Characterization of the cork oak transcriptome dynamics during acorn development

Background: Cork oak (Quercus suber L.) has a natural distribution across western Mediterranean regions and is a keystone forest tree species in these ecosystems. The fruiting phase is especially critical for its regeneration but the molecular mechanisms underlying the biochemical and physiological changes during cork oak acorn development are poorly understood. In this study, the transcriptome of the cork oak acorn, including the seed, was characterized in five stages of development, from early development to acorn maturation, to identify the dominant processes in each stage and reveal transcripts with important functions in gene expression regulation and response to water. Results: A total of 80,357 expressed sequence tags (ESTs) were de novo assembled from RNA-Seq libraries representative of the several acorn developmental stages. Approximately 7.6 % of the total number of transcripts present in Q. suber transcriptome was identified as acorn specific. The analysis of expression profiles during development returned 2,285 differentially expressed (DE) transcripts, which were clustered into six groups. The stage of development corresponding to the mature acorn exhibited an expression profile markedly different from other stages. Approximately 22 % of the DE transcripts putatively code for transcription factors (TF) or transcriptional regulators, and were found almost equally distributed among the several expression profile clusters, highlighting their major roles in controlling the whole developmental process. On the other hand, carbohydrate metabolism, the biological pathway most represented during acorn development, was especially prevalent in mid to late stages as evidenced by enrichment analysis. We further show that genes related to response to water, water deprivation and transport were mostly represented during the early (S2) and the last stage (S8) of acorn development, when tolerance to water desiccation is possibly critical for acorn viability. Conclusions: To our knowledge this work represents the first report of acorn development transcriptomics in oaks. The obtained results provide novel insights into the developmental biology of cork oak acorns, highlighting transcripts putatively involved in the regulation of the gene expression program and in specific processes likely essential for adaptation. It is expected that this knowledge can be transferred to other oak species of great ecological value.Fundação para a Ciência e a Tecnologi

Impact of primary kidney disease on the effects of empagliflozin in patients with chronic kidney disease: secondary analyses of the EMPA-KIDNEY trial

Background: The EMPA KIDNEY trial showed that empagliflozin reduced the risk of the primary composite outcome of kidney disease progression or cardiovascular death in patients with chronic kidney disease mainly through slowing progression. We aimed to assess how effects of empagliflozin might differ by primary kidney disease across its broad population. Methods: EMPA-KIDNEY, a randomised, controlled, phase 3 trial, was conducted at 241 centres in eight countries (Canada, China, Germany, Italy, Japan, Malaysia, the UK, and the USA). Patients were eligible if their estimated glomerular filtration rate (eGFR) was 20 to less than 45 mL/min per 1·73 m2, or 45 to less than 90 mL/min per 1·73 m2 with a urinary albumin-to-creatinine ratio (uACR) of 200 mg/g or higher at screening. They were randomly assigned (1:1) to 10 mg oral empagliflozin once daily or matching placebo. Effects on kidney disease progression (defined as a sustained ≥40% eGFR decline from randomisation, end-stage kidney disease, a sustained eGFR below 10 mL/min per 1·73 m2, or death from kidney failure) were assessed using prespecified Cox models, and eGFR slope analyses used shared parameter models. Subgroup comparisons were performed by including relevant interaction terms in models. EMPA-KIDNEY is registered with ClinicalTrials.gov, NCT03594110. Findings: Between May 15, 2019, and April 16, 2021, 6609 participants were randomly assigned and followed up for a median of 2·0 years (IQR 1·5–2·4). Prespecified subgroupings by primary kidney disease included 2057 (31·1%) participants with diabetic kidney disease, 1669 (25·3%) with glomerular disease, 1445 (21·9%) with hypertensive or renovascular disease, and 1438 (21·8%) with other or unknown causes. Kidney disease progression occurred in 384 (11·6%) of 3304 patients in the empagliflozin group and 504 (15·2%) of 3305 patients in the placebo group (hazard ratio 0·71 [95% CI 0·62–0·81]), with no evidence that the relative effect size varied significantly by primary kidney disease (pheterogeneity=0·62). The between-group difference in chronic eGFR slopes (ie, from 2 months to final follow-up) was 1·37 mL/min per 1·73 m2 per year (95% CI 1·16–1·59), representing a 50% (42–58) reduction in the rate of chronic eGFR decline. This relative effect of empagliflozin on chronic eGFR slope was similar in analyses by different primary kidney diseases, including in explorations by type of glomerular disease and diabetes (p values for heterogeneity all >0·1). Interpretation: In a broad range of patients with chronic kidney disease at risk of progression, including a wide range of non-diabetic causes of chronic kidney disease, empagliflozin reduced risk of kidney disease progression. Relative effect sizes were broadly similar irrespective of the cause of primary kidney disease, suggesting that SGLT2 inhibitors should be part of a standard of care to minimise risk of kidney failure in chronic kidney disease. Funding: Boehringer Ingelheim, Eli Lilly, and UK Medical Research Council

