The root pathogen Aphanomyces euteiches secretes modular proteases in pea apoplast during host infection.

To successfully colonize the host, phytopathogens have developed a large repertoire of components to both combat the host plant defense mechanisms and to survive in adverse environmental conditions. Microbial proteases are predicted to be crucial components of these systems. In the present work, we aimed to identify active secreted proteases from the oomycete Aphanomyces euteiches, which causes root rot diseases on legumes. Genome mining and expression analysis highlighted an overrepresentation of microbial tandemly repeated proteases, which are upregulated during host infection. Activity Based Protein Profiling and mass spectrometry (ABPP-MS) on apoplastic fluids isolated from pea roots infected by the pathogen led to the identification of 35 active extracellular microbial proteases, which represents around 30% of the genes expressed encoding serine and cysteine proteases during infection. Notably, eight of the detected active secreted proteases carry an additional C-terminal domain. This study reveals novel active modular extracellular eukaryotic proteases as potential pathogenicity factors in Aphanomyces genus.

Comparison of different methods to calculate the axial length measured by optical biometry.

PURPOSE: To compare axial length (AL) measurements in long eyes by 2 swept-source optical coherence tomography (SS-OCT) biometers, one based on the group refractive index (IOLMaster 700, Zeiss) and the other based on sum of segments (Argos, Movu Inc.), and compare these measurements with previously published methods to optimize AL. SETTING: G.B. Bietti Foundation I.R.C.C.S., Rome, Italy. DESIGN: Prospective case series. METHODS: AL was measured with both optical biometers in patients with myopia (AL > 24.0 mm) and compared with the values obtained with Wang-Koch adjustment, polynomial equations for the Holladay 1 and 2 formulas, and Cooke-modified AL (CMAL). RESULTS: In 102 eyes of 55 subjects, a statistically significant difference (P < .0001) was found among the 6 AL values. Posttest revealed that Argos measurements (26.90 ± 1.61 mm) were significantly lower compared with those provided by all methods (P < .001) but CMAL, whereas IOLMaster 700 measurements (27.01 ± 1.65) were higher (P < .001). No difference was found between the 2 Holladay equations. CMAL values did not reveal any difference compared with those of the Argos, but a proportional bias showed that in longer eyes, CMAL provided smaller values (P < .0001, r = -0.7221). AL overestimation by the IOLMaster 700 AL compared with the Argos was higher, the longer the eye was (P < .0001, r = 0.6959, r2 = 0.4842). CONCLUSIONS: The SS-OCT optical biometer based on the group refractive index overestimates AL compared with the device using segmented AL. CMAL provides the measurements closest to those of the device using segmented AL.

Autophagy Activation by Resveratrol Reduces Severity of Experimental Rheumatoid Arthritis.

SCOPE: Previous work reported that dietary supplementation with resveratrol lowers synovial hyperplasia, inflammatory and oxidative damage in an antigen-induced arthritis (AIA) model. Here, it is investigated whether resveratrol can regulate the abnormal synovial proliferation by inducing autophagy and controlling the associated inflammatory response. METHODS AND RESULTS: Animals treated with resveratrol 8 weeks before AIA induction show the highest significant signal for microtubule-associated protein 1 light chain 3 by confocal microscopy. Besides, resveratrol significantly reduces p62 expression, but it does not increase the signal of beclin-1. Also, active caspase-3 expression, as well as poly(ADP-ribose) polymerase, is upregulated in the AIA group, and is significantly reduced in resveratrol-treated AIA group. Resveratrol also mitigates angiopoietin-1 and vascular endothelial growth factor signals. Finally, resveratrol significantly reduces the serum levels of IL-1ß, C reactive protein, and prostaglandin E2, as well as nuclear factor κB synovial tissue expression, which shows a significant correlation with p62 expression. CONCLUSION: Dietary supplementation with resveratrol induces the noncanonical autophagy pathway and limits the cross-talk with inflammation, which in consequence modulates the synovial hyperplasia. Preventive strategies that incorporate dietary intervention with resveratrol may offer a potential therapeutic alternative to drugs to influence the risk of rheumatoid arthritis and influence its course.

