Transforming growth factor beta signaling is essential for the autonomous formation of cartilage-like tissue by expanded chondrocytes.

Cartilage is a tissue with limited self-healing potential. Hence, cartilage defects require surgical attention to prevent or postpone the development of osteoarthritis. For cell-based cartilage repair strategies, in particular autologous chondrocyte implantation, articular chondrocytes are isolated from cartilage and expanded in vitro to increase the number of cells required for therapy. During expansion, the cells lose the competence to autonomously form a cartilage-like tissue, that is in the absence of exogenously added chondrogenic growth factors, such as TGF-βs. We hypothesized that signaling elicited by autocrine and/or paracrine TGF-β is essential for the formation of cartilage-like tissue and that alterations within the TGF-β signaling pathway during expansion interfere with this process. Primary bovine articular chondrocytes were harvested and expanded in monolayer culture up to passage six and the formation of cartilage tissue was investigated in high density pellet cultures grown for three weeks. Chondrocytes expanded for up to three passages maintained the potential for autonomous cartilage-like tissue formation. After three passages, however, exogenous TGF-β1 was required to induce the formation of cartilage-like tissue. When TGF-β signaling was blocked by inhibiting the TGF-β receptor 1 kinase, the autonomous formation of cartilage-like tissue was abrogated. At the initiation of pellet culture, chondrocytes from passage three and later showed levels of transcripts coding for TGF-β receptors 1 and 2 and TGF-β2 to be three-, five- and five-fold decreased, respectively, as compared to primary chondrocytes. In conclusion, the autonomous formation of cartilage-like tissue by expanded chondrocytes is dependent on signaling induced by autocrine and/or paracrine TGF-β. We propose that a decrease in the expression of the chondrogenic growth factor TGF-β2 and of the TGF-β receptors in expanded chondrocytes accounts for a decrease in the activity of the TGF-β signaling pathway and hence for the loss of the potential for autonomous cartilage-like tissue formation.

Vital function for PRELI and essential requirement for its LEA motif

Proteins containing the late embryogenesis abundant (LEA) motif comprise an evolutionarily conserved family, long postulated to protect plant embryos from stress and death. However, the significance of LEA-containing proteins and the mechanisms behind their function remain undetermined. Here we show that PRELI, a mammalian protein that possesses tandem repeats of the LEA motif, can protect cells against staurosporine, TNF-α or UV irradiation-induced apoptosis. We found that key to PRELI-dependent mechanisms that promote cell resistance to death are the stabilization of the respiratory chain, upholding of mitochondrial membrane potential and retention of apoptogenic molecules. By in vitro and in vivo studies, we also show that the expression of mutant PRELI/LEA- proteins lacking the LEA motif, results in the complete loss of PRELI's anti-apoptotic functions. Collectively, our data uncover a new molecular player in the control of apoptosis and support the hypothesis that LEA-containing proteins are evolutionarily conserved cell protectors against stress and death. ^

Identification of essential apoptotic genes in Drosophila

Apoptosis is a normal physiological cell suicide process which is essential for tissue homeostasis and normal development of metazoans. Misregulation of apoptosis is associated with many developmental defects and human diseases. The genes involved in the regulation and execution of apoptosis are highly conserved in humans and flies. Caspases are the executioners of cell suicide. Because of the unavailability of specific fly mutants, the developmental function of many caspase genes and genetic relationship between caspases and apoptotic components were undefined in Drosophila. We isolated several mutant alleles of the initiator caspase gene dronc, the effector casase drICE, and the Mediator component Cyclin C from the GMR-hid eyFLP/FRT screens which is designed to isolate mutants of recessive cell death genes in Drosophila melanogaster. Characterization of these mutants defined that they are essential for developmental cell death in Drosophila. dronc is required for most, but not all, cell death in Drosophila. drICE is required for apoptosis in many cells and it shares redundancy with another effector caspase gene, dcp-1, in a subset of cells in Drosophila. The genetic relationship between caspases and other apoptotic components was established through mutant analysis. We found that the pro-apoptotic protein Hid induces transcription of the initiator caspase gene dronc and the GMR-induced dronc transcripts are dependent on activated effector casapses, revealing a novel regulatory mechanism to promote caspase activity in Drosophila. Cyclin C and its kinase partner Cdk8 are required for prompt transcriptional induction of dronc in cell killing contexts. In short, we define the essential pro-apoptoic function of dronc, drICE, and Cyclin C in Drosophila and reveal a novel mechanism for regulation of dronc transcription. In the long run, these studies will help us decipher the complicated regulatory mechanism of cell death in humans. ^

Analysis of VirB4, a membrane-associated ATPase subunit essential for T -complex transport in Agrobacterium tumefaciens

