Interference with plastome gene expression and Clp protease activity in Arabidopsis triggers a chloroplast unfolded protein response to restore protein homeostasis

Disruption of protein homeostasis in chloroplasts impairs the correct functioning of essential metabolic pathways, including the methylerythritol 4-phosphate (MEP) pathway for the production of plastidial isoprenoids involved in photosynthesis and growth. We previously found that misfolded and aggregated forms of the first enzyme of the MEP pathway are degraded by the Clp protease with the involvement of Hsp70 and Hsp100/ClpC1 chaperones in Arabidopsis thaliana. By contrast, the combined unfolding and disaggregating actions of Hsp70 and Hsp100/ClpB3 chaperones allow solubilization and hence reactivation of the enzyme. The repair pathway is promoted when the levels of ClpB3 proteins increase upon reduction of Clp protease activity in mutants or wild-type plants treated with the chloroplast protein synthesis inhibitor lincomycin (LIN). Here we show that LIN treatment rapidly increases the levels of aggregated proteins in the chloroplast, unleashing a specific retrograde signaling pathway that up-regulates expression of ClpB3 and other nuclear genes encoding plastidial chaperones. As a consequence, folding capacity is increased to restore protein homeostasis. This sort of chloroplast unfolded protein response (cpUPR) mechanism appears to be mediated by the heat shock transcription factor HsfA2. Expression of HsfA2 and cpUPR-related target genes is independent of GUN1, a central integrator of retrograde signaling pathways. However, double mutants defective in both GUN1 and plastome gene expression (or Clp protease activity) are seedling lethal, confirming that the GUN1 protein is essential for protein homeostasis in chloroplasts

Chloroplasts modulate elongation responses to canopy shade by retrograde pathways involving HY5 and abscisic acid

Plants use light as energy for photosynthesis but also as a signal of competing vegetation. Using different concentrations of norflurazon and lincomycin, we found that the response to canopy shade in Arabidopsis (Arabidopsis thaliana) was repressed even when inhibitors only caused a modest reduction in the level of photosynthetic pigments. High inhibitor concentrations resulted in albino seedlings that were unable to elongate when exposed to shade, in part due to attenuated light perception and signaling via phytochrome B and phytochrome-interacting factors. The response to shade was further repressed by a retrograde network with two separate nodes represented by the transcription factor LONG HYPOCOTYL 5 and the carotenoid-derived hormone abscisic acid. The unveiled connection among chloroplast status, light (shade) signaling, and developmental responses should contribute to achieve optimal photosynthetic performance under light-changing conditions

Specific Hsp100 chaperones determine the fate of the first enzyme of the plastidial isoprenoid pathway for either refolding or degradation by the stromal Clp protease in Arabidopsis

The lifespan and activity of proteins depend on protein quality control systems formed by chaperones and proteases that ensure correct protein folding and prevent the formation of toxic aggregates. We previously found that the Arabidopsis thaliana J-protein J20 delivers inactive (misfolded) forms of the plastidial enzyme deoxyxylulose 5-phosphate synthase (DXS) to the Hsp70 chaperone for either proper folding or degradation. Here we show that the fate of Hsp70-bound DXS depends on pathways involving specific Hsp100 chaperones. Analysis of individual mutants for the four Hsp100 chaperones present in Arabidopsis chloroplasts showed increased levels of DXS proteins (but not transcripts) only in those defec- tive in ClpC1 or ClpB3. However, the accumulated enzyme was active in the clpc1 mutant but inactive in clpb3 plants. Genetic evidence indicated that ClpC chaperones might be required for the unfolding of J20-delivered DXS protein coupled to degradation by the Clp protease. By contrast, biochemical and genetic approaches confirmed that Hsp70 and ClpB3 chaperones interact to collaborate in the refolding and activation of DXS. We conclude that specific J-proteins and Hsp100 chaperones act together with Hsp70 to recognize and deliver DXS to either reactivation (via ClpB3) or removal (via ClpC1) depending on the physiological status of the plastid

Thyroid diseases: Differences and controversies between the pathology report and clinical interpretation. PART II: Pathological aspects and diagnostic methods with therapeutic impact

