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Abstract Background Studying DNA methylation profiles in detail should be the first step in epigenetic research. Although sodium bisulfite modification of genomic DNA is the gold standard method for DNA methylation analysis, this method results in the loss of the majority of the DNA material. Whole genome amplification (WGA) of bisulfite-modified DNA is expected to provide a rich source of materials, but its validity has not been thoroughly evaluated. In this study, we evaluated the extent of biased amplification in the WGA of bisulfite-modified DNA and the reproducibility of independent WGA reactions. We performed the multiple displacement amplification-based WGA separately three times. Each experiment included two reactions using 10 or 50 ng of bisulfite-modified DNA as template. DNA methylation levels were compared between WGA products and original bisulfite-modified DNA at about 450,000 CpG sites. Results Using a sufficient amount of bisulfite-modified DNA for WGA was critical for downstream application. The considerable deviations from original bisulfite-modified DNA were found in the middle range of DNA methylation levels. Distribution of hyper- and hypomethylation were equal, which suggested that the deviation at each CpG site occurred randomly. Averaging the data from independently amplified WGA products dramatically improved the overall quality. Conclusions WGA of bisulfite-modified DNA could be a valuable tool for epigenetic research, but careful experimental design and data interpretation are required.

Abstract Background The early diagnosis of atherosclerotic disease is essential for developing preventive strategies in populations at high risk and acting when the disease is still asymptomatic. A low ankle-arm index (AAI) is a good marker of vascular events and may be diminished without presenting symptomatology (silent peripheral arterial disease). The aim of the PERART study (PERipheral ARTerial disease) is to determine the prevalence of peripheral arterial disease (both silent and symptomatic) in a general population of both sexes and determine its predictive value related to morbimortality (cohort study). Methods/Design This cross-over, cohort study consists of 2 phases: firstly a descriptive, transversal cross-over study to determine the prevalence of peripheral arterial disease, and secondly, a cohort study to evaluate the predictive value of AAI in relation to cardiovascular morbimortality. From September 2006 to June 2007, a total of 3,010 patients over the age of 50 years will be randomly selected from a population adscribed to 24 healthcare centres in the province of Barcelona (Spain). The diagnostic criteria of peripheral arterial disease will be considered as an AAI < 0.90, determined by portable Doppler (8 Mhz probe) measured twice by trained personnel. Cardiovascular risk will be calculated with the Framingham-Wilson tables, with Framingham calibrated by the REGICOR and SCORE groups. The subjects included will be evaluted every 6 months by telephone interview and the clnical history and death registries will be reviewed. The appearance of the following cardiovascular events will be considered as variables of response: transitory ischaemic accident, ictus, angina, myocardial infartction, symptomatic abdominal aneurysm and vascular mortality. Discussion In this study we hope to determine the prevalence of peripheral arterial disease, especially the silent forms, in the general population and establish its relationship with cardiovascular morbimortality. A low AAI may be a better marker of arterial disease than the classical cardiovascular risk factors and may, therefore, contribute to improving the predictive value of the equations of cardiovascular risk and thereby allowing optimisation of multifactorial treatment of atherosclerotic disease.

MicroRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression at the post-transcriptional level. In plants, miRNAs participate in diverse developmental processes and adaptive responses to biotic and abiotic stress. MiR827 has long been recognized to be involved in plant responses to phosphate starvation. In rice, the miR827 regulates the expression of OsSPX-MFS1 and OsSPX-MFS2, these genes encoding vacuolar phosphate transporters. In this study, we demonstrated that miR827 plays a role in resistance to infection by the fungus Magnaporthe oryzae in rice. We show that MIR827 overexpression enhances susceptibility to infection by M. oryzae which is associated to a weaker induction of defense gene expression during pathogen infection. Conversely, CRISPR/Cas9-induced mutations in the MIR827 gene completely abolish miR827 production and confer resistance to M. oryzae infection. This resistance is accompanied by a reduction of leaf Pi content compared to wild-type plants, whereas Pi levels increase in leaves of the blast-susceptible miR827 overexpressor plants. In wild-type plants, miR827 accumulation in leaves decreases during the biotrophic phase of the infection process. Taken together, our data indicates that silencing MIR827 confers resistance to M. oryzae infection in rice while further supporting interconnections between Pi signaling and immune signaling in plants. Unravelling the role of miR827 during M. oryzae infection provides knowledge to improve blast resistance in rice by CRISPR/Cas9-editing of MIR827., (© 2024. The Author(s).)

Background: To evaluate the association between diabetic foot disease (DFD) and the incidence of fatal and non-fatal events in individuals with type 2 diabetes (T2DM) from primary-care settings., Methods: We built a cohort of people with a first DFD episode during 2010-2015, followed up until 2018. These subjects were 1 to 1 propensity score matched to subjects with T2DM without DFD. The incidence of all-cause mortality, the occurrence of new DFD, amputations, cardiovascular diseases, or composite outcome, including all-cause mortality and/or cardiovascular events during the follow-up period, were calculated. A Cox proportional hazard analysis was conducted to evaluate the hazard ratios (HR) for different events., Results: Overall, 11,117 subjects with T2DM with a first episode of DFD were compared with subjects without DFD. We observed higher incidence rates (IRs) for composite outcome (33.9 vs. 14.5 IR per 100 person-years) and a new DFD episode event (22.2 vs. 1.1 IR per 100 person-years) in the DFD group. Compared to those without DFD, those with a first episode of DFD had a higher HR for all events, with excess rates particularly for amputation and new DFD occurrence (HR: 19.4, 95% CI: 16.7-22.6, HR: 15.1, 95% CI: 13.8-16.5, respectively) was found., Conclusions: Although DFD often coexists with other risk factors, it carries an intrinsic high risk of morbidity and mortality in individuals with T2DM. DFD should be regarded as a severe complication already at its onset, as it carries a poor clinical prognosis., (© 2024. The Author(s).)

