Reference Genes for Accurate Transcript Normalization in Citrus Genotypes under Different Experimental Conditions

Real-time reverse transcription PCR (RT-qPCR) has emerged as an accurate and widely used technique for expression profiling of selected genes. However, obtaining reliable measurements depends on the selection of appropriate reference genes for gene expression normalization. The aim of this work was to assess the expression stability of 15 candidate genes to determine which set of reference genes is best suited for transcript normalization in citrus in different tissues and organs and leaves challenged with five pathogens (Alternaria alternata, Phytophthora parasitica, Xylella fastidiosa and Candidatus Liberibacter asiaticus). We tested traditional genes used for transcript normalization in citrus and orthologs of Arabidopsis thaliana genes described as superior reference genes based on transcriptome data. geNorm and NormFinder algorithms were used to find the best reference genes to normalize all samples and conditions tested. Additionally, each biotic stress was individually analyzed by geNorm. In general, FBOX (encoding a member of the F-box family) and GAPC2 (GAPDH) was the most stable candidate gene set assessed under the different conditions and subsets tested, while CYP (cyclophilin), TUB (tubulin) and CtP (cathepsin) were the least stably expressed genes found. Validation of the best suitable reference genes for normalizing the expression level of the WRKY70 transcription factor in leaves infected with Candidatus Liberibacter asiaticus showed that arbitrary use of reference genes without previous testing could lead to misinterpretation of data. Our results revealed FBOX, SAND (a SAND family protein), GAPC2 and UPL7 (ubiquitin protein ligase 7) to be superior reference genes, and we recommend their use in studies of gene expression in citrus species and relatives. This work constitutes the first systematic analysis for the selection of superior reference genes for transcript normalization in different citrus organs and under biotic stress

Blood-Feeding Induces Reversible Functional Changes in Flight Muscle Mitochondria of Aedes aegypti Mosquito

Background: Hematophagy poses a challenge to blood-feeding organisms since products of blood digestion can exert cellular deleterious effects. Mitochondria perform multiple roles in cell biology acting as the site of aerobic energytransducing pathways, and also an important source of reactive oxygen species (ROS), modulating redox metabolism. Therefore, regulation of mitochondrial function should be relevant for hematophagous arthropods. Here, we investigated the effects of blood-feeding on flight muscle (FM) mitochondria from the mosquito Aedes aegypti, a vector of dengue and yellow fever. Methodology/Principal Findings: Blood-feeding caused a reversible reduction in mitochondrial oxygen consumption, an event that was parallel to blood digestion. These changes were most intense at 24 h after blood meal (ABM), the peak of blood digestion, when oxygen consumption was inhibited by 68%. Cytochromes c and a+a3 levels and cytochrome c oxidase activity of the electron transport chain were all reduced at 24 h ABM. Ultrastructural and molecular analyses of FM revealed that mitochondria fuse upon blood meal, a condition related to reduced ROS generation. Consistently, BF induced a reversible decrease in mitochondrial H2O2 formation during blood digestion, reaching their lowest values at 24 h ABM where a reduction of 51% was observed. Conclusion: Blood-feeding triggers functional and structural changes in hematophagous insect mitochondria, which may represent an important adaptation to blood feedin

Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study

Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe

Protein kinase D interacts with neuronal nitric oxide synthase and phosphorylates the activatory residue serine1412

