Cardiovascular disease (CVD) is a leading cause of death globally, with the prevalence projected to keep rising. Risk factors for adult CVD emerge at least as early as the prenatal period. Alterations in stress-responsive hormones in the prenatal period are hypothesized to contribute to CVD in adulthood, but little is known about relations between prenatal stress-responsive hormones and early precursors of CVD, such as cardiometabolic risk and health behaviors. The current review presents a theoretical model of the relation between prenatal stress-responsive hormones and adult CVD through cardiometabolic risk markers  and health behaviors . Emerging evidence in human and non-human animal literatures suggest that altered stress-responsive hormones during gestation predict higher cardiometabolic risk and poorer health behaviors in offspring. This review additionally highlights limitations of the current literature , and discusses future directions for this promising area of research. Alterations in stress responsive hormones are one plausible mechanism by which exposures in the prenatal period affect offspring health, as stress-responsive hormones are sensitive to prenatal perturbations and also associated with poorer birth outcomes (Cardiovascular disease (CVD) is a leading cause of death globally, killing 18.6 million people worldwide in 2019 . The Devoutcomes –20. HoweThe current paper will review associations between prenatal stress-responsive hormones and early risk factors for later CVD, which include both cardiometabolic risk markers  and health behaviors . We begin by providing a brief overview of the dynamic changes in prenatal stress-responsive hormones focusing on the hypothalamic-pituitary-adrenal (HPA) and placental axis. We then provide a theoretical model of the relation between prenatal stress-responsive hormones and adult CVD. We present the state of the literature testing this model in both animal models, where experimental evidence is robust, and in human research. We conclude by discussing important gaps  and highlighting future directions for this promising area of research.2Prenatal stress-responsive hormones such as placental corticotrophin-releasing hormone (CRH) and cortisol have been posited as prenatal influences that contribute to the programming of adult CVD , 22. ThiWhen an individual is confronted with a stressor, a cascade of physiological responses occurs to prepare the individual to cope with the stressor. When stressors are detected, neural signals are relayed to the paraventricular nucleus (PVN) in the hypothalamus, the amygdala, the hippocampus, and the locus coeruleus, which release the neuropeptides corticotropin-releasing hormone (CRH) and arginine-vasopressin (AVP) into the hypophyseal portal system –29. CRH Over the course of gestation, many changes occur in the functioning of the maternal and the developing fetal HPA axes, largely due to the growth of a new organ, the placenta , 34. TheMaternal cortisol levels also increase during pregnancy approximately three-to-five fold in comparison to pre-pregnancy levels , 48. OveAccelerated increases in placental CRH and maternal cortisol have a role in the development of physiological systems linked to CVD. As a result, it is plausible that these prenatal hormones predict offspring cardiometabolic risk. Prenatal stress-responsive hormones are indeed implicated in fetal development and may have far-reaching influences. Prior work has documented that placental CRH may alter development of physiological systems that contribute to CVD. Placental CRH is associated with altered HPA axis activity postnatally , 62, as in utero has been linked to altered postnatal HPA axis activity in offspring  and health behaviors . 4Researchers have begun to examine associations between prenatal stress-responsive hormones and offspring postnatal cardiometabolic risk markers . These cardiometabolic risk markers are robust predictors of adult CVD risk –78. A laThe relation between prenatal stress-responsive hormones and offspring cardiometabolic risk has also been examined in humans see Table 1.In this section, we will first describe research conducted examining the relation between prenatal stress-responsive hormones and catch-up growth (section 4.1), BMI and adiposity (section 4.2), blood pressure (section 4.3), and other cardiometabolic risk markers such as altered glucose, lipids, and metabolic hormones such as leptin and adiponectin (section 4.4). Within each section, experimental non-human animal model research will be described first, followed by observational research in humans. A summary of the literature on prenatal stress-responsive hormones and cardiometabolic risk markers in humans is summarized in 4.1Catch-up growth, which is a pattern of growth characterized by small size at birth followed by rapid weight gain, is a strong predictor of later obesity and cardiometabolic disease , 107. Onrapid-increase BMI trajectory, which was characterized by a low BMI percentile at birth and low birthweight followed by rapid increases in BMI percentile over the first year of life. The second is a delayed-increase BMI trajectory, which was characterized by a low birthweight, a subsequent reduction in BMI percentile over the first year, followed by a rapid increase in BMI percentile over the second year of life. Both of these growth profiles are indicative of catch-up growth, which is linked to an increased risk of obesity and metabolic diseases  with