Introduction: Women with type 2 diabetes mellitus (T2DM) face a greater risk of cardiovascular disease (CVD) and encounter challenges in managing cardiovascular risk factors (CVRF); however, limited data are available in individuals with newlydiagnosed T2DM., Methods: This study aimed to examine differences between women and men at the onset of T2DM in terms of clinical characteristics, glycaemic status, and CVRF management. This was a retrospective cohort study including subjects with newly-diagnosed T2DM from the System for the Development of Research in Primary Care (SIDIAP) database in Catalonia (Spain). Sex differences (Dif) were assessed at baseline and 1-year post-diagnosis, by calculating the absolute difference of means or proportions., Results: A total of 13,629 subjects with newly-diagnosed T2DM were analyzed. Women were older and had a higher BMI than men. At baseline, women had higher total cholesterol [Dif (95%CI) 10 mg/dL (9.1/10.8)] and low-density lipoprotein cholesterol (LDL-c) [Dif (95%CI) 7 mg/dL (6.3/7.7)], while men had higher rates of smoking and alcohol intake. Lipid target achievement was lower in women, in both primary prevention (LDL-c < 100 mg/dL) [Dif (95%CI) -7.3 mg/dL (-10.5/-4.1)] and secondary prevention (LDL-c < 70 mg/dL) [Dif (95%CI) -8.3 mg/dL (-17.3/0.7)], along with lower statin and antiplatelet prescriptions, especially one year after diagnosis. Changes in clinical and laboratory data one year post-diagnosis revealed that, in the primary prevention group, men experienced greater improvements in total cholesterol, LDL-c and triglycerides, while women had less success in achieving CVRF control targets compared to men. Additionally, cardiovascular events, such as coronary artery disease and peripheral artery disease increased more in men than in women within the first year of diagnosis, especially in primary prevention subjects., Conclusion: Differences between men and women CVRF are already apparent at the onset of T2DM, particularly in primary prevention, with notable differences in lipid profile and target level attainment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Ramírez-Morros, Franch-Nadal, Real, Miró-Catalina, Bundó, Vlacho and Mauricio.)

Rice is one of the most important crops in the world and a staple food for more than half of the world's population. At present, the blast disease caused by the fungus Magnaporthe oryzae poses a severe threat to food security through reduction of rice yields worldwide. High phosphate fertilization has previously been shown to increase blast susceptibility. At present, however, our knowledge on the mechanisms underpinning phosphate-induced susceptibility to M. oryzae infection in rice is limited. In this work, we conducted live cell imaging on rice sheaths inoculated with a M. oryzae strain expressing two fluorescently-tagged M. oryzae effectors. We show that growing rice under high phosphate fertilization, and subsequent accumulation of phosphate in leaf sheaths, promotes invasive growth of M. oryzae . Consistent with this, stronger expression of M. oryzae effectors and Pathogenicity Mitogen-activated Protein Kinase ( PMK1 ) occurs in leaf sheaths of rice plants grown under high a phosphate regime. Down-regulation of fungal genes encoding suppressors of plant cell death and up-regulation of plant cell death-inducing effectors also occurs in sheaths of phosphate over-accumulating rice plants. Treatment with high Pi causes alterations in the expression of fungal phosphate transporter genes potentially contributing to pathogen virulence. From the perspective of the plant, Pi accumulation in leaf sheaths prevents H 2 O 2 accumulation early during M. oryzae infection which was associated to a weaker activation of Respiratory Burst Oxidase Homologs ( RBOHs ) genes involved in reactive oxygen species (ROS) production. Further, a weaker activation of defense-related genes occurs during infection in rice plants over-accumulating phosphate. From these results, it can be concluded that phosphate fertilization has an effect on the two interacting partners, pathogen and host. Phosphate-mediated stimulation of fungal effector genes (e.g., potentiation of fungal pathogenicity) in combination with repression of pathogen-inducible immune responses (e.g., ROS accumulation, defense gene expression) explains higher colonization by M. oryzae in rice tissues accumulating phosphate. Phosphate content can therefore be considered as an important factor in determining the outcome of the rice/ M. oryzae interaction. As fertilizers and pesticides are commonly used in rice cultivation to maintain optimal yield and to prevent losses caused by pathogens, a better understanding of how phosphate impacts blast susceptibility is crucial for developing strategies to rationally optimize fertilizer and pesticide use in rice production., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Martín-Cardoso, Bundó, Val-Torregrosa and San Segundo.)

Background: Diabetic foot and lower limb complications are an important cause of morbidity and mortality among persons with diabetes mellitus. Very few studies have been carried out in the primary care settings. The main objective was to assess the prognosis of diabetic foot ulcer (DFU) in patients from primary care centers in Catalonia, Spain, during a 12-month follow-up period., Methods: We included participants with type 2 diabetes and a new DFU between February 2018 and July 2019. We estimated the incidence of mortality, amputations, recurrence and healing of DFU during the follow-up period. A multivariable analysis was performed to assess the association of these outcomes and risk factors., Results: During the follow-up period, 9.7% of participants died, 12.1% required amputation, 29.2% had a DFU recurrence, and 73.8% healed. Having a caregiver, ischemia or infection were associated with higher mortality risk (hazard ratio [HR]:3.63, 95% confidence interval [CI]:1.05; 12.61, HR: 6.41, 95%CI: 2.25; 18.30, HR: 3.06, 95%CI: 1.05; 8.94, respectively). Diabetic retinopathy was an independent risk factor for amputation events (HR: 3.39, 95%CI: 1.37; 8.39). Increasing age decreased the risk for a DFU recurrence, while having a caregiver increased the risk for this event (HR: 0.97, 95%CI: 0.94; 0.99). The need for a caregiver and infection decreased the probability of DFU healing (HR: 0.57, 95%CI: 0.39; 0.83, HR: 0.64, 95%CI: 0.42; 0.98, respectively). High scores for PEDIS (≥7) or SINBAD (≥3) were associated with an increased risk for DFU recurrence and a lower probability of DFU healing, respectively., Conclusions: We observed high morbidity among subjects with a new DFU in our primary healthcare facilities. Peripheral arterial disease, infection, and microvascular complications increased the risk of poor clinical outcomes among subjects with DFU., (© 2023. The Author(s).)

