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Communication between dying and neighbouring cells is vital to ensure appropriate processes such as tissue repair or inflammation are initiated in response to cell death. As a mechanism to aid intercellular communication, cells undergoing apoptosis can release membrane-bound extracellular vesicles (EVs) called apoptotic-cell-derived EVs (ApoEVs) that can influence downstream processes through biomolecules within or on ApoEVs. ApoEVs are broadly categorised based on size as either large ApoEVs known as apoptotic bodies (ApoBDs) or small ApoEVs (s-ApoEVs). Notably, the mechanisms of ApoBD and s-ApoEV formation are different, and the functions of these two ApoEV subsets are distinct. This Review focuses on the biogenesis and functional properties of both ApoBDs and s-ApoEVs, particularly in the context of cell clearance, cell signalling and disease progression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background and Aims: Atherosclerosis is the primary underlying cause of myocardial infarction and stroke, which are the major causes of death globally. Heparanase (Hpse) is a pro-inflammatory extracellular matrix degrading enzyme that has been implicated in atherogenesis. However, to date the precise roles of Hpse in atherosclerosis and its mechanisms of action are not well defined. This study aims to provide new insights into the contribution of Hpse in different stages of atherosclerosis in vivo., Methods: We generated Hpse gene-deficient mice on the atherosclerosis-prone apolipoprotein E gene knockout (ApoE -/- ) background to investigate the impact of Hpse gene deficiency on the initiation and progression of atherosclerosis after 6 and 14 weeks high-fat diet feeding, respectively. Atherosclerotic lesion development, blood serum profiles, lesion composition and aortic immune cell populations were evaluated., Results: Hpse-deficient mice exhibited significantly reduced atherosclerotic lesion burden in the aortic sinus and aorta at both time-points, independent of changes in plasma cholesterol levels. A significant reduction in the necrotic core size and an increase in smooth muscle cell content were also observed in advanced atherosclerotic plaques of Hpse-deficient mice. Additionally, Hpse deficiency reduced circulating and aortic levels of VCAM-1 at the initiation and progression stages of disease and circulating MCP-1 levels in the initiation but not progression stage. Moreover, the aortic levels of total leukocytes and dendritic cells in Hpse-deficient ApoE -/- mice were significantly decreased compared to control ApoE -/- mice at both disease stages., Conclusions: This study identifies Hpse as a key pro-inflammatory enzyme driving the initiation and progression of atherosclerosis and highlighting the potential of Hpse inhibitors as novel anti-inflammatory treatments for cardiovascular disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: Evaluate the effectiveness of the clinical decision support tools (CDSTs), POC Advisor (POCA), and Modified Early Warning System (MEWS) in identifying sepsis risk and influencing time to treatment for inpatients, comparing their respective alert mechanisms., Methods: This study was conducted at two academic university medical center hospitals. Data from adult inpatients in medical-surgical and telemetry units were analyzed from January 1, 2020, to December 31, 2020. Criteria included sepsis-related ICD-10 codes, antibiotic administration, and ordered sepsis labs. Subsequent statistical analyses utilized Fisher's exact test and Wilcoxon Rank Sum test, focusing on mortality differences by age, sex, and race/ethnicity., Results: Among 744 patients, 143 sepsis events were identified, with 83% already receiving treatment upon CDST alert. Group 1 (POCA alert) showed reduced response time compared with MEWS, while Group 3 (MEWS) experienced longer time to treatment. Group 4 included sepsis events missed by both systems. Mortality differences were not significant among the groups., Conclusion: While CDSTs play a role, nursing assessment and clinical judgment are crucial. This study recognized the potential for alarm fatigue due to a high number of CDST-driven alerts, while emphasizing the importance of a collaborative approach for prompt sepsis treatment and potential reduction in sepsis-related mortality., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Discovery of new small molecules that can activate distinct programmed cell death pathway is of significant interest as a research tool and for the development of novel therapeutics for pathological conditions such as cancer and infectious diseases. The small molecule raptinal was discovered as a pro-apoptotic compound that can rapidly trigger apoptosis by promoting the release of cytochrome c from the mitochondria and subsequently activating the intrinsic apoptotic pathway. As raptinal is very effective at inducing apoptosis in a variety of different cell types in vitro and in vivo, it has been used in many studies investigating cell death as well as the clearance of dying cells. While examining raptinal as an apoptosis inducer, we unexpectedly identified that in addition to its pro-apoptotic activities, raptinal can also inhibit the activity of caspase-activated Pannexin 1 (PANX1), a ubiquitously expressed transmembrane channel that regulates many cell death-associated processes. By implementing numerous biochemical, cell biological and electrophysiological approaches, we discovered that raptinal can simultaneously induce apoptosis and inhibit PANX1 activity. Surprisingly, raptinal was found to inhibit cleavage-activated PANX1 via a mechanism distinct to other well-described PANX1 inhibitors such as carbenoxolone and trovafloxacin. Furthermore, raptinal also interfered with PANX1-regulated apoptotic processes including the release of the 'find-me' signal ATP, the formation of apoptotic cell-derived extracellular vesicles, as well as NLRP3 inflammasome activation. Taken together, these data identify raptinal as the first compound that can simultaneously induce apoptosis and inhibit PANX1 channels. This has broad implications for the use of raptinal in cell death studies as well as in the development new PANX1 inhibitors., (© 2024. The Author(s).)

In this work, we investigate the intrinsic as well as modulated optical properties of the AB-stacking bilayer armchair graphene ribbons in the absence and presence of external electric fields. Single-layer ribbons are also considered for comparison. By using a tight-binding model in combination with the gradient approximation, we examine the energy bands, the density of states and the absorption spectra of the studied structures. Our results demonstrate that when external fields are not present, the low-frequency optical absorption spectra display numerous peaks and they vanish at the zero point. In addition, the number, the position, and the intensity of the absorption peaks are strongly associated with the ribbon width. With the wider ribbon width, more absorption peaks are present and a lower threshold absorption frequency is observed. Interestingly, in the presence of electric fields, bilayer armchair ribbons exhibit a lower threshold absorption frequency, more absorption peaks, and weaker spectral intensity. When increasing the strength of the electric field, the prominent peaks of the edge-dependent selection rules are lowered, and the sub-peaks satisfying the extra selection rules come to exist. The obtained results certainly provide a more comprehensive understanding of the correlation between the energy band transition and the optical absorption, in both single-layer and bilayer graphene armchair ribbons, and could provide new insights into developments of optoelectronic device applications based on graphene bilayer ribbons., (© 2023 IOP Publishing Ltd.)