Extracellular proteolytic cascade in tomato activates immune protease Rcr3.

Proteolytic cascades regulate immunity and development in animals, but these cascades in plants have not yet been reported. Here we report that the extracellular immune protease Rcr3 of tomato is activated by P69B and other subtilases (SBTs), revealing a proteolytic cascade regulating extracellular immunity in solanaceous plants. Rcr3 is a secreted papain-like Cys protease (PLCP) of tomato that acts both in basal resistance against late blight disease (Phytophthora infestans) and in gene-for-gene resistance against the fungal pathogen Cladosporium fulvum (syn. Passalora fulva) Despite the prevalent model that Rcr3-like proteases can activate themselves at low pH, we found that catalytically inactive proRcr3 mutant precursors are still processed into mature mRcr3 isoforms. ProRcr3 is processed by secreted P69B and other Asp-selective SBTs in solanaceous plants, providing robust immunity through SBT redundancy. The apoplastic effector EPI1 of P. infestans can block Rcr3 activation by inhibiting SBTs, suggesting that this effector promotes virulence indirectly by preventing the activation of Rcr3(-like) immune proteases. Rcr3 activation in Nicotiana benthamiana requires a SBT from a different subfamily, indicating that extracellular proteolytic cascades have evolved convergently in solanaceous plants or are very ancient in the plant kingdom. The frequent incidence of Asp residues in the cleavage region of Rcr3-like proteases in solanaceous plants indicates that activation of immune proteases by SBTs is a general mechanism, illuminating a proteolytic cascade that provides robust apoplastic immunity.

The mitochondrial inhibitor oligomycin induces an inflammatory response in the rat knee joint.

BACKGROUND: Recent findings support a connection between mitochondrial dysfunction and activation of inflammatory pathways in articular cells. This study investigates in vivo in an acute model whether intra-articular administration of oligomycin, an inhibitor of mitochondrial function, induces an oxidative and inflammatory response in rat knee joints. METHODS: Oligomycin was injected into the rat left knee joint on days 0, 2, and 5 before joint tissues were obtained on day 6. The right knee joint served as control. Results were evaluated by macroscopy and histopathology and by measuring cellular and mitochondrial reactive oxygen species (ROS), 4-hydroxy-2-nonenal (4-HNE, a marker of lipid peroxidation), nuclear factor erythroid 2-related factor 2 (Nrf2), and CD68 (macrophages) and chemokine levels. The marker of mitochondrial mass COX-IV was also evaluated. RESULTS: The macroscopic findings showed significantly greater swelling in oligomycin-injected knees than in control knees. Likewise, the histological score of synovial damage was also increased significantly. Immunohistochemical studies showed high expression of IL-8, coinciding with a marked infiltration of polymorphonuclears and CD68+ cells in the synovium. Mitochondrial mass was increased in the synovium of oligomycin-injected joints, as well as cellular and mitochondrial ROS production, and 4-HNE. Relatedly, expression of the oxidative stress-related transcription factor Nrf2 was also increased. As expected, no histological differences were observed in the cartilage; however, cytokine-induced neutrophil chemoattractant-1 mRNA and protein expression were up-regulated in this tissue. CONCLUSIONS: Mitochondrial failure in the joint is able to reproduce the oxidative and inflammatory status observed in arthritic joints.

Resveratrol lowers synovial hyperplasia, inflammatory markers and oxidative damage in an acute antigen-induced arthritis model.

