Epithelial–mesenchymal transition (EMT) and endothelial–mesenchymal transition (EndMT) are physiological processes required for normal embryogenesis. However, these processes can be hijacked in pathological conditions to facilitate tissue fibrosis and cancer metastasis. In the eye, EMT and EndMT play key roles in the pathogenesis of subretinal fibrosis, the end-stage of age-related macular degeneration (AMD) that leads to profound and permanent vision loss. Predominant in subretinal fibrotic lesions are matrix-producing mesenchymal cells believed to originate from the retinal pigment epithelium (RPE) and/or choroidal endothelial cells (CECs) through EMT and EndMT, respectively. Recent evidence suggests that EMT of RPE may also be implicated during the early stages of AMD. Transforming growth factor-beta (TGFβ) is a key cytokine orchestrating both EMT and EndMT. Investigations in the molecular mechanisms underpinning EMT and EndMT in AMD have implicated a myriad of contributing factors including signaling pathways, extracellular matrix remodelling, oxidative stress, inflammation, autophagy, metabolism and mitochondrial dysfunction. Questions arise as to differences in the mesenchymal cells derived from these two processes and their distinct mechanistic contributions to the pathogenesis of AMD. Detailed discussion on the AMD microenvironment highlights the synergistic interactions between RPE and CECs that may augment the EMT and EndMT processes in vivo. Understanding the differential regulatory networks of EMT and EndMT and their contributions to both the dry and wet forms of AMD can aid the development of therapeutic strategies targeting both RPE and CECs to potentially reverse the aberrant cellular transdifferentiation processes, regenerate the retina and thus restore vision. Subretinal fibrosis demarcates the end-stage of age-related macular degeneration (AMD), resulting in permanent vision loss . FibrosiPredominant in subretinal fibrotic lesions are myofibroblasts, which are not normally present in adult tissues and thus, their cellular origins remain an ongoing debate. Extensive literature supports the role of epithelial–mesenchymal transition (EMT) in myofibroblast production . In the Importantly, the process of EMT in RPE is not limited to subretinal fibrosis in AMD but rather, has also been extensively studied and described in the context of proliferative vitreoretinopathy (PVR) , epiretiIn this review, we explore the roles of EMT and EndMT in the pathogenesis of AMD . We provWhile EMT is a physiological process required for normal embryologic development, this process can be hijacked in pathological conditions to facilitate tissue fibrosis and cancer metastasis . Thus, tBoth EMT and EndMT follow a highly coordinated sequence of events. The first step in EMT involves downregulation of E-cadherin, a protein central to maintaining lateral contacts of neighboring epithelial cells through adherens junctions . IncreasAn analogous process occurs in EndMT, where endothelial cells lose their endothelial markers, including vascular endothelial-cadherin (VE-cadherin), CD31, platelet-endothelial cell adhesion molecule 1 (PECAM-1), tyrosine kinase with immunoglobulin-like and EGF-like domains  and von Willebrand Factor (vWF) . AdditioUltimately, in both EMT and EndMT, a dramatic cytoskeletal reorganization results in an elongated, spindle-shaped morphology characteristic of newly formed migratory mesenchymal cells. While both RPE and endothelial cells are highly differentiated cells, their ability to undergo such drastic morphological changes reveals immense cellular plasticity. The initial descriptions of EMT and EndMT suggested that this conversion from epithelial or endothelial cell into mesenchymal cell was a permanent conversion. However, more recently, these processes have lost the presumptions of directionality and permanence, now reflecting the reversibility and spectrum of intermediary states concomitant to these processes ,19. PartDuring angiogenic sprouting, it is important to note that while endothelial cells express many EndMT and ECM remodeling genes, they retain intercellular junctions and migrate as a connected sheet of cells rather than as individual cells . An emerMultiple extracellular ligands are involved in the initiation and progression of the EMT and EndMT programs . RobustlTGFβ belongs to the TGFβ superfamily of growth factors implicated in several physiological and pathological conditions. These span from embryological development to tumor metastasis, autoimmune diseases and fibrotic diseases of the eye ,24. TGFβLatent TGFβ can be activated in various ways such as heat, ultraviolet radiation, acidic pH, proteolytic cleavage by MMPs, reactive oxygen species (ROS) and mechanical shear stress . Once acOf the three described mammalian TGFβ isoforms (TGFβ1/2/3), all have been detected in the vitreous and aqueous humor of the human eye . ApproxiHistological studies on CNV membranes excised from patients with nAMD show a complex array of cells and proteins including RPE, vascular endothelial cells, macrophages, pericytes, fibroblastic cells, myofibroblasts and ECM ,40,41. DA key family of transcription factors extensively studied in both EMT  and EndMSnai1 is also prominently described for its key role in angiogenesis. Snai1 is expressed on sprouting vessels under both physiological conditions in the developing retinal vasculature and in pathological angiogenic models of laser photocoagulation-induced CNV and oxygen-induced retinopathy (OIR) mouse models . Snai1 oBoth EMT and EndMT are regulated through a diverse network of signaling pathways such as the canonical Smad and non-canonical TGFβ signaling pathways  Figure . Criticaβ-catenin is sequestered and maintained by cadherins at adherens junctional complexes . HoweverNuclear localization of β-catenin has been observed in RPE cells undergoing EMT and inhibition of β-catenin signaling prevented both EMT and proliferation ,61. BindThe Jagged/Notch pathway has also been implicated in both EMT and EndMT. During TGFβ2-induced EMT in RPE, a concomitant upregulation of Jagged-1, Notch-3 and their downstream target genes Hes-1 and Hey-1 was observed . KnockdoTGFβ pathway activators were highlighted in a transcriptome wide expression profile analysis in a RPE EMT model . Many RPA driving force for TGFβ in nAMD is inferred by animal models showing that TGFβ inhibition effectively blocks CNV formation ,72 and sBesides TGFβ, other growth factors also play a key role in the progression from neovascularization into fibrosis including VEGF and connective tissue growth factor (CTGF). The ratio of VEGF and CTGF levels in the retina drive a so-called “angio-fibrotic switch”, whereby increased levels of CTGF sequester VEGF. When the levels of CTGF sufficiently overcome VEGF levels, angiogenesis ceases and excess CTGF drives scar formation . CTGF coTargeting CTGF, a more specific downstream regulator of the pro-fibrotic activity of TGFβ may be a more feasible therapeutic option for eradicating fibrosis. In a rat model of diabetic retinopathy, Hu et al. (2014) combined anti-VEGF (bevacizumab) and anti-CTGF therapy (using CTGF shRNA) and showed that this dual-target intervention was more effective in improving microvessel ultrastructure compared to single-target intervention . While tThe transition from early to advanced AMD has many features consistent with an aberrant wound healing response resulting from underlying degeneration, oxidative stress and chronic inflammation. Drusen disrupts the normal retinal tissue architecture, impedes transport between the RPE and choroid and serves as sites of activation of the complement signaling cascade ,82. ImmuPro-inflammatory cytokines can propagate their signals by activating endothelial cells into mesenchymal cells through EndMT . Two sucAs described above, abnormal RPE cells in AMD have morphologic features characteristic of a type 2 EMT, a process that can be activated by oxidative stress-induced impairment of autophagy and lysosomal function ,91,92. EMechanistic insights into mitochondrial dysfunction in AMD have highlighted a protective role for proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in RPE during AMD ,108. RepCryba1 gene, is an important protein for lysosomal clearance in RPE [Cryba1 showed an AMD-like phenotype and also expressed key molecular markers of EMT [Lysosomal dysfunction has been identified as a major pathogenic process in AMD  and may e in RPE . Age-depe in RPE . Genetics of EMT . Autophas of EMT . Defectss of EMT . In cancs of EMT  and EndMs of EMT , warrantSandwiched between the RPE and choriocapillaris is Bruch’s membrane, a pentalaminar structure consisting of elastin- and collagen-rich ECM. Bruch’s membrane acts as a molecular sieve to regulate the reciprocal exchange of biomolecules, nutrients, oxygen and metabolic waste products between the retina and the general circulation. Since Bruch’s membrane is acellular, transport occurs primarily via passive diffusion and depends on the hydrostatic pressure on either side of the membrane. On the photoreceptor side of the RPE, the subretinal space is occupied by the interphotoreceptor matrix, a highly organized, hydrophilic matrix composed of large glycoproteins and proteoglycans that play a key role in retinal adhesion to the RPE and regulate nutrient transport .Due to its anatomical position and functional role in retinal homeostasis, the significance of Bruch’s membrane cannot be overlooked in AMD pathogenesis. The ECM acts as a supportive framework for RPE and CECs, creating an internal environment for signal transduction, nutrient transport, metabolism, structural integrity and scaffolding to regulate cellular adhesion, migration, proliferation and differentiation. A physiological balance exists between the synthesis and degradation of ECM components and any disruption of this homeostasis can initiate and propagate disease states. As a result of CNV, vessels from the choroid proliferate and penetrate through the ECM border, their immature vascular walls inducing an increase in leaks of serum, lipoproteins and hemorrhage into the extracellular space.One key event during EMT and EndMT is aberrant ECM remodeling and mounting evidence suggests that age- and/or disease-associated alterations in ECM composition act as driving forces of EMT and EndMT. RPE degeneration is preceded by age-dependent changes in Bruch’s membrane ,121, sucThis concept is supported by in vitro analysis showing that the culture of normal human RPE on Bruch’s membrane collected from aged or AMD patients drastically changes their behavior and gene expression profiles ,127,128.Further ECM alterations occur during both early and advanced stages of AMD as an imbalance of MMPs and TIMPs enhance pathological production, accumulation and degradation of ECM proteins by RPE and CECs . The mosAs noted, imbalances in MMP-2/9 and TIMP-1/2 in dysfunctional RPE play a key role in both early dry AMD and advanced wet AMD ,135. MMPAnother family of proteolytic enzymes, namely