Proline Metabolism and Its Functions in Development and Stress Tolerance

Proline takes an exceptional place among the proteinogenic amino acids by its specific accumulation in pollen and in response to multiple types of stress. Despite the more than 50 years of research, the biochemical pathways of proline biosynthesis and degradation still await their complete characterization in plants. Also, the molecular and physiological functions of proline metabolism in plant development and defense against stress are not yet fully understood. This chapter focuses on the current knowledge about the biochemical pathways of proline metabolism in plants, on its tissue-specific regulation and subcellular compartmentation, and on still open questions. Furthermore, we will summarize what is known about the influence of proline metabolism on plant development under optimal growth conditions and how it may support continued development despite the impact of stress. The cognate chapter “Regulation of Proline Accumulation and its molecular and physiological Functions in Stress Defense” will focus on the possible beneficial functions of proline metabolism and accumulation in the defense response against diverse stresses. With these two cohesive chapters, we aim to provide a comprehensive picture of the current knowledge and the open research questions in proline-dependent stress defense

Impact of primary kidney disease on the effects of empagliflozin in patients with chronic kidney disease: secondary analyses of the EMPA-KIDNEY trial

Background: The EMPA-KIDNEY trial showed that empagliflozin reduced the risk of the primary composite outcome of kidney disease progression or cardiovascular death in patients with chronic kidney disease mainly through slowing progression. We aimed to assess how effects of empagliflozin might differ by primary kidney disease across its broad population. Methods: EMPA-KIDNEY, a randomised, controlled, phase 3 trial, was conducted at 241 centres in eight countries (Canada, China, Germany, Italy, Japan, Malaysia, the UK, and the USA). Patients were eligible if their estimated glomerular filtration rate (eGFR) was 20 to less than 45 mL/min per 1·73 m2, or 45 to less than 90 mL/min per 1·73 m2 with a urinary albumin-to-creatinine ratio (uACR) of 200 mg/g or higher at screening. They were randomly assigned (1:1) to 10 mg oral empagliflozin once daily or matching placebo. Effects on kidney disease progression (defined as a sustained ≥40% eGFR decline from randomisation, end-stage kidney disease, a sustained eGFR below 10 mL/min per 1·73 m2, or death from kidney failure) were assessed using prespecified Cox models, and eGFR slope analyses used shared parameter models. Subgroup comparisons were performed by including relevant interaction terms in models. EMPA-KIDNEY is registered with ClinicalTrials.gov, NCT03594110. Findings: Between May 15, 2019, and April 16, 2021, 6609 participants were randomly assigned and followed up for a median of 2·0 years (IQR 1·5-2·4). Prespecified subgroupings by primary kidney disease included 2057 (31·1%) participants with diabetic kidney disease, 1669 (25·3%) with glomerular disease, 1445 (21·9%) with hypertensive or renovascular disease, and 1438 (21·8%) with other or unknown causes. Kidney disease progression occurred in 384 (11·6%) of 3304 patients in the empagliflozin group and 504 (15·2%) of 3305 patients in the placebo group (hazard ratio 0·71 [95% CI 0·62-0·81]), with no evidence that the relative effect size varied significantly by primary kidney disease (pheterogeneity=0·62). The between-group difference in chronic eGFR slopes (ie, from 2 months to final follow-up) was 1·37 mL/min per 1·73 m2 per year (95% CI 1·16-1·59), representing a 50% (42-58) reduction in the rate of chronic eGFR decline. This relative effect of empagliflozin on chronic eGFR slope was similar in analyses by different primary kidney diseases, including in explorations by type of glomerular disease and diabetes (p values for heterogeneity all >0·1). Interpretation: In a broad range of patients with chronic kidney disease at risk of progression, including a wide range of non-diabetic causes of chronic kidney disease, empagliflozin reduced risk of kidney disease progression. Relative effect sizes were broadly similar irrespective of the cause of primary kidney disease, suggesting that SGLT2 inhibitors should be part of a standard of care to minimise risk of kidney failure in chronic kidney disease. Funding: Boehringer Ingelheim, Eli Lilly, and UK Medical Research Council