OBJECTIVE: The present study aimed to determine the protective effects of dietary supplementation with resveratrol (RSV) in an acute antigen-induced arthritis (AIA) model. METHODS: Rats were randomly divided into three groups: control, AIA and RSV-treated AIA group. RSV (12.5 mg/kg/day) was given orally for 8 weeks before induction of AIA and until the end of the experiment (48 h after intra-articular injection). The control and AIA animals were administered 100 µl of water. Results were evaluated by macroscopic observation, histopathology and immunohistochemistry for anti-PCNA, macrophages (CD68), T lymphocytes (CD3), monocyte chemoattractant protein-1 and 8-oxo-7,8-dihydro-2'-deoxyguanine (a marker of DNA damage). Cytokine-induced neutrophil chemoattractant-1 in serum and peroxidase activity in synovial tissue were measured using commercial kits. RESULTS: At the end of the study, RSV significantly reduced knee swelling. Likewise, the histological score of synovial tissue also reduced significantly. The arthritis-protective effects were associated with a significant decrease in PCNA, CD68, CD3 and monocyte chemoattractant protein-1 staining, as well as a reduction in serum concentrations of cytokine-induced neutrophil chemoattractant-1. RSV treatment also decreased the level of the marker of DNA damage, 8-oxo-7,8-dihydro-2'-deoxyguanine. Accordingly, peroxidase activity in the synovial tissue was up-regulated. CONCLUSION: Dietary supplementation with RSV lowers the main pathological hallmarks of RA disease in an acute model of AIA. RSV may represent a promising strategy in controlling the severity of RA.

Exenteración pélvica total en el tratamiento de las neoplasias avanzadas, primarias o recurrentes, de vísceras pélvicas / Total pelvic exenteration for the treatment of advanced primary or recurrent pelvic neoplasia

INTRODUCCIÓN: La resección completa con márgenes libres en los tumores viscerales pélvicos localmente avanzados, primarios o recurrentes, requiere ocasionalmente de una exenteración pélvica total (EPT). MÉTODOS: Revisamos los resultados obtenidos con la EPT en 34 pacientes consecutivos operados entre junio de 2006 y diciembre de 2013. RESULTADOS: La mediana de edad fue de 62 (40-82) años; 24 (70%) eran varones. El origen tumoral más frecuente fue el avanzado y primitivo de recto (TAPR), con 19 casos (55,9%) y el tipo de exenteración, la supraelevadora (61,8%). Se logró una resección R0 en 24 (70,6%) pacientes y en 16 (85%) de los TAPR. Quince (79%) pacientes con TAPR tenían pT4, y 4 (20%) pN+. La continuidad intestinal y urinaria se realizó con 2 estomas en 17 casos (50%), Bricker y anastomosis colorrectal en 11 (32,3%) y colostomía húmeda double barreled en 6 (17,6%). No hubo mortalidad postoperatoria; 23 (67,5%) pacientes tuvieron complicaciones y 5 (14,6%) requirieron una reoperación en el postoperatorio. La mediana de seguimiento fue de 23 (13-45) meses. La supervivencia global (SG) y libre de enfermedad (SLE) a los 2 años fueron del 67% y 58% respectivamente, y la mediana de SG y SLE fue de 59 meses (IC 95%: 26 a 110) y de 39 meses (IC 95%: 14 a 64), respectivamente. La SLE de las R0 fue significativamente mejor (p = 0,003) que las R1. CONCLUSIONES: La EPT es un procedimiento potencialmente curativo para las neoplasias viscerales avanzadas de la pelvis, con una morbimortalidad similar a otras cirugías exeréticas mayores

