Noninvasive In Planta Live Measurements of H2O2 and Glutathione Redox Potential with Fluorescent roGFPs-Based Sensors.

In this protocol, we present a noninvasive in planta bioimaging technique for the analysis of hydrogen peroxide (H2O2) and glutathione redox potential in adult Arabidopsis thaliana plants. The technique is based on the use of stereo fluorescence microscopy to image A. thaliana plants expressing the two genetically encoded fluorescent sensors roGFP2-Orp1 and Grx1-roGFP2. We provide a detailed step-by-step protocol for performing low magnification imaging with mature plants grown in soil or hydroponic systems. This protocol aims to serve the scientific community by providing an accessible approach to noninvasive in planta bioimaging and data analysis.

Mitochondria-derived reactive oxygen species are the likely primary trigger of mitochondrial retrograde signaling in Arabidopsis.

Besides their central function in respiration, plant mitochondria play a crucial role in maintaining cellular homeostasis during stress by providing "retrograde" feedback to the nucleus. Despite the growing understanding of this signaling network, the nature of the signals that initiate mitochondrial retrograde regulation (MRR) in plants remains unknown. Here, we investigated the dynamics and causative relationship of a wide range of mitochondria-related parameters for MRR, using a combination of Arabidopsis fluorescent protein biosensor lines, in vitro assays, and genetic and pharmacological approaches. We show that previously linked physiological parameters, including changes in cytosolic ATP, NADH/NAD+ ratio, cytosolic reactive oxygen species (ROS), pH, free Ca2+, and mitochondrial membrane potential, may often be correlated with-but are not the primary drivers of-MRR induction in plants. However, we demonstrate that the induced production of mitochondrial ROS is the likely primary trigger for MRR induction in Arabidopsis. Furthermore, we demonstrate that mitochondrial ROS-mediated signaling uses the ER-localized ANAC017-pathway to induce MRR response. Finally, our data suggest that mitochondrially generated ROS can induce MRR without substantially leaking into other cellular compartments such as the cytosol or ER lumen, as previously proposed. Overall, our results offer compelling evidence that mitochondrial ROS elevation is the likely trigger of MRR.

AMEBaS: Automatic Midline Extraction and Background Subtraction of Ratiometric Fluorescence Time-Lapses of Polarized Single Cells.

Cell polarity is a macroscopic phenomenon established by a collection of spatially concentrated molecules and structures that culminate in the emergence of specialized domains at the subcellular level. It is associated with developing asymmetric morphological structures that underlie key biological functions such as cell division, growth, and migration. In addition, the disruption of cell polarity has been linked to tissue-related disorders such as cancer and gastric dysplasia. Current methods to evaluate the spatiotemporal dynamics of fluorescent reporters in individual polarized cells often involve manual steps to trace a midline along the cells' major axis, which is time consuming and prone to strong biases. Furthermore, although ratiometric analysis can correct the uneven distribution of reporter molecules using two fluorescence channels, background subtraction techniques are frequently arbitrary and lack statistical support. This manuscript introduces a novel computational pipeline to automate and quantify the spatiotemporal behavior of single cells using a model of cell polarity: pollen tube/root hair growth and cytosolic ion dynamics. A three-step algorithm was developed to process ratiometric images and extract a quantitative representation of intracellular dynamics and growth. The first step segments the cell from the background, producing a binary mask through a thresholding technique in the pixel intensity space. The second step traces a path through the midline of the cell through a skeletonization operation. Finally, the third step provides the processed data as a ratiometric timelapse and yields a ratiometric kymograph (i.e., a 1D spatial profile through time). Data from ratiometric images acquired with genetically encoded fluorescent reporters from growing pollen tubes were used to benchmark the method. This pipeline allows for faster, less biased, and more accurate representation of the spatiotemporal dynamics along the midline of polarized cells, thus advancing the quantitative toolkit available to investigate cell polarity. The AMEBaS Python source code is available at: https://github.com/badain/amebas.git.

Plant Ca2+-ATPases: From biochemistry to signalling.