Background: Rice is one of the most salt sensitive crops at seedling, early vegetative and reproductive stages. Varieties with salinity tolerance at seedling stage promote an efficient growth at early stages in salt affected soils, leading to healthy vegetative growth that protects crop yield. Saltol major QTL confers capacity to young rice plants growing under salt condition by maintaining a low Na + /K + molar ratio in the shoots., Results: Marker-assisted backcross (MABC) procedure was adopted to transfer Saltol locus conferring salt tolerance at seedling stage from donor indica IR64-Saltol to two temperate japonica varieties, Vialone Nano and Onice. Forward and background selections were accomplished using polymorphic KASP markers and a final evaluation of genetic background recovery of the selected lines was conducted using 15,580 SNP markers obtained from Genotyping by Sequencing. Three MABC generations followed by two selfing, allowed the identification of introgression lines achieving a recovery of the recurrent parent (RP) genome up to 100% (based on KASP markers) or 98.97% (based on GBS). Lines with highest RP genome recovery (RPGR) were evaluated for agronomical-phenological traits in field under non-salinized conditions. VN1, VN4, O1 lines were selected considering the agronomic evaluations and the RPGR% results as the most interesting for commercial exploitation. A physiological characterization was conducted by evaluating salt tolerance under hydroponic conditions. The selected lines showed lower standard evaluation system (SES) scores: 62% of VN4, and 57% of O1 plants reaching SES 3 or SES 5 respectively, while only 40% of Vialone Nano and 25% of Onice plants recorded scores from 3 to 5, respectively. VN1, VN4 and O1 showed a reduced electrolyte leakage values, and limited negative effects on relative water content and shoot/root fresh weight ratio., Conclusion: The Saltol locus was successfully transferred to two elite varieties by MABC in a time frame of three years. The application of background selection until BC 3 F 3 allowed the selection of lines with a RPGR up to 98.97%. Physiological evaluations for the selected lines indicate an improved salinity tolerance at seedling stage. The results supported the effectiveness of the Saltol locus in temperate japonica and of the MABC procedure for recovering of the RP favorable traits., (© 2023. The Author(s).)

Phosphorus is an important macronutrient required for plant growth and development. It is absorbed by the roots in the form of inorganic phosphate (Pi). Under Pi limiting conditions, plants activate the Phosphate Starvation Response (PSR) system to enhance Pi acquisition. The NITROGEN LIMITATION ADAPTION (NLA) gene is a component of the Arabidopsis PSR, and its expression is post-transcriptionally regulated by miR827. We show that loss-of-function of NLA and MIR827 overexpression increases Pi level and enhances resistance to infection by the fungal pathogen Plectosphaerella cucumerina in Arabidopsis. Upon pathogen infection, high Pi plants (e.g. nla plants and wild type plants grown under high Pi supply) showed enhanced callose deposition. High Pi plants also exhibited superinduction of camalexin biosynthesis genes which is consistent with increased levels of camalexin during pathogen infection. Pathogen infection and treatment with fungal elicitors, triggered up-regulation of MIR827 and down-regulation of NLA expression. Under non-infection conditions, the nla plants showed increased levels of SA and JA compared with wild type plants, their levels further increasing upon pathogen infection. Overall, the outcomes of this study suggest that NLA plays a role in Arabidopsis immunity, while supporting convergence between Pi signaling and immune signaling in Arabidopsis., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

In nature, plants are concurrently exposed to a number of abiotic and biotic stresses. Our understanding of convergence points between responses to combined biotic/abiotic stress pathways remains, however, rudimentary. Here we show that MIR399 overexpression, loss-of-function of PHOSPHATE2 (PHO2), or treatment with high phosphate (Pi) levels is accompanied by an increase in Pi content and accumulation of reactive oxygen species (ROS) in Arabidopsis thaliana. High Pi plants (e.g., miR399 overexpressors, pho2 mutants, and plants grown under high Pi supply) exhibited resistance to infection by necrotrophic and hemibiotrophic fungal pathogens. In the absence of pathogen infection, the expression levels of genes in the salicylic acid (SA)- and jasmonic acid (JA)-dependent signaling pathways were higher in high Pi plants compared to wild-type plants grown under control conditions, which is consistent with increased levels of SA and JA in non-infected high Pi plants. During infection, an opposite regulation in the two branches of the JA pathway (ERF1/PDF1.2 and MYC2/VSP2) occurs in high Pi plants. Thus, while pathogen infection induces PDF1.2 expression in miR399 OE and pho2 plants, VSP2 expression is downregulated by pathogen infection in these plants. This study supports the notion that Pi accumulation promotes resistance to infection by fungal pathogens in Arabidopsis, while providing a basis to better understand interactions between Pi signaling and hormonal signaling pathways for modulation of plant immune responses., (© 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Rice is the most salt sensitive cereal crop and its cultivation is particularly threatened by salt stress, which is currently worsened due to climate change. This study reports the development of salt tolerant introgression lines (ILs) derived from crosses between the salt tolerant indica rice variety FL478, which harbors the Saltol quantitative trait loci (QTL), and the salt-sensitive japonica elite cultivar OLESA. Genotyping-by-sequencing (GBS) and Kompetitive allele specific PCR (KASPar) genotyping, in combination with step-wise phenotypic selection in hydroponic culture, were used for the identification of salt-tolerant ILs. Transcriptome-based genotyping allowed the fine mapping of indica genetic introgressions in the best performing IL (IL22). A total of 1,595 genes were identified in indica regions of IL22, which mainly located in large introgressions at Chromosomes 1 and 3. In addition to OsHKT1;5 , an important number of genes were identified in the introgressed indica segments of IL22 whose expression was confirmed [e.g., genes involved in ion transport, callose synthesis, transcriptional regulation of gene expression, hormone signaling and reactive oxygen species (ROS) accumulation]. These genes might well contribute to salt stress tolerance in IL22 plants. Furthermore, comparative transcript profiling revealed that indica introgressions caused important alterations in the background gene expression of IL22 plants ( japonica cultivar) compared with its salt-sensitive parent, both under non-stress and salt-stress conditions. In response to salt treatment, only 8.6% of the salt-responsive genes were found to be commonly up- or down-regulated in IL22 and OLESA plants, supporting massive transcriptional reprogramming of gene expression caused by indica introgressions into the recipient genome. Interactions among indica and japonica genes might provide novel regulatory networks contributing to salt stress tolerance in introgression rice lines. Collectively, this study illustrates the usefulness of transcriptomics in the characterization of new rice lines obtained in breeding programs in rice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with one of the author, GS., (Copyright © 2022 Bundó, Martín-Cardoso, Pesenti, Gómez-Ariza, Castillo, Frouin, Serrat, Nogués, Courtois, Grenier, Sacchi and San Segundo.)