Crocodilians are an order of ancient reptiles that thrive in pathogen-rich environments. The ability to inhabit these harsh environments is indicative of a resilient innate immune system. Defensins, a family of cysteine-rich cationic host defence peptides, are a major component of the innate immune systems of all plant and animal species, however crocodilian defensins are poorly characterised. We now show that the saltwater crocodile defensin CpoBD13 harbors potent antifungal activity that is mediated by a pH-dependent membrane-targeting action. CpoBD13 binds the phospholipid phosphatidic acid (PA) to form a large helical oligomeric complex, with specific histidine residues mediating PA binding. The utilisation of histidine residues for PA engagement allows CpoBD13 to exhibit differential activity at a range of environmental pH values, where CpoBD13 is optimally active in an acidic environment., (© 2023. The Author(s).)

Extracellular vesicles (EVs) are membrane-bound particles released by cells in various (patho)physiological conditions. EVs can transfer effector molecules and elicit potent responses in recipient cells, making them attractive therapeutic agents and drug delivery platforms. In contrast to their tremendous potential, only a few EV-based therapies and drug delivery have been approved for clinical use, which is largely attributed to limited therapeutic loading technologies and efficiency. As EV cargo has major influence on their functionality, understanding and translating the biology underlying the packaging and transferring of biomolecule cargos (e.g. miRNAs, pathogen antigens, small molecule drugs) into EVs is key in harnessing their therapeutic potential. In this review, through recent insights into EVs' content packaging, we discuss different mechanisms utilized by EVs during cargo packaging, and how one might therapeutically exploit this process. Apart from the well-characterized EVs like exosomes and microvesicles, we also cover the less-studied and other EV subtypes like apoptotic bodies, large oncosomes, bacterial outer membrane vesicles, and migrasomes to highlight therapeutically-diverse opportunities of EV armoury., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ozkocak, Phan and Poon.)

Defensins are a class of host defence peptides (HDPs) that often harbour antimicrobial and anticancer activities, making them attractive candidates as novel therapeutics. In comparison with current antimicrobial and cancer treatments, defensins uniquely target specific membrane lipids via mechanisms distinct from other HDPs. Therefore, defensins could be potentially developed as therapeutics with increased selectivity and reduced susceptibility to the resistance mechanisms of tumour cells and infectious pathogens. In this review, we highlight recent advances in defensin research with a particular focus on membrane lipid-targeting in cancer and infection settings. In doing so, we discuss strategies to harness lipid-binding defensins for anticancer and anti-infective therapies., (© 2022 The Author(s).)

Defensins form an integral part of the cationic host defence peptide (HDP) family, a key component of innate immunity. Apart from their antimicrobial and immunomodulatory activities, many HDPs exert multifaceted effects on tumour cells, notably direct oncolysis and/or inhibition of tumour cell migration. Therefore, HDPs have been explored as promising anticancer therapeutics. Human β-defensin 2 (HBD-2) represents a prominent member of human HDPs, being well-characterised for its potent pathogen-killing, wound-healing, cytokine-inducing and leukocyte-chemoattracting functions. However, its anticancer effects remain largely unknown. Recently, we demonstrated that HBD-2 binds strongly to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ), a key mediator of defensin-induced cell death and an instructional messenger during cell migration. Hence, in this study, we sought to investigate the lytic and anti-migratory effects of HBD-2 on tumour cells. Using various cell biological assays and confocal microscopy, we showed that HBD-2 killed tumour cells via acute lytic cell death rather than apoptosis. In addition, our data suggested that, despite the reported PI(4,5)P 2 interaction, HBD-2 does not affect cytoskeletal-dependent tumour cell migration. Together, our findings provide further insights into defensin biology and informs future defensin-based drug development.

Apoptotic bodies (ApoBDs), which are large extracellular vesicles exclusively released by apoptotic cells, possess therapeutically exploitable properties including biomolecule loadability and transferability. However, current limited understanding of ApoBD biology has hindered its exploration for clinical use. Particularly, as ApoBD-accompanying cargoes (e.g., nucleic acids and proteins) have major influence on their functionality, further insights into the mechanism of biomolecule sorting into ApoBDs are critical to unleash their therapeutic potential. Previous studies suggested pannexin 1 (PANX1) channel, a negative regulator of ApoBD biogenesis, can modify synaptic vesicle contents. We also reported that trovafloxacin (a PANX1 inhibitor) increases proportion of ApoBDs containing DNA. Therefore, we sought to define the role of PANX1 in regulating the sorting of nuclear content into ApoBDs. Here, using flow cytometry and label-free quantitative proteomic analyses, we showed that targeting PANX1 activity during apoptosis, via either pharmacological inhibition or genetic disruption, resulted in enrichment of both DNA and nuclear proteins in ApoBDs that were unexpectedly smaller in size. Our data suggest that PANX1, besides being a key regulator of ApoBD formation, also functions as a negative regulator of nuclear content packaging and modulator of ApoBD size. Together, our findings provide further insights into ApoBD biology and form a novel conceptual framework for ApoBD-based therapies through pharmacologically manipulating ApoBD contents., (© 2021 Wiley-VCH GmbH.)

Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to biological functions, our knowledge regarding their composition and immunogenicity remains limited. Here we examine the morphology, contents and immunostimulatory functions of MVs produced by three Staphylococcus aureus strains; a methicillin resistant clinical isolate, a methicillin sensitive clinical isolate and a laboratory-adapted strain. We observed differences in the number and morphology of MVs produced by each strain and showed that they contain microbe-associated molecular patterns (MAMPs) including protein, nucleic acids and peptidoglycan. Analysis of MV-derived RNA indicated the presence of small RNA (sRNA). Furthermore, we detected variability in the amount and composition of protein, nucleic acid and peptidoglycan cargo carried by MVs from each S. aureus strain. S. aureus MVs activated Toll-like receptor (TLR) 2, 7, 8, 9 and nucleotide-binding oligomerization domain containing protein 2 (NOD2) signalling and promoted cytokine and chemokine release by epithelial cells, thus identifying that MV-associated MAMPs including DNA, RNA and peptidoglycan are detected by pattern recognition receptors (PRRs). Moreover, S. aureus MVs induced the formation of and colocalized with autophagosomes in epithelial cells, while inhibition of lysosomal acidification using bafilomycin A1 resulted in accumulation of autophagosomal puncta that colocalized with MVs, revealing the ability of the host to degrade MVs via autophagy. This study reveals the ability of DNA, RNA and peptidoglycan associated with MVs to activate PRRs in host epithelial cells, and their intracellular degradation via autophagy. These findings advance our understanding of the immunostimulatory roles of Gram-positive bacterial MVs in mediating pathogenesis, and their intracellular fate within the host., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Objective: To investigate the impact of limiting the definition of post-coronary artery bypass graft (CABG) atrial fibrillation (AF) to AF/flutter requiring treatment-as in the Society of Thoracic Surgeons' (STS) database- on the association with survival., Patients and Methods: We assessed in-hospital incidence of post-CABG AF in 7110 consecutive isolated patients with CABG without preoperative AF at 4 hospitals (January 1, 2004 to December 31, 2010). Patients with ≥1 episode of post-CABG AF detected via continuous in-hospital electrocardiogram (ECG)/telemetry monitoring documented by physicians were assigned to the following: Group 1, identified as having post-CABG AF in STS data and Group 2, not identified as having post-CABG AF in STS data. Patients without documented post-CABG AF constituted Group 3. Survival was compared via a Cox model, adjusted for STS risk of mortality and accounting for site differences., Results: Over 7 years' follow-up, 16.0% (295 of 1841) of Group 1, 18.7% (79 of 422) of Group 2, and 7.9% (382 of 4847) of Group 3 died. Group 2 had a significantly greater adjusted risk of death than both Group 1 (hazard ratio [HR]: 1.16; 95% confidence interval [CI], 1.02 to 1.33) and Group 3 (HR: 1.94; 95% CI, 1.69 to 2.22)., Conclusions: The statistically significant 16% higher risk of death for patients with AF post-CABG missed vs captured in STS data suggests treatment and postdischarge management should be investigated for differences. The historical misclassification of "missed" patients as experiencing no AF in the STS data weakens the ability to observe differences in risk between patients with and without post-CABG AF. Therefore, STS data should not be used for research examining post-CABG AF., (© 2020 Mayo Foundation for Medical Education and Research. Published by Elsevier Inc.)

Sepsis is a biphasic disease characterized by an acute inflammatory response, followed by a prolonged immunosuppressive phase. Therapies aimed at controlling inflammation help to reduce the time patients with sepsis spend in intensive care units, but they do not lead to a reduction in overall mortality. Recently, the focus has been on addressing the immunosuppressive phase, often caused by apoptosis of immune cells. However, molecular triggers of these events are not yet known. Using whole-genome CRISPR screening in mice, we identified a triggering receptor expressed on myeloid cells (TREM) family receptor, TREML4, as a key regulator of inflammation and immune cell death in sepsis. Genetic ablation of Treml4 in mice demonstrated that TREML4 regulates calcium homeostasis, the inflammatory cytokine response, myeloperoxidase activation, the endoplasmic reticulum stress response and apoptotic cell death in innate immune cells, leading to an overall increase in survival rate, both during the acute and chronic phases of polymicrobial sepsis.

Extracellular vesicles (EVs), membrane-bound vesicles that are naturally released by cells, have emerged as new therapeutic opportunities. EVs, particularly exosomes and microvesicles, can transfer effector molecules and elicit potent responses in recipient cells, making them attractive therapeutic targets and drug delivery platforms. Furthermore, containing predictive biomarkers and often being dysregulated in various disease settings, these EVs are being exploited for diagnostic purposes. In contrast, the therapeutic application of apoptotic bodies (ApoBDs), a distinct type of EVs released by cells undergoing a form of programmed cell death called apoptosis, has been largely unexplored. Recent studies have shed light on ApoBD biogenesis and functions, promisingly implicating their therapeutic potential. In this review, we discuss many strategies to develop ApoBD-based therapies as well as highlight their advantages and challenges, thereby positioning ApoBD for potential EV-based therapy., (© 2020 The Author(s).)

Mucosa-associated invariant T (MAIT) cells are abundant antimicrobial T cells in humans and recognize antigens derived from the microbial riboflavin biosynthetic pathway presented by the MHC-Ib-related protein (MR1). However, the mechanisms responsible for MAIT cell antimicrobial activity are not fully understood, and the efficacy of these mechanisms against antibiotic resistant bacteria has not been explored. Here, we show that MAIT cells mediate MR1-restricted antimicrobial activity against Escherichia coli clinical strains in a manner dependent on the activity of cytolytic proteins but independent of production of pro-inflammatory cytokines or induction of apoptosis in infected cells. The combined action of the pore-forming antimicrobial protein granulysin and the serine protease granzyme B released in response to T cell receptor (TCR)-mediated recognition of MR1-presented antigen is essential to mediate control against both cell-associated and free-living, extracellular forms of E. coli. Furthermore, MAIT cell-mediated bacterial control extends to multidrug-resistant E. coli primary clinical isolates additionally resistant to carbapenems, a class of last resort antibiotics. Notably, high levels of granulysin and granzyme B in the MAIT cell secretomes directly damage bacterial cells by increasing their permeability, rendering initially resistant E. coli susceptible to the bactericidal activity of carbapenems. These findings define the role of cytolytic effector proteins in MAIT cell-mediated antimicrobial activity and indicate that granulysin and granzyme B synergize to restore carbapenem bactericidal activity and overcome carbapenem resistance in E. coli., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: David P Fairle is an inventor on a patent application (PCT/AU2013/000742, WO2014005194), and Jeffrey YW Mak and David P Fairlie are inventors on another patent application (PCT/AU2015/050148, WO2015149130) involving MR1 ligands for MR1-restricted MAIT cells owned by University of Queensland, Monash University and University of Melbourne. The other authors declare no competing interests.

The disassembly of apoptotic cells into small membrane-bound vesicles termed apoptotic bodies (ApoBDs) is a hallmark of apoptosis; however, the functional significance of this process is not well defined. We recently discovered a new membrane protrusion (termed beaded apoptopodia) generated by apoptotic monocytes which fragments to release an abundance of ApoBDs. To investigate the function of apoptotic monocyte disassembly, we used influenza A virus (IAV) infection as a proof-of-concept model, as IAV commonly infects monocytes in physiological settings. We show that ApoBDs generated from IAV-infected monocytes contained IAV mRNA, protein and virions and consequently, could facilitate viral propagation in vitro and in vivo, and induce a robust antiviral immune response. We also identified an antipsychotic, Haloperidol, as an unexpected inhibitor of monocyte cell disassembly which could impair ApoBD-mediated viral propagation under in vitro conditions. Together, this study reveals a previously unrecognised function of apoptotic monocyte disassembly in the pathogenesis of IAV infections.