the urokinase-type plasminogen activator (uPA) and its receptor (uPAR) have been implicated in ECM remodeling during advanced nAMD . uPA is Plasminogen activator inhibitor type-1 (PAI-1) is the primary endogenous inhibitor of uPA. PAI-1 is expressed in human CNV specimens  and was Altered mechanical properties of the ECM such as matrix stiffness can drive fibrosis . Cells pClinically, excessive mechanical stress can be induced by the presence of epiretinal membranes (ERM) that may complicate AMD. ERM, a fibrocellular membrane that proliferates along the inner retinal surface at the macular region can exert tractional forces that lead to macular folds, macular edema and in advanced cases, foveal detachment . Co-exisWhile extensive literature has investigated the roles of RPE and CECs in AMD separately, the in vivo situation is far more complex. In the following section, we discuss how the microenvironment may influence the processes of EMT and EndMT in vivo through synergistic effects between RPE and CECs. We also discuss the promise of reversal and inhibition of EMT and EndMT as a therapeutic approach for AMD.During development, the RPE controls formation of the choriocapillaris and later its maintenance during adulthood. Development of the choroidal vasculature depends on proper RPE differentiation. The crucial relationship between RPE and CECs is not restricted to embryology but persists into adulthood. Histopathological studies in atrophic AMD patients shows that early damage to the RPE layer precedes atrophy of the choriocapillaris indicating that choriocapillaris loss is secondary to RPE dysfunction ,165,166.In both clinical and experimental settings, choroidal atrophy and/or ischemia is associated with corresponding regional RPE lesions and serous detachment ,171. CerOne potential unifying target is oxidized lipoproteins that act on both RPE and CECs. Oxidized low density lipoproteins (OxLDL) are known to preferentially accumulate in the macular region and are important players in AMD pathogenesis . OxLDL iWhether a similar crosstalk between RPE and CECs is involved in AMD progression by potentiating EMT or EndMT is unclear. Few studies have explored whether any factors are released by these cells to support the mesenchymal transdifferentiation of the other cell type. A recent study demonstrated that exosomes released from EMT-induced RPE cells are enriched in pro-angiogenic factors and promote endothelial cell migration and tube formation . WhetherOnce successfully restored, an organized and functional RPE monolayer has the capacity to produce a Bruch’s membrane-like matrix . ARPE-19Reversal of EMT and EndMT is known as mesenchymal–epithelial transition or mesenchymal–endothelial transition, herein collectively termed as MET. Ambitious as it may sound, activation of the MET program and restoration of a functional monolayer of epithelial or endothelial cells is a plausible therapeutic approach for combating AMD. In vitro studies show that RPE have the capacity to undergo MET depending on the culture conditions. High plating density, low passage number and reduced serum concentration are effective in limiting cell spreading and proliferation, thus re-establishing the epithelial monolayer ,186,187.Activation of EMT, EndMT, and MET are conserved embryological processes and as much as EMT and EndMT can be hijacked by pathological cells, so too can we harness the MET program to restore the epithelial or endothelial phenotype. Whether MET induces cells to return to their innate cell type or whether it can be induced to differentiate into another cell type based on the microenvironment is currently unknown. Complete reversal of TGFβ-induced EMT in renal tubular epithelial cells required inhibition of both Zeb expression and the Rho signaling pathway . ReversaIf reversal of EMT is to be a feasible therapeutic strategy for combating AMD, then measures to restore the microenvironment may also be required. Without a proper substrate to attach to, RPE cells undergo apoptosis , an impoThe inhibition of EMT and EndMT has primarily been explored in mechanistic experiments using in vitro cell culture and in vivo animal models with few drug candidates progressing to preclinical settings for AMD. Rather, most of the preclinical testing on blocking EMT of RPE has been pioneered in PVR. Methotrexate, a commonly used anti-cancer agent that inhibits de novo nucleotide synthesis, can also be used for its anti-inflammatory properties at low concentrations to treat rheumatoid arthritis and psoriasis . IntraocWhile EMT is a key process in both AMD and PVR, it is important to note that the two conditions represent vastly different timelines with AMD being a chronic condition that progresses at a slower rate compared to PVR that develops as a more acute response to the retinal trauma inflicted upon surgical repair of retinal detachment. Since subretinal fibrosis occurs in the end-stage of AMD, any potential intervention for this condition may need to be administered prophylactically over the course of a long period of time before the development of major visual symptoms, thus raising concerns regarding patient compliance and side effects.Extensive literature conclusively supports the importance of EMT and EndMT in AMD from in vitro experiments on RPE and ECs to in vivo animal studies and histopathological analyses of human AMD donor tissues. EndMT is still a relatively newly recognized type of cellular transdifferentiation and although it has been well accepted in the field of embryology, there has been some hesitation and controversy amongst the scientific community in recognizing EndMT as a pathogenic factor in pathological conditions in adult tissues. We hope that this review highlights the emerging involvement of both EMT and EndMT in AMD and its acceptance into the mainstream understanding of AMD pathogenesis. Many questions remain unanswered that will need to be addressed in future investigations, including the differential contributions of EMT and EndMT to the mesenchymal cell population in subretinal fibrosis, any potential synergistic activity between EMT and EndMT in AMD and the controversial dual role of TGFβ as both a pro- and anti-angiogenic factor in different clinical stages of AMD.Another key issue is the role of other cells in giving rise to the mesenchymal cell population in subretinal fibrotic membranes in AMD. In an experimental CNV model using irradiated mice engrafted with green fluorescent protein (GFP)-positive bone marrow cells, many of the α-SMA-positive cells were also GFP-positive suggesting a systemic source of mesenchymal cells in CNV lesions . MacrophIn order to adequately identify the cellular origins of the mesenchymal cell population, lineage tracing studies and genetic cell labeling need to be utilized to enhance our understanding. Tracking EMT, EndMT and MET in vivo in humans may also present an exciting possibility through improvement of positron emission tomography-magnetic resonance imaging (PET-MRI). The high resolution achieved through fluorodeoxyglucose-PET (FDG-PET) enables cell-tracing studies for a deeper analysis of fibrotic lesions as they develop in real-time.To date, the literature exploring EMT and EndMT has largely studied these processes in isolation. While it is important to narrow our focus to gain mechanistic insights in EMT and EndMT, it is equally important to understand their synergistic potential in vivo. The mesenchymal cell population in subretinal fibrotic lesions requires further characterization to determine their cellular source, but also to determine whether mesenchymal cells derived from EMT or EndMT are distinct and contribute uniquely to different stages of AMD pathogenesis. Excavating the missing pieces of the puzzle is essential in harnessing the power of MET. We are optimistic that this review will encourage further investigation into the burgeoning field of EMT and EndMT in AMD and thereby, enable the development of novel drugs to combat AMD.


Aversive or rewarding experiences are remembered better than those of lesser survival significance. These emotional memories, whether negative or positive, leave traces in the brain which can later be retrieved and strongly influence how we perceive, how we form associations with environmental stimuli and, ultimately, guide our decision-making. In this review aticle, we outline what constitutes an emotional memory by focusing on threat- and reward-related memories and describe how they are formed in the brain during learning and reformed during retrieval. Finally, we discuss how the field is moving from understanding emotional memory brain circuits separately, towards studying how these two opposing brain systems interact to guide choices during conflict. Here, we outline two novel tasks in rodents that model opposing binary choices (approach or avoid) guided by competing emotional memories. The prefrontal cortex (PFC) is a major integration hub of emotional information which is also known to be critical for decision-making. Consequently, brain circuits that involve this brain region may be key for understanding how the retrieval of emotional memories flexibly orchestrates adaptive choice behavior. Because several mental disorders  are characterized by deficits in decision-making in the face of conflicting emotional memories , the development of choice-based animal models for emotional regulation could give rise to new approaches for the treatment of these disorders in humans. An emotional memory represents the storage of information about a survival experience. Through associative mechanisms, neurons assign emotional significance to environmental stimuli, and these memories influence motivation to behave adaptively. Thus, individuals revisit places previously associated with reward, whereas they avoid ones previously associated with aversion.Emotional memories can be categorized along two dimensions: salience ranging from weak to strong, and valence ranging from negative (aversive) to positive (rewarding). The salience of an emotional memory represents the apparent impact of the experience, and it correlates with arousal intensity. Emotionally-charged memories are better retrieved than neutral ones  predicts a reward. When presented with reward, VTA neurons release dopamine in PFC and NAcc, which together with a concurrent surge of norepinephrine, stores the salient reward memory with positive valence. After learning, the reward memory is stored in the PFC and NAcc, and activation of the PFC-NAcc circuit that participated in storing the reward memory results in retrieval of the memory  task serves a model of risky-reward seeking, in which animals foraging reward in the corner of a behavioral box must go onto a safety platform on the opposite corner to avoid a tone-signaled foot-shock (Bravo-Rivera et al., Individuals weigh benefits of rewards against their associated costs to make more advantageous decisions Hu, . There aCB-R and FS-B wrote the manuscript.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.