Objective: To assess the trends in cardiovascular risk factor control and drug therapy from 2007 to 2018 in subjects with type 2 diabetes mellitus (T2DM)., Materials and Methods: Cross-sectional analysis using yearly clinical data and treatment obtained from the SIDIAP database. Patients aged ≥18 years with a diagnosis of T2DM seen in primary care in Catalonia, Spain., Results: The number of T2DM patients increased from 299,855 in 2007 to 394,266 in 2018. We also found an increasing prevalence of cardiovascular disease, heart failure, and chronic kidney disease (from 18.4 to 24.4%, from 4.5 to 7.3%, and from 20.2 to 31.3%, respectively). The achievement of glycemic targets (HbA1c<7%) scarcely changed (54.9% to 55.9%). Major improvements were seen in blood pressure (≤140/90 mmHg: from 55% to 71.8%), and in lipid control (low-density lipoprotein cholesterol <100 mg/dl: 33.4% to 48.4%), especially in people with established cardiovascular disease (48.8 to 69.7%). Simultaneous achievement of all three targets improved from 12.5% to 20.1% in the overall population and from 24.5% to 32.2% in those with cardiovascular disease but plateaued after 2013. There was an increase in the percentage of patients treated with any antidiabetic drug (70.1% to 81.0%), especially metformin (47.7% to 67.7%), and DPP4i (0 to 22.6%). The use of SGLT-2 and GLP-1ra increased over the years, but remained very low in 2018 (5.5% and 2.1% of subjects, respectively). There were also relevant increases in the use of statins (38.0% to 49.2%), renin-angiotensin system (RAS) drugs (52.5% to 57.2%), and beta-blockers (14.3% to 22.7%)., Conclusions: During the 2007-2018 period, relevant improvements in blood pressure and lipid control occurred, especially in people with cardiovascular disease. Despite the increase in the use of antidiabetic and cardiovascular drugs, the proportion of patients in which the three objectives were simultaneously achieved is still insufficient and plateaued after 2013. The use of antidiabetic drugs with demonstrated cardio renal benefits (SGLT-2 and GLP-1ra) increased over the years, but their use remained quite low., Competing Interests: MM-C has received advisory honorarium from Astra-Zeneca, Bayer, Boehringer Ingelheim, GSK, Lilly, MSD, Novartis, Novo Nordisk, and Sanofi; he has received speaker honorarium from Astra-Zeneca, Bayer, Boehringer Ingelheim, GSK, Lilly, Menarini, MSD, Novartis, Novo Nordisk, and Sanofi; he has received research grants to the institution from Astra-Zeneca, GSK, Lilly, MSD, Novartis, Novo Nordisk and Sanofi. JF-N has received advisory and or speaking fees from Astra-Zeneca, Ascensia, Boehringer Ingelheim, GSK, Lilly, MSD, Novartis, Novo Nordisk, and Sanofi; he has received research grants to the institution from Astra-Zeneca. GSK, Lilly, MSD, Novartis, Novo Nordisk, Sanofi, and Boehringer. DM has received advisory and/or speaking fees from Astra-Zeneca, Ascensia, Boehringer Ingelheim, GSK, Lilly, MSD, Novartis, Novo Nordisk, and Sanofi; he has received research grants to the institution from Astra-Zeneca, GSK, Lilly, MSD, Novartis, Novo Nordisk, Sanofi, and Boehringer. MB has received advisory and speaking fees from MSD. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mata-Cases, Vlacho, Real, Puig-Treserra, Bundó, Franch-Nadal and Mauricio.)

Aims: The main objective was to assess the incidence of diabetic foot ulcers in type 2 diabetes individuals from primary care centres in Catalonia, Spain., Methods: Prospective observational study in 36 Primary Care centres in Catalonia during February 2018 and July 2019 was conducted. We included participants with type 2 diabetes and a new foot ulcer. We estimated the annual foot ulcer incidence and described the characteristics: presence of comorbidities, clinical parameters and the characteristics of the diabetic foot ulcers (DFU) at inclusion in the study., Results: The incidence of a new DFU during the 12-month recruitment period was 0.42%. The mean age of the participants was 72.2 years (± 12.7), the majority of them were males (n = 178; 69.5%). Overall, 43.8% of DFUs were located on the dorsal aspect of toes or interdigital spaces. A percentage of 43.4% of the participants had ulcers of less than 1 cm2 surface. Further, 44.1% of the participants had a neuroischemic, 20.3% a neuropathic, 20.3% an ischemic ulcer. A 25.3% of ulcers with a concomitant peripheral artery disease were infected., Conclusions: Although the incidence found was low, our study shows the great complexity of patients with foot ulcers treated in primary care., (Copyright © 2021 Primary Care Diabetes Europe. Published by Elsevier Ltd. All rights reserved.)