Background: New-onset atrial fibrillation (AF) after coronary artery bypass graft surgery (CABG) is associated with poor outcomes, but data on the effects of its characteristics are lacking and conflicting. We examined the effect number of post-CABG AF events has on long-term mortality risk, and whether this is sex dependent., Methods: Routinely collected Society of Thoracic Surgeons (STS) data were supplemented with details on new-onset post-CABG AF (detected in-hospital by continuous electrocardiogram/telemetry monitoring) and long-term survival for 9203 consecutive patients with isolated-CABG (2002-2010). With the use of Cox regression, we determined the propensity-adjusted (STS-recognized risk factors) effect of number of AF events on survival, testing for effect modification by sex and controlling for AF duration., Results: AF occurred in 739 women (29.4%) and 2157 men (32.3%) (P < .001). Adjusted results showed 2 or more AF events significantly (P < .001) increased 5-year mortality risk, independently of total AF duration. However, mortality risk differed between the sexes (P < .001): women with 2 AF episodes had the greatest increase (hazard ratio [HR] = 2.98; 95% confidence interval [CI], 1.43-4.83; versus women without AF), followed by women and men with 4 or more AF events (HR = 2.76 [95% CI, 1.27-4.55] and HR = 2.73 [95% CI, 2.30-3.19], respectively). A single post-CABG AF episode was not associated with increased mortality risk., Conclusions: Both men and women who experienced 2 or more post-CABG AF episodes showed increased risk of 5-year mortality, independent of total AF duration. Although men's risk increased as the number of AF events increased, women's risk peaked at 2 AF events. Future research needs to determine whether this divergence stems from differences in treatment/management or underlying biology., (Copyright © 2020 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: We sought to fill important gaps in the existing evidence regarding new-onset atrial fibrillation (AF) after coronary artery bypass graft surgery (CABG) by comparing the incidence, characteristics, and effect on long-term survival between men and women., Methods: Nine thousand two hundred three consecutive patients without preoperative AF underwent isolated CABG from 2002 to 2010 at 3 US academic medical centers and 1 high-volume specialty cardiac hospital. Detailed data on CABG AF events detected via continuous in-hospital electrocardiogram/telemetry monitoring were supplemented with Society of Thoracic Surgeons data, and survival data, censored at October 31, 2011, using a copy of the Social Security Death Master File archived before state-owned data were removed (November 1, 2011)., Results: Propensity-adjusted (Society of Thoracic Surgeons-recognized risk factors) incidence of post-CABG AF was 31.5% overall, 32.8% in men, and 27.4% in women. Over the 9-year study period, women had a significantly lower risk of post-CABG AF (absolute difference, -5.3% [95% confidence interval (CI), -10.5% to -0.6%]), and significantly shorter first (-2.9 hours; 95% CI, -5.8 to 0.0), and longest (-4.3 hours; 95% CI, -8.3 to -0.3) AF duration. Post-CABG AF was associated with significantly increased risk of long-term mortality (overall hazard ratio [HR], 1.56; 95% CI, 1.45-1.67; men HR, 1.57; 95% CI, 1.49-1.65; women HR, 1.54; 95% CI, 1.14-2.07)., Conclusions: In our study, women had lower adjusted risk of post-CABG AF and experienced shorter episodes. The adjusted risk of long-term mortality was 56% greater among patients who developed post-CABG AF compared with those who did not. The effect of post-CABG AF on long-term survival did not differ between the sexes., (Copyright © 2019. Published by Elsevier Inc.)

A self-powered and low pressure loss gas flowmeter is presently proposed and developed based on a membrane's flutter driven triboelectric nanogenerator (TENG). Such a flowmeter, herein named "TENG flowmeter", is made of a circular pipe in which two copper electrodes are symmetrically fixed and a nonconductive, thin membrane is placed in the middle plane of the pipe. When a gas flows through the pipe at a sufficiently high speed, the membrane will continuously oscillate between the two electrodes, generating a periodically fluctuating electric voltage whose frequency can be easily measured. As demonstrated experimentally, the fluctuation frequency ( f F ) relates linearly with the pipe flow mean velocity ( U m ), i.e., f F U m ; therefore, the volume flow rate Q (= U m A ) = C 1 f F + C 2 , where C 1 and C 2 are experimental constants and A is the pipe cross-sectional area. That is, by the TENG flowmeter, the pipe flow rate Q can be obtained by measuring the frequency f F . Notably, the TENG flowmeter has several advantages over some commercial flowmeters (e.g., vortex flowmeter), such as considerable lower pressure loss, higher sensitiveness of the measured flow rate, and self-powering. In addition, the effects of membrane material and geometry as well as flow moisture on the flowmeter are investigated. Finally, the performance of the TENG flowmeter is demonstrated., Competing Interests: The authors declare no conflict of interest.

Many cell types are known to undergo a series of morphological changes during the progression of apoptosis, leading to their disassembly into smaller membrane-bound vesicles known as apoptotic bodies (ApoBDs). In particular, the formation of circular bulges called membrane blebs on the surface of apoptotic cells is a key morphological step required for a number of cell types to generate ApoBDs. Although apoptotic membrane blebbing is thought to be regulated by kinases including ROCK1, PAK2 and LIMK1, it is unclear whether these kinases exhibit overlapping roles in the disassembly of apoptotic cells. Utilising both pharmacological and CRISPR/Cas9 gene editing based approaches, we identified ROCK1 but not PAK2 or LIMK1 as a key non-redundant positive regulator of apoptotic membrane blebbing as well as ApoBD formation. Functionally, we have established an experimental system to either inhibit or enhance ApoBD formation and demonstrated the importance of apoptotic cell disassembly in the efficient uptake of apoptotic materials by various phagocytes. Unexpectedly, we also noted that ROCK1 could play a role in regulating the onset of secondary necrosis. Together, these data shed light on both the mechanism and function of cell disassembly during apoptosis.

