Exo84c-regulated degradation is involved in the normal self-incompatible response in Brassicaceae.

The self-incompatibility system evolves in angiosperms to promote cross-pollination by rejecting self-pollination. Here, we show the involvement of Exo84c in the SI response of both Brassica napus and Arabidopsis. The expression of Exo84c is specifically elevated in stigma during the SI response. Knocking out Exo84c in B. napus and SI Arabidopsis partially breaks down the SI response. The SI response inhibits both the protein secretion in papillae and the recruitment of the exocyst complex to the pollen-pistil contact sites. Interestingly, these processes can be partially restored in exo84c SI Arabidopsis. After incompatible pollination, the turnover of the exocyst-labeled compartment is enhanced in papillae. However, this process is perturbed in exo84c SI Arabidopsis. Taken together, our results suggest that Exo84c regulates the exocyst complex vacuolar degradation during the SI response. This process is likely independent of the known SI pathway in Brassicaceae to secure the SI response.

Regional Anesthesia for Arthroscopic Knee Repair in a Patient With Hypertrophic Obstructive Cardiomyopathy (HOCM) Under Monitored Anesthesia Care With Dexmedetomidine Infusion.

Patients with hypertrophic obstructive cardiomyopathy (HOCM) who are scheduled for elective, noncardiac surgery present a distinctive challenge for perioperative healthcare providers. The use of general anesthesia and neuraxial anesthesia carries the risk of unpredictable hemodynamic changes and potential complications. Regional anesthesia (RA) emerges as a prudent and effective option for HOCM patients. RA provides advantages such as minimizing hemodynamic fluctuations, avoiding intubation, reducing pharmacologic side effects, facilitating enhanced recovery after surgery, and contributing to greater patient satisfaction. We share the case of a 15-year-old individual diagnosed with HOCM and exercise intolerance, undergoing arthroscopic repair for right patellar instability. In this instance, the patient received preoperative peripheral nerve blocks for surgical anesthesia and underwent repair utilizing monitored anesthesia care (MAC) with a dexmedetomidine (DEX) infusion.

Mitophagy in plants: Emerging regulators of mitochondrial targeting for selective autophagy.

The degradation and turnover of mitochondria is fundamental to Eukaryotes and is a key homeostatic mechanism for maintaining functional mitochondrial populations. Autophagy is an important pathway by which mitochondria are degraded, involving their sequestration into membrane-bound autophagosomes and targeting to lytic endosomal compartments (the lysosome in animals, the vacuole in plants and yeast). Selective targeting of mitochondria for autophagy, also known as mitophagy, distinguishes mitochondria from other cell components for degradation and is necessary for the regulation of mitochondria-specific cell processes. In mammals and yeast, mitophagy has been well characterised and is regulated by numerous pathways with diverse and important functions in the regulation of cell homeostasis, metabolism and responses to specific stresses. In contrast, we are only just beginning to understand the importance and functions of mitophagy in plants, chiefly as the proteins that target mitochondria for autophagy in plants are only recently emerging. Here, we discuss the current progress of our understanding of mitophagy in plants, the importance of mitophagy for plant life and the regulatory autophagy proteins involved in mitochondrial degradation. In particular, we will discuss the recent emergence of mitophagy receptor proteins that selectively target mitochondria for autophagy, and discuss the missing links in our knowledge of mitophagy-regulatory proteins in plants compared to animals and yeast.

Studying Plant ER-PM Contact Site Localized Proteins Using Microscopy.

As in most eukaryotic cells, the plant endoplasmic reticulum (ER) network is physically linked to the plasma membrane (PM), forming ER-PM contact sites (EPCS). The protein complex required for maintaining the EPCS is composed of ER integral membrane proteins (e.g., VAP27, synaptotagmins), PM-associated proteins (e.g., NET3C), and the cytoskeleton. Here, we describe methods for studying EPCS structures and identifying possible EPCS-associated proteins. These include using artificially constructed reporters, GFP tagged protein expression followed by image analysis, and immunogold labelling at the ultrastructural level. In combination, these methods can be used to identify the location of putative EPCS proteins, which can aid in predicting their potential subcellular function.