Metabolic syndrome (MetS) is characterized by hyperglycemia, hypertension, dyslipidemia and abdominal obesity. Patients with MetS or other metabolic disorders are more susceptible to cancer development and recurrence and have a worse long-term prognosis. Moreover, the metabolic reprogramming observed in cancer cells has also been described as one of the new hallmarks of cancer. Thus, aberrant metabolism has been proposed as an important risk factor for cancer. Chronic inflammation, reactive oxygen species (ROS), and oncogenic signaling pathways are considered as main potential triggers. Considering the strong association between metabolism and cancer, metabolism-modulating drugs, including metformin and statins, as well as adopting a healthy lifestyle, have been extensively investigated as strategies to combat cancer. Furthermore, strategies that interfere with the metabolic rewiring of cells may also have potent anti-cancer effects. In this article, we provide a comprehensive review of current knowledge on the relationship between aberrant metabolism and cancer and discuss the potential use of metabolism-targeting strategy for the treatment of cancer. Metabolic Syndrome (MetS) is a constellation of metabolic risk factors and a significant cause of morbidity and mortality. The five main components of MetS are abdominal obesity, hyperglycemia, high blood pressure, hypertriglyceridemia, and low high-density lipoprotein (HDL)-cholesterol levels , 2. AlthSeveral studies have suggested that people who suffer from MetS have higher chances of developing cancer; the rate of cancer recurrence and mortality are also higher. A recent study has shown that people diagnosed with MetS have a 33% higher cancer mortality rate compared to patients who have no metabolic disorders . FurtherThe identification of metabolic reprogramming and metabolic syndrome as important regulators of cancer development and progression has provided a rationale for the development of metabolism-targeting therapies as a promising therapeutic approach for cancer. Herein, we provide a comprehensive review of the current understanding related to the association between metabolism and cancer, the potential underlying mechanisms, and emerging metabolism-targeting anticancer therapies.The rapidly dividing cancer cells have high demands for energy and nutrients to meet their high metabolic needs. Consequently, the cellular metabolism undergoes a rewiring during malignant transformation. Importantly, cancer cells switch from oxidative phosphorylation to aerobic glycolysis to generate ATP (adenosine triphosphate), a phenomenon commonly referred to as the Warburg effect . SeveralApart from cell-autonomous mechanisms, extrinsic factors have also been identified as driving metabolic reprogramming in cancer cells. The tumor microenvironment is often hypoxic in solid cancers, leading to the activation of HIF-1α. The transcriptional activity of HIF-1α inhibits mitochondrial respiratory chains and induces glycolysis, among other things. Several types of stromal cells, including tumor-associated macrophages (TAM), have also been implicated in establishing a hypoxic tumor microenvironment and promoting aerobic glycolysis , subsequIncreased uptake of glutamine and enhanced glutaminolysis have been identified as a hallmark of cancer cells. Glutamine is essential in cancer cells and has been implicated in cancer progression, as it serves as a substrate for oxidative metabolism, which generates more than half of the ATP that is required by malignant cells . As wellde novo synthesis of fatty acids have been described as a frequent event in cancer cells , has been linked to enhanced metastatic ability and resistance to radiotherapy  . The serotherapy .Inflammatory immune responses protect organisms from external pathological insults, including mechanical trauma and infectious microorganisms. In contrast to acute inflammation that has protective roles, chronic inflammation has been linked to numerous diseases, including cancer . ConditiEpidemiological studies have shown that approximately 15–20% of all cancers can be attributed to chronic inflammation or chronic infections . Inflamm2O2), and hydroxyl radical (HO∙) are the most important ROS and are primarily produced in mitochondria by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) (Reactive oxygen species (ROS) are chemically reactive molecules that are produced during oxygen metabolism. Superoxide anion (s (NOXs) . ROS sers (NOXs) , 65.Detection of a high ROS level in many cancer cells suggests that ROS play an essential role in cancer initiation and progression  Table 1Table 1. Mutagenic signaling pathways like Mitogen-activated protein kinase (MAPK), Wnt, TGF-β, and JAK/STAT have been well-demonstrated to be critical for cancer initiation and progression . AccumulSeveral epidemiological studies have suggested the potential anti-cancer effects of the antidiabetic agent metformin, especially in colorectal cancer  and endoStatins are lipid-lowering agents commonly prescribed in patients with hyperlipidemia. Numerous epidemiological studies have suggested that statins may be useful in lowering the risk of cancer development. A prospective cohort study has demonstrated that statins can improve cancer patient survival and decrease the rate of cancer-related mortality in postmenopausal women . PatientExtensive multicenter cohort studies have demonstrated that an unhealthy lifestyle is a strong risk factor for all-cause mortality , includiAs indicated by numerous epidemiological studies, along with changes in the pace and patterns of modern lifestyles, an increasing number of people are suffering from metabolic syndrome and cancer. A positive association between metabolic syndrome and cancer has been established . MetabolIn conclusion, metabolic syndrome has been established as an important risk factor for cancer development; hence, cancer patients or individuals who are at high risk of cancer development are likely to benefit from the prevention and treatment of metabolic diseases. Further efforts are required for the development of therapeutic interventions that target both aberrant metabolism and cancer.YY and LG wrote the review, were responsible for figure and legend and final editing. JY contributes to defining the topic, analyzing article and preparing for submission. All the authors have approved for the final version of manuscript.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.


Extracellular vesicles (EVs) are emerging as promising nanoscale therapeutics due to their intrinsic role as mediators of intercellular communication, regulating tissue development and homeostasis. The low immunogenicity and natural cell-targeting capabilities of EVs has led to extensive research investigating their potential as novel acellular tools for tissue regeneration or for the diagnosis of pathological conditions. However, the clinical use of EVs has been hindered by issues with yield and heterogeneity. From the modification of parental cells and naturally-derived vesicles to the development of artificial biomimetic nanoparticles or the functionalisation of biomaterials, a multitude of techniques have been employed to augment EVs therapeutic efficacy. This review will explore various engineering strategies that could promote EVs scalability and therapeutic effectiveness beyond their native utility. Herein, we highlight the current state-of-the-art EV-engineering techniques with discussion of opportunities and obstacles for each. This is synthesised into a guide for selecting a suitable strategy to maximise the potential efficacy of EVs as nanoscale therapeutics. There is a strong incentive to develop acellular technologies capable of circumventing the regulatory hurdles associated with cell-based therapies . One sucThree subtypes of EVs have been identified, which can be classified based on their biogenesis, composition and diameter . ExosomeAs EVs are secreted by all cells, they can be isolated from various tissues as well as from bodily fluids, including plasma, urine, breast milk, saliva and amniotic fluid . These nMany of the beneficial effects once accredited to cell-based therapies are now believed to be attributed in some degree to the paracrine cargo packaged within EVs ,16. ThisDespite growing promise, limitations associated with the scalability of EV manufacturing, purity and their therapeutic potency have hindered the clinical translation of these naturally-derived nanoparticles . As suchRe-engineering of the parental cell is achieved either through genome modification, stimulation with exogenous biomolecules, exposure to fusogenic liposomes or specific environmental factors with the aim of modulating de novo EV yield and therapeutic efficacy A 28]..28].Modification of the parental cell genome allows the production of EVs enriched specifically with a desired therapeutic cargo. This method is thought to enhance the loading efficiency without compromising EV integrity or the functionality of the loaded compound ,30. The Due to issues regarding EV loading efficiency, several approaches have been investigated to further control the enrichment of the target molecules by harnessing the machinery involved in vesicle biogenesis. Li et al. engineered EVs for RNA loading by fusing the RNA binding protein human antigen R (HuR) with the tetraspanin membrane protein CD9. This successfully enriched miR-155 into EVs, with the functionally-intact miRNA efficiently delivered to recipient cells C 34]. M. M34]. MOverall, the identification of proteins modulating EV content may provide a useful approach for the artificial, selective enrichment of clinically useful macromolecules within secreted EVs. Although this strategy has shown increased loading efficiency, a potential hurdle could be the ineffectual release of the tethered therapeutic molecule at the recipient cell . ConsequIn addition to altering EV content, genetic modification of the parental cell has been investigated as a mean to modulate yield. Due to our increased understanding of the pathways involved in EV biogenesis, researchers have identified key components involved in their secretion. Several studies have reported the essential role of Rab proteins that regulate intracellular vesicle trafficking ,40. SimiAlthough these studies demonstrate the promise of genetic modification, issues remain with regard to the transduction efficiency. Therefore, this approach is time-consuming and expensive compared to the direct modification of isolated EVs; also concerns over the safety of the process may complicate clinical translation ,43. MoreExogenous stimulation provides an alternative strategy in modifying EVs efficacy whilst overcoming limitations associated with genetic manipulation. This approach involves introducing a non-native bioactive molecule  into the cell culture medium, essentially appropriating the cellular biosynthesis machinery to manufacture and enrich EVs with the therapeutic molecule of interest. For example, MSCs cultured with erythropoietin (100 IU/mL) led to a 33% increased EV yield when compared to untreated cells, in addition to the vesicles being enriched with miRNAs, such as miR-299, miR-499, miR-302 and miRNA-200, leading to a more potent anti-apoptotic effect by exhibiting better restorative efficacy on TGF-β1-induced fibrosis in human renal proximal tubular epithelial cells in vitro and greater recovery in mice from renal injury of unilateral ureteral obstruction in vivo . SimilarThis strategy is, however dependent on the amount of the exogenous compound that is delivered to the cell, dictated by the strength of the compound–cell interaction . Hence, Fusogenic liposomes are synthetic vesicles used as delivery vehicles to cells and they have been readily explored, due to the simplicity in functionalising and encapsulating both hydrophobic and hydrophilic molecules . This apAs research continues to foray into the utilisation of 3D culture environments, there also emerges the potential of harnessing environmental stimulation to scale-up EVs manufacturing for clinical applications. Several approaches have been explored to expand the synthesis of EVs by introducing external stimulation, modifying the culture platform and the use of bioreactors A.2) which increased EV synthesis 8-fold from human vascular endothelial cells when compared to normoxic conditions (21% O2), which was found to be mediated by the transcription factor hypoxia-inducible factor-1α  whiDue to the limitations that 2D culture systems infer on EV production, modification of the platform used to culture parental cells has been investigated to improve scalability and reproducibility. 