Endocytic trafficking promotes vacuolar enlargements for fast cell expansion rates in plants.

The vacuole has a space-filling function, allowing a particularly rapid plant cell expansion with very little increase in cytosolic content (Löfke et al., 2015; Scheuring et al., 2016; Dünser et al., 2019). Despite its importance for cell size determination in plants, very little is known about the mechanisms that define vacuolar size. Here, we show that the cellular and vacuolar size expansions are coordinated. By developing a pharmacological tool, we enabled the investigation of membrane delivery to the vacuole during cellular expansion. Our data reveal that endocytic membrane sorting from the plasma membrane to the vacuole is enhanced in the course of rapid root cell expansion. While this 'compromise' mechanism may theoretically at first decelerate cell surface enlargements, it fuels vacuolar expansion and, thereby, ensures the coordinated augmentation of vacuolar occupancy in dynamically expanding plant cells.

New fluorescent auxin probes visualise tissue-specific and subcellular distributions of auxin in Arabidopsis.

In a world that will rely increasingly on efficient plant growth for sufficient food, it is important to learn about natural mechanisms of phytohormone action. In this work, the introduction of a fluorophore to an auxin molecule represents a sensitive and non-invasive method to directly visualise auxin localisation with high spatiotemporal resolution. The state-of-the-art multidisciplinary approaches of genetic and chemical biology analysis together with live cell imaging, liquid chromatography-mass spectrometry (LC-MS) and surface plasmon resonance (SPR) methods were employed for the characterisation of auxin-related biological activity, distribution and stability of the presented compounds in Arabidopsis thaliana. Despite partial metabolisation in vivo, these fluorescent auxins display an uneven and dynamic distribution leading to the formation of fluorescence maxima in tissues known to concentrate natural auxin, such as the concave side of the apical hook. Importantly, their distribution is altered in response to different exogenous stimuli in both roots and shoots. Moreover, we characterised the subcellular localisation of the fluorescent auxin analogues as being present in the endoplasmic reticulum and endosomes. Our work provides powerful tools to visualise auxin distribution within different plant tissues at cellular or subcellular levels and in response to internal and environmental stimuli during plant development.

A role for the auxin precursor anthranilic acid in root gravitropism via regulation of PIN-FORMED protein polarity and relocalisation in Arabidopsis.

distribution of auxin within plant tissues is of great importance for developmental plasticity, including root gravitropic growth. Auxin flow is directed by the subcellular polar distribution and dynamic relocalisation of auxin transporters such as the PIN-FORMED (PIN) efflux carriers, which can be influenced by the main natural plant auxin indole-3-acetic acid (IAA). Anthranilic acid (AA) is an important early precursor of IAA and previously published studies with AA analogues have suggested that AA may also regulate PIN localisation. Using Arabidopsis thaliana as a model species, we studied an AA-deficient mutant displaying agravitropic root growth, treated seedlings with AA and AA analogues and transformed lines to over-produce AA while inhibiting its conversion to downstream IAA precursors. We showed that AA rescues root gravitropic growth in the AA-deficient mutant at concentrations that do not rescue IAA levels. Overproduction of AA affects root gravitropism without affecting IAA levels. Treatments with, or deficiency in, AA result in defects in PIN polarity and gravistimulus-induced PIN relocalisation in root cells. Our results revealed a previously unknown role for AA in the regulation of PIN subcellular localisation and dynamics involved in root gravitropism, which is independent of its better known role in IAA biosynthesis.

Selective auxin agonists induce specific AUX/IAA protein degradation to modulate plant development.

Auxin phytohormones control most aspects of plant development through a complex and interconnected signaling network. In the presence of auxin, AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) transcriptional repressors are targeted for degradation by the SKP1-CULLIN1-F-BOX (SCF) ubiquitin-protein ligases containing TRANSPORT INHIBITOR RESISTANT 1/AUXIN SIGNALING F-BOX (TIR1/AFB). CULLIN1-neddylation is required for SCFTIR1/AFB functionality, as exemplified by mutants deficient in the NEDD8-activating enzyme subunit AUXIN-RESISTANT 1 (AXR1). Here, we report a chemical biology screen that identifies small molecules requiring AXR1 to modulate plant development. We selected four molecules of interest, RubNeddin 1 to 4 (RN1 to -4), among which RN3 and RN4 trigger selective auxin responses at transcriptional, biochemical, and morphological levels. This selective activity is explained by their ability to consistently promote the interaction between TIR1 and a specific subset of AUX/IAA proteins, stimulating the degradation of particular AUX/IAA combinations. Finally, we performed a genetic screen using RN4, the RN with the greatest potential for dissecting auxin perception, which revealed that the chromatin remodeling ATPase BRAHMA is implicated in auxin-mediated apical hook development. These results demonstrate the power of selective auxin agonists to dissect auxin perception for plant developmental functions, as well as offering opportunities to discover new molecular players involved in auxin responses.

New fluorescently labeled auxins exhibit promising anti-auxin activity.