offspring BMI trajectories over the first 24 postnatal months . Higher diseases . The assdiseases . This stdiseases .4.2The majority of rodent animal work has examined offspring body mass and adiposity, finding an overall pattern where dams who were administered prenatal glucocorticoids had adult offspring with lower total body mass, but higher adiposity (fat mass), suggesting a profile of lower-weight animals with higher body fat . SimilarIn parallel with the non-human animal model work, the majority of human research assessing the relation between prenatal stress-responsive hormones and cardiometabolic risk has examined offspring body mass and adiposity. One study conducted by Gillman and colleagues, examined the relation between placental CRH at 27 gestational weeks and both BMI and central adiposity when offspring were three years of age . They foA few studies have tested the relation between endogenous maternal cortisol and offspring body mass and adiposity. One study found that higher maternal cortisol at the end of the first trimester  was linked to higher offspring fat mass in females, but lower fat mass in males at 5 years of age . ExtendiLastly, in parallel to the literature examining endogenous cortisol in humans, there is one study that assessed the link between glucocorticoid administration during pregnancy and offspring overweight status between 5-8 years of age using cohort data from Denmark . They fo4.3Fewer studies have examined effects of prenatal stress-responsive hormones on blood pressure in offspring. Studies reviewed in the meta-analysis of rodent model glucocorticoid administration revealed that prenatal maternal glucocorticoid administration led to higher systolic blood pressure in adult offspring . ResearcWhile the link in humans between prenatal CRH and blood pressure has not yet been examined, there are studies testing the relations between both endogenous maternal cortisol and glucocorticoid administration with offspring blood pressure. Rondó and colleagues found that higher maternal cortisol in late pregnancy predicted lower arterial elasticity when offspring were 5-7 years of age , indicat4.4Other cardiometabolic risk markers, such as metabolic hormones and altered blood glucose and lipids have also been examined. Work reviewed in the rodent animal model meta-analysis documents that glucocorticoid administration caused higher levels of the metabolic hormone leptin, which is involved in the regulation of hunger . There wIn humans, one study has investigated the cardiometabolic hormones adiponectin and leptin in 3-year-old children in relation to placental CRH levels . They foOne study examined an alternative way of operationalizing cardiometabolic risk in humans. In this study, the researchers examined the relation between endogenous maternal cortisol levels in the third trimester of pregnancy and risk for developing coronary heart disease in the next 10 years  when offspring were 42 years of age . They fo4.5The literature reviewed in this section indicates that prenatal stress-responsive hormones such as glucocorticoids and placental CRH are implicated in cardiometabolic risk . These signals indicate that offspring exposed to altered levels of prenatal stress-responsive hormones have higher cardiometabolic risk. The small human literature is bolstered by the robust animal model literature, where glucocorticoid levels can be manipulated through glucocorticoid administration and potential confounding variables can be tightly controlled. Further, prenatal stress-responsive hormones are strongly implicated in offspring birth outcomes, and the current literature points to a relation between offspring birth outcomes and cardiometabolic risk. As a result, this work lends further support to the hypothesis that prenatal stress-responsive hormones contribute to CVD later in life. Most of the studies described in this section examine outcomes in infancy and early childhood –88, 91, 5As described above, growing evidence from both research with humans and non-human animal models suggests there is an association between prenatal stress-responsive hormones and offspring cardiometabolic risk markers. However, much less is known about potential behavioral risk factors that may mediate the association between prenatal stress-responsive hormones and adult CVD. Health behaviors such as substance use, sleep, obesogenic eating behaviors, diet, and physical activity, are robust predictors of CVD –131. As 5.1There is evidence from non-human animal research that substance use might be a risk factor that mediates the relation between prenatal stress-responsive hormones and cardiovascular risk. Substance use behaviors such as cigarette smoking , 142, exThe literature testing the relation between stress-responsive hormones and substance use in humans is smaller but consistent with the experimental animal work , 138. InWhile the literature examining the relation between prenatal stress-responsive hormones and substance use is small, there is a larger number of studies aimed at establishing the association between prenatal exposures such as maternal stress, with offspring substance use in rodents –154. As 5.2Poorer sleep across the lifespan is related to higher CVD risk –123, 156Although the prenatal stress-responsive hormone and sleep literature is small, there is a much larger literature that aims to determine the association between prenatal exposures such as maternal stress, depression, and anxiety with