3D culture systems have been favoured in recent years as a more representative model of in vivo conditions, including extracellular matrix composition, heterogeneous cell–cell interactions and 3D tissue architecture . By confCha et al. cultured MSCs as spheroids within a microwell array system and reported a 100-fold increase in EV yield, when compared to cells grown on 2D surfaces, in addition to an increased therapeutic efficacy through the stimulation of angiogenic and neurogenic in vitro differentiation C 18]. M. M18]. MBioreactors have been extensively utilised for the large-scale synthesis of clinical-grade cells ,66. ReceThe strategy of bioengineering the parental cell predictably loads only a small proportion of the modified content into the secreted EVs, as the nanoparticle’s cargo represents a small portion of the parental cell cytoplasm, making this approach time- and cost-intensive. Direct modification of purified EVs may provide a more efficient therapeutic functionalisation strategy to enrich the surface membrane or cargo of these nanoparticles.Co-incubation is the simplest method to insert a cargo within EVs. Hydrophobic molecules can interact with the lipid membrane and diffuse into the aqueous cavity following the cargo concentration gradient . Sun et This method relies on the application of an electrical field that permeates the EV-membrane, allowing for the diffusion of a water-soluble load into the vesicle . After rEV loading via sonication utilises ultrasound, rather than electricity, to permeabilise the membrane ,77. SoniThis approach involves extruding a mixture of the EVs and the cargo of interest through a membrane with pore sizes ranging from 100–400 nm, at a set temperature . PerforaLoading EVs via freeze-thawing involves combining vesicles and the cargo at ambient temperature, freezing this mixture at −80 °C or in liquid nitrogen, then thawing back to ambient temperature; these cycles are repeated several times, ultimately allowing for the loading of the cargo through membrane deformation . AlthougThe surfactant saponin can disrupt the EVs membrane via its interactions with cholesterol; this increases membrane permeability, leading to high loading efficiencies ,80. HoweAlthough these loading approaches have demonstrated an increased degree of cargo internalisation, the development of clinically relevant EVs is limited by issues concerning the robustness, reproducibility and scalability of currently available manufacturing methods . MoreoveThe surface of EVs is integral in determining the biodistribution, cell-targeting and therapeutic capabilities of these naturally derived nanoparticles. Therefore, as with loading of therapeutic cargo in the EVs cavity, numerous strategies have been investigated to functionalise the surface including: hydrophobic insertion, synthetic lipid nanoparticle fusion and other covalent and non-covalent methods.The hydrophobic nature of the EVs membrane allows for the direct insertion of hydrophobic molecules on the nanoparticle’s surface. Endowing EVs with increased targeting capabilities has been extensively investigated for cancer therapeutics. For example, Huang et al. conjugated the aptamer AS1411 to cholesterol on the EVs membrane, which resulted in enhanced cell death (~2.8-fold compared to unmodified EVs) via increased delivery of vesicle-encapsulated miR-21 to leukaemia cells . SimilarNu mice bearing A431 human squamous carcinoma cell xenografts. Utilising this approach may augment the natural composition of the vesicle surface, which is integral to many of its homing capabilities and cellular interactions.The use of fusogenic liposomes has been investigated for the surface modification of isolated EVs. For instance, Piffoux et al. fused liposomes with EVs via a polyethylene glycol (PEG) mediated reaction . The hybClick-chemistry is an example of covalent modification successfully utilised to functionalise the surface of EVs. The click reaction occurs when an azide chemical group reacts with an alkyne group forming a triazole bond . This reNotably for these approaches, given the altered surface composition, it is important to explore any effects this functionalisation may have on therapeutic efficacy of the modified EVs.Non-covalent approaches to functionalise the EV membrane, such as receptor–ligand binding and electrostatic interactions, have also been reported in the literature . Qi et aElectrostatic interactions rely on the interaction of exogenous cationic species with the negatively charged groups on the EV membrane. For example, Nakase and Futaki utilised such an approach to attach cationic lipids and a pH-sensitive fusogenic peptide (GALA) to the surface of HeLa-derived EVs, producing positively-charged surfaces, which enhanced cell attachment and subsequent uptake . It is hDespite the progress made in EV surface engineering, realising efficient nanoparticle modification is not trivial. It is important to strictly control reaction conditions, such as temperature or pH, to avoid disruption to EV integrity and aggregation ,82. NoneThe development of artificial EVs presents an opportunity to overcome the potential safety issues associated with those derived from allogenic sources. In addition, this approach circumvents the low procurement and extensive time associated with acquiring autologous EVs. The manufacturing of such systems is already well studied, with two approaches dominating the literature, (1) cell-derived nanovesicles (CDNs) and (2) EV-inspired liposomes (EVLs). Notably, use of microfluidics to increase manufacturing scale for either method is a rapidly growing research area.CDNs represent a new class of bioinspired drug delivery systems, overcoming the extensive purification steps and low procurement yields of current EV-manufacturing methods . Whole cIt has been reported that during plasma membrane self-assembly, CDNs can encapsulate and deliver exogenous therapeutic molecules to targets cells. This suggests a role for these vesicles as viable drug carriers for the treatment of various diseases . The nanSuccessful utilisation of CDNs has also been demonstrated in the field of regenerative medicine. Wu et al. reported that CDNs derived from hepatocytes stimulated hepatocyte proliferation in vitro and liver regeneration in vivo by enriching target cells with sphingosine kinase 2 C 106]. . 106]. HBiomimicry of naturally-derived EV composition will likely result in replicating the therapeutic effects of these nanoparticles while overcoming issues regarding native EV purity and scalability. To facilitate this approach, it is vital to define and characterise the key therapeutic components within naturally-derived EVs  A. FollowAlthough the use of EVLs is still in its infancy, a growing number of studies have demonstrated the potential of this approach. For example, Lu et al. fabricated EVLs /Sphingomyelin (SM)/Cholesterol (Chol)/1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS)/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)) ) to deliver an anti-VEGF siRNA to A549 cancer cells . CompareDe La Pena et al. developed artificial vesicles mimicking the composition of dendritic cell-derived EVs, which are capable of directing T cell activation and modulating the immune response to primary, metastatic and relapsed tumours B. LiposoChallenges presented with the use of EVLs, is to determine the influence of artificial surface compositions on the biodistribution and cell-targeting capabilities when compared to native-EVs . OverallResearchers have investigated the therapeutic efficacy of EVs in vivo via intravenous delivery into the systemic circulation. Studies have reported systemically injected EVs are rapidly taken up by macrophages in the reticuloendothelial system and cleared from the body . Hence, The ideal biomaterial for administering EVs should control the release kinetics of these nanoparticles to sufficiently recruit endogenous cells of interest. It should also provide a suitable mechanical environment to support the defect site and a degradation rate that matches de novo tissue growth. Biomaterials are generally derived from natural or synthetic sources. Natural biomaterials provide a biomimetic environment and specific sites for cellular attachment , however, they are often associated with biological variability and limited mechanical strength ,120. SynControlling the porosity of the biomaterial, along with its degradation rate, will ultimately affect the EVs release rate and favouring nanosized-pores can help prevent the bulk release observed following local delivery . Howevero-nitrobenzyl alcohol moieties-modified hyaluronic acid and gelatin) for articular cartilage regeneration [Zhang et al. investigated delivering human placental MSC-derived EVs embedded within a chitosan hydrogel for muscle regeneration , which wneration . They deAlthough numerous studies have demonstrated the efficacy of EV retention within biomaterials for tissue regeneration, there are a lack of systems that offer modifiable release kinetics that could be tailored to a specific clinical application. Nikravesh et al. developed two technologies for controlling the structure of alginate-based microgel suspensions, which demonstrated augmented EV release kinetics . MicropaIn recent years, EVs have emerged as essential regulators of normal physiological processes and pathological conditions, and present exciting opportunities for development as novel acellular tools for regenerative medicine and as diagnostic markers ,138. HowUnderstanding the biology of EVs by analysing their composition is important for the translation of any cell modification techniques of these nanoparticles. However, the majority of studies elucidating EVs composition and hence their mechanism of action, isolate these nanoparticles from 2D TCP, a highly artificial system compared to the cells’ native microenvironment . For exaIn addition to the production of more physiologically relevant EVs, the large-scale manufacture of therapeutically viable EVs is essential to meet clinical demand . SeveralThe clinical translation of EVs has been limited due to issues associated with manufacturing scalability, purity and therapeutic potency . This haFinally, the growing use of biomaterials to control the delivery and retention of EVs for different clinical needs have shown promise in the literature ,131,133.In this review we highlighted the current state of the art EV-engineering techniques with discussion of opportunities and obstacles for each. This is synthesised into a guide for selecting a suitable strategy to maximise the potential efficacy of EVs as nanoscale therapeutics . When seAlthough great strides have been made in understanding the physiological utility of EVs, clinical translational has been limited by a number of formulation challenges. This has resulted in intensive research into methods to enhance EVs potency and yield. In this review, key areas, including the procurement of EVs from physiologically relevant environments, scalability of manufacturing, the fabrication of artificial nanoparticles and controlling in vivo release kinetics, have been identified as being crucial to the development of a clinically-viable EV product. With continued efforts, these strategies present an exciting platform to engineer EVs beyond their native function, thereby enhancing their potential clinical efficacy as acellular nanoscale therapeutics.