NET4 and RabG3 link actin to the tonoplast and facilitate cytoskeletal remodelling during stomatal immunity.

Members of the NETWORKED (NET) family are involved in actin-membrane interactions. Here we show that two members of the NET family, NET4A and NET4B, are essential for normal guard cell actin reorganization, which is a process critical for stomatal closure in plant immunity. NET4 proteins interact with F-actin and with members of the Rab7 GTPase RABG3 family through two distinct domains, allowing for simultaneous localization to actin filaments and the tonoplast. NET4 proteins interact with GTP-bound, active RABG3 members, suggesting their function being downstream effectors. We also show that RABG3b is critical for stomatal closure induced by microbial patterns. Taken together, we conclude that the actin cytoskeletal remodelling during stomatal closure involves a molecular link between actin filaments and the tonoplast, which is mediated by the NET4-RABG3b interaction. We propose that stomatal closure to microbial patterns involves the coordinated action of immune-triggered osmotic changes and actin cytoskeletal remodelling likely driving compact vacuolar morphologies.

Exo84c interacts with VAP27 to regulate exocytotic compartment degradation and stigma senescence.

In plants, exocyst subunit isoforms exhibit significant functional diversity in that they are involved in either protein secretion or autophagy, both of which are essential for plant development and survival. Although the molecular basis of autophagy is widely reported, its contribution to plant reproduction is not very clear. Here, we have identified Exo84c, a higher plant-specific Exo84 isoform, as having a unique function in modulating exocytotic compartment degradation during stigmatic tissue senescence. This process is achieved through its interaction with the ER localised VAP27 proteins, which regulate the turnover of Exo84c through the autophagy pathway. VAP27 recruits Exo84c onto the ER membrane as well as numerous ER-derived autophagosomes that are labelled with ATG8. These Exo84c/exocyst and VAP27 positive structures are accumulated in the vacuole for degradation, and this process is partially perturbed in the exo84c knock-out mutants. Interestingly, the exo84c mutant showed a prolonged effective pollination period with higher seed sets, possibly because of the delayed stigmatic senescence when Exo84c regulated autophagy is blocked. In conclusion, our studies reveal a link between the exocyst complex and the ER network in regulating the degradation of exocytosis vesicles, a process that is essential for normal papilla cell senescence and flower receptivity.

Use of Dexmedetomidine During an Emergent Exploratory Laparotomy in a High-Risk Cardiac Patient With an Intra-aortic Balloon Pump.

An intra-aortic balloon pump (IABP) may be placed preoperatively for high-risk patients with reduced ejection fraction or multivessel coronary disease undergoing non-cardiac surgery. Dexmedetomidine (DEX) has both anesthetic and cardioprotective effects, and little evidence is present on its effect on minimum alveolar concentration (MAC) and bispectral index (BIS). We present the case of a high-risk cardiac patient who was admitted and required fluid optimization prior to coronary artery bypass grafting (CABG). An IABP was placed after failure to tolerate intermittent hemodialysis (iHD). Bowel ischemia complicated this patient's course, necessitating an urgent exploratory laparotomy with the IABP in place. DEX and 0.3-MAC sevoflurane were successfully used without perioperative cardiac complications. Continuous BIS monitoring was performed to maintain an adequate level of anesthesia. DEX should be considered as an alternative anesthetic adjuvant in high-risk and medically complex patients.

Tacrolimus-Induced Akinetic Mutism or Epidural Catheter Migration: A Case Report.

