmisión, Difracción de Rayos X, Espectroscopía de fluorescencia, Espectroscopía de Absorción Atómica y Resonancia Magnética Nuclear. Una aplicación importante de las nanopartículas y ensambles de oro es el uso de éstos materiales para tratamientos contra el cáncer de mama, lo cual primordialmente se prueba en las líneas celulares de las cuales se origina éste cáncer (MCF-12F línea celular normal, MCF-7 carcinoma, MDA-MB-231 carcinoma metastásico), en algunos estudios se utilizan adicionalmente fármacos ligados al nanomaterial para combatir de doble manera el cáncer, es decir, por la liberación del medicamento que va ligado a su superficie y por la generación de calor de los nanomateriales de oro, debido a las radiaciones incidentes por quimioterapia para tratar el cáncer de mama. Ahora bien, una alternativa eficaz contra este tipo de enfermedad es utilizar cisplatino como fármaco de tratamiento, por su rápida eliminación del cuerpo y por su alta efectividad contra diversos tipos de cáncer; el problema es que, al ser administrado al paciente, éste fármaco destruye tanto las células normales como las cancerígenas, provocando daños secundarios tales como lesiones en el ADN, enfermedades renales, ototoxicidad, etc. La solución a éste problema es ligar mediante las sustancias orgánicas provenientes del extracto de la planta T. officinale lo cual se podría realizar en estudios futuros a la presente investigación.

Instituto de Investigaciones Agropecuarias y Forestales. Instituto de Investigaciones sobre los Recursos Naturales. Instituto de Investigaciones Químico Biológicas. Facultad de Biología. Facultad de Medicina Veterinaria y Zootecnia. Facultad de Químico Farmacobiología. Programa Institucional de Doctorado en Ciencias Biológicas The aim of this study was to evaluate the effect of melengestrol acetate (MGA) on follicular dynamics, estrus and fertility in ewes. Two experiments were performed. Experiment 1: 33 non-pregnant ewes with ovarian activity were used; with a body condition of 3.07±0.3 points (scale 1-5), 40.3±4.9 kg of weight, 4.6±1.7 years of age and 4.5±2.4 birth. Ewes were divided into three groups in a design stratified. Group 1 was administered a dose of 0.11 mg de MGA/ewe/day, group 2 a dose of 0.22 mg de MGA/ewe/day and group 3 a dose of 0.5 mg de MGA/ewe/day. In all three groups the treatment was administered for a period of 17 days. During treatment and post-treatment until ovulation or atresia of the dominant follicle in all the ewes follicular dynamics were followed by transrectal ultrasound. Follicles were measured by placing electronic calibrators in the boundary between the follicular wall and the ovarian stroma. In group 1 all ewes developed persistent follicles in both ovaries. The diameter of the persistent follicles was 5.8 ± 0.6 mm. In group 2, 75% of the sheep presented 3 follicular waves and the remaining 25% presented 2 waves. In the ewes that manifested three waves the emergence of the second wave was to the 5.8±1.0 and of the third was the 12.5±1.7 days. In the ewes that presented two waves the emergence of the second wave was at 7.6±1.8 days. The dominant follicles observed had a range of 5.2 a 5.7 mm of diameter. In group 3 81.81% of the ewes presented 3 follicular waves and the remaining 18.18% presented two waves. In the ewes that presented three waves, it was observed that the emergence of the second wave was at 8.7±1.4 and from the third to the 15.6±2.8 days. In those that presented two waves the emergence of the second wave was to the 12.2±2.0 days. El objetivo de este trabajo fue evaluar el efecto del acetato de melengestrol (MGA) en la dinámica folicular, estro y fertilidad en borregas. Se realizaron dos experimentos. Experimento 1: se utilizaron 33 borregas no gestantes y con actividad ovárica; con una condición corporal de 3.07±0.3 puntos (escala de 1 a 5), 40.3±4.9 kg de peso, 4.6±1.7 años de edad y 4.5±2.4 partos. Las borregas fueron distribuidas en tres grupos mediante un diseño estratificado; al grupo 1 se le administro una dosis de 0.11 mg de MGA/borrega/día, al grupo 2 una dosis de 0.22 mg de MGA/borrega/día y el grupo 3 una dosis de 0.5 mg de MGA/borrega/día. En los tres grupos el tratamiento se administró por un periodo de 17 días. Durante el tratamiento y pos-tratamiento hasta la ovulación o atresia del folículo dominante en todas las borregas se realizó un seguimiento de la dinámica folicular por ecografía transrectal. Los folículos se midieron colocando los calibradores electrónicos en el límite ubicado entre la pared folicular y el estroma ovárico. En el grupo 1 todas las borregas desarrollaron folículos persistentes en los dos ovarios. El diámetro de los folículos persistentes fue de 5.8±0.6 mm. En el grupo 2 el 75% de las borregas presentaron 3 ondas foliculares y el 25% restante presento 2 ondas. En las borregas que manifestaron tres ondas el surgimiento de la segunda onda fue a los 5.8±1.0 y de la tercera fue a los 12.5±1.7 días. En las borregas que presentaron dos ondas el surgimiento de la segunda onda fue a los 7.6±1.8 días. Los folículos dominantes observados tuvieron un rango de 5.2 a 5.7 mm de diámetro. En el grupo 3 el 81.81% de las borregas presentaron 3 ondas foliculares y el 18.18% restante presento dos ondas. En las borregas que presentaron tres ondas se observó que el surgimiento de la segunda onda fue a los 8.7±1.4 y de la tercera a los 15.6±2.8 días.