Agrobacterium tumefaciens is a plant pathogen with the unique ability to export oncogenic DNA-protein complexes (T-complexes) to susceptible plant cells and cause crown gall tumors. Delivery of the T-complexes across the bacterial membranes requires eleven VirB proteins and VirD4, which are postulated to form a transmembrane transporter. This thesis examines the subcellular localization and oligomeric structure of the 87-kDa VirB4 protein, which is one of three essential ATPases proposed to energize T-complex transport and/or assembly. Results of subcellular localization studies showed that VirB4 is tightly associated with the cytoplasmic membrane, suggesting that it is a membrane-spanning protein. The membrane topology of VirB4 was determined by using a nested deletion strategy to generate random fusions between virB4 and the periplasmically-active alkaline phosphatase, $\sp\prime phoA$. Analysis of PhoA and complementary $\beta$-galactosidase reporter fusions identified two putative periplasmically-exposed regions in VirB4. A periplasmic exposure of one of these regions was further confirmed by protease susceptibility assays using A. tumefaciens spheroplasts. To gain insight into the structure of the transporter, the topological configurations of other VirB proteins were also examined. Results from hydropathy analyses, subcellular localization, protease susceptibility, and PhoA reporter fusion studies support a model that all of the VirB proteins localize at one or both of the bacterial membranes. Immunoprecipitation and Co$\sp{2+}$ affinity chromatography studies demonstrated that native VirB4 (87-kDa) and a functional N-terminally tagged HIS-VirB4 derivative (89-kDa) interact and that the interaction is independent of other VirB proteins. A $\lambda$ cI repressor fusion assay supplied further evidence for VirB4 dimer formation. A VirB4 dimerization domain was localized to the N-terminal third of the protein, as judged by: (i) transdominance of an allele that codes for this region of VirB4; (ii) co-retention of a His-tagged N-terminal truncation derivative and native VirB4 on Co$\sp{2+}$ affinity columns; and (iii) dimer formation of the N-terminal third of VirB4 fused to the cI repressor protein. Taken together, these findings are consistent with a model that VirB4 is topologically configured as an integral cytoplasmic membrane protein with two periplasmic domains and that VirB4 assembles as homodimers via an N-terminal dimerization domain. Dimer formation is postulated to be essential for stabilization of VirB4 monomers during T-complex transporter assembly. ^

Proposal for the inclusion of misoprostol in the WHO model list of essential medicines

Gynuity

Essential elements of postabortion care: origins, evolution and future directions

In this comment, we chronicle the development and expansion of a postabortion care model designed to promote interventions that address abortion-related public health concerns even when abortion laws and policies are restrictive. We review years of program experience with the original model, which led to the development of an expanded and updated model, Essential Elements of Postabortion Care (PAC). Implementing the model challenges global public health leaders, donors, technical assistance agencies and ministries of health to work with communities to ensure that all women who want to prevent or space pregnancies can obtain contraceptive services; that all women have access to services to manage complications from abortion, whether induced or spontaneous; and that all women receiving treatment also receive counseling and the reproductive and other health services they need at the treatment visit, as well as follow-up care and contraceptive resupply

Territory and Infrastructure : Strategic Identification and Project Pondering

El Territorio hoy es visto como una totalidad organizada que no puede ser pensada separando cada uno de los elementos que la componen; cada uno de ellos es definido por su relación con los otros elementos. Así, un pensamiento que integra diferentes disciplinas y saberes comienza a manejar una realidad que lejos está de definir certezas inamovibles, y comienza a vislumbrar horizontes estratégicos. La adaptación a la no linealidad de las relaciones que se dan sobre el territorio, y la diferencia de velocidades en las que actúan los distintos actores, nos exige hacer de la flexibilidad una característica esencial de la metodología de planificación estratégica. La multi-causalidad de los fenómenos que estructuran el territorio nos obliga a construir criterios cualitativos, entendiendo que nos es imposible la medición de estas cadenas causales y su reconstrucción completa en el tiempo; sin dejar por ello de edificar un marco profundo de acción y transformación que responda a una realidad cierta y veraz. Los fenómenos producidos sobre el territorio nunca actúan de manera aislada, lo que implica una responsabilidad a la hora de comprender las sinergias y la restricción que afectan los resultados de los procesos desatados. La presente ponencia corresponde a la Segunda Fase del proceso de identificación estratégica de los proyectos Plan Estratégico Territorial (PET) que se inició en el año 2005; dicho Plan es llevada a cabo por la Subsecretaría de Planificación Territorial del Ministerio de Planificación Federal y fue abordado sobre la base de tres pretensiones: institucionalizar el ejercicio del pensamiento estratégico, fortalecer la metodología de trabajo transdisciplinaria y multisectorial, y diseñar un sistema de ponderación de proyectos estratégicos de infraestructura, tanto a nivel provincial como nacional, con una fuerte base cualitativa. Este proceso dio como resultado una cartera ponderada de proyectos de infraestructura conjuntamente con una metodología que permitió consolidar los equipos provinciales de planificación, tanto en su relación con los decisores políticos como con los actores de los múltiples sectores del gobierno, y en estos resultados consolidar y reforzar una cultura del pensamiento estratégico sobre el territorio