INTRODUCTION: Complete resection with clear margins in locally advanced pelvic visceral tumors, primary or recurrent, occasionally requires total pelvic exenteration (TPE). METHODS: We reviewed the results of EFA in 34 consecutive patients operated on between June 2006 and December 2013.ResultsMedian age was 62 (40-82) years; 24 (70%) were male. The tumor origin most frequent was advanced primary rectal tumor (APRT), with 19 cases (55.9%) and most common type of exenteration was supraelevator (61.8%). R0 resection was achieved in 24 (70.6%) patients and in 16 (85%) of the APRT. Fifteen (79%) patients had pT4 APRT, and 4 (20%) pN +. Reconstruction of the bowel and bladder was performed with two stomas in 17 cases (50%), colorectal anastomosis and Bricker in 11 (32.3%) and wet double barreled colostomy in 6 (17.6%). There was no postoperative mortality; 23 (67,5%) patients had complications, and 5 (14.6%) required a postoperative reoperation to solve them. Median follow-up was 23 (13-45) months. Overall survival (OS) and disease free survival (DFS) at 2 years were 67% and 58% respectively, and the median OS and DFS was 59 months (95% CI 26-110) and 39 months (95% CI 14-64), respectively. The DFS of R0 was significantly better (p = 0.003) than R1. CONCLUSIONS: TPE is a potentially curative procedure for advanced pelvic visceral malignancies with similar morbi-mortality than other extended excisional surgery

Total pelvic exenteration for the treatment of advanced primary or recurrent pelvic neoplasia.

INTRODUCTION: Complete resection with clear margins in locally advanced pelvic visceral tumors, primary or recurrent, occasionally requires total pelvic exenteration (TPE). METHODS: We reviewed the results of EFA in 34 consecutive patients operated on between June 2006 and December 2013. RESULTS: Median age was 62 (40-82) years; 24 (70%) were male. The tumor origin most frequent was advanced primary rectal tumor (APRT), with 19 cases (55.9%) and most common type of exenteration was supraelevator (61.8%). R0 resection was achieved in 24 (70.6%) patients and in 16 (85%) of the APRT. Fifteen (79%) patients had pT4 APRT, and 4 (20%) pN +. Reconstruction of the bowel and bladder was performed with two stomas in 17 cases (50%), colorectal anastomosis and Bricker in 11 (32.3%) and wet double barreled colostomy in 6 (17.6%). There was no postoperative mortality; 23 (67,5%) patients had complications, and 5 (14.6%) required a postoperative reoperation to solve them. Median follow-up was 23 (13-45) months. Overall survival (OS) and disease free survival (DFS) at 2 years were 67% and 58% respectively, and the median OS and DFS was 59 months (95% CI 26-110) and 39 months (95% CI 14-64), respectively. The DFS of R0 was significantly better (p=0.003) than R1. CONCLUSIONS: TPE is a potentially curative procedure for advanced pelvic visceral malignancies with similar morbi-mortality than other extended excisional surgery.

Mitochondrial dysfunction promotes and aggravates the inflammatory response in normal human synoviocytes.

OBJECTIVES: In RA, synoviocytes cause increased oxidative stress, leading to mitochondrial alterations that may participate in the pathogenesis of RA. Here we investigated whether mitochondrial dysfunction induces inflammatory responses in cultured normal human synoviocytes, a hallmark of RA. METHODS: Mitochondrial dysfunction was induced with the inhibitor oligomycin. The effects of mitochondrial dysfunction on cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) and IL-8 expression; cellular and mitochondrial reactive oxygen species (ROS) production; nuclear factor-κB (NF-κB) activation and p65 translocation were studied. ROS scavengers (N-acetylcysteine and mitoTEMPO) and an NF-κB inhibitor (BAY-117085) were used to investigate the pathways involved. The natural anti-inflammatory antioxidant resveratrol was also tested. RESULTS: Mitochondrial dysfunction per se significantly stimulated mitochondrial ROS production as well as low-grade expressions of COX-2, PGE2 and IL-8. Interestingly, mitochondrial dysfunction induced by pretreatment of synoviocytes with oligomycin synergized with IL-1ß to increase the expression of these inflammatory mediators. The inflammatory effects of mitochondrial damage appeared to be dependent on ROS production and NF-κB activation since the inflammatory response was counteracted by both N-acetylcysteine and mitoTEMPO and it was also reduced by BAY-117085. Antimycin A and paraquat (inhibitors of mitochondrial function) also induced inflammatory responses. Furthermore, resveratrol significantly reduced the inflammatory response by decreasing ROS production and NF-κB activation. CONCLUSION: These data suggest that mitochondrial dysfunction could induce an inflammatory response in normal human synoviocytes and sensitize these cells, causing a significant amplification of the inflammatory response induced by IL-1ß. Resveratrol may represent a promising strategy in controlling the synovial inflammatory response.