Phosphorus (P) is an essential nutrient for plant growth and productivity. Due to soil fixation, however, phosphorus availability in soil is rarely sufficient to sustain high crop yields. The overuse of fertilizers to circumvent the limited bioavailability of phosphate (Pi) has led to a scenario of excessive soil P in agricultural soils. Whereas adaptive responses to Pi deficiency have been deeply studied, less is known about how plants adapt to Pi excess and how Pi excess might affect disease resistance. We show that high Pi fertilization, and subsequent Pi accumulation, enhances susceptibility to infection by the fungal pathogen Magnaporthe oryzae in rice. This fungus is the causal agent of the blast disease, one of the most damaging diseases of cultivated rice worldwide. Equally, MIR399f overexpression causes an increase in Pi content in rice leaves, which results in enhanced susceptibility to M. oryzae. During pathogen infection, a weaker activation of defence-related genes occurs in rice plants over-accumulating Pi in leaves, which is in agreement with the phenotype of blast susceptibility observed in these plants. These data support that Pi, when in excess, compromises defence mechanisms in rice while demonstrating that miR399 functions as a negative regulator of rice immunity. The two signalling pathways, Pi signalling and defence signalling, must operate in a coordinated manner in controlling disease resistance. This information provides a basis to understand the molecular mechanisms involved in immunity in rice plants under high Pi fertilization, an aspect that should be considered in management of the rice blast disease., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

The aim of this study was to assess the co-digestion of residual biomass flows generated in microalgae-based wastewater treatment plants: microalgae, primary sludge and fat, oil and grease (FOG), with and without microalgae thermal pretreatment. The results evidenced the high methane yield of FOG (563 mL CH 4 /g VS) as compared to microalgae (140 mL CH 4 /gVS) and sludge (299 mL CH 4 /g VS). The methane yield of microalgae and sludge co-digestion (50-50% VS) was increased by 25 and 42% by adding 10 and 20% VS of FOG, respectively. Moreover, co-digestion trials improved the anaerobic digestion first-order kinetics by up to 67%. Regarding the thermal pretreatment, it increased the methane yield of microalgae by 60%, and 15% upon co-digestion with sludge and FOG. Therefore, co-digestion of microalgae, primary sludge and FOG appears as a promising strategy to enhance the biogas production, hence bioenergy recovery from wastewater, even without pretreatment., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

PAFs are short cationic and tryptophan-rich synthetic peptides with cell-penetrating antifungal activity. They show potent and selective killing activity against major fungal pathogens and low toxicity to other eukaryotic and bacterial cells. These properties make them a promising alternative to fulfill the need of novel antifungals with potential applications in crop protection, food preservation, and medical therapies. However, the difficulties of cost-effective manufacturing of PAFs by chemical synthesis or biotechnological production in microorganisms have hampered their development for practical use. This work explores the feasibility of using rice seeds as an economical and safe production system of PAFs. The rationally designed PAF102 peptide with improved antifungal properties was selected for assessing PAF biotechnological production. Two different strategies are evaluated: (1) the production as a single peptide targeted to protein bodies and (2) the production as an oleosin fusion protein targeted to oil bodies. Both strategies are designed to offer stability to the PAF peptide in the host plant and to facilitate its downstream purification. Our results demonstrate that PAF does not accumulate to detectable levels in rice seeds when produced as a single peptide, whereas it is successfully produced as fusion protein to the Oleosin18, up to 20 μg of peptide per gram of grain. We show that the expression of the chimeric Ole18-PAF102 gene driven by the Ole18 promoter results in the specific accumulation of the fusion protein in the embryo and aleurone layer of the rice seed. Ole18-PAF102 accumulation has no deleterious effects on seed yield, germination capacity, or seedling growth. We also show that the Oleosin18 protein serves as carrier to target the fusion protein to oil bodies facilitating PAF102 recovery. Importantly, the recovered PAF102 is active against the fungal phytopathogen Fusarium proliferatum . Altogether, our results prove that the oleosin fusion technology allows the production of PAF bioactive peptides to assist the exploitation of these antifungal compounds.

Rice ( Oryza sativa ) stands among the world's most important crop species. Rice is salt sensitive, and the undue accumulation of sodium ions (Na + ) in shoots has the strongest negative correlation with rice productivity under long-term salinity. The plasma membrane Na + /H + exchanger protein Salt Overly Sensitive 1 (SOS1) is the sole Na + efflux transporter that has been genetically characterized to date. Here, the importance of SOS1-facilitated Na + flux in the salt tolerance of rice was analyzed in a reverse-genetics approach. A sos1 loss-of-function mutant displayed exceptional salt sensitivity that was correlated with excessive Na + intake and impaired Na + loading into the xylem, thus indicating that SOS1 controls net root Na + uptake and long-distance Na + transport to shoots. The acute Na + sensitivity of sos1 plants at low NaCl concentrations allowed analysis of the transcriptional response to sodicity stress without effects of the osmotic stress intrinsic to high-salinity treatments. In contrast with that in the wild type, sos1 mutant roots displayed preferential down-regulation of stress-related genes in response to salt treatment, despite the greater intensity of stress experienced by the mutant. These results suggest there is impaired stress detection or an inability to mount a comprehensive response to salinity in sos1 In summary, the plasma membrane Na + /H + exchanger SOS1 plays a major role in the salt tolerance of rice by controlling Na + homeostasis and possibly contributing to the sensing of sodicity stress., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Microalgal-based wastewater treatment plants are conceived as low cost and low energy consuming systems. The operation of these plants involves the management of primary sludge and microalgal biomass. The aim of this study is to analyse the anaerobic co-digestion of both by-products in terms of biogas production and contaminants of emerging concern removal. The co-digestion of microalgae and primary sludge (25/75% on a volatile solids basis) was investigated in continuous reactors and compared to microalgae mono-digestion at a hydraulic retention time of 20days. Results showed how the co-digestion enhanced the anaerobic digestion of microalgal biomass, since primary sludge is a more readily biodegradable substrate, which increased the methane production by 65% and reduced the risk of ammonia toxicity. Regarding the contaminants, musk fragrances (galaxolide and tonalide) and triclosan showed the highest abundance on primary sludge (0.5-25μg/g TS), whereas caffeine, methyl dihydrojasmonate and triphenyl phosphate were barely detected in both substrates (<0.1μg/g TS). The removal of these contaminants was compound-depending and ranged from no removal to up to 90%. On the whole, microalgae mono-digestion resulted in a higher removal of selected contaminants than the co-digestion with primary sludge., (Copyright © 2019. Published by Elsevier B.V.)