During apoptosis, dying cells undergo dynamic morphological changes that ultimately lead to their disassembly into fragments called apoptotic bodies (ApoBDs). Reorganisation of the cytoskeletal structures is key in driving various apoptotic morphologies, including the loss of cell adhesion and membrane bleb formation. However, whether cytoskeletal components are also involved in morphological changes that occur later during apoptosis, such as the recently described generation of thin apoptotic membrane protrusions called apoptopodia and subsequent ApoBD formation, is not well defined. Through monitoring the progression of apoptosis by confocal microscopy, specifically focusing on the apoptopodia formation step, we characterised the presence of F-actin and microtubules in a subset of apoptopodia generated by T cells and monocytes. Interestingly, targeting actin polymerisation and microtubule assembly pharmacologically had no major effect on apoptopodia formation. These data demonstrate apoptopodia as a novel type of membrane protrusion that could be formed in the absence of actin polymerisation and microtubule assembly.

Billions of cells undergo apoptosis daily and often fragment into small, membrane-bound extracellular vesicles termed apoptotic bodies (ApoBDs). We demonstrate that apoptotic monocytes undergo a highly coordinated disassembly process and form long, beaded protrusions (coined as beaded apoptopodia), which fragment to release ApoBDs. Here, we find that the protein plexin B2 (PlexB2), a transmembrane receptor that regulates axonal guidance in neurons, is enriched in the ApoBDs of THP1 monocytes and is a caspase 3/7 substrate. To determine whether PlexB2 is involved in the disassembly of apoptotic monocytes, we generate PlexB2-deficient THP1 monocytes and demonstrate that lack of PlexB2 impairs the formation of beaded apoptopodia and ApoBDs. Consequently, the loss of PlexB2 in apoptotic THP1 monocytes impairs their uptake by both professional and non-professional phagocytes. Altogether, these data identify PlexB2 as a positive regulator of apoptotic monocyte disassembly and demonstrate the importance of this process in apoptotic cell clearance., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Pathogens and tumor cells have adopted various adept strategies to evade immunosurveillance and promote their growth and survival. There has been substantial evidence demonstrating phosphoinositide lipids and their modifying enzymes as essential host targets that are often hijacked by pathogens and tumor cells. The common dependence of pathogen virulence and tumor progression on phosphoinositides presents an exciting disease-combating potential, particularly combinatorial therapeutics. While traditional approaches to pharmacologically inhibit phosphoinositide-metabolizing enzymes has shown some promise, the direct targeting of phosphoinositides has recently emerged as a novel therapeutic strategy. Our review provides a current picture of the role of phosphoinositides during pathogen virulence and tumorigenesis as well as a thorough discussion on promises, challenges, and new perspectives of phosphoinositide-targeting drug development., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Extracellular vesicles (EVs) are an important class of membrane-bound structures that have been widely investigated for their roles in intercellular communication in the contexts of tumor progression, vascular function, immunity and regenerative medicine. Much of the current knowledge on the functions of EVs pertains to those derived from viable cells (e.g. exosomes and microvesicles) or apoptotic cells (e.g. apoptotic bodies) whilst the generation of EVs from dying cells under non-apoptotic conditions remains poorly characterized. Herein, the release of EVs from THP-1 monocytes under conditions of primary necrosis, secondary necrosis and pyroptosis, was investigated. A comprehensive analysis of THP-1-derived EVs revealed that cells undergoing lytic forms of cell death generated a high number of EVs compared with viable or apoptotic cells in vitro. Differential centrifugation via 16,000 g and 100,000 g revealed that dying THP-1 cells release both medium and small EVs, respectively, consistent with the known characteristics of microvesicles and/or exosomes. In addition, large EVs isolated via 2000 g centrifugation were also present in all samples. These findings suggest that lytic cell death under both sterile and non-sterile inflammatory conditions induces monocytes to generate EVs, which could potentially act as mediators of cell-to-cell communication.

Phosphorylated phosphatidylinositol lipids, or phosphoinositides, critically regulate diverse cellular processes, including signalling transduction, cytoskeletal reorganisation, membrane dynamics and cellular trafficking. However, phosphoinositides have been inadequately investigated in the context of cell death, where they are mainly regarded as signalling secondary messengers. However, recent studies have begun to highlight the importance of phosphoinositides in facilitating cell death execution. Here, we cover the latest phosphoinositide research with a particular focus on phosphoinositides in the mechanisms of cell death. This progress article also raises key questions regarding the poorly defined role of phosphoinositides, particularly during membrane-associated events in cell death such as apoptosis and secondary necrosis. The review then further discusses important future directions for the phosphoinositide field, including therapeutically targeting phosphoinositides to modulate cell death.

Defensins are an extensive family of host defense peptides found ubiquitously across plant and animal species. In addition to protecting against infection by pathogenic microorganisms, some defensins are selectively cytotoxic toward tumor cells. As such, defensins have attracted interest as potential antimicrobial and anticancer therapeutics. The mechanism of defensin action against microbes and tumor cells appears to be conserved and involves the targeting and disruption of cellular membranes. This has been best defined for plant defensins, which upon binding specific phospholipids, such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid, form defensin-lipid oligomeric complexes that destabilize membranes, leading to cell lysis. In this study, to further define the anticancer and therapeutic properties of plant defensins, we have characterized a novel plant defensin, Nicotiana occidentalis defensin 173 (NoD173), from N. occidentalis. NoD173 at low micromolar concentrations selectively killed a panel of tumor cell lines over normal primary cells. To improve the anticancer activity of NoD173, we explored increasing cationicity by mutation, with NoD173 with the substitution of Q22 with lysine [NoD173(Q22K)], increasing the antitumor cell activity by 2-fold. NoD173 and the NoD173(Q22K) mutant exhibited only low levels of hemolytic activity, and both maintained activity against tumor cells in serum. The ability of NoD173 to inhibit solid tumor growth in vivo was tested in a mouse B16-F1 model, whereby injection of NoD173 into established subcutaneous tumors significantly inhibited tumor growth. Finally, we showed that NoD173 specifically targets PIP2 and determined by X-ray crystallography that a high-resolution structure of NoD173, which forms a conserved family-defining cysteine-stabilized-αβ motif with a dimeric lipid-binding conformation, configured into an arch-shaped oligomer of 4 dimers. These data provide insights into the mechanism of how defensins target membranes to kill tumor cells and provide proof of concept that defensins are able to inhibit tumor growth in vivo .-Lay, F. T., Ryan, G. F., Caria, S., Phan, T. K., Veneer, P. K., White, J. A., Kvansakul, M., Hulett M. D. Structural and functional characterization of the membrane-permeabilizing activity of Nicotiana occidentalis defensin NoD173 and protein engineering to enhance oncolysis.