Arabidopsis thaliana is the ideal system to obtain insights into fundamental key parameters and molecular players involved in underlying regulatory circuits of root growth, particularly in responses to environmental stimuli. Root gravitropism, directional growth along the gravity, in particular represents a highly sensitive readout, suitable to study adjustments in polar auxin transport and to identify molecular determinants involved. This review strives to summarize and give an overview into the function of PIN-FORMED auxin transport proteins, emphasizing on their sorting and polarity control. As there already is an abundance of information, the focus lies in integrating this wealth of information on mechanisms and pathways. This overview of a highly dynamic and complex field highlights recent developments in understanding the role of auxin in higher plants. Specifically, it exemplifies, how analysis of a single, defined growth response contributes to our understanding of basic cellular processes in general.Root architecture and growth are decisive for crop performance and yield, and thus a highly topical research field in plant sciences. The root system of the model plant  Arabidopsis thaliana . . Lo) phaesponses . SA alsoesponses . A closeesponses . In thisesponses . A relatesponses  . IAA, on the other hand, seems to antagonize these NPA effects, hinting at mechanisms, by which substrate-controlled adjustments in PIN quaternary structure act to regulate auxin transport activity [Structural insights into PIN conformation either in the mobile, dispersed or less mobile, clustered fraction are urgently needed, in order to understand function of and interplay between these differing states. Remarkably, there is strong evidence for hetero- and homo-oligomerization of different plasma membrane-resident activity . A relatactivity .pin2 null allele [Whilst these two reports represent an important step forward towards elucidating the native PIN configuration and its relationship to auxin transport, we can only guess about intrinsic determinants, participating in the control of PIN oligomerization. Comparison of PIN conformation under reducing vs. non-reducing conditions, indicated disulfide bridge formation between PIN monomers as such a determinant  Figure C. This hl allele . Howeverl allele  (Figure By exploiting root gravitropism as a simple but highly sensitive readout for monitoring variations in auxin fluxes, stimulating novel insights into the function of PIN proteins have been established in recent years. This involves major advances in our understanding of intracellular protein sorting processes, modes of polar protein targeting as well as first insights into the native conformation of PINs and its relationships to activity in polar auxin transport. However, and as always when it comes to outstanding research, emerging new questions posit demanding challenges for designing future experiments. This relates to systemic approaches, trying to integrate and to resolve the interplay between those various pathways and switches that jointly affect auxin fluxes via controlling PINs. Elucidation of the native PIN conformation in further detail represents another important issue, as are attempts aiming at unraveling composition and function of the enigmatic PIN clusters at the plasma membrane. Ultimately, only a PIN structure at a reasonably high resolution will provide valid answers to at least some of these questions.


Ubiquitous worldwide, coronaviruses are known to cause serious and economically damaging respiratory, enteric and neurologic diseases in animals such as cattle, pigs and fowl. Since the mid-1960s, they are recognized human respiratory pathogens involved in up to a third of infections of the upper respiratory tract, of the common cold type. Even though they are suspected to cause more serious infections in humans, affecting the lower respiratory and gastro-intestinal tracts, as well as the central nervous system (CNS), this remained subject of investigations in only a few laboratories. This changed dramatically in 2002–2003, when the Severe Acute Respiratory Syndrome (SARS) epidemic swept the world, and was linked to a coronavirus (SARS-CoV) that evolved in bats, before being transmitted to humans in Southeast Asia, with some reports of neurological involvement.We have accumulated evidence that human coronaviruses (HCoV) may cause neurologic disease after CNS neuroinvasion and persistence. Strain OC43 readily established persistent infections of neural cell lines and acquired mutations in the gene encoding the viral surface protein S, which interacts with cellular receptors. Biologically cloned emerging virus variants had mutations in the putative S receptor-binding domain, which correlated with the ability to infect not only neurons, the main target cell type, but also astrocytes. Four reproducibly observed S mutations were introduced into an infectious cDNA clone (rOC/ATCC) to yield rOC/U and the two recombinant viruses were compared for the induced neuropathogenesis in BALB/c mice. We found that susceptibility to rOC/U infection was enhanced and mice showed hind limb paralysis associated with spinal cord demyelination, whereas rOC/ATCC induced encephalitis.These results suggest that acquisition of discrete S mutations after persistence of HCoV-OC43 in neural cells results in modulation of neurovirulence and modified neuropathology. This may have implications in our understanding of coronavirus pathogenesis in humans, and points to the need for global research, prevention and intervention efforts targeting various infectious pathogens that could unexpectedly cause a global threat.Funded by the Canadian Institutes of Health Research, Canada Research Chair.


Emerging disease epidemics often result from a pathogen establishing transmission in a novel host species. However, for reasons that remain poorly understood, most cross-species transmissions fail to establish in the newly infected species. Examining experimental cross-species inoculations of rabies virus, we show that host and viral factors predict differences in disease progression in ways that are expected to impact the likelihood of onward transmission. Disease progression was accelerated and virus excretion decreased when the reservoir and novel host were physiologically or genetically more dissimilar. These insights may help to explain and predict host shifts in rabies and other zoonotic viruses and highlight meta-analyses of experimental inoculation data as a powerful and generalizable approach for understanding the dynamics of index infections. Whether a pathogen entering a new host species results in a single infection or in onward transmission, and potentially an outbreak, depends upon the progression of infection in the index case. Although index infections are rarely observable in nature, experimental inoculations of pathogens into novel host species provide a rich and largely unexploited data source for meta-analyses to identify the host and pathogen determinants of variability in infection outcomes. We analyzed the progressions of 514 experimental cross-species inoculations of rabies virus, a widespread zoonosis which in nature exhibits both dead-end infections and varying levels of sustained transmission in novel hosts. Inoculations originating from bats rather than carnivores, and from warmer- to cooler-bodied species caused infections with shorter incubation periods that were associated with diminished virus excretion. Inoculations between distantly related hosts tended to result in shorter clinical disease periods, which are also expected to impede onward transmission. All effects were modulated by infection dose. Taken together, these results suggest that as host species become more dissimilar, increased virulence might act as a limiting factor preventing onward transmission. These results can explain observed constraints on rabies virus host shifts, describe a previously unrecognized role of host body temperature, and provide a potential explanation for host shifts being less likely between genetically distant species. More generally, our study highlights meta-analyses of experimental infections as a tractable approach to quantify the complex interactions between virus, reservoir, and novel host that shape the outcome of cross-species transmission. Cross-species transmission is an important source of emerging and endemic disease. Viruses such as West Nile virus, rabies virus, and Lassa virus cause tens of thousands of human infections annually through transmission from animal reservoirs –3. Cross5Rabies lyssavirus (family Rhabdoviridae) as a model pathogen for understanding cross-species transmission  and the duration of clinical signs prior to death  of infected hosts. Longer incubation periods are associated with greater distribution of virus through the central nervous system , spread 141)Features of exposed host species (host effects), irrespective of the infecting virus. For example, larger-bodied species may be more resistant to infection and thus require either higher infectious doses or a longer period of virus replication before symptoms become apparent. More generally, evolutionarily conserved similarities in host physiology mean that groups of related taxa might have similar susceptibility or clinical outcomes of infection .2)Features inherent to the virus lineage involved (virus effects), irrespective of the infected host, likely due to adaptation of individual lineages to reservoir host species. Although rabies virus has shifted multiple times within and between bats and carnivores, only a handful of amino acid changes have been linked to host adaptation , 20, 21.3)Host–virus interactions. Both initial cross-species transmission and successful establishment occur most often between closely related hosts, often referred to as the phylogenetic distance effect , 23, 24.Based on previous work on rabies virus and in other host–pathogen systems, several mechanisms are hypothesized to influence infection dynamics and the outcome of cross-species transmission :1)FeaturHere, we test these hypotheses by conducting a meta-analysis of individual-level data from 514 published experimental cross-species infections involving rabies virus. We show that features of the virus and of the inoculated host species interact with the initial conditions of exposure to influence the outcome of cross-species transmission in ways expected to affect the likelihood of onward transmission in the novel host species.Our meta-analysis of cross-species inoculation experiments recorded results from 65 experiments in total. In agreement with observations from natural infections , 28, notn = 443 inoculations) and clinical period (n = 178) and the amount of virus excreted (n = 278). These experiments involved 20 mammal species , inoculated with 39 unique inocula from seven reservoir species in the orders Carnivora and Chiroptera .We modeled incubation period duration using log-normal generalized linear mixed models (GLMMs), correcting for phylogenetic nonindependence among inoculated species and among reservoir species, as well as for clustering within experiments A and potSI Appendix, Fig. S1). Models fitting effects for inoculated and reservoir species body temperature separately allowed us to explore this temperature effect further. Regardless of viral origin, inoculated species with higher typical body temperatures tended to have longer incubation periods . However, viruses from reservoirs with higher body temperatures were associated with shorter incubation periods across all inoculated hosts , suggesting that these viruses had adapted to counteract any losses in efficiency caused by the body temperature of their reservoir host. Many bat species have lower body temperatures than most carnivore species, but the distributions of body temperatures across bats and carnivores show considerable overlap . Importantly, there was little correlation between phylogenetic distance and body temperature difference among species , indicating that the observed temperature effects were not explainable by the level of taxonomic relatedness among species. All fixed effects combined explained 19.2% of the variation in incubation period durations (HPD: 2.8 to 40.2%).The difference in typical body temperature between the virus reservoir and the inoculated species had a more marked effect on incubation periods A. The onOnce symptoms appeared, the median time to death  was 2 d, ranging from <1 d to 8 d. We modeled clinical period duration using log-normal GLMMs, correcting for phylogenetic clustering among inoculated species and for clustering within experiments. As observed for incubation periods, increasing relative distance between the inoculation site and the brain increased clinical period durations (HPD: 0.131 to 1.221), while dose interacted with a range of other factors A. Cross-n = 80). The dose-dependent body temperature difference effect observed for incubation periods was poorly estimated in the salivary gland titer model, with a large posterior median effect size (−2.445) but high levels of uncertainty , and others have shown that body temperatures are clustered primarily at higher taxonomic levels , consistent with infection progression being matched to each host species. Given this host adaptation and poor correspondence between body temperature differences and phylogenetic distance, the observed temperature effect may help explain rabies virus host shifts across large phylogenetic distances. For example, despite host shifts from bats into carnivores being generally very rare, rabies virus has shifted repeatedly from big brown bats  to striped skunks [Mephitis mephitis, 36.45 °C  in sympatric frugivorous bats [Artibeus lituratus, 37.3 °C . Alternatively, since bats are considerably smaller than known carnivore reservoirs and likely transfer much smaller volumes of saliva during transmission, bat-associated rabies viruses may be adapted to transmit at lower doses. Although