Calcium (Ca2+)-ATPases are ATP-dependent enzymes that transport Ca2+ ions against their electrochemical gradient playing the fundamental biological function of keeping the free cytosolic Ca2+ concentration in the submicromolar range to prevent cytotoxic effects. In plants, type IIB autoinhibited Ca2+-ATPases (ACAs) are localised both at the plasma membrane and at the endomembranes including endoplasmic reticulum (ER) and tonoplast and their activity is primarily regulated by Ca2+-dependent mechanisms. Instead, type IIA ER-type Ca2+-ATPases (ECAs) are present mainly at the ER and Golgi Apparatus membranes and are active at resting Ca2+. Whereas research in plants has historically focused on the biochemical characterization of these pumps, more recently the attention has been also addressed on the physiological roles played by the different isoforms. This review aims to highlight the main biochemical properties of both type IIB and type IIA Ca2+ pumps and their involvement in the shaping of cellular Ca2+ dynamics induced by different stimuli.

Long-distance turgor pressure changes induce local activation of plant glutamate receptor-like channels.

In Arabidopsis thaliana, local wounding and herbivore feeding provoke leaf-to-leaf propagating Ca2+ waves that are dependent on the activity of members of the glutamate receptor-like channels (GLRs). In systemic tissues, GLRs are needed to sustain the synthesis of jasmonic acid (JA) with the subsequent activation of JA-dependent signaling response required for the plant acclimation to the perceived stress. Even though the role of GLRs is well established, the mechanism through which they are activated remains unclear. Here, we report that in vivo, the amino-acid-dependent activation of the AtGLR3.3 channel and systemic responses require a functional ligand-binding domain. By combining imaging and genetics, we show that leaf mechanical injury, such as wounds and burns, as well as hypo-osmotic stress in root cells, induces the systemic apoplastic increase of L-glutamate (L-Glu), which is largely independent of AtGLR3.3 that is instead required for systemic cytosolic Ca2+ elevation. Moreover, by using a bioelectronic approach, we show that the local release of minute concentrations of L-Glu in the leaf lamina fails to induce any long-distance Ca2+ waves.

Long-Term Outcome After Out-of-Hospital Cardiac Arrest: An Utstein-Based Analysis.

Background: No data are available regarding long-term survival of out-of-hospital cardiac arrest (OHCA) patients based on different Utstein subgroups, which are expected to significantly differ in terms of survival. We aimed to provide the first long-term survival analysis of OHCA patients divided according to Utstein categories. Methods: We analyzed all the 4,924 OHCA cases prospectively enrolled in the Lombardia Cardiac Arrest Registry (Lombardia CARe) from 2015 to 2019. Pre-hospital data, survival, and cerebral performance category score (CPC) at 1, 6, and 12 months and then every year up to 5 years after the event were analyzed for each patient. Results: A decrease in survival was observed during the follow-up in all the Utstein categories. The risk of death of the "all-EMS treated" group exceeded the general population for all the years of follow-up with standardized mortality ratios (SMRs) of 23 (95%CI, 16.8-30.2), 6.8 (95%CI, 3.8-10.7), 3.8 (95%CI, 1.7-6.7), 4.05 (95%CI, 1.9-6.9), and 2.6 (95%CI, 1.03-4.8) from the first to the fifth year of follow-up. The risk of death was higher also for the Utstein categories "shockable bystander witnessed" and "shockable bystander CPR": SMRs of 19.4 (95%CI, 11.3-29.8) and 19.4 (95%CI, 10.8-30.6) for the first year and of 6.8 (95%CI, 6.6-13) and 8.1 (95%CI, 3.1-15.3) for the second one, respectively. Similar results were observed considering the patients discharged with a CPC of 1-2. Conclusions: The mortality of OHCA patients discharged alive from the hospital is higher than the Italian standard population, also considering those with the most favorable OHCA characteristics and those discharged with good neurological outcome. Long-term follow-up should be included in the next Utstein-style revision.

A preprocedural risk score predicts acute kidney injury following primary percutaneous coronary intervention.

BACKGROUND: Reliable preprocedural risk scores for the prediction of Contrast-Induced Acute Kidney Injury (CI-AKI) following Percutaneous Coronary Intervention (pPCI) in patients with ST-elevation myocardial infarction (STEMI) are lacking. Aim of this study was to derive and validate a preprocedural Risk Score in this setting. METHODS: Two prospectively enrolled patient cohorts were used for derivation and validation (n = 3,736). CI-AKI was defined as creatinine increase ≥0.5 mg/dl <72 h postpPCI. Odds ratios from multivariable logistic regression model were converted to an integer, whose sum represented the Risk Score. RESULTS: Independent CI-AKI predictors were: diabetes, Killip class II-III (2 points each), age > 75 years, anterior MI (3 points), Killip class IV (4 points), estimated GFR < 60 ml/min/1.73m2 (5 points). The Risk Score c-statistic was 0.84 in both cohorts. Compared with patients with Risk Score ≤ 4, the relative risks of CI-AKI among patients scoring 5-9 were 6.2 (derivation cohort) and 7.1 (validation cohort); among patients scoring ≥10, 19.8, and 21.4, respectively. CONCLUSIONS: Among STEMI patients, a simple preprocedural Risk Score accurately and reproducibly predicted the risk of CI-AKI, identifying » of patients with a seven-fold risk and 1/10 of patients with a 20-fold risk. This knowledge may help tailored strategies, including delaying revascularization of nonculprit vessels in patients at high risk of CI-AKI.

