Mouse models to investigate in situ cell fate decisions induced by p53.

Investigating how transcription factors control complex cellular processes requires tools that enable responses to be visualised at the single-cell level and their cell fate to be followed over time. For example, the tumour suppressor p53 (also called TP53 in humans and TRP53 in mice) can initiate diverse cellular responses by transcriptional activation of its target genes: Puma to induce apoptotic cell death and p21 to induce cell cycle arrest/cell senescence. However, it is not known how these processes are regulated and initiated in different cell types. Also, the context-dependent interaction partners and binding loci of p53 remain largely elusive. To be able to examine these questions, we here developed knock-in mice expressing triple-FLAG-tagged p53 to facilitate p53 pull-down and two p53 response reporter mice, knocking tdTomato and GFP into the Puma/Bbc3 and p21 gene loci, respectively. By crossing these reporter mice into a p53-deficient background, we show that the new reporters reliably inform on p53-dependent and p53-independent initiation of both apoptotic or cell cycle arrest/senescence programs, respectively, in vitro and in vivo.

The small molecule raptinal can simultaneously induce apoptosis and inhibit PANX1 activity.

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.

GOLDEN2-like1 is sufficient but not necessary for chloroplast biogenesis in mesophyll cells of C4 grasses.

Chloroplasts are the site of photosynthesis. In land plants, chloroplast biogenesis is regulated by a family of transcription factors named GOLDEN2-like (GLK). In C4 grasses, it has been hypothesized that genome duplication events led to the sub-functionalization of GLK paralogs (GLK1 and GLK2) to control chloroplast biogenesis in two distinct cell types: mesophyll and bundle sheath cells. Although previous characterization of golden2 (g2) mutants in maize has demonstrated a role for GLK2 paralogs in regulating chloroplast biogenesis in bundle sheath cells, the function of GLK1 has remained elusive. Here we show that, contrary to expectations, GLK1 is not required for chloroplast biogenesis in mesophyll cells of maize. Comparisons between maize and Setaria viridis, which represent two independent C4 origins within the Poales, further show that the role of GLK paralogs in controlling chloroplast biogenesis in mesophyll and bundle sheath cells differs between species. Despite these differences, complementation analysis revealed that GLK1 and GLK2 genes from maize are both sufficient to restore functional chloroplast development in mesophyll and bundle sheath cells of S. viridis mutants. Collectively our results suggest an evolutionary trajectory in C4 grasses whereby both orthologs retained the ability to induce chloroplast biogenesis but GLK2 adopted a more prominent developmental role, particularly in relation to chloroplast activation in bundle sheath cells.

A common human MLKL polymorphism confers resistance to negative regulation by phosphorylation.

Across the globe, 2-3% of humans carry the p.Ser132Pro single nucleotide polymorphism in MLKL, the terminal effector protein of the inflammatory form of programmed cell death, necroptosis. Here we show that this substitution confers a gain in necroptotic function in human cells, with more rapid accumulation of activated MLKLS132P in biological membranes and MLKLS132P overriding pharmacological and endogenous inhibition of MLKL. In mouse cells, the equivalent Mlkl S131P mutation confers a gene dosage dependent reduction in sensitivity to TNF-induced necroptosis in both hematopoietic and non-hematopoietic cells, but enhanced sensitivity to IFN-ß induced death in non-hematopoietic cells. In vivo, MlklS131P homozygosity reduces the capacity to clear Salmonella from major organs and retards recovery of hematopoietic stem cells. Thus, by dysregulating necroptosis, the S131P substitution impairs the return to homeostasis after systemic challenge. Present day carriers of the MLKL S132P polymorphism may be the key to understanding how MLKL and necroptosis modulate the progression of complex polygenic human disease.

A systematic review of the symptomatic management of Lhermitte's phenomenon.

INTRODUCTION: Lhermitte's phenomenon (LP) is a transient shock-like sensation that radiates down the spine into the extremities, usually with neck flexion. The potential efficacy and tolerability of various symptomatic therapies in the management of LP have not been systematically reviewed previously. METHOD: A systematic review was conducted using PubMed, EMBASE, and the Cochrane Library from inception to August 2022 for peer-reviewed articles describing the treatment of patients with Lhermitte's phenomenon. The review adheres to the PRISMA guidelines and was registered on PROSPERO. RESULTS: This systematic review included sixty-six articles, which included 450 patients with LP. Treatment of the underlying cause varied by aetiology. Whilst LP is most commonly considered in the context of structural pathology of the cervical cord, medication-induced LP was a common theme in the literature. The most common cause of medication-induced LP was platinum-based chemotherapy agents such as cisplatin and oxaliplatin. In medication-induced LP, symptoms typically resolved with cessation of the causative agent. Non-pharmacological treatment options were associated with mild-moderate symptomatic improvement. The most commonly used agents to treat patients with LP were carbamazepine and gabapentin, which resulted in variable degrees of symptomatic benefit. CONCLUSIONS: No randomised studies currently exist to support the use of symptomatic therapies to treat LP. Observational data suggest that some therapies may yield a symptomatic benefit in the management of LP. However, this systematic review identified a significant paucity of evidence in the literature, which suggests that further controlled studies are needed to investigate the optimal management of this common neurologic phenomenon.