it may be expected that smaller animals would succumb faster to a given dose, we found no evidence that the interaction between the body mass of inoculated species and dose affects the duration of incubation or clinical periods , and future infection studies should aim to utilize doses closer to those of natural exposures. Following cross-species transmission, rabies virus shows increased virulence  in more distantly related species, to the point that opportunities for transmission are likely to be markedly reduced. At the same time, a mismatch in host physiological features  can alter both infectivity and disease progression, with implications for onward transmission. Thus, the picture that emerges is one of a potential virulence mismatch in index infections, that may partially explain why—despite having the ability to infect all mammals and frequent involvement in cross-species transmission events—rabies virus remains restricted to a relatively small number of species-specific maintenance cycles.The experimental data analyzed here offer a unique view on index infection dynamics following cross-species transmission. By revealing the complex links between dose, physiological differences between hosts, disease progression, and virus excretion, our analyses bring us closer to being able to model and predict the process of disease emergence and host shifts. The large dataset of controlled infections further enabled us to generate disease progression parameter distributions for all observed combinations of within- and cross-species transmissions which can be directly applied in future efforts to model rabies transmission dynamics . It is oWhile the determinants of cross-species transmission have been the subject of intense research , and right-censored observations . Right-censored observations were rare, however, and the final dataset of cross-species inoculations analyzed here contained only exact and interval-censored observations. In particular, all animals for which clinical period durations were available had been observed until rabies-induced death and with only one exception stem from studies published between 1958 and 1995 .https://www.itis.gov/). Data from our meta-analysis were supplemented with species-level data from the PanTHERIA database, along with body-temperature data from the AnAge database . Further data cleaning and validation steps are described in SI Appendix, Supplementary Information Text.Taxonomic classifications were updated to match ref. database , 39. BecThe durations of incubation and clinical periods were modeled using independent GLMMs on the censored event times, in this context more frequently termed accelerated failure time models. These models perform a regression on the waiting time to some specific event , with coefficients acting to increase or decrease the time to the event. We assumed a log-normal distribution for the event times Thus, the duration of the incubation period of each individual where Phylogenetic random effects were drawn from a multivariate normal distribution taking the form0 is a vector of zeros . Exact observations were recorded by setting To accommodate censoring, the vector of event times, In an independent model, the amount of virus detected in salivary glands postmortem was modeled jointly with incubation period durations, to allow estimation of the amount of residual correlation between incubation period duration and the amount of virus in the salivary glands. Several authors have noted a link between these measures –16, but This regression was similar to the model above, except that the normal distribution on log(observations) was replaced with a multivariate normal distribution. Thus, in the full model, the observed value where In these models, where timetree.org used above. As above, similar results were obtained when using the mammalian supertree of ref. Variables measuring differences between the reservoir and the inoculated species were included to assess the influence of previous virus adaptation on the outcome of infection in heterologous host species. These included the phylogenetic distance between the reservoir and the inoculated species, measured as patristic distances along the same composite time-scaled phylogeny generated by Features of the inoculated host species where accommodated primarily through random effects for species and inoculated species phylogeny. However, we also included a measure of the typical body mass of the inoculated species, since larger species may be proportionally more resistant to the effects of a given dose of virus. Because only some species maintain rabies virus transmission endemically, for reasons that are not well understood, a binary variable distinguishing known reservoirs of rabies virus from other inoculated species was also included.SI Appendix, Table S3). Such proportional scaling means this variable is independent of the differing body sizes of the inoculated species, which allowed us to account for inoculation site and inoculated species body size in the same model. This, in turn, allowed us to include an interaction term between dose and the typical body mass of each inoculated species, to capture potential differences in dose–response between species of different sizes. Including a similar interaction between our inoculation distance measure and body mass caused identifiability issues, with the model unable to distinguish between the main effect of inoculation distance and the effect of this interaction. We therefore concluded that such an interaction was not needed here. Finally, because larger doses may compensate for any decreases in infectivity caused by features of the inoculated species and/or physiological differences between the inoculated species and the reservoir to which the virus was adapted, we also included interactions between dose and all other host and virus effects above.Differences between experiments were accommodated by including variables for dose, the inoculation site, and whether the inoculum consisted of brain material or was derived from salivary glands/saliva, along with a random effect distinguishing between experiments to accommodate any remaining differences. Because the doses encountered in these experiments differed over several orders of magnitude and the effects of increasing dose is assumed to decrease (saturate) at very large doses, this variable was included in its log-transformed form. The varying inoculation sites encountered were summarized as a “proportional inoculation distance,” representing the relative distance between the inoculation site and the brain (the primary site of rabies virus replication). This distance was calculated by classifying inoculation sites by body part  and depth  and was expressed as a proportion, where 1 indicates the furthest and shallowest possible inoculation site relative to the brain (subcutaneous inoculation of a limb), while 0 indicates intracerebral inoculation (Models were fit using version 2.25 of the MCMCglmm package in R version 3.5.1 , 45. AllSupplementary File


The new disease COVID-19 pandemic has completely modified our lifestyle, changing our personal habits and daily activities and strongly our professional activity.Following World Health Organization (WHO) and health care authorities around the World recommendations, all elective surgeries from benign diagnose procedures must be postponed and imperatively continue working on emergent and oncological urgent pathologies. Surgical elective treatment of benign prostatic hyperplasia (BPH) is not considered as a priority.During BPH endoscopic surgeries, urine and blood are mixed with the irrigation liquid implying a risk of viral presence. Furthermore, a steam and smoke bubble is being accumulated inside the bladder implying the risk of splashing and aerosols. The risks of other viral infections have been identified during endourological procedures and they are related to splashing events. Several studies observed 33-100% of splashing on goggles.All BPH endoscopic procedures must be postponed. In case of complete urinary obstruction, this event can be adequately treated by urethral or suprapubic catheter under local anesthesia.As soon as local COVID-19 prevalence decreases, endourological procedures could be restarted. As protocols are being validating around the World to redeem elective surgeries, a symptomatic obstructed patient could be operated knowing his COVID-19 status with a molecular PCR, a cleaned epidemiological interview with a normal preoperative protocol.If patient is COVID-19+, surgery must be delayed until complete recovery, because mortality could increase as Lei from Wuhan describes.Informed consent must include risks of complications related to COVID-19 disease. Surgery must be performed by an experienced surgeon in order to avoid increase of operating time and risks of complications.Surgical approach of BPH must be considered depending on availability of disposable material, infrastructure, and the epidemiological COVID-19 status of your area. The main aim is patients and healthcare staff safety. The coronavirus named SARS-CoV-2 is causing an outbreak of a new disease COVID-19. This pandemic has completely modified our lifestyle, changing our personal habits and daily activities and strongly our professional activity. At this point, current medical data and its clinical applications as suggested by conventional guidelines has had to be adapted to the feasibility of this new state of affairs.Nowadays, we face a new disease on Earth and there are not high level of evidence recommendations to deal with. Furthermore, during this pandemic, we have to follow expert recommendations modifying our urological indications and out-patient and in-patient schedules prioritizing emergent and urgent procedures in order to avoid a life-threatening situations or disease progression and/or maintain patient and sanitary staff safety with novels biosecurity protocols.Following World Health Organization (WHO) and health care authorities around the World recommendations, all elective surgeries from benign diagnose procedures must be postponed and imperatively continue working on emergent and oncological urgent pathologies. Unfortunately, in this context, surgical elective treatment of benign prostatic hyperplasia (BPH) is not considered as a priority.However, this is a dynamic situation and information is evolving rapidly, so it is important to consider the status of your institution and locality to continue or recover your usual practice as proposed by the American College of Surgeons   .SARS-CoV-2 is a RNA virus belonging to the beta coronavirus family able to infect the upper respiratory tract. Spread mechanisms have been studied and identified from human-to-human respiratory interactions to airborne and fomites transmissions   .In a hospital setting, knowing the extent of environmental contamination of SARS-CoV-2 in hospital wards (HW), operating room (OR) and intensive care units (ICU) is critical for improving safety practices for sanitary staff. Airborne and touching-fomites transmission have been observed in COVID dedicated ICUs and HWs   .Some specific aerosol-producing procedures as endotracheal intubation, bronchoscopy, open suctioning, administration of nebulized treatment, manual ventilation before intubation, turning the patient to the prone position, disconnecting the patient from the ventilator, non-invasive positive-pressure ventilation, tracheostomy, and cardiopulmonary resuscitation, are considered high-risk situations   , 6 . Fucorynebacterium , tuberculosis, human immunodeficiency virus (HIV), human papillomavirus (HPV) and Hepatitis B have been described in different studies related to surgical smoke ( Carcinogenetic hazard and risk of transmission of several bacteria and virus as l smoke   – 13 . Al smoke   .Some risks during endourological procedures have been identified and they are related to splashing events and smoke/aerosolization producing procedures. SARS-CoV-2 RNA has been found also in blood, feces and urine implying a potential risk of transmission during endoscopic surgeries   – 21 .All surgeons are aware of risks of viral transmission in blood splashing, however they are underestimated. Those who use routinely goggles notice these droplets over them. Several studies observed 33-100% of splashing on goggles   – 24  asIn relation to aerosolization and smoke produced, the hazard in endoscopic procedures are moderate compared with the biological and carcinogenetic risks observed in open and laparoscopic surgeries   . LaparoSince hospitals will face a huge demand of resources to fight against a possible surge of COVID-19 cases, elective surgeries of benign pathology have been recommended to be delayed until the strain on the hospital system from COVID-19 decreases. Following this rules, all BPH procedures as TURP, HoLEP, ThuLEP, PVP, etc. must be postponed. In case of complete urinary obstruction, this event can be adequately treated by urethral or suprapubic catheter under local anesthesia   , 26 .As soon as local COVID-19 prevalence decreases   , endourIncluding non COVID-19, all patients must be considered as suspected ones. At this point, surgical staff including scrub nurse and anesthesiologist must wear a level 2 or 3 PPE. Initially, only the anesthesiologist and the assistant must be inside the OR with the patient. Once the patient is anesthetized, surgical staff can enter into the OR. It is important to minimize the number of strictly needed people and to count with all required materials in order to avoid frequent door openings   . SurgerEndoscopic procedures apparently are not as hazardous as open or laparoscopic approaches because aerosolization and smoke carcinogenetic risks and viral transmission are less frequent.Surgical approach of BPH must be considered depending on availability of disposable material, infrastructure, and the epidemiological COVID-19 status of your area. The main aim is patients and healthcare staff safety.