Apoptosis is a form of programmed cell death that occurs throughout life as part of normal development as well as pathologic processes including chronic inflammation and infection. Although the death of a cell is often considered as the only biological outcome of a cell committed to apoptosis, it is becoming increasingly clear that the dying cell can actively communicate with other cells via soluble factors as well as membrane-bound extracellular vesicles (EVs) to regulate processes including cell clearance, immunity and tissue repair. Compared to EVs generated from viable cells such as exosomes and microvesicles, apoptotic cell-derived EVs (ApoEVs) are less well defined and the basic criteria for ApoEV characterization have not been established in the field. In this study, we will examine the current understanding of ApoEVs, in particular, the ApoEV subtype called apoptotic bodies (ApoBDs). We described that a subset of ApoBDs can be larger than 5 μm and smaller than 1 μm based on flow cytometry and live time-lapse microscopy analysis, respectively. We also described that a subset of ApoBDs can expose a relatively low level of phosphatidylserine on its surface based on annexin A5 staining. Furthermore, we characterized the presence of caspase-cleaved proteins (in particular plasma membrane-associated or cytoplasmic proteins) in samples enriched in ApoBDs. Lastly, using a combination of biochemical-, live imaging- and flow cytometry-based approaches, we characterized the progressive lysis of ApoBDs. Taken together, these results extended our understanding of ApoBDs.

Pathogenic microbes are developing resistance to established antibiotics, making the development of novel antimicrobial molecules paramount. One major resource for discovery of antimicrobials is the arsenal of innate immunity molecules that are part of the first line of pathogen defense in many organisms. Gene encoded cationic antimicrobial peptides are a major constituent of innate immune arsenals. Many of these peptides exhibit potent antimicrobial activity in vitro . However, a major hurdle that has impeded their development for use in the clinic is the loss of activity at physiological salt concentrations, attributed to weakening of the electrostatic interactions between the cationic peptide and anionic surfaces of the microbial cells in the presence of salt. Using plant defensins we have investigated the relationship between the charge of an antimicrobial peptide and its activity in media with elevated salt concentrations. Plant defensins are a large class of antifungal peptides that have remarkable stability at extremes of pH and temperature as well as resistance to protease digestion. A search of a database of over 1200 plant defensins identified ZmD32, a defensin from Zea mays , with a predicted charge of +10.1 at pH 7, the highest of any defensin in the database. Recombinant ZmD32 retained activity against a range of fungal species in media containing elevated concentrations of salt. In addition, ZmD32 was active against Candida albicans biofilms as well as both Gram negative and Gram-positive bacteria. This broad spectrum antimicrobial activity, combined with a low toxicity on human cells make ZmD32 an attractive lead for development of future antimicrobial molecules.

Objective: Studies with authors trained in research methods are of higher quality than those without. We examined inclusion of authors with master's or doctoral degrees incorporating advanced research methods training on original research articles in high-impact journals, investigating differences between journals and by first-author sex., Methods: Using all original research articles from 1 issue of The New England Journal of Medicine ( NEJM ), Journal of the American Medical Association ( JAMA ), Annals of Internal Medicine ( Annals ), and JAMA-Internal Medicine/Archives of Internal Medicine ( Archives ) every alternate month, February 1994 to October 2016, we assessed the prevalence of articles listing authors with master's/doctoral research degrees and its adjusted associations with time of publication, journal, and first-author sex via multivariable logistic regression models (accounting for number of authors, study type, specialty/topic, and continent and for interactions between journal and time of publication, study type, and continent)., Results: Of 3009 articles examined, 84.4% (n=2539) had authors listing research degrees. After adjustment, the prevalence of such articles increased from 1994 to 2016 ( P <.001), but patterns differed among journals. Annals and NEJM increased to approximately100% by 2016; JAMA and Archives peaked around 2010 to 2011, then declined. Articles with female first authors were more likely to list authors with research degrees (adjusted odds ratio=1.66; 95% CI, 1.29-2.13; P <.001)., Conclusion: The prevalence of original research articles listing authors trained in research methods in high-impact journals increased significantly but is now declining at some journals, with potential effects on quality. The greater prevalence among female first-authored articles suggests possible sex differences in structuring/crediting research teams or subconscious sex bias during review.

During the progression of necroptosis and pyroptosis, the plasma membrane will become permeabilized through the activation of mixed lineage kinase domain like pseudokinase (MLKL) or gasdermin D (GSDMD), respectively. Recently, the progression of apoptotic cells into secondary necrotic cells following membrane lysis was shown to be regulated by gasdermin E (GSDME, or DFNA5), a process dependent on caspase 3-mediated cleavage of GSDME. Notably, GSDME was also proposed to negatively regulate the disassembly of apoptotic cells into smaller membrane-bound vesicles known as apoptotic bodies (ApoBDs) by promoting earlier onset of membrane permeabilisation. The presence of a process downstream of caspase 3 that would actively drive cell lysis and limit cell disassembly during apoptosis is somewhat surprising as this could favor the release of proinflammatory intracellular contents and hinder efficient clearance of apoptotic materials. In contrast to the latter studies, we present here that GSDME is not involved in regulating secondary necrosis in human T cells and monocytes, and also unlikely in epithelial cells. Furthermore, GSDME is evidently not a negative regulator of apoptotic cell disassembly in our cell models. Thus, the function of GSDME in regulating membrane permeabilization and cell disassembly during apoptosis may be more limited.

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. It has been proposed that ApoBDs can aid cell clearance as well as intercellular communication through trafficking biomolecules. Conventional approaches used for the identification and isolation of ApoBDs are often limited by the lack of accurate quantification and low sample purity. Here, we describe a workflow to confirm the induction of apoptosis, validate ApoBD formation, and isolate ApoBDs to high purity. We will also outline and compare fluorescence-activated cell sorting (FACS) and differential centrifugation based approaches to isolate ApoBDs. Furthermore, the purity of isolated ApoBDs will be confirmed using a previously establish flow cytometry-based staining and analytical method. Taken together, using the described approach, THP-1 monocyte apoptosis and apoptotic cell disassembly was induced and validated, and ApoBD generated from THP-1 monocytes were isolated to a purity of 97-99%.