Como revisamos en la primera parte1 hay algunos conceptos que deben ser considerados para el adecuado manejo médico por parte del equipo clínico que recibe el reporte de patología, de esta misma manera existen aspectos por parte del patólogo que este debe conocer, los cuales tienen impacto terapéutico. En esta segunda parte queremos revisar algunos conceptos que son de importancia por parte del patólogo que aplican directamente sobre la interpretación del clínico, como: procesamiento macroscópico; estudio de la citología por aspiración con aguja fina (FNA) vs. biopsia trucut, utilidad de la biopsia por congelación y de la inmunohistoquímica, así como los métodos e imágenes diagnósticas.As was reviewed in the first part, there are some concepts that should be considered for the appropriate medical management by the medical team that receives the pathology report. Similarly, there are some aspects that the pathologist should know, such as those that can have therapeutic impact. In this second part, a short review is presented on some of the concepts that are of importance to the pathologist that apply directly to the interpretation by the clinician, such as the macroscopic processing, the cytology study of the fine-needle aspirate (FNA) vs. “tru-cut” biopsy, the usefulness of the frozen biopsy, and immunohistochemistry, as well as diagnostic methods and diagnostic images

Interference with Clp protease impairs carotenoid accumulation during tomato fruit ripening

Profound metabolic and structural changes are required for fleshy green fruits to ripen and become colorful and tasty. In tomato (Solanum lycopersicum), fruit ripening involves the differentiation of chromoplasts, specialized plastids that accumulate carotenoid pigments such as β-carotene (pro-vitamin A) and lycopene. Here, we explored the role of the plastidial Clp protease in chromoplast development and carotenoid accumulation. Ripening-specific silencing of one of the subunits of the Clp proteolytic complex resulted in β-carotene-enriched fruits that appeared orange instead of red when ripe. Clp-defective fruit displayed aberrant chromoplasts and up-regulated expression of nuclear genes encoding the tomato homologs of Orange (OR) and ClpB3 chaperones, most probably to deal with misfolded and aggregated proteins that could not be degraded by the Clp protease. ClpB3 and OR chaperones protect the carotenoid biosynthetic enzymes deoxyxylulose 5-phosphate synthase and phytoene synthase, respectively, from degradation, whereas OR chaperones additionally promote chromoplast differentiation by preventing the degradation of carotenoids such as β-carotene. We conclude that the Clp protease contributes to the differentiation of chloroplasts into chromoplasts during tomato fruit ripening, acting in co-ordination with specific chaperones that alleviate protein folding stress, promote enzyme stability and accumulation, and prevent carotenoid degradation

Protein requirement in masculinized and non-masculinized juveniles of Bay Snook Petenia splendida

The effect of the dietary protein level on growth and total body chemical composition of the native cichlid Bay snook (Petenia splendida), masculinized and non-masculinized, was studied. Five semi-purified diets with protein levels 35, 40, 45, 50 and 55% crude protein (CP) were formulated and evaluated by triplicate. Groups of 50 juveniles were each stocked in 70 L tanks and fed to apparent satiation for 42 days trial. At the end, weight gain (WG) (403.41%), body length (BL) (6.58 ± 0.10 cm) and specific growth rate (SRG) (1.67%/day) of the masculinized fish were obtained with the 45% CP diet, and they were significantly different (p = 0.002) from the other treatments. In the case of non-masculinized fish, the 45 and 55% CP treatments showed significant differences (p = 0.00001), with respect to other treatments, with a WG of 398 and 394%, SGR of 1.66 and 1.63%/day, protein productive value (PPV) of 28.91 and 29.21%, and feed conversion rate (FCR) of 1.23 and 1.08 respectively. Protein body composition for masculinized fish was different (p = 0.0001) only for fish fed 35% CP compared with fish at the beginning of the experiment. We conclude that the optimum protein requirement, estimated by the broken-line model for masculinized and non-masculinized P. splendida was 45 and 44.8% PC respectivel

Cause of Death and Predictors of All-Cause Mortality in Anticoagulated Patients With Nonvalvular Atrial Fibrillation : Data From ROCKET AF