Moreover, JNK has at least three isoforms with different functions in cancer development and identifying specific selective inhibitors is crucial for the development of targeted therapy in cancer. Some selective inhibitors of JNK are identified; however, their clinical studies in cancer are relatively less conducted. In this review, we first summarised the function of JNK signalling in cancer progression; there is a focus on the discussion of the novel selective JNK inhibitors as potential targeting therapy in cancer. Finally, we have offered a future perspective of the selective JNK inhibitors in the context of cancer therapies. We hope this review will help to further understand the role of JNK in cancer progression and provide insight into the design of novel selective JNK inhibitors in cancer treatment.c-Jun N-terminal kinase (JNK) signalling regulates both cancer cell apoptosis and survival. Emerging evidence show that JNK promoted tumour progression is involved in various cancers, that include human pancreatic-, lung-, and breast cancer. The pro-survival JNK oncoprotein functions in a cell context- and cell type-specific manner to affect signal pathways that modulate tumour initiation, proliferation, and migration. JNK is therefore considered a potential oncogenic target for cancer therapy. Currently, designing effective and specific JNK inhibitors is an active area in the cancer treatment. Some ATP-competitive inhibitors of JNK, such as SP600125 and AS601245, are widely used  Therefore, JNK is an attractive target for therapeutic intervention with small molecule kinase inhibitors. Actually some ATP-competitive (e.g. SP600125) and ATP-non-competitive inhibitors have been developed; however, some limitations are noted in these inhibitors. For example, cell toxicity and a lack of specificity are observed due to their indiscriminately inhibition of the phosphorylation of all JNK substratesIn this review, we primarily summarise the function of JNK in cancer cell survival; our main goal was to mainly discuss the development of JNK inhibitors and especially the selective JNK inhibitors. Finally, we also cast a future perspective of the selective JNK inhibitors in cancer treatment. We hope this review will help to further understand the function of JNK in cancer development and provide some new lights on the development of selective JNK inhibitors in the cancer therapy.2.,,,,Studies indicate that JNK is a mediator of cancer cell apoptosis and cell death. JNK activation promotes gastric cancer cell apoptosis,,,,JNK activation is involved in tumorigenesis in liver-, breast-, and skin cancers, brain tumours, leukaemia, multiple myeloma, and lymphomaA unique pro-survival role of JNK was demonstrated in a B-lymphoma model. Blocking the JNK pathway inhibits the proliferation of murine and human B-lymphoma cells. JNK inhibition downregulates the early growth response gene-1 (Egr-1) protein and Egr-1 overexpression partially rescues lymphoma cell apoptosiscIAP-2. In the absence of activated NF-κB, the JNK pathway promotes an apoptotic responseJNK1 acts synergistically with Bcl-2 to promote prolonged cell survival in the absence of IL-3 or in response to different stressesin vitro studies and additional in vivo studies using animal cancer models are necessary to clarify the function of JNK. In summary, the tumour suppressive or oncogenic role of JNK is likely to depend on cancer/cell type-specific differences, the tumour microenvironment, and crosstalk with other signalling pathways. The molecular profiles associated with the role of JNK in promoting cancer cell survival are shown in Together, JNK activation can promote cancer cell survival and plays an important role in cancer cell proliferation, migration, and invasion. JNK associates with other regulators, including MKK, c-Jun, Slug, and Egr-1 and this interaction promotes cancer cell survival. JNK is therefore a potential oncogenic target for cancer treatment. However, these findings are mostly based on 3.,,Earlier studies indicate that the cancer cell survival or death function of JNK depends on functional time. In 2002, LinLin4.,,Due to the important role of JNK in the cancer development, designing effective and specific JNK inhibitors is a very active filed of research in different academic and industrial laboratories in the world. Currently, the clinical success of selective kinase inhibitors, such as imatinib and erlotinib, as therapeutic agents for several human cancers has prompted substantial interest in the development and clinical testing of such inhibitors for a wide variety of malignanciesin vitro and in vivo studies,,,,In vivo, AS602801 inhibits CSCs in established xenograft tumours when administered at a dose and schedule that does not adversely affect the health of tumour-bearing miceSP600125that belongs to the ATP-competitive inhibitor, is the most commonly used JNK inhibitor in many Hepatic ischemia/reperfusion (I/R), which is characterised by severe inflammation and cell death, causes significant liver damage and hepatic cancerHowever, JNK has at least three isoforms (JNK1–3) with different functions in cancer development. These inhibitors lack specificity and selectivity for the different JNK isoforms,Several peptide inhibitors have been developed, such as JNKi-1The studies summarised above indicate that selective JNK inhibitors are important for the treatment of cancer. However, compared with other pathway inhibitors, there are relatively less JNK inhibitors for clinical application. This could be partly attributed to our poor understanding of the complex functions of JNK, as JNK not only functions in cancer development, but also contributes to cancer cell death and is involved in many physiological processes,The role of JNK as a target for cancer treatment needs to be further explored to develop substrate-specific inhibitors, this may contribute to the alleviation of different types of cancer5.,,’s tyrosine kinase (BTK) inhibitor ibrutinib for treatment of chronic lymphocyte leukaemia; this prompted researchers to conduct “me too/me better” investigationsDevelopment of selective JNK inhibitors that target specific JNK-mediated cellular events is challenging. Currently, numerous selective pathway inhibitors have entered the clinical and market stages as anticancer therapies. For example, STAT3 inhibitors OPB-31121 and OPB-51602 have completed Phase I and II studies for treatment of various cancers, including advanced solid tumours, non-Hodgkin’s lymphoma, and liver cancerCurrently, an ideal JNK-specific inhibitor has not been identified yet, and many reported JNK inhibitors lack specificity for JNK with different conformations. Highly specific JNK inhibitors can be developed by chemical synthesis, by screening natural products that specifically inhibit the JNK pathways or by chemically modifying these natural products. Of course, development of JNK inhibitors for cancer therapy requires a systemic approach that requires consideration of metabolism, toxicity, drug resistance, side effects, and price. Development of substrate- or isoform-specific inhibitors and their clinical application may represent an innovative therapeutic approach to prevention and treatment of some cancers.Development of novel JNK inhibitors specific for different tumour types should be encouraged. However, considering the dual roles of JNK in cancer, treatment with a JNK inhibitor during chemotherapy would require careful planning. It is very possible that the mechanism underlying JNK-mediated cell survival and even chemoresistance is not the same for different cancers. We think that, as a first step, we may do not need to clarify all JNK-mediated resistance mechanisms for all cancers. Rather, we can try to develop a single specific JNK inhibitor for one or some, typical cancers once we understand the JNK-mediated mechanisms relevant to that cancer. This process will make development of JNK inhibitors much faster and we believe that more specific JNK inhibitors will be tested in many clinical studies in the near future.6.JNK signalling is a crucial oncogenic target that raises many researcher’s interest. Uncovering highly efficient selective JNK inhibitors is a hot topic of the last decade. Currently, some selective JNK inhibitors have been developed; however, more clinical studies of these inhibitors should be tested. Moreover, clinical studies of JNK inhibitors should determine which JNK inhibitor is most effective against cancer therapy. We should make further efforts to understand which JNK proteins are beneficial targets and should also uncover the potential mechanisms of the inhibitors on the various physiological process. The particular use of JNK inhibitors should be considered as the genetic background and the precise signalling pathway that directs the carcinogenic properties of the cells for a given cancer type needs to be noted. In conclusion, we believe that there is a bright future of JNK inhibitors and their use in cancer therapies should be tested in more clinical trials.