Differential diagnosis of the underlying cause of new-onset total body paralysis can be challenging and unsatisfying. In akinetic mutism, a rare side effect of tacrolimus, patients become apathetic, mute, and lose voluntary muscle movement. Epidural subarachnoid migration can present with similar symptoms. Delayed emergence/paralysis after anesthesia can include the common culprits of residual operative medications, stroke, as well as tacrolimus-induced akinetic mutism and thoracic epidural migration. We present a case of new-onset total body paralysis, presenting on postoperative day 1 following a double-lung transplant in a patient started on tacrolimus with a thoracic epidural catheter in place.

A Case Report of Spinal Anesthesia for Cerclage Placement in the Setting of Severe Hypertrophic Obstructive Cardiomyopathy.

Neuraxial anesthesia is preferred over general anesthesia in obstetric patients to avoid airway manipulation, aspiration, and maternal-fetal transfer of medications; however, a sudden sympathetic block is generally avoided in patients with hypertrophic obstructive cardiomyopathy (HOCM). The case of a 31-year-old G2P0010 with HOCM with severe resting left ventricular outflow tract (LVOT) obstruction and systolic anterior motion of the mitral valve undergoing a cerclage under choroprocaine spinal anesthesia is presented. Risks and benefits of general versus neuraxial anesthesia, and epidural versus spinal anesthesia, in this specific setting are reviewed.

Investigating Plant Protein-Protein Interactions Using FRET-FLIM with a Focus on the Actin Cytoskeleton.

The study of protein-protein interactions is fundamental to understanding how actin-dependent processes are controlled through the regulation of actin-binding proteins by their interactors. FRET-FLIM (Förster resonance energy transfer-fluorescence lifetime imaging microscopy) is a sensitive bioimaging method to detect protein-protein interactions in living cells through measurement of FRET, facilitated by the interactions of fluorophore-tagged fusion protein. As a sensitive and noninvasive method for the spatiotemporal visualization of dynamic protein-protein interactions, FRET-FLIM holds several advantages over other methods of protein interaction assays. FRET-FLIM has been widely employed to characterize many plant protein interactions, including interactions between actin-regulatory proteins and their binding partners. As we increasingly understand the plant actin cytoskeleton to coordinate a diverse number of complex functions, the study of actin-regulatory proteins and their interactors becomes increasingly technically challenging. Sophisticated and sensitive in vivo methods such as FRET-FLIM are likely to be crucial to the study of protein-protein interactions as more complex and challenging hypotheses are addressed.

Keep in contact: multiple roles of endoplasmic reticulum-membrane contact sites and the organelle interaction network in plants.

Functional regulation and structural maintenance of the different organelles in plants contribute directly to plant development, reproduction and stress responses. To ensure these activities take place effectively, cells have evolved an interconnected network amongst various subcellular compartments, regulating rapid signal transduction and the exchange of biomaterial. Many proteins that regulate membrane connections have recently been identified in plants, and this is the first step in elucidating both the mechanism and function of these connections. Amongst all organelles, the endoplasmic reticulum is the key structure, which likely links most of the different subcellular compartments through membrane contact sites (MCS) and the ER-PM contact sites (EPCS) have been the most intensely studied in plants. However, the molecular composition and function of plant MCS are being found to be different from other eukaryotic systems. In this article, we will summarise the most recent advances in this field and discuss the mechanism and biological relevance of these essential links in plants.

TraB, a novel plant ER-mitochondrial contact site protein functions as a mitophagy receptor in plants.

Autophagic degradation of mitochondria (known as mitophagy) is known to occur in all eukaryotes, and is important for the turnover of damaged mitochondria and recycling of nutrients during starvation. Targeting of mitochondria for autophagic degradation is regulated by recognition of mitochondrial-localized mitophagy receptors by the autophagy adaptor protein, ATG8, which regulates the formation of phagophore membranes to encapsulate mitochondrial cargo. Mitophagy receptor proteins have been well characterized in animals and yeast; however, proteins that function as mitophagy receptors in plants have not been discovered until now. We have recently characterized the plant TraB-family proteins AT1G05270/TRB1 and AT2G32340/TRB2, as novel mitophagy receptors, elucidating novel mechanisms of mitophagy in plants.