Effects of empagliflozin on progression of chronic kidney disease: a prespecified secondary analysis from the empa-kidney trial

Background: Sodium-glucose co-transporter-2 (SGLT2) inhibitors reduce progression of chronic kidney disease and the risk of cardiovascular morbidity and mortality in a wide range of patients. However, their effects on kidney disease progression in some patients with chronic kidney disease are unclear because few clinical kidney outcomes occurred among such patients in the completed trials. In particular, some guidelines stratify their level of recommendation about who should be treated with SGLT2 inhibitors based on diabetes status and albuminuria. We aimed to assess the effects of empagliflozin on progression of chronic kidney disease both overall and among specific types of participants in the EMPA-KIDNEY trial. Methods: EMPA-KIDNEY, a randomised, controlled, phase 3 trial, was conducted at 241 centres in eight countries (Canada, China, Germany, Italy, Japan, Malaysia, the UK, and the USA), and included individuals aged 18 years or older with an estimated glomerular filtration rate (eGFR) of 20 to less than 45 mL/min per 1·73 m2, or with an eGFR of 45 to less than 90 mL/min per 1·73 m2 with a urinary albumin-to-creatinine ratio (uACR) of 200 mg/g or higher. We explored the effects of 10 mg oral empagliflozin once daily versus placebo on the annualised rate of change in estimated glomerular filtration rate (eGFR slope), a tertiary outcome. We studied the acute slope (from randomisation to 2 months) and chronic slope (from 2 months onwards) separately, using shared parameter models to estimate the latter. Analyses were done in all randomly assigned participants by intention to treat. EMPA-KIDNEY is registered at ClinicalTrials.gov, NCT03594110. Findings: Between May 15, 2019, and April 16, 2021, 6609 participants were randomly assigned and then followed up for a median of 2·0 years (IQR 1·5-2·4). Prespecified subgroups of eGFR included 2282 (34·5%) participants with an eGFR of less than 30 mL/min per 1·73 m2, 2928 (44·3%) with an eGFR of 30 to less than 45 mL/min per 1·73 m2, and 1399 (21·2%) with an eGFR 45 mL/min per 1·73 m2 or higher. Prespecified subgroups of uACR included 1328 (20·1%) with a uACR of less than 30 mg/g, 1864 (28·2%) with a uACR of 30 to 300 mg/g, and 3417 (51·7%) with a uACR of more than 300 mg/g. Overall, allocation to empagliflozin caused an acute 2·12 mL/min per 1·73 m2 (95% CI 1·83-2·41) reduction in eGFR, equivalent to a 6% (5-6) dip in the first 2 months. After this, it halved the chronic slope from -2·75 to -1·37 mL/min per 1·73 m2 per year (relative difference 50%, 95% CI 42-58). The absolute and relative benefits of empagliflozin on the magnitude of the chronic slope varied significantly depending on diabetes status and baseline levels of eGFR and uACR. In particular, the absolute difference in chronic slopes was lower in patients with lower baseline uACR, but because this group progressed more slowly than those with higher uACR, this translated to a larger relative difference in chronic slopes in this group (86% [36-136] reduction in the chronic slope among those with baseline uACR <30 mg/g compared with a 29% [19-38] reduction for those with baseline uACR ≥2000 mg/g; ptrend<0·0001). Interpretation: Empagliflozin slowed the rate of progression of chronic kidney disease among all types of participant in the EMPA-KIDNEY trial, including those with little albuminuria. Albuminuria alone should not be used to determine whether to treat with an SGLT2 inhibitor. Funding: Boehringer Ingelheim and Eli Lilly