Clathrin-mediated endocytosis facilitates the internalization of Magnaporthe oryzae effectors into rice cells.

Fungi and oomycetes deliver effectors into living plant cells to suppress defenses and control plant processes needed for infection. Little is known about the mechanism by which these pathogens translocate effector proteins across the plasma membrane into the plant cytoplasm. The blast fungus Magnaporthe oryzae secretes cytoplasmic effectors into a specialized biotrophic interfacial complex (BIC) before translocation. Here, we show that cytoplasmic effectors within BICs are packaged into punctate membranous effector compartments that are occasionally observed in the host cytoplasm. Live cell imaging with fluorescently labeled proteins in rice (Oryza sativa) showed that these effector puncta colocalize with the plant plasma membrane and with CLATHRIN LIGHT CHAIN 1, a component of clathrin-mediated endocytosis (CME). Inhibiting CME using virus-induced gene silencing and chemical treatments resulted in cytoplasmic effectors in swollen BICs lacking effector puncta. By contrast, fluorescent marker colocalization, gene silencing, and chemical inhibitor studies failed to support a major role for clathrin-independent endocytosis in effector translocation. Effector localization patterns indicated that cytoplasmic effector translocation occurs underneath appressoria before invasive hyphal growth. Taken together, this study provides evidence that cytoplasmic effector translocation is mediated by CME in BICs and suggests a role for M. oryzae effectors in coopting plant endocytosis.

Dynamic metabolic modelling of overproduced protein secretion in Streptomyces lividans using adaptive DFBA.

BACKGROUND: Streptomyces lividans is an appealing host for the production of proteins of biotechnological interest due to its relaxed exogenous DNA restriction system and its ability to secrete proteins directly to the medium through the major Sec or the minor Tat routes. Often, protein secretion displays non-uniform time-dependent patterns. Understanding the associated metabolic changes is a crucial step to engineer protein production. Dynamic Flux Balance Analysis (DFBA) allows the study of the interactions between a modelled organism and its environment over time. Existing methods allow the specification of initial model and environment conditions, but do not allow introducing arbitrary modifications in the course of the simulation. Living organisms, however, display unexpected adaptive metabolic behaviours in response to unpredictable changes in their environment. Engineering the secretion of products of biotechnological interest has systematically proven especially difficult to model using DFBA. Accurate time-dependent modelling of complex and/or arbitrary, adaptive metabolic processes demands an extended approach to DFBA. RESULTS: In this work, we introduce Adaptive DFBA, a novel, versatile simulation approach that permits inclusion of changes in the organism or the environment at any time in the simulation, either arbitrary or interactively responsive to environmental changes. This approach extends traditional DFBA to allow steering arbitrarily complex simulations of metabolic dynamics. When applied to Sec- or Tat-dependent secretion of overproduced proteins in S. lividans, Adaptive DFBA can overcome the limitations of traditional DFBA to reproduce experimental data on plasmid-free, plasmid bearing and secretory protein overproducing S. lividans TK24, and can yield useful insights on the behaviour of systems with limited experimental knowledge such as agarase or amylase overproduction in S. lividans TK21. CONCLUSIONS: Adaptive DFBA has allowed us to overcome DFBA limitations and to generate more accurate models of the metabolism during the overproduction of secretory proteins in S. lividans, improving our understanding of the underlying processes. Adaptive DFBA is versatile enough to permit dynamical metabolic simulations of arbitrarily complex biotechnological processes.

Lytic reactions of drugs with lipid membranes.