TraB family proteins are components of ER-mitochondrial contact sites and regulate ER-mitochondrial interactions and mitophagy.

ER-mitochondrial contact sites (EMCSs) are important for mitochondrial function. Here, we have identified a EMCS complex, comprising a family of uncharacterised mitochondrial outer membrane proteins, TRB1, TRB2, and the ER protein, VAP27-1. In Arabidopsis, there are three TraB family isoforms and the trb1/trb2 double mutant exhibits abnormal mitochondrial morphology, strong starch accumulation, and impaired energy metabolism, indicating that these proteins are essential for normal mitochondrial function. Moreover, TRB1 and TRB2 proteins also interact with ATG8 in order to regulate mitochondrial degradation (mitophagy). The turnover of depolarised mitochondria is significantly reduced in both trb1/trb2 and VAP27 mutants (vap27-1,3,4,6) under mitochondrial stress conditions, with an increased population of dysfunctional mitochondria present in the cytoplasm. Consequently, plant recovery after stress is significantly perturbed, suggesting that TRB1-regulated mitophagy and ER-mitochondrial interaction are two closely related processes. Taken together, we ascribe a dual role to TraB family proteins which are component of the EMCS complex in eukaryotes, regulating both interaction of the mitochondria to the ER and mitophagy.

Membrane contact sites and cytoskeleton-membrane interactions in autophagy.

In Eukaryotes, organelle interactions occur at specialised contact sites between organelle membranes. Contact sites are regulated by specialised tethering proteins, which bring organelle membranes into close proximity, and facilitate functional crosstalk between compartments. While contact site proteins are well characterised in mammals and yeast, the regulators of plant contact site formation are only now beginning to emerge. Having unique subcellular structures, plants must also utilise unique mechanisms of organelle interaction to regulate plant-specific functions. The recently characterised NETWORKED proteins are the first dedicated family of plant-specific contact site proteins. Research into the NET proteins and their interacting partners continues to uncover plant-specific mechanisms of organelle interaction and the importance of these organelle contacts to plant life. Moreover, it is becoming increasingly apparent that organelle interactions are fundamental to autophagy in plants. Here, we will present recent developments in our understanding of the mechanisms of plant organelle interactions, their functions, and emerging roles in autophagy.

Echocardiography for extracorporeal membrane oxygenation.

Extracorporeal membrane oxygenation (ECMO) provides advanced cardiopulmonary life support for patients in cardiac and/or respiratory failure. Echocardiography provides essential diagnostic and anatomic information prior to ECMO initiation, allows for safe and efficient ECMO cannula positioning, guides optimization of flow, provides a modality for rapid troubleshooting and patient evaluation, and facilitates decision-making for eventual weaning of ECMO support. Currently, guidelines for echocardiographic assessment in this clinical context are lacking. In this review, we provide an overview of echocardiographic considerations for advanced imagers involved in the care of these complex patients. We focus predominately on new cannulas and complex cannulation techniques, including a special focus on double lumen cannulas and a section discussing indirect left ventricular venting. Echocardiography is tremendously valuable in providing optimal care in these challenging clinical situations. It is imperative for imaging physicians to understand the pertinent anatomic considerations, the often complicated physiological and hemodynamic context, and the limitations of the imaging modality.

Need for preoperative anemia management clinics in Japan: initiatives at a university hospital in the USA.

Untreated preoperative anemia increases the risk of morbidity and mortality and there is increasing evidence that early intervention for preoperative anemia improves outcomes after major surgery. Accordingly, anemia management clinics have been established in various institutions in the USA. As an example, the University of Iowa Hospitals and Clinics outpatient clinic treats pre-surgical anemic patients, who undergo major surgery with anticipated blood loss of more than 500 mL, by providing effective standardized care in a timely manner. This standardized care is an integral part of patient blood management to reduce perioperative blood transfusion and improve patient outcomes. The importance of preoperative anemia management has not yet been sufficiently recognized in Japan. Timely intervention for preoperative anemia should be incorporated into routine pre-surgical patient care in Japan.