Territory and Infrastructure : Strategic Identification and Project Pondering

El Territorio hoy es visto como una totalidad organizada que no puede ser pensada separando cada uno de los elementos que la componen; cada uno de ellos es definido por su relación con los otros elementos. Así, un pensamiento que integra diferentes disciplinas y saberes comienza a manejar una realidad que lejos está de definir certezas inamovibles, y comienza a vislumbrar horizontes estratégicos. La adaptación a la no linealidad de las relaciones que se dan sobre el territorio, y la diferencia de velocidades en las que actúan los distintos actores, nos exige hacer de la flexibilidad una característica esencial de la metodología de planificación estratégica. La multi-causalidad de los fenómenos que estructuran el territorio nos obliga a construir criterios cualitativos, entendiendo que nos es imposible la medición de estas cadenas causales y su reconstrucción completa en el tiempo; sin dejar por ello de edificar un marco profundo de acción y transformación que responda a una realidad cierta y veraz. Los fenómenos producidos sobre el territorio nunca actúan de manera aislada, lo que implica una responsabilidad a la hora de comprender las sinergias y la restricción que afectan los resultados de los procesos desatados. La presente ponencia corresponde a la Segunda Fase del proceso de identificación estratégica de los proyectos Plan Estratégico Territorial (PET) que se inició en el año 2005; dicho Plan es llevada a cabo por la Subsecretaría de Planificación Territorial del Ministerio de Planificación Federal y fue abordado sobre la base de tres pretensiones: institucionalizar el ejercicio del pensamiento estratégico, fortalecer la metodología de trabajo transdisciplinaria y multisectorial, y diseñar un sistema de ponderación de proyectos estratégicos de infraestructura, tanto a nivel provincial como nacional, con una fuerte base cualitativa. Este proceso dio como resultado una cartera ponderada de proyectos de infraestructura conjuntamente con una metodología que permitió consolidar los equipos provinciales de planificación, tanto en su relación con los decisores políticos como con los actores de los múltiples sectores del gobierno, y en estos resultados consolidar y reforzar una cultura del pensamiento estratégico sobre el territorio

Territory and Infrastructure : Strategic Identification and Project Pondering

El Territorio hoy es visto como una totalidad organizada que no puede ser pensada separando cada uno de los elementos que la componen; cada uno de ellos es definido por su relación con los otros elementos. Así, un pensamiento que integra diferentes disciplinas y saberes comienza a manejar una realidad que lejos está de definir certezas inamovibles, y comienza a vislumbrar horizontes estratégicos. La adaptación a la no linealidad de las relaciones que se dan sobre el territorio, y la diferencia de velocidades en las que actúan los distintos actores, nos exige hacer de la flexibilidad una característica esencial de la metodología de planificación estratégica. La multi-causalidad de los fenómenos que estructuran el territorio nos obliga a construir criterios cualitativos, entendiendo que nos es imposible la medición de estas cadenas causales y su reconstrucción completa en el tiempo; sin dejar por ello de edificar un marco profundo de acción y transformación que responda a una realidad cierta y veraz. Los fenómenos producidos sobre el territorio nunca actúan de manera aislada, lo que implica una responsabilidad a la hora de comprender las sinergias y la restricción que afectan los resultados de los procesos desatados. La presente ponencia corresponde a la Segunda Fase del proceso de identificación estratégica de los proyectos Plan Estratégico Territorial (PET) que se inició en el año 2005; dicho Plan es llevada a cabo por la Subsecretaría de Planificación Territorial del Ministerio de Planificación Federal y fue abordado sobre la base de tres pretensiones: institucionalizar el ejercicio del pensamiento estratégico, fortalecer la metodología de trabajo transdisciplinaria y multisectorial, y diseñar un sistema de ponderación de proyectos estratégicos de infraestructura, tanto a nivel provincial como nacional, con una fuerte base cualitativa. Este proceso dio como resultado una cartera ponderada de proyectos de infraestructura conjuntamente con una metodología que permitió consolidar los equipos provinciales de planificación, tanto en su relación con los decisores políticos como con los actores de los múltiples sectores del gobierno, y en estos resultados consolidar y reforzar una cultura del pensamiento estratégico sobre el territorio