Propranolol is shown to undergo lipidation reactions in three types of lipid membrane: (1) synthetic single-component glycerophospholipid liposomes; (2) liposomes formed from complex lipid mixtures extracted from E. coli or liver cells; and (3) in cellulo in Hep G2 cells. Fourteen different lipidated propranolol homologues were identified in extracts from Hep G2 cells cultured in a medium supplemented with propranolol. This isolation of lipidated drug molecules from liver cells demonstrates a new drug reactivity in living systems. Acyl transfer from lipids to the alcoholic group of propranolol was favoured over transfer to the secondary amine. Migration of acyl groups from the alcohol to the amine was diminished. Other drugs that were examined did not form detectable levels of lipidation products, but many of these drugs did affect the lysolipid levels in model membranes. The propensity for a compound to induce lysolipid formation in a model system was found to be a predictor for phospholipidosis activity in cellulo.

Short-Term Exposure to High CO2 and O2 Atmospheres to Inhibit Postharvest Gray Mold of Pomegranate Fruit.

The effect of short-term exposure to high CO2 or O2 atmospheres, alone or in combination with heat (35°C), for the control of postharvest gray mold was evaluated on 'Mollar de Elche' pomegranate fruit artificially inoculated with Botrytis cinerea and stored at 20 or 5°C. Exposure to high CO2 for 48 h at 20°C effectively reduced gray mold on pomegranate fruit incubated at 20°C for 5 days in a concentration-based manner. Furthermore, gaseous treatments with partial pressures of 95 kPa CO2 or 30 kPa O2 + 70 kPa CO2 for 48 h significantly reduced gray mold incidence and severity on fruit regardless of storage temperature. Moreover, for fruit receiving gaseous treatments at 20°C then cold stored for 12 weeks, there were no apparent negative effects of the treatments on their quality (weight loss, skin color, maturity index, pH, and sensory quality). In general, compared with those applied at 20°C, treatments at 35°C did not improve gray mold inhibition and adversely affected some quality parameters. In conclusion, 48-h exposures to specific atmospheres at 20°C may be a suitable treatment to extend pomegranate storage life and could be part of integrated control programs to control postharvest decay.

Incidence and Etiology of Postharvest Fungal Diseases of Persimmon (Diospyros kaki Thunb. cv. Rojo Brillante) in Spain.

'Rojo Brillante' is currently the most important persimmon cultivar in Spain. The incidence and etiology of postharvest diseases affecting this cultivar were determined under local conditions. Latent and wound pathogens were assessed for two consecutive seasons on commercially grown persimmons from two orchards. Healthy persimmons were either surface-disinfested or artificially wounded on the rind and placed in humid chambers at 20 or 25°C for up to 9 weeks. Additionally, decay was assessed on commercially handled persimmons stored at 1°C for up to 20 weeks. In all cases, the most frequent disease was alternaria black spot (ABS) caused by Alternaria alternata and an ABS severity index specific for 'Rojo Brillante' persimmons was established. Other minor pathogens causing latent infections, mostly stem-end rots, included Botrytis cinerea, Lasiodiplodia theobromae, Neofusicoccum spp., Pestalotiopsis clavispora, and Colletotrichum gloeosporioides. Penicillium expansum and, to much a lesser extent, Cladosporium cladosporioides were other pathogens causing wound infections. These two fungi and A. alternata and B. cinerea were also isolated from cold-stored fruit. Common isolates were identified by macroscopic and microscopic morphology and/or DNA amplification and sequencing. Pathogenicity of selected isolates was demonstrated by fulfilling Koch's postulates. Disease development at 20 and 5°C was characterized on artificially inoculated persimmons.

Puntos gatillo miofasciales, dolor, discapacidad y calidad del sueño en pacientes con cefalea de tensión crónica: estudio piloto / Myofascial trigger points, pain, disability and quality of sleep in patients with chronic tension-type headache: a pilot study