The early eukaryotic evolution was deeply influenced by the acquisition of two endosymbiotic organelles - the mitochondrion and the chloroplast. Here we discuss the possibly important role of reactive oxygen species in these processes. When trying to reconstruct the evolutionary trajectories during early eukaryogenesis, one is struck by clear differences in the developments of two organelles of endosymbiotic origin: the mitochondrion and the chloroplast. From a symbiogenic perspective, eukaryotic development can be interpreted as a process in which many of the defining eukaryotic characteristics arose as a result of mutual adaptions of both prokaryotes (an archaeon and a bacterium) involved. This implies that many steps during the bacterium-to-mitochondrion transition trajectory occurred in an intense period of dramatic and rapid changes. In contrast, the subsequent cyanobacterium-to-chloroplast development in a specific eukaryotic subgroup, leading to the photosynthetic lineages, occurred in a full-fledged eukaryote. The commonalities and differences in the two trajectories shed an interesting light on early, and ongoing, eukaryotic evolutionary driving forces, especially endogenous reactive oxygen species (ROS) formation. Differences between organellar ribosomes, changes to the electron transport chain (ETC) components, and mitochondrial codon reassignments in nonplant mitochondria can be understood when mitochondrial ROS formation, e.g., during high energy consumption in heterotrophs, is taken into account. The concept emerged in the context of the hydrogen  that eukaryotes seem an even mixture of archaeal and bacterial characteristics , 17, 18,before the uptake of a primordial photosynthetic cyanobacterium (the chloroplast-to-be), which gave rise to the plastid-bearing eukaryotes -associated degradation (ERAD) transport systems are also used for mitochondria-associated degradation (MAD) and peroxisomal import (and possibly export) , evoluti25–2-.) and hydroxyl radicals (.OH) formed in mitochondria.Though the question of how the merger between an archaeon and a bacterium, giving rise to the mitochondrion, occurred is hotly debated, we do not discuss it here. Also, whether the uptake of the progenitor of chloroplasts was accidental or occurred using a (newly available) phagocytic process by a “Cryptista-like” cell  is not cPaulinella species , with oxygenic photosynthesis relying on PS I and II in chloroplasts and the respiratory chain consuming molecular oxygen in mitochondria. Both are capable of substantial ROS formation. It is difficult to compare relative contributions, as measurements include different organisms, tissues, and kinds of ROS, as well as an abundance of diverse antioxidant mechanisms, as illustrated by the alternative plant oxidase mentioned above. Additionally, most reviews tend to be either plant-oriented  or anima34– species , 39. The species . One canears ago , yet we more resistant to some forms of ROS stress (In metazoan and some yeast lineages (which are closely related from a eukaryotic vantage point), ROS may have led to the antioxidant codon reassignment of Ile → Met (codon AUA) in the mitochondria . This seS stress . This agS stress .But why would this potentially beneficial feature be restricted to animals and yeasts? Metazoans are prime examples of high-energy-consuming heterotrophs and yeasts can have lifestyles leading to increased levels of ROS formation. Of note, mitochondrial codes in plants, along with their resident chloroplasts, do not display this antioxidant codon reassignment, which seems to indicate that local oxygen production as such does not determine ROS levels. Thus, this arrangement could be an example of high local ETC ROS pressure leaving evolutionary markers. Obviously, these codon reassignments developed after LECA formation and are not informative when it comes to the pressures encountered by the merged set of prokaryotes during the evolution toward LECA. However, important insights might be gained through comparison of mitochondria and chloroplasts regarding this transition, as well.exterior of the respective complexes. The authors then demonstrate that in both the mitoribosomal RNAs and hydrophobic core subunits of the OXPHOS complexes, destabilizing mutations are compensated by extra protein subunits. This process was completed in LECA for all universally conserved supernumerary proteins (∼75 novel subunits). This conclusion is borne out by the reconstructed mitoproteome (from a draft nuclear genome sequence) of the jakobid Andalucia godoyi  restricted to metazoans, a reductive phase occurred, resulting in a gradual length reduction of the mitochondrion-encoded rRNAs and OXPHOS proteins, causing further intrinsic destabilization compensated by (extra) lineage-specific supernumerary proteins . That thibosomes .As discussed above, the authors argue for structural compensation of the mutationally destabilized mitochondrion-encoded components by the newly recruited, nuclear-encoded ribosomal and OXPHOS subunits. However, we can observe a gulf in this respect between chloroplasts and mitochondria. Despite seemingly low mutation rates, chloroplast genomes have likewise almost completely migrated to the nucleus but their ribosomes remained “bacterial” in structure, without destabilizing mutations and with only a few supernumerary subunits , 51.both chloroplasts and mitochondria form extremely small populations inside cells, which partly explains their accelerated genomic evolution rates. Both additionally possess membrane-bound redox complexes that likely form the basis for their genome retention, though the specific reasons why are debated  makes the highly efficient ATP generation in mitochondria, characteristic of many heterotrophs, less crucial. That plants are not limited at the energy level in the way of metazoans is nicely illustrated by calculations for the illuminated leaves of C3 plants. Here, mitochondrial ATP synthase is predicted to contribute less than 1/5 of total ATP, the rest coming from chloroplast thylakoid ATP synthase  in response to ROS production can be implemented . This disynthase . Accordisynthase .Thus, the synergistic combination of the two driving forces of highly efficient mitochondrial ATP generation plus initially enhanced endogenous ROS formation gave rise to the accelerated evolution of eukaryotes. This explains the constructive evolution stage. Such accelerated evolution might also have contributed to the fact that mitochondrial proteomes are largely encoded by genes without identifiable archaeal or alphaproteobacterial provenance , 60, altIn conclusion, timing of the encounter, and the nature of the two organisms involved, is reflected in many aspects of present-day cellular architecture. The original merger between equals seems highly unlikely to succeed. However, when it succeeded, it turned out to be incredibly fruitful, as eukaryotes attest. All subsequent fascinating  acquisitions , even th


Purpura fulminans (PF) is a life-threatening disease of cutaneous microvascular thrombosis and hemorrhagic necrosis. Linear IgA bullous disease (LABD) is an autoimmune disease of subepidermal blistering. We present the first known case of PF following a toxic epidermal necrolysis-like presentation of spontaneous LABD in a 70-year-old female. Purpura fulminans (PF) is characterized by cutaneous microvascular thrombosis and hemorrhagic necrosis secondary to quantitative or functional deficiency of proteins C, S, and antithrombin III. In acute infectious PF, the most common type, infection attenuates thrombomodulin by cleaving it from the endothelial surface. This impairs protein C activation and leads to disseminated intravascular coagulation (DIC) ,2.Linear IgA bullous disease (LABD) is characterized by subepidermal blisters and linear deposition of immunoglobulin A (IgA) along the basement membrane zone (BMZ) on direct immunofluorescence (DIF). It is divided into a spontaneous and drug-induced form, the latter of which is most frequently seen with vancomycin. Though usually associated with polycyclic lesions in children, LABD has a much more variable presentation in adults, even mimicking toxic epidermal necrolysis (TEN) ,4.PF has been noted following TEN . We presA 70-year-old female was brought to the Emergency Department (ED) with altered mental status after an unwitnessed fall, of which she had no recollection. The patient complained of bilateral leg pain from blistering lesions on her lower extremities. She denied any recent history of new drugs in the past year, and had long been taking apixaban, furosemide, and valsartan. No other collateral information was available. She was intubated for airway protection after workup revealed cardiogenic and septic shock. She was given vancomycin and aztreonam and admitted to the cardiac care unit along with a dermatology consult.Klebsiella pneumoniae, Citrobacter youngae, and Enterococcus faecalis.On examination, she was found to have several areas of full thickness epidermolysis limited to lower extremities without mucocutaneous involvement or Nikolsky’s sign Figure . Two punOn day 3, large tense fluid-filled bullae surrounded by retiform purpura were abruptly noted on both legs, all the way up to the pelvic rim Figure . NikolskTwo separate instances of histopathology at our institution showed subepidermal blisters with mixed infiltrate and rare necrotic keratinocytes. DIF showed linear staining for IgA at the BMZ. No staining was seen for IgG, IgM, C3, or fibrin. At the burn center, biopsies were done again due to the second episode of PF. Histopathology here showed pauci-inflammatory thrombogenic vasculopathy and necrosis of the epidermis and eccrine coil, with resultant subepidermal bullae and no sign of IgA deposition or any other autoimmune process.The classic presentation of PF includes erythema, petechiae, and ecchymosis that evolve into painful retiform purpura, usually on the lower extremities and moving proximally. Our patient presented with bullous lesions and denudation with minimal erythema or purpura, making differentiation from TEN challenging.Clinical progression eventually revealed the classic presentation of PF, but the initial presentation and histopathology complicated the final diagnosis. Linear staining for IgA at the BMZ on DIF suggested the presence of an inflammatory process; however, the third biopsy weeks later showed pauci-inflammatory infiltrate. Given the initial presentation and the stark differences in the disease processes between LABD and PF, we suggest that the patient had an initial instance of spontaneous TEN-like LABD that led to a gram negative sepsis and eventual PF. We feel LABD is the most likely explanation for her sepsis as she lacked any of the common risk factors for gram negative sepsis, such as prior hospitalization, indwelling lines or catheters, or immunosuppression. LABD and its accompanied loss of skin barrier function represents the most likely avenue of infection. While drug-induced LABD is far more likely to cause a TEN-like presentation , she wasOur report seeks to draw attention to the phenomenon of TEN-like presentations for LABD as well as the severe consequences of such widespread skin barrier dysfunction. This patient was extremely unfortunate to sequentially acquire two rare and ultimately lethal disease processes.