offspring sleep –165. Two5.3Dietary quality and obesogenic eating behaviors are robust predictors of CVD –127, andA related yet small human literature on other prenatal exposures related to alterations in prenatal stress-responsive hormones is consistent with this non-human animal work. Prenatal factors like maternal stress, depression, and anxiety activate prenatal stress-responsive hormones , 172, wh5.4Low physical activity and greater sedentary behavior are well-established predictors of CVD –131. HowThere is almost no research on the links between prenatal stress-responsive hormones and physical activity, but there are good reasons to believe that prenatal stress-responsive hormones may predict physical activity levels. High levels of stress and stress-responsive hormones are typically related to lower physical activity when they are measured concurrently –177. Add5.5The body of literature covered in this section illustrates that we know very little about the role of prenatal stress-responsive hormones in the development of each of these health behaviors . What we do know indicates that offspring exposed to altered levels of prenatal stress-responsive hormones may be more likely to engage in substance use, poorer sleep, poorer diet, obesogenic eating behaviors, and lower physical activity. However, the very small number of studies in this area indicates a huge gap in the literature and opportunity for exciting further work to elucidate the developmental pathways to CVD. Due to suggestive research on prenatal hormones and other prenatal risk factors  predicting each of these health behaviors, we argue that this area of research presents many opportunities to test the role of these behavioral risk factors in the development of adult CVD. Further, it is likely that these health behaviors are related. For example, there is robust evidence of a bidirectional association between sleep and physical activity . Therefo6As stated throughout this review, much more research is needed to fully understand the role of prenatal stress-responsive hormones in the development of CVD. While there is a more robust animal literature, there are a small number of studies on the relation between prenatal stress-responsive hormones and cardiometabolic risk in humans. The behavioral pathways to CVD have been overlooked, as there are even fewer studies examining health behaviors as potential mediators between prenatal stress-responsive hormones and offspring cardiometabolic risk. This area presents a rich potential for future work to fully characterize these possible developmental mechanisms.There are many unanswered questions regarding the role of prenatal stress-responsive hormones in the development of CVD. We highlight here several needed directions for future research. Studies that examine trajectories of stress-responsive hormones across pregnancy rather than single timepoints are needed. Recent work documents that there are distinct trajectories of stress-responsive hormones and that these trajectories better predict offspring outcomes than single timepoint measures , 155. AdThere is a critical gap in our understanding of sex differences in the impact of prenatal experiences on development of CVD. There are known, dramatic sex differences in CVD risk and CVD-related mortality, such that while males and females develop CVD at the same rate, males are more likely to develop and die from CVD earlier in life , 186. AdAdditionally, it is imperative that future work in humans is conducted in diverse samples. The overwhelming majority of the current human literature reviewed in this paper was conducted in WEIRD countries. When studies did report race/ethnicity, the participants were overwhelmingly White. Only a few of the studies reviewed included racially and ethnically diverse populations , 84, 91.The literature could also benefit from a cross-species approach. Cross-species research allows for the parallel examination of experimental animal work and observational human work. This approach allows for the disentanglement of many factors that cannot be controlled in human research, such as shared genes, other prenatal influences than stress-responsive hormones, and postnatal influences such as parenting. This approach has been impactful in other areas –200 and While the focus of the current paper was to review the potential influences of prenatal stress-responsive hormones in the development of offspring CVD, we recognize that these hormones do not exert their influence in isolation. It is likely that prenatal exposures such as environmental toxin exposure –203, mat7Converging research across non-human animal and human research indicates that prenatal stress-responsive hormones have a role in the development of offspring CVD, potentially through intermediary cardiovascular risk and altered health behaviors. Altered levels of prenatal stress-responsive hormones are implicated in higher offspring cardiometabolic risk, and potentially, CVD-related health behaviors such as substance use, poor sleep, poor diet and eating behaviors, and physical activity. Continued research examining trajectories of prenatal hormones and offspring outcomes in diverse samples is an exciting opportunity for future research.LKD, EPD, and JRD conceptualized the review. LKD, CS, NAT, EPD, and JRD drafted the manuscript. LKD and NAT drafted the figures and LKD and CS drafted the tables. All authors contributed to the article and approved the submitted version.