This study aims at optimizing the anaerobic digestion (AD) of biomass in microalgal-based wastewater treatment systems. It comprises the co-digestion of microalgae with primary sludge, the thermal pretreatment (75 °C for 10 h) of microalgae and the role of the hydraulic retention time (HRT) in anaerobic digesters. Initially, a batch test comparing different microalgae (untreated and pretreated) and primary sludge proportions showed how the co-digestion improved the AD kinetics. The highest methane yield was observed by adding 75% of primary sludge to pretreated microalgae (339 mL CH₄/g VS). This condition was then investigated in mesophilic lab-scale reactors. The average methane yield was 0.46 L CH₄/g VS, which represented a 2.9-fold increase compared to pretreated microalgae mono-digestion. Conversely, microalgae showed a low methane yield despite the thermal pretreatment (0.16 L CH₄/g VS). Indeed, microscopic analysis confirmed the presence of microalgae species with resistant cell walls (i.e., Stigioclonium sp. and diatoms). In order to improve their anaerobic biodegradability, the HRT was increased from 20 to 30 days, which led to a 50% methane yield increase. Overall, microalgae AD was substantially improved by the co-digestion with primary sludge, even without pretreatment, and increasing the HRT enhanced the AD of microalgae with resistant cell walls.

In this study, microalgae digestate and secondary effluent were used to grow microalgae in a tertiary wastewater treatment, and then, the biomass was co-digested for biogas generation. A 30L closed-photobioreactor was used for microalgae cultivation. The biomass, mainly composed by Scenedesmus sp., reached and maintained a concentration of 1.1gTSS/L during 30days. A complete removal of N-NH 4 + and P-PO 4 3- and high nitrates and organic matter removals were achieved (58% N-NO 3 - and 70% COD) with 8d of HRT. The potential biogas production of the cultivated microalgae was determined in batch tests. To improve their biodegradability, a novel method combining their co-digestion with activated sludge after a simultaneous autohydrolysis co-pretreatment was evaluated. After the co-pretreatment, the methane yield increased by 130%. Thus, integrating microalgae tertiary treatment into activated sludge systems is a promising and feasible solution to recover energy and nutrients from waste, improving wastewater treatment plants sustainability., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This study aimed at analyzing the anaerobic co-digestion of microalgal biomass grown in wastewater and wheat straw. To this end, Biochemical Methane Potential (BMP) tests were carried out testing different substrate proportions (20-80, 50-50 and 80-20%, on a volatile solid basis). In order to improve their biodegradability, the co-digestion of both substrates was also evaluated after applying a thermo-alkaline pretreatment (10% CaO at 75°C for 24h). The highest synergies in degradation rates were observed by adding at least 50% of wheat straw. Therefore, the co-digestion of 50% microalgae - 50% wheat straw was investigated in mesophilic lab-scale reactors. The results showed that the methane yield was increased by 77% with the co-digestion as compared to microalgae mono-digestion, while the pretreatment only increased the methane yield by 15% compared to the untreated mixture. Thus, the anaerobic co-digestion of microalgae and wheat straw was successful even without applying a thermo-alkaline pretreatment., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Plant growth and productivity is negatively affected by different stresses. Most stresses trigger calcium signals that initiate acclimation responses in plants. The multigene family of plant calcium-dependent protein kinases (CPKs) functions in multiple stress responses by transducing calcium signals into phosphorylation events. This work reports that the OsCPK10 isoform positively mediates tolerance to different stresses in rice plants by enhancing their antioxidant capacity and protecting them from reactive oxygen species (ROS) damage, with the uncontrolled generation of ROS being a common feature of these stresses. Here, we show that the constitutive accumulation of an HA-tagged OsCPK10 full-length protein enhances the hydrogen peroxide detoxifying capacity of rice plants during desiccation. This is achived by modulating the accumulation of catalase proteins, which reduces the extent of lipid peroxidation and protects the integrity of cell membranes, resulting in drought tolerance. OsCPK10HA accumulation also confers blast disease resistance by interfering with fungal necrotrophic growth via a reduction in the accumulation of hydrogen peroxide. Furthermore, we show by bimolecular complementation assays that OsCPK10 is a plasma membrane protein that physically interacts in vivo with catalase A. OsCPK10 therefore appears to be a good molecular target to improve tolerance to abiotic stresses as well as to blast disease, which limit rice crop productivity., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Microalgae anaerobic digestion produces biogas along with a digestate that may be reused in agriculture. However, the properties of this digestate for agricultural reuse have yet to be determined. The aim of this study was to characterise digestates from different microalgae anaerobic digestion processes (i.e. digestion of untreated microalgae, thermally pretreated microalgae and thermally pretreated microalgae in co-digestion with primary sludge). The main parameters evaluated were organic matter, macronutrients and heavy metals content, hygenisation, potential phytotoxicity and organic matter stabilisation. According to the results, all microalgae digestates presented suitable organic matter and macronutrients, especially organic and ammonium nitrogen, for agricultural soils amendment. However, the thermally pretreated microalgae digestate was the least stabilised digestate in comparison with untreated microalgae and co-digestion digestates. In vivo bioassays demonstrated that the digestates did not show residual phytotoxicity when properly diluted, being the co-digestion digestate the one which presented less phytotoxicity. Heavy metals contents resulted far below the threshold established by the European legislation on sludge spreading. Moreover, low presence of E. coli was observed in all digestates. Therefore, agricultural reuse of thermally pretreated microalgae and primary sludge co-digestate through irrigation emerges a suitable strategy to recycle nutrients from wastewater., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: BP178 peptide is a synthetic BP100-magainin derivative possessing strong inhibitory activity against plant pathogenic bacteria, offering a great potential for future applications in plant protection and other fields. Here we report the production and recovery of a bioactive BP178 peptide using rice seeds as biofactories., Results: A synthetic gene encoding the BP178 peptide was prepared and introduced in rice plants. The gene was efficiently expressed in transgenic rice under the control of an endosperm-specific promoter. Among the three endosperm-specific rice promoters (Glutelin B1, Glutelin B4 or Globulin 1), best results were obtained when using the Globulin 1 promoter. The BP178 peptide accumulated in the seed endosperm and was easily recovered from rice seeds using a simple procedure with a yield of 21 μg/g. The transgene was stably inherited for at least three generations, and peptide accumulation remained stable during long term storage of transgenic seeds. The purified peptide showed in vitro activity against the bacterial plant pathogen Dickeya sp., the causal agent of the dark brown sheath rot of rice. Seedlings of transgenic events showed enhanced resistance to the fungal pathogen Fusarium verticillioides, supporting that the in planta produced peptide was biologically active., Conclusions: The strategy developed in this work for the sustainable production of BP178 peptide using rice seeds as biofactories represents a promising system for future production of peptides for plant protection and possibly in other fields.