M. Kaste on työryhmän ROCKET AF Steering Comm jäsen.Background-Atrial fibrillation is associated with higher mortality. Identification of causes of death and contemporary risk factors for all-cause mortality may guide interventions. Methods and Results-In the Rivaroxaban Once Daily Oral Direct Factor Xa Inhibition Compared with Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation (ROCKET AF) study, patients with nonvalvular atrial fibrillation were randomized to rivaroxaban or dose-adjusted warfarin. Cox proportional hazards regression with backward elimination identified factors at randomization that were independently associated with all-cause mortality in the 14 171 participants in the intention-to-treat population. The median age was 73 years, and the mean CHADS(2) score was 3.5. Over 1.9 years of median follow-up, 1214 (8.6%) patients died. Kaplan-Meier mortality rates were 4.2% at 1 year and 8.9% at 2 years. The majority of classified deaths (1081) were cardiovascular (72%), whereas only 6% were nonhemorrhagic stroke or systemic embolism. No significant difference in all-cause mortality was observed between the rivaroxaban and warfarin arms (P=0.15). Heart failure (hazard ratio 1.51, 95% CI 1.33-1.70, P= 75 years (hazard ratio 1.69, 95% CI 1.51-1.90, P Conclusions-In a large population of patients anticoagulated for nonvalvular atrial fibrillation, approximate to 7 in 10 deaths were cardiovascular, whereasPeer reviewe

EDUCACIÓN AMBIENTAL Y SOCIEDAD. SABERES LOCALES PARA EL DESARROLLO Y LA SUSTENTABILIDAD

Este texto contribuye al análisis científico de varias áreas del conocimiento como la filosofía social, la patología, la educación para el cuidado del medio ambiente y la sustentabilidad que inciden en diversas unidades de aprendizaje de la Licenciatura en Educación para la Salud y de la Maestría en Sociología de la SaludLas comunidades indígenas de la sierra norte de Oaxaca México, habitan un territorio extenso de biodiversidad. Sin que sea una área protegida y sustentable, la propia naturaleza de la región ofrece a sus visitantes la riqueza de la vegetación caracterizada por sus especies endémicas que componen un paisaje de suma belleza

Unveiling the role of DXS-Interacting (DXI) proteins in the regulation of plastidial isoprenoid biosynthesis