Introducción. El dolor referido inducido por puntos gatillo miofasciales (PGM) y las alteraciones del sueño pueden serfactores contribuyentes en la cefalea tensional crónica. Objetivo. Determinar la relación entre los PGM, intensidad del dolor, discapacidad y calidad del sueño en personas con cefalea tensional crónica. Sujetos y métodos. Participaron 16 pacientes con cefalea tensional crónica y 15 controles sanos. Se utilizó una escala analógica visual para la intensidad del dolor, el cuestionario de discapacidad de cuello y el cuestionario de calidad del sueñode Pittsburgh. Se exploraron PGM en los músculos temporal, masetero, trapecio superior, suboccipitales, esternocleidomastoideo, esplenio de la cabeza, semiespinoso de la cabeza y digástrico anterior por un evaluador ciego. Resultados. Los sujetos con cefalea tensional crónica tenían mayor discapacidad cervical (p < 0,001) que los controles, mientras que la calidad del sueño mostró una tendencia (p = 0,092). Se encontró una correlación positiva entre peor dolorde la semana pasada con el cuestionario de Pittsburgh (r = 0,631; p = 0,009) y la discapacidad (r = 0,521; p = 0,046), y una correlación positiva entre la discapacidad y calidad del sueño (r = 0,815; p < 0,001). Los pacientes con cefalea mostraron mayor número de PGM que los sujetos sanos (p < 0,001), siendo la presencia de PGM activos exclusiva en los pacientes. No se encontró asociación entre el número de PGM con la intensidad del dolor, discapacidad o calidad del sueño.Conclusiones. La calidad del sueño y los PGM activos pueden ser diferentes factores contribuyentes en la cefalea tensional crónica. No obstante, la presencia de PGM podría ser también un epifenómeno del dolor (AU)

Introduction. The referred pain induced by myofascial trigger points (MTP) and sleep disorders can be factors that contributeto chronic tension-type headache.Aim. To determine the relationship between MTP, intensity of pain, disability and quality of sleep in people with chronic tension-type headache. subjects and methods. Participants in the study consisted of 16 patients with chronic tension-type headache and 15 healthy controls. A visual analogue scale was used to measure the intensity of the pain, and the neck disability questionnaire and thePittsburgh (quality of sleep) questionnaire were also employed. MTP were explored in the temporal, masseter, upper trapezius,suboccipital, sternocleidomastoid, splenius capitis, semispinalis capitis and anterior digastric muscles by a blind evaluator. Results. The subjects with chronic tension-type headache had greater cervical disability (p < 0.001) than the controls, whereas the quality of sleep showed a tendency (p = 0.092). A positive correlation was found between the worst pain lastweek with the Pittsburgh cuestionnaire (r = 0.631; p = 0.009) and disability (r = 0.521; p = 0.046), as well as a positive correlation between disability and quality of sleep (r = 0.815; p < 0.001). The patients with headache displayed a higher number of MTP than the healthy controls (p < 0.001), the presence of active MTP being found exclusively in the patients. No association was found between the number of MTP and intensity of pain, disability or quality of sleep.Conclusions. Quality of sleep and active MTP can be different factors contributing to chronic tension-type headache. Nevertheless, the presence of MTP could also be an epiphenomenon of the pain (AU)

[Myofascial trigger points, pain, disability and quality of sleep in patients with chronic tension-type headache: a pilot study]. / Puntos gatillo miofasciales, dolor, discapacidad y calidad del sueno en pacientes con cefalea de tension cronica: estudio piloto.

INTRODUCTION. The referred pain induced by myofascial trigger points (MTP) and sleep disorders can be factors that contribute to chronic tension-type headache. AIM. To determine the relationship between MTP, intensity of pain, disability and quality of sleep in people with chronic tension-type headache. SUBJECTS AND METHODS. Participants in the study consisted of 16 patients with chronic tension-type headache and 15 healthy controls. A visual analogue scale was used to measure the intensity of the pain, and the neck disability questionnaire and the Pittsburgh (quality of sleep) questionnaire were also employed. MTP were explored in the temporal, masseter, upper trapezius, suboccipital, sternocleidomastoid, splenius capitis, semispinalis capitis and anterior digastric muscles by a blind evaluator. RESULTS. The subjects with chronic tension-type headache had greater cervical disability (p < 0.001) than the controls, whereas the quality of sleep showed a tendency (p = 0.092). A positive correlation was found between the worst pain last week with the Pittsburgh questionnaire (r = 0.631; p = 0.009) and disability (r = 0.521; p = 0.046), as well as a positive correlation between disability and quality of sleep (r = 0.815; p < 0.001). The patients with headache displayed a higher number of MTP than the healthy controls (p < 0.001), the presence of active MTP being found exclusively in the patients. No association was found between the number of MTP and intensity of pain, disability or quality of sleep. CONCLUSIONS. Quality of sleep and active MTP can be different factors contributing to chronic tension-type headache. Nevertheless, the presence of MTP could also be an epiphenomenon of the pain.