Rice is the most important staple food for more than half of the human population, and blast disease is the most serious disease affecting global rice production. In this work, the isoform OsCPK4 of the rice calcium-dependent protein kinase family is reported as a regulator of rice immunity to blast fungal infection. It shows that overexpression of OsCPK4 gene in rice plants enhances resistance to blast disease by preventing fungal penetration. The constitutive accumulation of OsCPK4 protein prepares rice plants for a rapid and potentiated defence response, including the production of reactive oxygen species, callose deposition and defence gene expression. OsCPK4 overexpression leads also to constitutive increased content of the glycosylated salicylic acid hormone in leaves without compromising rice yield. Given that OsCPK4 overexpression was known to confer also salt and drought tolerance in rice, the results reported in this article demonstrate that OsCPK4 acts as a convergence component that positively modulates both biotic and abiotic signalling pathways. Altogether, our findings indicate that OsCPK4 is a potential molecular target to improve not only abiotic stress tolerance, but also blast disease resistance of rice crops., (© 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

Cecropin A is a natural antimicrobial peptide that exhibits fast and potent activity against a broad spectrum of pathogens and neoplastic cells, and that has important biotechnological applications. However, cecropin A exploitation, as for other antimicrobial peptides, is limited by their production and purification costs. Here, we report the efficient production of this bioactive peptide in rice bran using the rice oleosin 18 as a carrier protein. High cecropin A levels were reached in rice seeds driving the expression of the chimeric gene by the strong embryo-specific oleosin 18 own promoter, and targeting the peptide to the oil body organelle as an oleosin 18-cecropin A fusion protein. The accumulation of cecropin A in oil bodies had no deleterious effects on seed viability and seedling growth, as well as on seed yield. We also show that biologically active cecropin A can be easily purified from the transgenic rice seeds by homogenization and simple flotation centrifugation methods. Our results demonstrate that the oleosin fusion technology is suitable for the production of cecropin A in rice seeds, which can potentially be extended to other antimicrobial peptides to assist their exploitation.

Background: Cecropin A is a natural antimicrobial peptide that exhibits rapid, potent and long-lasting lytic activity against a broad spectrum of pathogens, thus having great biotechnological potential. Here, we report a system for producing bioactive cecropin A in rice seeds., Results: Transgenic rice plants expressing a codon-optimized synthetic cecropin A gene drived by an endosperm-specific promoter, either the glutelin B1 or glutelin B4 promoter, were generated. The signal peptide sequence from either the glutelin B1 or the glutelin B4 were N-terminally fused to the coding sequence of the cecropin A. We also studied whether the presence of the KDEL endoplasmic reticulum retention signal at the C-terminal has an effect on cecropin A subcellular localization and accumulation. The transgenic rice plants showed stable transgene integration and inheritance. We show that cecropin A accumulates in protein storage bodies in the rice endosperm, particularly in type II protein bodies, supporting that the glutelin N-terminal signal peptides play a crucial role in directing the cecropin A to this organelle, independently of being tagged with the KDEL endoplasmic reticulum retention signal. The production of cecropin A in transgenic rice seeds did not affect seed viability or seedling growth. Furthermore, transgenic cecropin A seeds exhibited resistance to infection by fungal and bacterial pathogens (Fusarium verticillioides and Dickeya dadantii, respectively) indicating that the in planta-produced cecropin A is biologically active., Conclusions: Rice seeds can sustain bioactive cecropin A production and accumulation in protein bodies. The system might benefit the production of this antimicrobial agent for subsequent applications in crop protection and food preservation.