Human defensins belong to a subfamily of the cationic antimicrobial peptides and act as a first line of defense against invading microbes. Their often broad-spectrum antimicrobial and antitumor activities make them attractive for therapeutic development; however, their precise molecular mechanism(s) of action remains to be defined. We show that human β-defensin 2 (HBD-2) permeabilizes Candida albicans cell membranes via a mechanism targeting the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ). We determined the structure of HBD-2 bound to PIP 2 , which revealed two distinct PIP 2 -binding sites, and showed, using functional assays, that mutations in these sites ablate PIP 2 -mediated fungal growth inhibition by HBD-2. Our study provides the first insight into lipid-mediated human defensin membrane permeabilization at an atomic level and reveals a unique mode of lipid engagement to permeabilize cell membranes.

Objectives: Postoperative atrial fibrillation (AF) following coronary artery bypass graft surgery (CABG) is significantly associated with reduced survival, but poor characterisation and inconsistent definitions present barriers to developing effective prophylaxis and management. We sought to address this knowledge gap., Methods: From 2002 to 2010, 11 239 consecutive patients without AF underwent isolated CABG at five sites. Clinical data collected for the Society of Thoracic Surgeons (STS) Database were augmented with details on AF detected via continuous in-hospital ECG/telemetry monitoring to assess new-onset post-CABG AF (adjusted for STS risk of mortality); time to first AF; durations of first and longest AF episodes; total in-hospital time in AF; number of in-hospital AF episodes; operative mortality; stroke; discharge in AF; and length of stay (LOS)., Results: Unadjusted incidence of new-onset post-CABG AF was 29.5%. Risk-adjusted incidence was 33.1% and varied little over time (P=0.139). Among 3312 patients with post-CABG AF, adjusted median time to first AF was 52 (IQR: 48-55) hours; mean (SD) duration of first and longest events were 7.2 (5.3,9.1) and 13.1 (10.4,15.9) hours, respectively, and adjusted median total time in AF was 22 (IQR: 18-26) hours. Adjusted rates of operative mortality, stroke and discharge in AF did not vary significantly over time (P=0.156, P=0.965 and P=0.347, respectively). LOS varied (P=0.035), but in no discernible pattern., Conclusions: Each year, ~800 000 people undergo CABG worldwide; >264 000 will develop post-CABG AF. Onset is typically 2-3 days post-CABG and episodes last, on average, several hours. Effective prophylaxis and management is urgently needed to reduce associated risks of adverse outcomes., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Defensins are cationic antimicrobial peptides expressed throughout the plant and animal kingdoms as a first line of defense against pathogens. Membrane targeting and disruption is a crucial function of many defensins, however the precise mechanism remains unclear. Certain plant defensins form dimers that specifically bind the membrane phospholipids phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphosphate, thereby triggering the assembly of defensin-lipid oligomers that permeabilize cell membranes. To understand this permeabilization mechanism, here we determine the crystal structure of the plant defensin NaD1 bound to PA. The structure reveals a 20-mer that adopts a concave sheet- or carpet-like topology where NaD1 dimers form one face and PA acyl chains form the other face of the sheet. Furthermore, we show that Arg39 is critical for PA binding, oligomerization and fungal cell killing. These findings identify a putative defensin-phospholipid membrane attack configuration that supports a longstanding proposed carpet mode of membrane disruption.

Host defense peptides (HDPs) are well-characterized for their antimicrobial activities but also variously display potent immunomodulatory effects. Human β-defensin 3 (HBD-3) belongs to a well-known HDP family known as defensins and is able to induce leukocyte chemotactic recruitment, leukocyte activation/maturation, proinflammatory cytokine release, and co-stimulatory marker expression. HBD-3-stimulated cytokine induction is NF-κB-dependent and was initially suggested to act via G protein-coupled C-C chemokine receptor phospholipase C (PLC) and/or Toll-like receptor signaling. Subsequent pharmacological inhibition, however, revealed that NF-κB activation by HBD-3 is receptor-independent and instead involves the phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) pathway, the mechanism of which remains undetermined. Recently, we have shown that HBD-3 can enter mammalian cells and bind to inner membrane phosphoinositide 4,5-bisphosphate [PI(4,5)P 2 ], an important second lipid messenger of PLC and PI3K-Akt pathways. In this study, we report that the interaction of HBD-3 with PI(4,5)P 2 is important for PI3K-Akt-NF-κΒ-mediated induction of tumor necrosis factor and interleukin-6. These data provide insights into the mechanism of immunomodulation by HBD-3, and more generally, highlight the complex multifaceted signaling roles of HDPs in innate defense. Furthermore, it is suggested that the proposed mode of action may be conserved in other HDPs., (© 2017 Australasian Society for Immunology Inc.)

Background: New-onset atrial fibrillation (AF) after coronary artery bypass graft (CABG) operation is associated with poorer survival. Blanket prophylaxis efforts have not appreciably decreased incidence, making targeted prevention for high-risk patients desirable. We compared predictive abilities of existing scores developed/used to predict adverse CABG outcomes (Society of Thoracic Surgeons' [STS] risk of mortality) or AF not associated with cardiac operation (the Cohorts for Heart and Aging Research in Genomic Epidemiology [CHARGE]-AF score, the CHA 2 DS 2 -VASc score), and a risk model for predicting postoperative AF following cardiac operations (POAF score), with age (the most consistently identified post-CABG AF risk factor)., Methods: Data submitted to the STS Adult Cardiac Surgery Database were used to assess new-onset AF in 8,976 consecutive patients without preoperative AF undergoing isolated CABG from 2004 to 2010 at five participating centers. Five logistic regression models (for CHA 2 DS 2 -VASc score, CHARGE-AF score, POAF score, STS risk score, and age, respectively, all modeled with restricted cubic splines) with a random effect for site were fitted to predict post-CABG AF. Estimates were used to compute and compare receiver operating characteristic (ROC) areas., Results: New-onset AF occurred in 2,141 patients (23.9%). The ROC area was greatest for CHARGE-AF (0.68, 95% confidence interval [CI]: 0.67-0.69), followed by age (0.66, 95% CI: 0.65-0.68), POAF score (0.65, 95% CI: 0.64-0.66), CHA 2 DS 2 -VASc (0.59, 95% CI: 0.58 to 0.60), and STS risk of mortality (0.58, 95% CI: 0.56-0.59). CHARGE-AF was significantly more predictive than age (p < 0.0001); the other scores were significantly less predictive (p < 0.0001)., Conclusions: Only CHARGE-AF performed better than age alone. Its performance was moderate and comparable with published risk models specifically targeted at new-onset post-isolated CABG AF. Future research should continue to focus on developing better predictive models., (Copyright © 2018 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Over 200 billion cells undergo apoptosis every day in the human body in order to maintain tissue homeostasis. Increased apoptosis can also occur under pathological conditions including infection and autoimmune disease. During apoptosis, cells can fragment into subcellular membrane-bound vesicles known as apoptotic bodies (ApoBDs). We recently developed a flow cytometry-based method to accurately differentiate ApoBDs from other particles (e.g. cells and debris). In the present study, we aim to further characterize subsets of ApoBDs based on intracellular contents and cell type-specific surface markers. Utilizing a flow cytometry-based approach, we demonstrated that intracellular contents including nuclear materials and mitochondria are distributed to some, but not all ApoBDs. Interestingly, the mechanism of ApoBD formation could affect the distribution of intracellular contents into ApoBDs. Furthermore, we also showed that ApoBDs share the same surface markers as their cell of origin, which can be used to distinguish cell type-specific ApoBDs from a mixed culture. These studies demonstrate that ApoBDs are not homogeneous and can be divided into specific subclasses based on intracellular contents and cell surface markers. The described flow cytometry-based method to study ApoBDs could be used in future studies to better understand the function of ApoBDs.