Cot/tpl2-MKK1/2-Erk1/2 controls mTORC1-mediated mRNA translation in Toll-like receptor-activated macrophages.

Cot/tpl2 is the only MAP3K that activates MKK1/2-Erk1/2 in Toll-like receptor-activated macrophages. Here we show that Cot/tpl2 regulates RSK, S6 ribosomal protein, and 4E-BP phosphorylation after stimulation of bone marrow-derived macrophages with lipopolysaccharide (LPS), poly I:C, or zymosan. The dissociation of the 4E-BP-eIF4E complex, a key event in the cap-dependent mRNA translation initiation, is dramatically reduced in LPS-stimulated Cot/tpl2-knockout (KO) macrophages versus LPS-stimulated wild-type (Wt) macrophages. Accordingly, after LPS activation, increased cap-dependent translation is observed in Wt macrophages but not in Cot/tpl2 KO macrophages. In agreement with these data, Cot/tpl2 increases the polysomal recruitment of the 5´ TOP eEF1α and eEF2 mRNAs, as well as of inflammatory mediator gene-encoding mRNAs, such as tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and KC in LPS-stimulated macrophages. In addition, Cot/tpl2 deficiency also reduces total TNFα, IL-6, and KC mRNA expression in LPS-stimulated macrophages, which is concomitant with a decrease in their mRNA half-lives. Macrophages require rapid fine control of translation to provide an accurate and not self-damaging response to host infection, and our data show that Cot/tpl2 controls inflammatory mediator gene-encoding mRNA translation in Toll-like receptor-activated macrophages.

Short Exposure to High CO2 and O2 at Curing Temperature to Control Postharvest Diseases of Citrus Fruit.

Curing of citrus fruit at 30 to 37°C and 90 to 98% relative humidity for 65 to 72 h is an effective alternative to fungicides to control postharvest green and blue molds caused by Penicillium digitatum and P. italicum, respectively. However, commercial adoption is limited because treatment is long and it may harm fruit quality. In order to improve the feasibility of curing, short CO2 or O2 exposures at curing temperature were evaluated on 'Nadorcott', 'Clemenules', and 'Ortanique' mandarin fruit and 'Valencia' orange. Fruit were artificially inoculated, exposed 24 h later to air (control); CO2 at 15, 30, 50, or 95 kPa; or O2 at 30 or 45 kPa at 20 or 33°C for 8, 24, or 48 h and incubated at 20°C for 4, 7, or 15 days. Exposure at 33°C with CO2 at 15 kPa for 24 h or O2 at 30 kPa for 48 h effectively controlled both green and blue molds after 7 days of incubation at 20°C; however, control of both diseases was minimal after 15 days. To assess potential induction of disease resistance, fruit were treated as described above, then inoculated after 1, 2, or 5 days at 20°C and evaluated after 3 and 6 more days at 20°C. All of the treatments were ineffective in inducing fruit resistance. Short exposures of citrus fruit to high CO2 or O2 at curing temperatures may be part of a control program alternative to synthetic fungicides, especially for organic fruit markets.

Intensidad de cuidados enfermeros: ¿cargas de trabajo o complejidad individual? / Intensity of nursing care: workloads or individual complexity?