Background and Objective: We aimed to know the prevalence of abnormal toe-brachial index (TBI) in a group of patients with type 2 diabetes mellitus (DM2). We also aimed to know the correlation between TBI and the ankle-brachial index (ABI) in these patients and determine the abnormal value of TBI., Patients and Methods: Descriptive cross-sectional study conducted in primary care. DM2 patients over 50 years were included. Systolic blood pressure at the arm, ankle and toe were determined to calculate the ABI and the TBI., Results: We studied 175 patients (350 limbs) of a mean age of 67 years (SD=9), 53.7% were women and 72.3% had hypertension. The ABI was abnormal (≤ 0.90) in 8.3% of the limbs and 8.6% met criteria of calcification (≥ 1.40). The TBI was abnormal (≤ 0.60) in 18.6% of cases. Of the 291 limbs with a normal ABI, 34 (11.7%) had an abnormal value of TBI and the 30 limbs with a calcified ABI, 6 (20%) had an abnormal value of TBI. There was a positive linear correlation between ABI and TBI (r = 0.395, P < .001)., Conclusions: Determination of TBI provides additional information to ABI determination and exploration can be very useful in cases of arterial calcification. The value ≤ 0.60 is the best one to consider an abnormal TBI., (Copyright © 2011 Elsevier España, S.L. All rights reserved.)

Background: The removal of mercury sphygmomanometers from health centers requires the validation of other instruments to measure blood pressure in the limbs to calculate the ankle-brachial index (ABI)., Methods: Descriptive cross-sectional study of agreement between two measurement methods in type 2 diabetes patients from three urban primary healthcare centres in the Barcelonès Nord i Maresme area (Catalonia, Spain)., Results: 211 patients were included, from these, 421 limbs were available for study. The mean age of the participants was 67 years (SD = 10), 51.7% were women., Conclusion: The combination of a Doppler device with the hybrid sphygmomanometer is a simple and reliable method to measure ABI showing that hybrid sphygmomanometer is a good alternative to the use of mercury sphygmomanometers.

Background: Our aim was to evaluate the relationship between asymptomatic peripheral arterial disease, diagnosed only by the ankle brachial index (ABI), and cardiovascular disease (CVD) after a 10-year follow-up period in patients with type 2 diabetes., Methods: In 1996, the ankle brachial index was measured in 262 patients with type 2 diabetes. During the 10-year follow-up period (mean follow-up time, 7.7 years), all nonfatal cardiovascular events and mortality were recorded., Results: A total of 52 patients died during the follow-up time. The mortality of the patients with normal (0.91-1.24) and abnormal ABI (≤0.90) at the beginning of the study was 16.8% and 52.8%, respectively (p < 0.05). The incidence rate of fatal and nonfatal CVD was 26.9 (95% confidence intervals [CI]: 20.7-37.3) for the patients with a normal baseline ABI and 81.9 (95% CI: 50.9-131.8) for those with an abnormal baseline ABI. An abnormal baseline ABI was associated with a greater risk of CVD (hazard ratio = 2.32; 95% CI: 1.27-4.22)., Conclusion: ABI values ≤0.9 in patients with type 2 diabetes mellitus and no history of intermittent vascular claudication or rest pain were associated with a higher risk of coronary or cerebrovascular morbidity and mortality., (Copyright © 2010 Annals of Vascular Surgery Inc. Published by Elsevier Inc. All rights reserved.)

Background: The early diagnosis of atherosclerotic disease is essential for developing preventive strategies in populations at high risk and acting when the disease is still asymptomatic. A low ankle-arm index (AAI) is a good marker of vascular events and may be diminished without presenting symptomatology (silent peripheral arterial disease). The aim of the PERART study (PERipheral ARTerial disease) is to determine the prevalence of peripheral arterial disease (both silent and symptomatic) in a general population of both sexes and determine its predictive value related to morbimortality (cohort study)., Methods/design: This cross-over, cohort study consists of 2 phases: firstly a descriptive, transversal cross-over study to determine the prevalence of peripheral arterial disease, and secondly, a cohort study to evaluate the predictive value of AAI in relation to cardiovascular morbimortality. From September 2006 to June 2007, a total of 3,010 patients over the age of 50 years will be randomly selected from a population adscribed to 24 healthcare centres in the province of Barcelona (Spain). The diagnostic criteria of peripheral arterial disease will be considered as an AAI < 0.90, determined by portable Doppler (8 Mhz probe) measured twice by trained personnel. Cardiovascular risk will be calculated with the Framingham-Wilson tables, with Framingham calibrated by the REGICOR and SCORE groups. The subjects included will be evaluted every 6 months by telephone interview and the clnical history and death registries will be reviewed. The appearance of the following cardiovascular events will be considered as variables of response: transitory ischaemic accident, ictus, angina, myocardial infartction, symptomatic abdominal aneurysm and vascular mortality., Discussion: In this study we hope to determine the prevalence of peripheral arterial disease, especially the silent forms, in the general population and establish its relationship with cardiovascular morbimortality. A low AAI may be a better marker of arterial disease than the classical cardiovascular risk factors and may, therefore, contribute to improving the predictive value of the equations of cardiovascular risk and thereby allowing optimisation of multifactorial treatment of atherosclerotic disease.

Objectives: To find the prevalence of peripheral arteriopathy (PA) in type 2 diabetics registered at a Health Centre, to detect their main risk factors and examine the usefulness of the portable Doppler in Primary Care consultations., Design: A crossover descriptive study., Setting: Urban Health Centre., Patients: All the type 2 diabetics registered., Measurements and Results: Anamnesis, physical examination, analysis and base ECG were performed. A portable Doppler determined systolic blood pressure in the lower extremities (foot and posterior tibial arteries) and brachial arteries in order to calculate the ankle/arm index (AAI). PA well recorded in the clinical history and AAI < or = 0.90 were considered criteria of PA. Diabetics with AAI > or = 1.25 were analysed separately. 289 patients with an average age of 65.3 (+/- 10.8 SD) were studied. 45.7% were men and 67% had Diabetes Mellitus for less than 10 years. 37.4% followed a dietary treatment and 21.1% were treated with insulin. Multivariate analysis showed that risk factors were: tobacco dependency, age, Hypertension and the type of treatment for the DM., Conclusions: The prevalence of PA found (21.4%) was very much higher than what had been previously diagnosed (6.9%). Identified risk factors were tobacco dependency, age, Hypertension and the type of DM treatment. A portable Doppler is easy to handle and allows peripheral arteriopathy to be diagnosed at an early stage.