In particular, the injectable hydrogels, which can be employed to deliver cells and therapeutics to stimulate in-situ regeneration, are actively being developed -exposed encapsulated macrophages exhibited elongated morphology and out-migration capability from microspheres. This study shows the feasibility of polymer-encapsulated cell delivery to repair pulmonary damage and for general tissue repair.Sun et al. present an injectable, adhesive, and self-healing composite hydrogel system loaded with oxybutynin hydrochloride and evaluate its function in the treatment of overactive bladder (OAB). Authors found that the oxybutynin hydrogel could affect the expression of Orail and STIM1 and change the intracellular calcium concentration to improve the progression of the overactive bladder. In this study, authors demonstrated that dextran oxidized by periodate could be used as a macromolecular cross-linking agent of oxybutynin hydrogel. This system regulated Oxybutynin hydrochloride hydrogel mediated Ca2+ entry by increasing the relative expressions of Orail mRNA, STIM1 mRNA, and protein in OAB rats thus improving their therapeutic effect when compared with oxybutynin sustained–release tablets and oxybutynin hydrochloride solution. This exciting research could lay a foundation for improved OAB management and repair.Injectable, and simultaneously, tissue adhesive hydrogels are exciting hosting materials to carry and deliver, cells, drugs, and biomolecules such as proteins. In this article, Zhou et al. produced lactobionic acid (LA) modified chitosan and cinnamaldehyde (CA) modified chitosan with improved bioactivity and target specificity for reactive oxygen species (ROS)-induced apoptosis of cancer cells. Authors produced nanoparticles from modified chitosan and loaded anticancer drug doxorubicin (DOX). One of the major challenges in DOX delivery is its half-life and associated stability in physiological environment. This study demonstrated that drug-loaded chitosan-LA and chitosan-CA showed improved anti-tumor ability most probably due the fact that DOX’s half-life improved when loaded inside nanoparticles. Target specificity was significantly improved as well due to LA modification. Additionally, nanoparticles are known to exhibit enhanced permeability and retention (EPR) effect which helped improve DOX delivery when compared to free DOX solution.Chitosan is a common biodegradable, naturally occurring material used to prepare a variety of hydrogels. In this article, Liu et al. explored alternate crosslinking options to simultaneously stabilize three major components to enhance their long-term durability. They use Neomycin Trisulfate, Polyethylene glycol diglycidyl ether and Tannic acid as a substitute for glutaraldehyde, with reduced calcification, degradation, and inflammation. Hybrid crosslinking had multiple benefits including improved hydrophilicity, thermodynamics stability, cytocompatibility, resistance to enzymatic hydrolysis among others in subcutaneous implants in rat. These exciting results are a step forward towards producing highly useful biomaterials with non-toxic crosslinkers which can have far reaching applications in tissue engineering.Heart valves are among the tissues undergoing enormous shear, tensile and flexural stresses due to their dynamic function. Calcified heart valves reduce their dynamic functionality and can lead to heart failures. Current method to treat such conditions involve heart replacement however such replacements involve use of glutaraldehyde as crosslinking agent. Glutaraldehyde is known to have long term residual toxic effects. In this study, Khan et al. reported synthesis and characterization of acrylic acid/hydroxypropyl methylcellulose superporous hydrogels using gas blowing method, crosslinked via glycerol. Extensive testing was carried out to evaluate physical properties, in-vitro gelling capacity, void fraction examination and in vivo analgesic assessment. Furthermore, hydrogels were used to deliver mefenamic acid. Improved mechanical properties, gelling ability, high porosity and low density make these hydrogels potentially exciting material for sustained delivery of small molecular drugs.Drug delivery and release from hydrogels is governed by a number of factors including polymeric matrix’s chemical and physical structure, porosity, degradability, morphology, pore structure and crosslinking density. Superporous hydrogels are exciting biomaterials for use as drug carriers. Xu et al. discuss progress in materials, crosslinkers, and fabrication techniques of hydrogels and provide a cohesive analysis of the design criteria to improve the clinical translation of hydrogel scaffolds. Authors address key design challenges in fabricating microporous scaffolds to combine hydrogels and live cells in a single step with special focus on scale up, sterility and useability in clinical settings. They describe advantages and outstanding challenges involved in commonly used scaffolds fabrication techniques in detail and suggest potential solutions to overcome these obstacles. Moreover, Im et al. summarize the advances in gelatin methacrylate (GelMA) hydrogels for use in tissue engineering and regenerative medicine. These hydrogels have attracted enormous attention due to their ease of fabrication, tunable crosslinking and extracellular matrix-like physical structure, to name a few. Authors focus on applicability of GelMA hydrogels in bioengineering human vascular network in-vitro and in-vivo and suggest ways to leverage the properties of these hydrogels to improve the vascularization, which is critically needed to improve engineered tissues.In a review article, Collectively these research studies provide a snapshot of the exciting progress being made in developing new injectable hydrogel formulations and crosslinking strategies. Future work focusing on improving the viability of encapsulated cells by reducing the mechanical shear during injection , and the