Neuronal Nitric Oxide Synthase (nNOS) is the biosynthetic enzyme responsible for nitric oxide (·NO) production in muscles and in the nervous system. This constitutive enzyme, unlike its endothelial and inducible counterparts, presents an N-terminal PDZ domain known to display a preference for PDZ-binding motifs bearing acidic residues at -2 position. In a previous work, we discovered that the C-terminal end of two members of protein kinase D family (PKD1 and PKD2) constitutes a PDZ-ligand. PKD1 has been shown to regulate multiple cellular processes and, when activated, becomes autophosphorylated at Ser 916, a residue located at -2 position of its PDZ-binding motif. Since nNOS and PKD are spatially enriched in postsynaptic densities and dendrites, the main objective of our study was to determine whether PKD1 activation could result in a direct interaction with nNOS through their respective PDZ-ligand and PDZ domain, and to analyze the functional consequences of this interaction. Herein we demonstrate that PKD1 associates with nNOS in neurons and in transfected cells, and that kinase activation enhances PKD1-nNOS co-immunoprecipitation and subcellular colocalization. However, transfection of mammalian cells with PKD1 mutants and yeast two hybrid assays showed that the association of these two enzymes does not depend on PKD1 PDZ-ligand but its pleckstrin homology domain. Furthermore, this domain was able to pull-down nNOS from brain extracts and bind to purified nNOS, indicating that it mediates a direct PKD1-nNOS interaction. In addition, using mass spectrometry we demonstrate that PKD1 specifically phosphorylates nNOS in the activatory residue Ser 1412, and that this phosphorylation increases nNOS activity and ·NO production in living cells. In conclusion, these novel findings reveal a crucial role of PKD1 in the regulation of nNOS activation and synthesis of ·NO, a mediator involved in physiological neuronal signaling or neurotoxicity under pathological conditions such as ischemic stroke or neurodegeneration.This work was supported by the Ministerio de Economía y Competitividad [SAF2011-26233 to T.I., BFU2009-10442 and BFU2012-37934 to I.R-C.]; Comunidad de Madrid [S2010/BMD-2331-Neurodegmodels-CM to T.I.]; and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas – CIBERNED, Instituto de Salud Carlos III, to T.I. Postdoctoral fellows L.S-R. and L.G-G. have been funded by research contracts from CIBERNED; Clara Aicart-Ramos is a recipient of a FPU predoctoral fellowship from Ministerio de Economía y Competitividad.Peer Reviewe

Avaliação artroscópica e macroscópica do complexo da fibrocartilagem triangular do punho. Estudo em cadáveres Arthroscopic and gross evaluation of the triangular fibrocartilage complex of the wrist: a cadaver-based study

O complexo da fibrocartilagem triangular tem importante papel na biomecânica do punho. O diagnóstico preciso das lesões é fundamental para se obter sucesso no tratamento. Há controvérsias acerca da especificidade e sensibilidade dos métodos de imagem empregados atualmente. A artroscopia de punho é um método pouco empregado para o diagnóstico das lesões do CFCT em nosso meio, embora apresente grandes vantagens, como possibilidade de visão direta das lesões e tratamento no mesmo tempo cirúrgico. O objetivo deste estudo foi o de avaliar o papel da artroscopia de punho na inspeção do CFCT, bem como na detecção de possíveis lesões, comparando os dados com a dissecção macroscópica. Foram avaliados 15 punhos de cadáveres sexo masculino, média de idade de 56,1 anos. A artroscopia demonstrou presença de lesões em 33,3% dos punhos avaliados. Estes achados foram coincidentes após estudo anatômico com ampla dissecção. Concluímos que houve absoluta correlação entre o exame artroscópico e a dissecção macroscópica na detecção de lesões do CFCT.<br>The triangular fibrocartilage complex plays a key role on wrist biomechanics. An accurate injuries diagnosis is paramount for a successful treatment. There are controversies regarding specificity and sensitiveness of imaging methods employed today. Wrist arthroscopy is a method uncommonly used for diagnosing TFCC injuries in our environment, although it presents good advantages, such as the potential of direct viewing injuries, and treatment at the same surgical time. The objective of this study was to evaluate the role of wrist arthroscopy for inspecting TFCC, as well as for detecting potential injuries, comparing those data to gross dissection. Fifteen wrists of male cadavers (mean age: 56.1 years old) were assessed. Arthroscopy showed the presence of injuries in 33.3% of the assessed wrists. Those findings showed consistency after an anatomical study with broad dissection. We concluded that an absolute correlation existed between arthroscopic test and gross dissection in detecting TFCC injuries