On nonlinearity in neural encoding models applied to the primary visual cortex

Within the regression framework, we show how different levels of nonlinearity influence the instantaneous firing rate prediction of single neurons. Nonlinearity can be achieved in several ways. In particular, we can enrich the predictor set with basis expansions of the input variables (enlarging the number of inputs) or train a simple but different model for each area of the data domain. Spline-based models are popular within the first category. Kernel smoothing methods fall into the second category. Whereas the first choice is useful for globally characterizing complex functions, the second is very handy for temporal data and is able to include inner-state subject variations. Also, interactions among stimuli are considered. We compare state-of-the-art firing rate prediction methods with some more sophisticated spline-based nonlinear methods: multivariate adaptive regression splines and sparse additive models. We also study the impact of kernel smoothing. Finally, we explore the combination of various local models in an incremental learning procedure. Our goal is to demonstrate that appropriate nonlinearity treatment can greatly improve the results. We test our hypothesis on both synthetic data and real neuronal recordings in cat primary visual cortex, giving a plausible explanation of the results from a biological perspective.

Seasonal Variation of Essential Oil Yield and Composition of Sage (Salvia officinalis L.) Grown in Castilla - La Mancha (Central Spain)

Links between phenology, yield and composition of the essential oil of common sage, Salvia officinalis L., grown in Guadalajara (Central Spain) were determined in the different phases of the biological cycle during one year. Data showed an average yield about 1.0%. The analysis of the oil components was carried out by GC-FID and GC/MS. The main oil constituent was alpha thujone (40.1 - 46.5%). Other identified compounds are beta pinene (2.6 - 4.5%), cineole (3.5 - 8.7%), beta thujone (4.1 - 5.6%), camphor (4.1 - 8.0%), borneol (1.3 - 3.7%), alpha humulene (3.8 - 7.3%), viridiflorol (3.4-12.6%) and manool (0.1-4.5%). The highest yield of oil was obtained in the period of full flowering and the highest concentration of alpha thujone in the period of initial flowering.

Transport of Pru p 3 across gastrointestinal epithelium - an essential step towards the induction of food allergy?

Background Since intestinal absorption of food protein can trigger an allergic reaction, the effect of plant food allergen on intestinal epithelial cell permeability and its ability to cross the epithelial monolayer was evaluated. Objective To study the interaction of Pru p 3 with intestinal epithelium, its natural entrance, analyzing transport kinetics and cellular responses that trigger. Methods This was achieved using Pru p 3, the peach LTP, as a model. Enterocytic monolayers were established by culturing Caco 2 cells, as a model of enterocytes, on permeable supports that separate the apical and basal compartments. Pru p 3 was added to the apical compartment, the transepithelial resistance (TEER) was measured, and the transport was quantified. Results The peach allergen that crossed the cell monolayer was detected in the cell fraction and in the basal medium by immunodetection with specific antibodies and the quantity was measured by ELISA assay. Pru p 3 was able to cross the monolayer without disturbing the integrity of the tight junctions. This transport was significantly higher than that of a non-allergenic peach LTP, LTP1, and occurred via lipid raft pathway. The incubation of Caco 2 cells with Pru p 3 and LTP1 produced the expression of epithelial-specific cytokines TSLP, IL33 and IL25. Conclusion These results suggest that Pru p 3 was able to cross the cell monolayer by the transcellular route and then induce the production of Th2 cytokines. The results of the present study represent a step towards clarifying the importance of Pru p 3 as a sensitizer. Clinical relevance The capacity of food allergens to cross the intestinal monolayer could explain their high allergenic capacity and its fast diffusion through the body associating to severe symptoms.

Quorum Sensing is essential for an effective symbiosis in R. leguminosarum UPM791.

The implications of Quorum Sensing in the establishment of a successful symbiosis of Rhizobium leguminosarum bv. viciae (Rlv) with legume plants are discussed in this work. In order to analyze the significance and regulation of the production of AHL signal molecules, mutants deficient in each of the two QS systems present in Rlv UPM791 were constructed. A detailed analysis of the effect of these mutations on growth, AHL production, biofilm formation and symbiosis with pea, vetch and lentil plants has been carried out.

Native bradyrhizobia isolated from Lupinus mariae-josephae possess an essential T3SS for symbiosis

Analysis of the genome sequence of bradyrhizobia strains isolated from root nodules of Lupinus mariae-josephae revealed the presence of a type III secretion system (T3SS). Mutagenesis of ttsI gene that codes for the transcriptional activator (TtsI) resulted in the formation of white, non-fixing nodules in L. mariae-josephae. The T3SS cluster includes a gene coding for a NopE-like protein with an autocleavage motif. The NopE protein is an effector in the Bradyrhizobium-soybean symbiosis (Wenzel et al., 2010). The autocatalytic properties of the purified NopE-like protein have been studied.