The plant hormone auxin is a key player in the regulation of plant growth and development. Despite numerous studies devoted to understanding its role in a wide spectrum of physiological processes, full appreciation of its function is linked to a comprehensive determination of its spatio-temporal distribution, which plays a crucial role in its mode of action. Conjugation of fluorescent tracers to plant hormones enables sensitive and specific visualization of their subcellular and tissue-specific localization and transport in planta, which represents a powerful tool for plant physiology. However, to date, only a few fluorescently labeled auxins have been developed. We report the synthesis of four novel fluorescently labeled derivatives of indole-3-acetic acid (IAA) in the form of a conjugate with a nitrobenzoxadiazole (NBD) fluorophore together with validation of their biological activity. These compounds, unlike other previously reported auxins fluorescently labeled at N1 position (nitrogen of the indole ring), do not possess auxin activity but rather show dose-dependent inhibition of auxin-induced effects, such as primary root growth inhibition, root hair growth and the auxin reporter DR5::GUS expression. Moreover, the study demonstrates the importance of the character of the linker and optimal choice of the labeling site in the preparation of fluorescently labeled auxins as important variables influencing their biological activity and fluorescent properties.

Retrieval team-initiated early donor management increases the donor heart utilization rate for transplantation.

OBJECTIVES: Heart transplantation represents the most effective therapy that is currently available for end-stage heart failure. Despite the shortage of organ donors, many donor hearts are not accepted for transplantation due to poor function. Targeted donor management may increase the donor heart utilization rate. The aim of this study is to analyse a 2-year experience of early donor management through the 'scout programme' by a high-volume national cardiothoracic organ retrieval team. METHODS: A prospective cohort study was carried out between 2013 and 2015 on consecutive donation from brain-dead donors. A member of the cardiothoracic retrieval team travelled to the intensive care unit of the donor hospital to assist with early management. RESULTS: One hundred and seventy-eight cardiac donors were enrolled; 106 (59.5%) were 'scouted', and 72 (40.5%) were 'non-scouted'. Donor heart utilization rate in the 'scouted' group was 47.2% (50/106) compared with 30.6% (22/72) in the 'non-scouted' group (P = 0.03). On logistic regression analysis, early donor management by the scouts independently predicted donor heart utilization. The time in the operating theatre from donor arrival to skin incision was significantly reduced in the 'scouted' group. No differences were found in the 30-day graft failure rate or the 30-day, 1-year and 2-year survival rates of the recipients between the 2 groups. CONCLUSIONS: Early donor management delivered by the cardiothoracic retrieval team significantly increased the donor heart utilization rate from existing donors. Moreover, the time in the operating theatre from donor heart arrival to skin incision was significantly reduced.

Human heart transplantation from donation after circulatory-determined death donors using normothermic regional perfusion and cold storage.

Heart transplantation from donation after circulatory-determined-death (DCD) donors is emerging as an additional avenue to increase heart transplant activity. Previous methods of DCD heart retrieval include direct procurement and cold storage, direct procurement, and machine perfusion and normothermic regional perfusion, followed by machine perfusion during transportation. Herein we report a further technique resulting in successful DCD heart transplantation utilizing normothermic regional perfusion and permitting functional assessment followed by cold storage.

What Has Been Seen Cannot Be Unseen-Detecting Auxin In Vivo.

Auxins mediate various processes that are involved in plant growth and development in response to specific environmental conditions. Its proper spatio-temporal distribution that is driven by polar auxin transport machinery plays a crucial role in the wide range of auxins physiological effects. Numbers of approaches have been developed to either directly or indirectly monitor auxin distribution in vivo in order to elucidate the basis of its precise regulation. Herein, we provide an updated list of valuable techniques used for monitoring auxins in plants, with their utilities and limitations. Because the spatial and temporal resolutions of the presented approaches are different, their combination may provide a comprehensive outcome of auxin distribution in diverse developmental processes.

Outcome after heart transplantation from donation after circulatory-determined death donors.

BACKGROUND: The requirement for heart transplantation is increasing, vastly outgrowing the supply of hearts available from donation after brain death (DBD) donors. Transplanting hearts after donation after circulatory-determined death (DCD) may be a viable additive alternative to DBD donors. This study compared outcomes from the largest single-center experience of DCD heart transplantation against matched DBD heart transplants. METHODS: DCD hearts were retrieved using normothermic regional perfusion (NRP) or direct procurement and perfusion (DPP). During NRP, perfusion was restored to the arrested heart within the donor with the exclusion of the cerebral circulation, whereas DPP hearts were removed directly. All hearts were maintained on machine perfusion during transportation. A retrospective cohort of DBD heart transplants, matched for donor and recipient characteristics, was used as a comparison group. The primary outcome measure of this study (set by the United Kingdom regulatory body) was 90-day survival. RESULTS: There were 28 DCD heart transplants performed during the 25-month study period. Survival at 90 days was not significantly different between DCD and matched DBD transplant recipients (DCD, 92%; DBD, 96%; p = 1.0). Hospital length of stay, treated rejection episodes, allograft function, and 1-year survival (DCD, 86%; DBD, 88%; p = 0.98) were comparable between groups. The method of retrieval (NRP or DPP) was not associated with a difference in outcome. CONCLUSIONS: These results suggest that heart transplantation from DCD heart donation provides comparable short-term outcomes to traditional DBD heart transplants and can serve to increase heart transplant activity in well-selected patients.