Immunocompromised patients with acute respiratory distress syndrome : Secondary analysis of the LUNG SAFE database

The aim of this study was to describe data on epidemiology, ventilatory management, and outcome of acute respiratory distress syndrome (ARDS) in immunocompromised patients. Methods: We performed a post hoc analysis on the cohort of immunocompromised patients enrolled in the Large Observational Study to Understand the Global Impact of Severe Acute Respiratory Failure (LUNG SAFE) study. The LUNG SAFE study was an international, prospective study including hypoxemic patients in 459 ICUs from 50 countries across 5 continents. Results: Of 2813 patients with ARDS, 584 (20.8%) were immunocompromised, 38.9% of whom had an unspecified cause. Pneumonia, nonpulmonary sepsis, and noncardiogenic shock were their most common risk factors for ARDS. Hospital mortality was higher in immunocompromised than in immunocompetent patients (52.4% vs 36.2%; p < 0.0001), despite similar severity of ARDS. Decisions regarding limiting life-sustaining measures were significantly more frequent in immunocompromised patients (27.1% vs 18.6%; p < 0.0001). Use of noninvasive ventilation (NIV) as first-line treatment was higher in immunocompromised patients (20.9% vs 15.9%; p = 0.0048), and immunodeficiency remained independently associated with the use of NIV after adjustment for confounders. Forty-eight percent of the patients treated with NIV were intubated, and their mortality was not different from that of the patients invasively ventilated ab initio. Conclusions: Immunosuppression is frequent in patients with ARDS, and infections are the main risk factors for ARDS in these immunocompromised patients. Their management differs from that of immunocompetent patients, particularly the greater use of NIV as first-line ventilation strategy. Compared with immunocompetent subjects, they have higher mortality regardless of ARDS severity as well as a higher frequency of limitation of life-sustaining measures. Nonetheless, nearly half of these patients survive to hospital discharge. Trial registration: ClinicalTrials.gov, NCT02010073. Registered on 12 December 2013

Immunocompromised patients with acute respiratory distress syndrome: Secondary analysis of the LUNG SAFE database

Background: The aim of this study was to describe data on epidemiology, ventilatory management, and outcome of acute respiratory distress syndrome (ARDS) in immunocompromised patients. Methods: We performed a post hoc analysis on the cohort of immunocompromised patients enrolled in the Large Observational Study to Understand the Global Impact of Severe Acute Respiratory Failure (LUNG SAFE) study. The LUNG SAFE study was an international, prospective study including hypoxemic patients in 459 ICUs from 50 countries across 5 continents. Results: Of 2813 patients with ARDS, 584 (20.8%) were immunocompromised, 38.9% of whom had an unspecified cause. Pneumonia, nonpulmonary sepsis, and noncardiogenic shock were their most common risk factors for ARDS. Hospital mortality was higher in immunocompromised than in immunocompetent patients (52.4% vs 36.2%; p &lt; 0.0001), despite similar severity of ARDS. Decisions regarding limiting life-sustaining measures were significantly more frequent in immunocompromised patients (27.1% vs 18.6%; p &lt; 0.0001). Use of noninvasive ventilation (NIV) as first-line treatment was higher in immunocompromised patients (20.9% vs 15.9%; p = 0.0048), and immunodeficiency remained independently associated with the use of NIV after adjustment for confounders. Forty-eight percent of the patients treated with NIV were intubated, and their mortality was not different from that of the patients invasively ventilated ab initio. Conclusions: Immunosuppression is frequent in patients with ARDS, and infections are the main risk factors for ARDS in these immunocompromised patients. Their management differs from that of immunocompetent patients, particularly the greater use of NIV as first-line ventilation strategy. Compared with immunocompetent subjects, they have higher mortality regardless of ARDS severity as well as a higher frequency of limitation of life-sustaining measures. Nonetheless, nearly half of these patients survive to hospital discharge. Trial registration: ClinicalTrials.gov, NCT02010073. Registered on 12 December 2013