Peripartum considerations for women with cardiac disease.

PURPOSE OF REVIEW: The aim of this review of cardiac disease in pregnancy is to delineate current best practices and highlight emerging themes in the literature. RECENT FINDINGS: Cardiovascular disease is the leading cause of death among pregnant women in the United States. Many clinicians and institutions have developed care pathways to approach care in these high-risk patients including highly coordinated multidisciplinary teams. The diagnosis of pulmonary hypertension is the greatest risk factor for an adverse event in pregnant women. Vaginal delivery, with good neuraxial anesthesia, is usually the preferred mode of delivery in women with cardiac disease, although the rate of cesarean delivery is higher among women with heart disease. SUMMARY: The leading cause of morbidity and mortality in pregnant women is cardiac disease. Preconception counseling is useful for optimizing patients for pregnancy and setting appropriate expectations about care and outcomes. Ensuring that women are cared for in centers with appropriate multidisciplinary resources is key for improving outcomes for cardio-obstetric patients.

NETWORKED2-subfamily proteins regulate the cortical actin cytoskeleton of growing pollen tubes and polarised pollen tube growth.

We have recently characterised NET2A as a pollen-specific actin-binding protein that binds F-actin at the plasma membrane of growing pollen tubes. However, the role of NET2 proteins in pollen development and fertilisation have yet to be elucidated. To further characterise the role of Arabidopsis NET2 proteins in pollen development and fertilisation, we analysed the subcellular localisation of NET2A over the course of pollen grain development and investigated the role of the NET2 family using net2 loss-of-function mutants. We observed NET2A to localise to the F-actin cytoskeleton in developing pollen grains as it underwent striking structural reorganisations at specific stages of development and during germination and pollen tube growth. Furthermore, net2 loss-of-function mutants exhibited striking morphological defects in the early stages of pollen tube growth, arising from frequent changes to pollen tube growth trajectory. We observed defects in the cortical actin cytoskeleton and actin-driven subcellular processes in net2 mutant pollen tubes. We demonstrate that NET2 proteins are essential for normal actin-driven pollen development highlighting an important role for the NET2 family members in regulating pollen tube growth during fertilisation.

A novel plant actin-microtubule bridging complex regulates cytoskeletal and ER structure at ER-PM contact sites.

In plants, the cortical endoplasmic reticulum (ER) network is connected to the plasma membrane (PM) through the ER-PM contact sites (EPCSs), whose structures are maintained by EPCS resident proteins and the cytoskeleton.1-7 Strong co-alignment between EPCSs and the cytoskeleton is observed in plants,1,8 but little is known of how the cytoskeleton is maintained and regulated at the EPCS. Here, we have used a yeast-two-hybrid screen and subsequent in vivo interaction studies in plants by fluorescence resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM) analysis to identify two microtubule binding proteins, KLCR1 (kinesin-light-chain-related protein 1) and IQD2 (IQ67-domain 2), that interact with the actin binding protein NET3C and form a component of plant EPCS that mediates the link between the actin and microtubule networks. The NET3C-KLCR1-IQD2 module, acting as an actin-microtubule bridging complex, has a direct influence on ER morphology and EPCS structure. Their loss-of-function mutants, net3a/NET3C RNAi, klcr1, or iqd2, exhibit defects in pavement cell morphology, which we suggest is linked to the disorganization of both actin filaments and microtubules. In conclusion, our results reveal a novel cytoskeletal-associated complex, which is essential for the maintenance and organization of cytoskeletal structure and ER morphology at the EPCS and for normal plant cell morphogenesis.