A Study of R-R Interval Transition Matrix Features for Machine Learning Algorithms in AFib Detection.

Atrial Fibrillation (AFib) is a heart condition that occurs when electrophysiological malformations within heart tissues cause the atria to lose coordination with the ventricles, resulting in "irregularly irregular" heartbeats. Because symptoms are subtle and unpredictable, AFib diagnosis is often difficult or delayed. One possible solution is to build a system which predicts AFib based on the variability of R-R intervals (the distances between two R-peaks). This research aims to incorporate the transition matrix as a novel measure of R-R variability, while combining three segmentation schemes and two feature importance measures to systematically analyze the significance of individual features. The MIT-BIH dataset was first divided into three segmentation schemes, consisting of 5-s, 10-s, and 25-s subsets. In total, 21 various features, including the transition matrix features, were extracted from these subsets and used for the training of 11 machine learning classifiers. Next, permutation importance and tree-based feature importance calculations determined the most predictive features for each model. In summary, with Leave-One-Person-Out Cross Validation, classifiers under the 25-s segmentation scheme produced the best accuracies; specifically, Gradient Boosting (96.08%), Light Gradient Boosting (96.11%), and Extreme Gradient Boosting (96.30%). Among eleven classifiers, the three gradient boosting models and Random Forest exhibited the highest overall performance across all segmentation schemes. Moreover, the permutation and tree-based importance results demonstrated that the transition matrix features were most significant with longer subset lengths.

Phagocytic clearance of apoptotic, necrotic, necroptotic and pyroptotic cells.

Although millions of cells in the human body will undergo programmed cell death each day, dying cells are rarely detected under homeostatic settings in vivo. The swift removal of dying cells is due to the rapid recruitment of phagocytes to the site of cell death which then recognise and engulf the dying cell. Apoptotic cell clearance - the engulfment of apoptotic cells by phagocytes - is a well-defined process governed by a series of molecular factors including 'find-me', 'eat-me', 'don't eat-me' and 'good-bye' signals. However, in recent years with the rapid expansion of the cell death field, the removal of other necrotic-like cell types has drawn much attention. Depending on the type of death, dying cells employ different mechanisms to facilitate engulfment and elicit varying functional impacts on the phagocyte, from wound healing responses to inflammatory cytokine secretion. Nevertheless, despite the mechanism of death, the clearance of dying cells is a fundamental process required to prevent the uncontrolled release of pro-inflammatory mediators and inflammatory disease. This mini-review summarises the current understandings of: (i) apoptotic, necrotic, necroptotic and pyroptotic cell clearance; (ii) the functional consequences of dying cell engulfment and; (iii) the outstanding questions in the field.

Apoptotic Bodies: Mechanism of Formation, Isolation and Functional Relevance.

In the final stages of apoptosis, apoptotic cells can generate a variety of membrane-bound vesicles known as apoptotic extracellular vesicles (ApoEVs). Apoptotic bodies (ApoBDs), a major subset of ApoEVs, are formed through a process termed apoptotic cell disassembly characterised by a series of tightly regulated morphological steps including plasma membrane blebbing, apoptotic membrane protrusion formation and fragmentation into ApoBDs. To better characterise the properties of ApoBDs and elucidate their function, a number of methods including differential centrifugation, filtration and fluorescence-activated cell sorting were developed to isolate ApoBDs. Furthermore, it has become increasingly clear that ApoBD formation can contribute to various biological processes such as apoptotic cell clearance and intercellular communication. Together, recent literature demonstrates that apoptotic cell disassembly and thus, ApoBD formation, is an important process downstream of apoptotic cell death. In this chapter, we discuss the current understandings of the molecular mechanisms involved in regulating apoptotic cell disassembly, techniques for ApoBD isolation, and the functional roles of ApoBDs in physiological and pathological settings.

Spontaneous fundal uterine rupture in a non-labouring 31-week twin pregnancy and unknown previous hysteroscopic adhesiolysis: A case report.

A 46-year-old woman presented at 31 weeks of gestation with a twin pregnancy (dichorionic, diamniotic) and with mild abdominal pain, not in labour, leading to complete spontaneous fundal uterine rupture. She underwent prompt surgical intervention and resuscitation with packed red cells, cell-salvage blood and fresh frozen plasma (FFP). Twin 1 survived and twin 2 died. Risk factors for fundal uterine rupture were multiple pregnancy and hysteroscopic adhesiolysis, which was unknown during antenatal care. The mother and twin 1 made excellent progress post-operatively. This case highlights the importance of swift intervention to minimise maternal and perinatal morbidity and mortality.

Distinguishing gastroesophageal reflux disease and eosinophilic esophagitis in adults: The role of esophageal mucosal immunoglobulin G4.