Objetivo: explorar la identificación de los factores individuales de complejidad de cuidados en enfermos hospitalizados. Método: se empleó un diseño cualitativo de investigación-acción participativa con el método de análisis directo de contenido de los breves relatos de los participantes, enfermeras y enfermeros de los hospitales del Instituto Catalán de la Salud, sobre casos en los que hubieran experimentado situaciones de complejidad. La suficiencia muestral se estableció a partir del criterio de saturación de la información. Los participantes fueron invitados a formar parte de una ronda de talleres/sesiones de discusión que se hicieron durante18 meses. Uno de los investigadores recogía por escrito las opiniones de los participantes, mientras el otro moderaba el debate y realizaba preguntas reflexivas sobre el contenido de la propuesta. Posteriormente se invitaba a los participantes a organizarse en pequeños grupos para discutir y registrar en un formulario individual, breves narrativas sobre casos en los que hubieran experimentado situaciones de complejidad. Resultados: el número de relatos breves incluidos en el análisis final fue de 287. Los factores individuales de complejidad de cuidados incluye cinco dominios:(1) evolutivo, (2) mental y cognitivo, (3) psicoemocional, (4) sociocultural y (5) comorbilidad y complicaciones. La complejidad individual de cuidados se estructura fuentes, factores y especificaciones.Conclusiones: de los cinco ejes de complejidad identificados en el Modelo Vectorial de Complejidad de Safford, cuatro coinciden parcialmente con el análisis presentado. La arquitectura de la complejidad de cuidados debería incluir una consideración multiperspectiva, incluyendo los ejes de complejidad individual, terapéutica-procedimental y organizativa (AU)

Objective: to explore the identification of individual care complexity factors in hospitalized patients.Method: a qualitative design of participative research-action was employedwith the direct analysis method for the content of brief stories of participants and nurses of the hospitals of the Catalan Institute of Health, on casesin which they had experienced situations of complexity. Sample sufficiency was established based on the information’s saturation criterion. Participants were offered the chance to participate in a series of workshops/discussion sessions that were carried out over the course of 18 months. One of the researchers collected participants’ opinions in written form, whilethe other one moderated the debate and asked reflexive questions on the proposal’s content. Later, participants were asked to form small groups inorder discuss and record in an individual form brief stories of cases in which they had experienced situations of complexity.Results: 287 brief stories were included in the final analysis. The individualfactors of care complexity include five domains: (1) evolutionary, (2) mental and cognitive, (3) psychoemotional, (4) sociocultural and (5) comorbidity and complications. The individual complexity of care is structured insources, factors and specifications.Conclusions: of the five complexity axes identified in Safford’s Vectorial Method of Complexity, four coincide in part with the analysis presented.The architecture of care complexity should include a multiperspective consideration,including the domains of individual, therapeutic-procedural and organizational complexity axes (AU)

Potassium sorbate residue levels and persistence in citrus fruit as detected by a simple colorimetric method.

A colorimetric method that employed extraction of the macerated fruit, followed by a reaction with 2-thiobarbituric acid, was used to quantify potassium sorbate residues in citrus fruit. A recovery of more than 90% in oranges and lemons was obtained. Potassium sorbate residues determined by this method and a standard high-performance liquid chromatography (HPLC) method were similar. Residues were proportionate to the potassium sorbate concentration in the treatment solution. In oranges stored at 15 degrees C, following the potassium sorbate treatments, residues declined initially rapidly and later more slowly, until residues stopped declining after 6 days. A brief double-dip rinse in tap water applied immediately after immersion of lemons in a 2% (wt/vol) potassium sorbate removed more than 90% of the potassium sorbate residue. The influence of high-pressure water washing (HPWW) on potassium sorbate residues in potassium-sorbate-treated fruit was determined. Potassium sorbate residues were more effectively reduced by rinsing oranges than lemons.