Functional assessment and transplantation of the donor heart after circulatory death.

BACKGROUND: After a severe shortage of brain-dead donors, the demand for heart transplantation has never been greater. In an attempt to increase organ supply, abdominal and lung transplant programs have turned to the donation after circulatory-determined death (DCD) donor. However, because heart function cannot be assessed after circulatory death, DCD heart transplantation was deemed high risk and never adopted routinely. We report a novel method of functional assessment of the DCD heart resulting in a successful clinical program. METHODS: Normothermic regional perfusion (NRP) was used to restore function to the arrested DCD heart within the donor after exclusion of the cerebral circulation. After weaning from support, DCD hearts underwent functional assessment with cardiac-output studies, echocardiography, and pressure-volume loops. In the feasibility phase, hearts were transported perfused before evaluation of function in modified working mode extracorporeally. After the establishment of a reliable assessment technique, hearts with demonstrable good function were then selected for clinical transplantation. RESULTS: NRP was instituted in 13 adult DCD donors, median age of 33 years (interquartile range [IQR], 28-38 years), after a median ischemic time from withdrawal to perfusion of 24 minutes (IQR, 21-29; range, 17-146 minutes). Two of 4 hearts in the feasibility phase were unsuitable for transplantation after functional assessment. Nine DCD hearts were transplanted in the clinical phase, with 100% survival. The median intensive care duration was 5 days (IQR, 4-5 days), with 2 patients requiring mechanical support. There were no episodes of rejection (total, 1,436 patient-days; range, 48-297). During the same period, we performed 20 standard heart transplants using brain-dead donors. CONCLUSIONS: NRP allows rapid reperfusion and functional assessment of the DCD donor heart, ensuring only viable hearts are selected for transplantation. This technique minimizes the risk of primary graft dysfunction and maximizes confidence in DCD heart transplantation, realizing a 45% increase in our heart transplant activity.

2,4-D and IAA Amino Acid Conjugates Show Distinct Metabolism in Arabidopsis.

The herbicide 2,4-D exhibits an auxinic activity and therefore can be used as a synthetic and traceable analog to study auxin-related responses. Here we identified that not only exogenous 2,4-D but also its amide-linked metabolite 2,4-D-Glu displayed an inhibitory effect on plant growth via the TIR1/AFB auxin-mediated signaling pathway. To further investigate 2,4-D metabolite conversion, identity and activity, we have developed a novel purification procedure based on the combination of ion exchange and immuno-specific sorbents combined with a sensitive liquid chromatography-mass spectrometry method. In 2,4-D treated samples, 2,4-D-Glu and 2,4-D-Asp were detected at 100-fold lower concentrations compared to 2,4-D levels, showing that 2,4-D can be metabolized in the plant. Moreover, 2,4-D-Asp and 2,4-D-Glu were identified as reversible forms of 2,4-D homeostasis that can be converted to free 2,4-D. This work paves the way to new studies of auxin action in plant development.

Extracorporeal membrane oxygenation for the treatment of adult sickle cell acute chest syndrome.

Sickle cell disease (SCD) is a hereditary haemoglobinopathy that results in polymerization of haemoglobin molecules and subsequent vaso-occlusion. A common cause of death in adults is acute chest syndrome (AChS) with resulting hypoxemic respiratory failure.Veno-venous extracorporeal membrane oxygenation (VV-ECMO) has been used successfully in acutely reversible respiratory failure when conventional mechanical ventilation has been unable to adequately oxygenate and ventilate in a lung-protective fashion.We present an adult SCD patient with severe respiratory failure due to AChS, successfully treated with VV-ECMO. We also discuss some of the technical challenges and considerations when using ECMO in the SCD patient.

Perioperative transoesophageal echocardiography.

New technologies are being introduced every day in the perioperative setting and perioperative practitioners will need to become increasingly familiar with transoesophageal echocardiography (TOE). TOE is used as a diagnostic tool during cardiac surgery in the operating theatre to direct the surgical procedure and to detect acute complications. TOE is also used to monitor cardiac function in patients undergoing non-cardiac surgery and in the intensive care unit (ICU) (Figure 1). The use of TOE is increasing in the cardiac catheterisation laboratory for percutaneous transcatheter procedures such as patent foramen ovale (PFO) closure, atrial septal defect (ASD) closure, aortic and mitral valvuloplasty, aortic valve implantation and mitral valve repair. Since the oesophagus is located directly behind the heart, TOE provides better image quality than transthoracic imaging for the assessment of posterior cardiac structures such as the mitral valve, left atrium and pulmonary veins.