[eng] Chloroplasts provide plants with metabolic pathways that are unique among eu- karoytes, including the methylerythritol 4-phosphate pathway (MEP) for the pro- duction of isoprenoids essential for photosynthesis and plant growth. The first re- action of the MEP pathway involves the synthesis of deoxyxylulose 5-phosphate (DXP) from the central metabolic intermediates glyceraldehyde 3-phosphate (GAP) and pyruvate catalyzed by DXP synthase (DXS). DXS has a major role in regulating the MEP pathway flux, but little is known about how its levels and activity are regu- lated. It has been shown that DXS stability and enzymatic activity can be modulated by interaction with other plastidial DXS-interacting (DXI) proteins. The goal of this thesis work has been to characterize the physiological role of DXS-DXI interactions and the molecular pathways leading from the interactions to the eventual biological effects in the model plant Arabidopsis thaliana. To investigate whether loss of DXI function in mutants impacted DXS activity, we analyzed their resistance to clomazone (CLM), a DXS-specific inhibitor. Most of the loss-of-function mutants tested did not show resistance or sensitivity to this in- hibitor. However, two mutant alleles for the gene SBP, which encodes the Calvin cycle enzyme sedoheptulose 1,7-bisphosphatase (SBPase), showed an increased re- sistance to CLM. In contrast, overproduction of SBPase in transgenic Arabidopsis plants resulted in reduced CLM resistance. Strikingly, we found that DXS protein levels or activity did not change when SBPase levels are altered in plants. Although co-immunoprecipitation assays were unable to confirm the interaction DXS-SBPase, our results do show a functional relationship between the Calvin cycle and the MEP pathway. We propose that excess GAP in the sbp mutant is diverted into the MEP pathway, preventing CLM binding to DXS. The second part of the thesis continued previous work with DXI1, a DXS-interacting J-protein that facilitates the recognition of inactive DXS forms to deliver them to eventual reactivation or degradation pathways. In particular, we focused on inves- tigating the molecular components involved in these two opposite pathways. By bioinformatic and experimental approaches, we confirmed that DXS is prone to ag- gregate in the chloroplast and associate to insoluble (membrane) fractions in an in- active form. These inactive forms of the enzyme were found to overaccumulate in plants defective in DXI1 (renamed J20). J20 is an adaptor of the Hsp70 chaperone. We demonstrated that the DXS-Hsp70 complex interacts with the Hsp100/ClpC1 vi chaperone to unfold DXS for delivery into the proteolytic chamber of the Clp pro- teolytic complex. On the other hand, correct folding of DXS is achieved with the contribution of Hsp100/ClpB3. Our work suggests that degradation or activation of DXS might depend mostly on changes in ClpB3 levels. This disaggregase accumulates when the MEP pathway flux is decreased and in situations causing protein folding stress. Through molecular, genetic and pharmacological approaches, we demonstrated that this accumulation depends on a mechanism called chloroplast Unfolded Protein Response (cpUPR). Elicitation of this cpUPR by inhibition of protein synthesis in the chloroplast led to increased expression of nuclear genes encoding ClpB3 and other chloroplast chap- erones, eventually causing a stress acclimation response. We further demonstrated that cpUPR is independent of GUN1, an integrator of retrograde signaling, since we observed that chaperones accumulate in both wild-type and gun1 mutant plants. However, GUN1-defective plants were unable to develop the acclimation response. Our data therefore confirm that GUN1 is a central integrator of different pathways controlling chloroplast protein homeostasis beyond the control of nuclear gene ex- pression. Our results will contribute to taking more informed decisions on future approaches to manipulate levels of chloroplast isoprenoids of interest (such as vitamins, biofuels or drugs against cancer and malaria) in crop plants.[spa] Los cloroplastos tienen vías metabólicas únicas entre los eucariontes, incluyendo la vía del metileritritol 4-fosfato (vía MEP) para la producción de isoprenoides. La primera reacción de la vía MEP es la síntesis de desoxixilulosa 5-fosfato (DXP) a partir de gliceraldehído 3-fostato (GAP) y piruvato, catalizada por la enzima DXP sintasa (DXS). DXS tiene un papel central en regular el flujo de la vía MEP, pero aún se sabe relativamente poco acerca de cómo se regulan sus niveles y actividad enz- imática. En esta tesis se ha estudiado cómo la interacción de DXS con otras proteínas regula su función en Arabidopsis thaliana. De entre estas proteínas interactoras, solo la pérdida de función de la enzima sedoheptulosa 1,7-bifosfatasa generó un fenotipo de resistencia a la inhibición de DXS. Los resultados de la tesis permiten concluir que existe un vínculo funcional entre el ciclo de Calvin y la vía MEP, seguramente medi- ado por la disponibilidad de GAP. Por otro lado, se ha observado que DXS es un en- zima propenso a agregarse. Estas formas inactivas interaccionan con la proteína J20, un adaptador de la chaperona Hsp70. El complejo DXS-Hsp70 a su vez interactúa con ClpC1 para degradar DXS mediante la proteasa Clp. Por otro lado, la interacción del complejo con ClpB3 pliega correctamente y activa DXS. El destino de DXS podría depender mayoritariamente de los cambios en los niveles de ClpB3. Un menor flujo de vía MEP, la pérdida de la homeostasis proteica en el cloroplasto, o la expresión defectuosa del plastoma causan la acumulación de ClpB3 y de otras chaperonas me- diante la activación de la expresión de los correspondientes genes nucleares. Esta respuesta no requiere de la actividad de GUN1, un nodo central en la comunicación cloroplasto-núcleo. Sin embargo, GUN1 es esencial para la posterior respuesta de aclimatación que permite a las plantas soportar mejor otros tipos de estrés como los causados por la inhibición de la síntesis de isoprenoides. Nuestros resultados pueden contribuir a tomar decisiones más informadas para manipular los niveles de isoprenoides cloroplastídicos de interés (como vitaminas, biocombustibles o fárma- cos contra el cáncer y la malaria) en plantas de cultivo