Establishment of cell suspension cultures of two Costa Rican Jatropha species (Euphorbiaceae).

J. curcas has been studied in different countries and some interesting agronomic, pharmacological and industrial properties have been reported. More recently, it has been considered an important alternative source for biofuel production. The objective of this study was to establish a long-term method for the maintenance of calli and cell suspension cultures of the local species J. curcas and J. gossypifolia, in order to allow future studies for novel compounds with pharmaceutical or industrial applications. For this, friable calli were successfully induced from hypocotyl segments of.. curcas and J. gossypifolia that were cultured in semisolid MS media supplemented with 1.5 mg/L, and 0.5 mg/L of 2,4-D, respectively. Cell suspension cultures of J. curcas were established using 1 g of 35 and 60-day calli, in 50 mL of liquid MS media supplied with 1.5 mg/L of 2,4-D; sucrose and maltose were additionally evaluated as carbon sources. After 35 days, cell suspension cultures initiated with 35-day calli, showed greater cell growth with a maximum biomass of 194.9 g/L fresh weight, 6.59 g/L dry weight and 17.3% packed volume. The exponential phase ended at day 35 for cultures initiated with 35-day calli, and at day 21 for cultures initiated with 60-day calli. Higher biomass production was obtained with sucrose. Cell cultures were established with 35-day calli in MS media with the same 2,4-D concentration used for calli induction and 30g/L sucrose. This medium was considered optimum for the maintenance and growth of cell suspensions for both species, with sub-cultures every 20 days. The biotechnological potential for the production of bioactive compounds in these species for pharmacological, agricultural and industrial applications is being evaluated.

Establishment of cell suspension cultures of two Costa Rican Jatropha species (Euphorbiaceae) / Establecimiento de suspensiones celulares de dos especies Jatropha (Euphorbiaceae) de Costa Rica

J. curcas has been studied in different countries and some interesting agronomic, pharmacological and industrial properties have been reported. More recently, it has been considered an important alternative source for biofuel production. The objective of this study was to establish a long-term method for the maintenance of calli and cell suspension cultures of the local species J. curcas and J. gossypifolia, in order to allow future studies for novel compounds with pharmaceutical or industrial applications. For this, friable calli were successfully induced from hypocotyl segments of J. curcas and J. gossypifolia that were cultured in semisolid MS media supplemented with 1.5mg/L, and 0.5mg/L of 2,4-D, respectively. Cell suspension cultures of J. curcas were established using 1g of 35 and 60-day calli, in 50mL of liquid MS media supplied with 1.5mg/L of 2,4-D; sucrose and maltose were additionally evaluated as carbon sources. After 35 days, cell suspension cultures initiated with 35-day calli, showed greater cell growth with a maximum biomass of 194.9g/L fresh weight, 6.59g/L dry weight and 17.3% packed volume. The exponential phase ended at day 35 for cultures initiated with 35-day calli, and at day 21 for cultures initiated with 60-day calli. Higher biomass production was obtained with sucrose. Cell cultures were established with 35-day calli in MS media with the same 2,4-D concentration used for calli induction and 30g/L sucrose. This medium was considered optimum for the maintenance and growth of cell suspensions for both species, with sub-cultures every 20 days. The biotechnological potential for the production of bioactive compounds in these species for pharmacological, agricultural and industrial applications is being evaluated.