Instituto de Investigaciones Metalúrgicas. Doctorado en Ciencias en Metalurgia y Ciencias de los Materiales Nowadays, there is a great variety of formulations for the synthesis of nanostructured materials based on iron oxide with biomedical applications. The aim of the present work was to design and synthesize nanostructured macro-mesoporous hierarchical spherical aggregates of barium hexagonal ferrite BaFe12O19 using the sol-gel method through molecular self-assembly and spraydrying. Therefore, using a 2f factorial experimental design, the concentration (wt%) of template and the parameters of temperature (T) and pressure (P) in the spray drying process were evaluated to determine the effect of these variables on the aggregate size of BaFe12O19 and the size of macropore. Characterization techniques such as XRD, SEM, and TEM determined the structure, morphology and composition of the BaFe12O19 powders obtained after the spray-drying process. Spherical aggregates of 1.7 μm were obtained with a meso-macroporous structure determined from SEM and BET. The macropore size was about 223 nm and the mesopore size varied between 3.4 nm and 12 nm. Hysteresis loops were obtained from VSM technique and the magnetic properties of BaFe12O19 aggregates were determined. At a calcination temperature of 700 ° C a material with semi-hard behavior was obtained, whose saturation magnetization and coercive field values were, Ms = 14.24 emu/g and Hc = 194.65 Oe, respectively. Finally, the BaFe12O19 aggregates were tested as carriers of biologically active molecules. For this purpose, the peptide DS (Hairless suppressor binding domain [Su (H)] was used as the template drug, synthesized from the plasmids pF1A T7 Flexi® and pFN21K from the T7 strain of Escherichia coli The loading capacity and release profile of the DS peptide was determined by the SDSPAGE, FTIR, Wester blot and UV-vis techniques. Hoy en día existe una gran variedad de formulaciones para la síntesis de materiales nanoestructurados a base de óxido de hierro para aplicaciones biomédicas. El objetivo del presente trabajo fue diseñar y sintetizar agregados nanoestructurados de hexaferrita de bario (BaFe12O19) de tamaño micrométrico con jerarquía meso-macroporosa usando el método sol-gel y secado por aspersión. Usando un diseño experimental factorial 2f, se evaluaron la concentración (% peso) de agente porógeno y los parámetros de temperatura (T) y presión (P) en el proceso de secado por aspersión para determinar el efecto de estas variables sobre el tamaño de agregado de BaFe12O19 y el tamaño de macroporo. A parir de técnicas de caracterización tales como XRD, SEM y TEM se determinó la estructura, morfología y composición de los polvos de BaFe12O19. Se obtuvieron agregados esféricos de 1.7 μm de diámetro con una estructura meso-macroporosa determinada a partir de SEM y BET. El tamaño de macroporo fue de aproximadamente 223 nm y el tamaño de mesoporo varió entre 3.4 nm y 12 nm. Así mismo, mediante VSM se obtuvieron curvas de histéresis a partir de las cuales se determinó las propiedades magnéticas de los agregados de BaFe12O19. A una temperatura de calcinación de 700 °C se obtuvo un material con comportamiento semiduro, cuyos valores de magnetización de saturación y campo coercitivo fueron, Ms = 14.24 emu/g y Hc = 194.65 Oe, respectivamente. Finalmente, los agregados de BaFe12O19 fueron probados como portadores de moléculas con actividad biológica, para ello, se usó el péptido DS como fármaco modelo, sintetizado a partir de los plásmidos pF1A T7 Flexi® y pFN21K a partir de la cepa T7 de Escherichia coli. Se determinó la capacidad de carga y perfil de liberación del péptido DS mediante las técnicas de SDS-PAGE, FTIR, análisis tipo Wester blot y UV-vis.