BACKGROUND AND AIM: Eosinophilic esophagitis (EoE) and gastroesophageal reflux disease (GERD) can be difficult to distinguish as many of their clinical and histological features overlap. Preliminary data suggest a potential association between EoE and immunoglobulin G4 (IgG4) but not GERD. This study aimed to examine the role of esophageal mucosal IgG4 staining when differentiating EoE from GERD. METHODS: Esophageal biopsy specimens from patients with proven EoE and GERD were evaluated, and immunohistochemical staining for IgG4 was performed by an experienced gastrointestinal pathologist blinded to the clinical and endoscopic data. The results on IgG4 staining were then correlated with clinical, endoscopic, and histological features. RESULTS: Sixty patients were included in the study, with 30 EoE (38.8 ± 12.8 years, 23 M:7 F) and 30 GERD (50.7 ± 14.3 years, 14 M:16 F) patients. The prevalence of a positive intercellular IgG4 stain was significantly higher in the EoE patients than those with GERD (23/29 vs 2/30; P < 0.0001). Positive IgG4 stain had the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 77%, 93%, 92%, and 80% for predicting the diagnosis of EoE, respectively. In both EoE and GERD patients, correlation was found between positive IgG4 staining and food bolus obstruction, dysphagia to solids, reflux, fixed rings, Barrett's esophagus, hiatus hernia, and esophagitis. In EoE patients, positive IgG4 staining was not correlated with the type of symptoms, endoscopic findings, histological findings, proton pump inhibitor therapy, or history of allergy/atopy. CONCLUSION: Given the high specificity and PPV of positive IgG4 staining in esophageal biopsies for EoE, this can be a useful marker to distinguish the disease from GERD.

Monocyte apoptotic bodies are vehicles for influenza A virus propagation.

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.

Effects of 5-HT2C, 5-HT1A receptor challenges and modafinil on the initiation and persistence of gambling behaviours.

RATIONALE: Problematic patterns of gambling are characterised by loss of control and persistent gambling often to recover losses. However, little is known about the mechanisms that mediate initial choices to begin gambling and then continue to gamble in the face of losing outcomes. OBJECTIVES: These experiments first assessed gambling and loss-chasing performance under different win/lose probabilities in C57Bl/6 mice, and then investigated the effects of antagonism of 5-HT2CR with SB242084, 5-HT1AR agonism with 8-OH-DPAT and modafinil, a putative cognitive enhancer. RESULTS: As seen in humans and other species, mice demonstrated the expected patterns of behaviour as the odds for winning were altered increasing gambling and loss-chasing when winning was more likely. SB242084 decreased the likelihood to initially gamble, but had no effects on subsequent gambling choices in the face of repeated losses. In contrast, 8-OH-DPAT had no effects on choosing to gamble in the first place, but once started 8-OH-DPAT increased gambling choices in a dose-sensitive manner. Modafinil effects were different to the serotonergic drugs in both decreasing the propensity to initiate gambling and chase losses. CONCLUSIONS: We present evidence for dissociable effects of systemic drug administration on different aspects of gambling behaviour. These data extend and reinforce the importance of serotonergic mechanisms in mediating discrete components of gambling behaviour. They further demonstrate the ability of modafinil to reduce gambling behaviour. Our work using a novel mouse paradigm may be of utility in modelling the complex psychological and neurobiological underpinnings of gambling problems, including the analysis of genetic and environmental factors.

Real-time neurochemical measurement of dynamic metabolic events during cardiac arrest and resuscitation in a porcine model.

This work describes a fully-integrated portable microfluidic analysis system for real-time monitoring of dynamic changes in glucose and lactate occurring in the brain as a result of cardiac arrest and resuscitation. Brain metabolites are sampled using FDA-approved microdialysis probes and coupled to a high-temporal resolution 3D printed microfluidic chip housing glucose and lactate biosensors. The microfluidic biosensors are integrated with a wireless 2-channel potentiostat forming a compact analysis system that is ideal for use in a crowded operating theatre. Data are transmitted to a custom-written app running on a tablet for real-time visualisation of metabolic trends. In a proof-of-concept porcine model of cardiac arrest, the integrated analysis system proved reliable in a challenging environment resembling a clinical setting; noise levels were found to be comparable with those seen in the lab and were not affected by major clinical interventions such as defibrillation of the heart. Using this system, we were able, for the first time, to measure changes in brain glucose and lactate levels caused by cardiac arrest and resuscitation; the system was sensitive to clinical interventions such as infusion of adrenaline. Trends suggest that cardiopulmonary resuscitation alone does not meet the high energy demands of the brain as metabolite levels only return to their values preceding cardiac arrest upon return of spontaneous circulation.

ROCK1 but not LIMK1 or PAK2 is a key regulator of apoptotic membrane blebbing and cell disassembly.

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.

Plexin B2 Is a Regulator of Monocyte Apoptotic Cell Disassembly.

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.

Correction to: Defining the role of cytoskeletal components in the formation of apoptopodia and apoptotic bodies during apoptosis.

The original version of the article unfortunately contained a typo in the fourth author name. The author name was incorrectly listed as Rochelle Tixeria. The correct name should be Rochelle Tixeira. The original article has been corrected.

Defining the role of cytoskeletal components in the formation of apoptopodia and apoptotic bodies 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.