Objective: Inconsistent definitions of atrial fibrillation after coronary artery bypass grafting have caused uncertainty about its incidence and risk. We examined the extent to which limiting the definition to post-coronary artery bypass grafting atrial fibrillation events requiring treatment underestimates its incidence and impact on 30-day mortality., Methods: We assessed in-hospital atrial fibrillation and 30-day mortality in 9268 consecutive patients without preoperative atrial fibrillation who underwent isolated coronary artery bypass grafting at 5 US hospitals (2004-2010). Patients who experienced 1 or more episode of post-coronary artery bypass grafting atrial fibrillation detected via continuous in-hospital electrocardiogram/telemetry monitoring were divided into those for whom Society of Thoracic Surgeons data (applying the definition "atrial fibrillation/flutter requiring treatment") also indicated atrial fibrillation versus those for whom it did not. Risk-adjusted 30-day mortality was compared between these 2 groups and with patients without post-coronary artery bypass grafting atrial fibrillation., Results: Risk-adjusted incidence of post-coronary artery bypass grafting atrial fibrillation incidence was 33.4% (27.0% recorded in Society of Thoracic Surgeons data, 6.4% missed). Patients with post-coronary artery bypass grafting atrial fibrillation missed by Society of Thoracic Surgeons data had a significantly greater risk of 30-day mortality (odds ratio, 2.08, 95% confidence interval, 1.17-3.69) than those captured. By applying the significant underestimation of post-coronary artery bypass grafting atrial fibrillation incidence we observed (odds ratio [Society of Thoracic Surgeons vs missed], 0.78; 95% confidence interval, 0.72-0.83) to the approximately 150,000 patients undergoing isolated coronary artery bypass grafting in the United States each year estimates this increased risk of mortality is carried by 9600 patients (95% confidence interval, 9420-9780) annually., Conclusions: Defining post-coronary artery bypass grafting atrial fibrillation as episodes requiring treatment significantly underestimates incidence and misses patients at a significantly increased risk for mortality. Further research is needed to determine whether this increased risk carries over into long-term outcomes and whether it is mediated by differences in treatment and management., (Copyright © 2017 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

Defensins are a well-characterised group of small, disulphide-rich, cationic peptides that are produced by essentially all eukaryotes and are highly diverse in their sequences and structures. Most display broad range antimicrobial activity at low micromolar concentrations, whereas others have other diverse roles, including cell signalling (e.g. immune cell recruitment, self/non-self-recognition), ion channel perturbation, toxic functions, and enzyme inhibition. The defensins consist of two superfamilies, each derived from an independent evolutionary origin, which have subsequently undergone extensive divergent evolution in their sequence, structure and function. Referred to as the cis- and trans-defensin superfamilies, they are classified based on their secondary structure orientation, cysteine motifs and disulphide bond connectivities, tertiary structure similarities and precursor gene sequence. The utility of displaying loops on a stable, compact, disulphide-rich core has been exploited by evolution on multiple occasions. The defensin superfamilies represent a case where the ensuing convergent evolution of sequence, structure and function has been particularly extreme. Here, we discuss the extent, causes and significance of these convergent features, drawing examples from across the eukaryotes.

Apoptotic bodies (ApoBDs) are membrane-bound extracellular vesicles that can mediate intercellular communication in physiological and pathological settings. By combining recently developed analytical strategies with fluorescence-activated cell sorting (FACS), we have developed a method that enables the isolation of ApoBDs from cultured cells to 99% purity. In addition, this approach also enables the identification and isolation of cell type-specific ApoBDs from tissue, bodily fluid and blood-derived samples.

Background: New-onset atrial fibrillation (AF) after coronary artery bypass graft surgery (CABG) is associated with increased morbidity and poorer long-term survival. Although many studies show differences in outcome in women versus men after CABG, little is known about the sex-specific incidence and characteristics of post-CABG AF., Methods and Results: Overall, 11 236 consecutive patients without preoperative AF underwent isolated CABG from 2002 to 2010 at 4 US academic medical centers and 1 high-volume specialty cardiac hospital. Data routinely collected for the Society of Thoracic Surgeons database were augmented with details on new-onset post-CABG AF events detected via continuous in-hospital ECG/telemetry monitoring. Unadjusted incidence of post-CABG AF was 29.5% (3312/11 236) overall, 30.2% (2485/8214) in men, and 27.4% (827/3022) in women. After adjustment for Society of Thoracic Surgeons-recognized risk factors, women had significantly lower risk for post-CABG AF (odds ratio [95% confidence interval]=0.75 [0.64-0.89]), shorter first, longest, and total duration of AF episodes (mean difference [95% confidence interval]=-2.7 [-4.7 to -0.8] hours; -4.1 [-6.9 to -1.2] hours; -2.4 [-2.5 to -2.3] hours, respectively). At 48 hours, AF-free probabilities were 77% for women and 72% for men ( P <0.001). Number of episodes ( P =0.18), operative mortality ( P =0.048), stroke ( P =0.126), and discharge in AF ( P =0.234) did not differ significantly by sex., Conclusions: These novel data on sex-specific characteristics of new-onset AF after isolated CABG show that women had lower adjusted risk for post-CABG AF and experienced shorter episodes. Investigation of sex-specific impacts on outcomes is needed to identify optimal strategies for prevention and management to ensure all patients achieve the best possible outcomes., (© 2016 American Heart Association, Inc.)