Instituto de Investigaciones Agropecuarias y Forestales. Facultad de Biología. Facultad de Medicina Veterinaria y Zootecnia. Facultad de Agrobiología. Facultad de Químico Farmacobiología. Programa Institucional de Maestría en Ciencias Biológicas Staphylococcus aureus is a bacterium with a worldwide distribution, which is responsible for a number of infectious diseases found in various hosts. Protein A is one of the many virulence factors of S. aureus. The polymorphic nature of protein A is used for molecular characterization of S. aureus. Several functions of protein A have been described such as activation of interferon gene expression and tumor necrosis factor α1 receptor (TNFR1). In this work we were able to establish an association between protein A polymorphisms and the source of the S. aureus strain. These data allowed us to determine an evolutive trend of SpA. In addition, a new criteria for SpA characterization and acceptance is proposed, which is based on single nucleotide polymorphisms (SNP’s) identification in the signature sequences that flank the Xr region of SpA Staphylococcus aureus es una bacteria de distribución mundial que es responsable de una amplia gama de enfermedades infecciosas en diversos hospederos. La proteína A, uno de los múltiples factores de virulencia de S. aureus, es de naturaleza polimórfica y en epidemiología molecular se utiliza para la caracterización de S. aureus. Se han descrito diversas funciones que la proteína A ejerce sobre células del hospedero como la activación de la expresión de interferón y del receptor al factor de necrosis tumoral alfa 1 (TNFR1). En este trabajo se logró establecer una asociación entre el polimorfismo de la proteína A y el origen de la cepa de S. aureus a la que pertenece, lo cual permitió determinar una tendencia evolutiva de la proteína A de S. aureus. Además, se identificaron Polimorfismos de nucleótido único (SNP´s) en las secuencias “firma” que delimitan la región Xr del gen de la proteína A, por lo que se propone un criterio nuevo de aceptación.

Instituto de Investigaciones Agropecuarias y Forestales. Facultad de Biología. Facultad de Medicina Veterinaria y Zootecnia. Facultad de Agrobiología. Facultad de Químico Farmacobiología. Programa Institucional de Maestría en Ciencias Biológicas The functions of different types of cells that constitute a multicellular organism are regulated by signal transduction pathways, whose final goal is target genes regulation. The Notch signaling pathway is mainly involved in early and late embryo development, and recently, has been described its participation in the adult organism proper function. Hairless, a protein member of the Notch signaling pathway, modulates negatively this transduction signal pathway. Despite efforts to detect Hairless in higher animals, it has not been able to identify a homologue protein. The identification of a homologue protein is essential for the understanding of the regulatory processes during embryo development and other pathological processes where Notch pathway is involved. The Hairless counterparts described in insects have at least three highly conserved domains, where the suppressor of Hairless binding domain is the most conserved. Using analysis in silico and molecular biology techniques was identified and delimited a potentially immunogenic peptide from the Suppressor of Hairless binding domain of Hairless protein from Drosophila melanogaster. This peptide was used as antigen to generate polyclonal antibodies in rabbits and chickens. The sera were tested against total protein extract from embryos of the fruitfly. La vía de señalización Notch es una de las rutas de comunicación celular, que se utiliza principalmente durante el desarrollo temprano de los individuos, ya que participa en la diferenciación celular y por lo tanto en la organogénesis, y en conjunto con otras vías de transducción de señales, permite las complejas organizaciones espacio-temporales que orquestan y diseñan a los organismos multicelulares. La proteína Hairless es el principal regulador negativo de esta vía y por lo tanto funciona como un antagonista de la misma. Si bien ha sido caracterizada en Drosophila melanogaster y otros insectos, esta proteína de vital importancia para la vía, no cuenta con un homólogo reportado en mamíferos. Hairless cuenta con varios dominios conservados entre los diferentes homólogos conocidos en insectos y uno de estos dominios en particular, le sirve como sitio de enlace a Supresor de Hairless, factor transcripcional de la vía, que cuenta con un homólogo en mamíferos, CBF-1 (RBPJκ). Mediante PCR se amplificó el dominio de unión a Supresor a Hairless de la proteína Hairless de Drosophila melanogaster para producir un péptido in vitro que se usó como antígeno en la inmunización de conejos y gallinas y producir anticuerpos policlonales contra esta región. Posteriormente los sueros se probaron en proteína total de Drosophila melanogaster para detectar Hairless mediante Western Blot. Estos anticuerpos serán probados en proteína total de mamífero con la intención de identificar alguna proteína que conserve la función de Hairless de Drosophila melanogaster o se una a los homólogos de Supresor de Hairless conocidos en estas especies.