Load distributing band device for mechanical chest compressions: An Utstein-categories based analysis of survival to hospital discharge.

PURPOSE: The role of load distributing band device (LDB, AutoPulse®, Zoll Medical Corporation, Chelmsford, MA, USA) in out-of-hospital cardiac arrest is still a matter of debate, with few studies reaching conflicting results available in literature. We sought to assess whether the use of the LBD device could affect survival to hospital discharge in the different Utstein categories. MATERIALS AND METHODS: All consecutive patients enrolled in our provincial cardiac arrest registry (Pavia CARe) from January 2015 to December 2017 were included and pre-hospital data were computed as well as survival to hospital discharge. RESULTS: Among 1401 resuscitation attempts, the LDB device was used in 235 (17%) patients. The LDB device was significantly more used for shockable cardiac arrest (42.6% vs 13.7%, p < 0.001). The rate of ROSC and of survival to hospital discharge in the LDB group compared to the manual group was 40% vs 17% (p < 0.001) and 10% vs 7% (p = 0.2), respectively. However, after correction for independent predictors of LDB use, LDB device was a strong independent predictor of survival to hospital discharge only for non-shockable witnessed OHCA [n = 624/1401, OR 11.9 (95% CI 1.5-95.2), p = 0.02]. In this categories of patients LDB group showed longer resuscitation time [49.3 min (IQR 37-71) vs 23.6 (IQR 15-35), p < 0.001] and a higher rate of conversion to a shockable rhythm (33/83 = 40% vs 29/541 = 5%, p < 0.001). CONCLUSION: Utstein categories-based analysis showed that the LDB device positively affect survival to hospital discharge for non-shockable witnessed cardiac arrests with a neutral effect for shockable rhythms.

Acute Kidney Injury Definition and In-Hospital Mortality in Patients Undergoing Primary Percutaneous Coronary Intervention for ST-Segment Elevation Myocardial Infarction.

BACKGROUND: Acute kidney injury (AKI) has been associated with increased mortality in ST-segment elevation myocardial infarction. We compared the mortality predictive accuracy of the 3 AKI definitions used most widely for patients with ST-segment elevation myocardial infarction undergoing primary percutaneous coronary intervention. METHODS AND RESULTS: We included 3771 patients with ST-segment elevation myocardial infarction treated with primary percutaneous coronary intervention at 2 Italian hospitals. AKI incidence was evaluated according to creatinine increases of ≥25% (AKI-25), ≥0.3 mg/dL (AKI-0.3), and ≥0.5 mg/dL (AKI-0.5). The primary end point was in-hospital mortality. Overall, 557 (15%), 522 (14%), and 270 (7%) patients developed AKI-25, AKI-0.3, and AKI-0.5, respectively (P<0.01). All AKI definitions independently predicted in-hospital mortality (adjusted odds ratio 4.9 [95% CI 3.1-7.8], 5.4 [95% CI 3.3-8.6], and 8.3 [95% CI 5.1-13.3], respectively; P<0.01 for all). At receiver operating characteristic analysis, the addition of each AKI definition to combined clinical predictors of mortality (age, sex, left ventricular ejection fraction, admission creatinine, creatine kinase-MB peak) found at stepwise analysis significantly improved mortality prognostication (area under the curve increased from 0.89 for clinical predictor combination alone to 0.92 for AKI-25, 0.92 for AKI-0.3, and 0.93 for AKI-0.5; P<0.01 for all). At reclassification analysis, AKI-0.5 added to clinical predictors, provided the highest score in mortality (net reclassification improvement +10% versus AKI-0.3 [P=0.01] and +8% versus AKI-25 [P=0.05]). CONCLUSIONS: Each AKI definition significantly improved the mortality prediction beyond major clinical variables. AKI-0.5 showed a mortality discrimination advantage, suggesting it should be the preferred definition in studies addressing ST-segment elevation myocardial infarction and focusing on short-term mortality.