J. curcas es un importante recurso alternativo de biocombustible. Por otro lado, propiedades de interés agronómico, farmacológico e industrial han sido reportadas para esta especie. El objetivo de este estudio fue el establecimiento y mantenimiento a largo plazo de callos y cultivos celulares en suspensión de J. curcas y J. gossypifolia, con el objetivo de permitir futuros estudios para nuevos compuestos con aplicaciones farmaceúticas e industriales. Los callos friables fueron exitosamente inducidos a partir de segmentos de hipocótilos J. curcas and J. gossypifolia cultivados en medio MS semisólido suplementado con 1.5mg/L y 0.5mg/L of 2,4-D, respectivamente. Los cultivos celulares en suspensión de J. curcas fueron establecidos utilizando 1g de callos de 35 y 60 días de edad en 50mL de medio MS líquido adicionado con 1.5mg/L de 2,4-D. Después de 35 días, los cultivos en suspensión celular iniciados con callos de 35 días, mostraron mayor crecimiento celular con una biomasa máxima de 194.9g/L de peso fresco y 6.59g/L de peso seco y 17.3% de volumen empacado. La fase exponencial finalizó al día 35 en los cultivos iniciados con callos de 35 días, y al día 21 en los cultivos iniciados con callos de 60 días. Dos fuentes de carbono fueron evaluadas: sacarosa y maltosa. La producción de mayor biomasa fue obtenida con sacarosa. Los cultivos celulares se establecieron con callos de 35 días cultivados en medio MS con la misma concentración de 2,4-D utilizada para la inducción de callos y 30g/L de sacarosa. Este medio fue considerado el óptimo para el mantenimiento y crecimiento de suspensiones celulares en ambas especies con subcultivos cada 20 días. El potencial biotecnológico para la producción de compuestos bioactivos en estas especies, para aplicaciones farmacológicas, agrícolas e industriales está siendo evaluado.

Mitochondrial dysfunction increases inflammatory responsiveness to cytokines in normal human chondrocytes.

OBJECTIVE: Alterations in mitochondria play a key role in the pathogenesis of osteoarthritis (OA). The role of inflammation in the progression of OA has also acquired important new dimensions. This study was undertaken to evaluate the potential role of mitochondrial dysfunction in increasing the inflammatory response of normal human chondrocytes to cytokines. METHODS: Mitochondrial dysfunction was induced by commonly used inhibitors. Interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNFα) were used as inflammatory mediators. IL-8 and cyclooxygenase 2 (COX-2) protein and messenger RNA (mRNA) expression and prostaglandin E(2) (PGE(2) ) levels were assessed. The chemotactic activity of neutrophils was assayed. Additionally, inhibitors of reactive oxygen species (ROS) and NF-κB were used to identify possible inflammatory response pathways induced by mitochondrial dysfunction, and the effects of the natural antioxidant resveratrol were tested. RESULTS: Pretreatment with antimycin A or oligomycin (inhibitors of mitochondrial respiratory chain complexes III and V, respectively) triggered a strong potentiation of IL-1ß-induced IL-8 mRNA and protein expression (mean ± SEM at 18 hours 5,932 ± 1,995 pg/50,000 cells for IL-1ß alone versus 16,241 ± 5,843 pg/50,000 cells for antimycin A plus IL-1ß and 20,087 ± 5,407 pg/50,000 cells for oligomycin plus IL-1ß; P < 0.05). Similar results were observed with TNFα or when expression of the inflammatory mediator COX-2 or PGE(2) production was assessed. Mitochondrial dysfunction increased the chemotactic activity induced by cytokines, and ROS and NF-κB inhibitors decreased the production of IL-8. Resveratrol significantly reduced the inflammatory response. CONCLUSION: Our findings indicate that mitochondrial dysfunction could amplify the responsiveness to cytokine-induced chondrocyte inflammation through ROS production and NF-κB activation. This pathway might lead to the impairment of cartilage and joint function in OA.