In recent years, research on phytochemicals has underscored pleiotropic actions and medicinal and health-promoting properties which certainly deserve serious attention. Natural-derived molecules, such as phytohormones, glycosides, terpenoids, alkaloids, and phenolic compounds, offer a protective or preventative shield against many several pathological conditions such as aging, cardiovascular diseases, diabetes, obesity, cancer, asthma, and neurodegenerative disorders ,2. On thIn the light of these considerations, we put together this Special Issue, titled “Advances in the Astonishing World of Phytochemicals: State-of-the-Art for Antioxidants”, containing seventeen papers .The scientific evidence reported in the SI analyzed both distribution and pleiotropic beneficial effects  of some bioactive compounds with antioxidant properties. However, it also has to be considered that the relative abundance as well as the distribution of phytochemicals in plants or fruits is consistently affected by different parameters, such as environmental edaphic conditions, ripeness degree of fruits, and right season harvest .Tasmannia lanceolata), rosella (Hibiscus sabdariffa), lemon aspen (Acronychia acidula), and strawberry gum  represent a rich reservoir of bioactive phenolic metabolites . Among these, the analysis provided evidence that both Eucalyptus olida and Tasmannia lanceolata possess the highest antioxidant and antidiabetic potential [In their contribution, Ali et al., for example, demonstrated that Australian fruits and spices such as mountain pepper berries  were identified, and the development of phyto-engineered carrier systems capable of merging the biological properties of horseradish and kaolinite was proposed [Studies performed on Romanian proposed . As a whDryopteris juxtapostia, a species belonging to the Dryopteris genus growing in the states of the north temperate zone. The study demonstrated that both extracts exerted radical-scavenging and anti-inflammatory and antitumoral effects in vitro as well as hepatoprotective actions in rats. D. juxtapostia root dichloromethane extracts exhibited the highest biological potential compared to other extracts, thus demonstrating the importance of using dichloromethane to obtain extracts enriched in phenolic components [In another study, Rani et al. explored the biological potential of dichloromethane and methanol root and shoot extracts of mponents .E. purpurea, a plant whose extracts are traditionally used to treat cold and flu. The study compared the effects of dichloromethanolic and ethanolic root and flowers extracts with alkylamide-rich extracts obtained by using the accelerated solvent extractor system, a green and innovative extraction technique. The authors concluded that all the extracts were capable of reducing the IL-6 levels as well as the intracellular levels of ROS/RNS in lipopolysaccharide-stimulated human-monocyte-derived macrophages. However, the alkylamide fractions possessed the strongest anti-inflammatory effects, thus evidencing these compounds as the main active extract components [In addition, Vieira et al. demonstrated the anti-inflammatory effects of roots and flowers extracts of mponents . Lycopersicon esculentum Mill.), a food largely consumed for its nutritive and nutraceutical properties [A fruit particularly rich in phytocompounds is tomato , Matricaria chamomilla (MC), and Murraya koenigii (MK), traditionally used in Ayurvedic medicine as nerve relaxants and cognition enhancers [Drosophila melanogaster. The study evidenced the ability of plant extracts to exert neuroprotective effects on Drosophila melanogaster by alleviating rotenone-induced oxidative stress, enhancing locomotion, and restoring acetylcholine levels, thus suggesting a potential use of these extracts to treat neurological diseases. Of course, the right recovery of phytochemicals also depends either on the type of extraction techniques or solvents applied in the extraction procedure. This aspect was clearly addressed by Boyadzhieva et al., demonstrating a good recovery efficiency of phytochemicals from different parts  of Gnaphalium viscosum . Interestingly, for the first time, this study also demonstrated the presence in this species of leontopodic acids A and B, two highly potent antioxidants derived from glucaric acid [The biological properties of nhancers , were evnhancers . Consideric acid .Achillea millefolium L.), a flowering plant commonly used in folk medicine to alleviate symptoms related to gastrointestinal discomfort [Helicobacter pylori strains. Furthermore, the extracted fractions exerted antioxidant and anti-inflammatory effects in Helicobacter pylori-infected human gastric AGS cells. From this study, we can conclude that yarrow extracts can be useful against Helicobacter pylori infection. The Villalva’s data have been criticized by Franski and Beszterda-Buszczak [In a study performed in yarrow , green (Chlorophyta), and brown algae (Phaeophyta), which are not included in the diet of the Western world, are widely spread in Asian and Chinese nutrition for their high-quality profile in bioactive molecules as phenolic compounds, vitamins, pigments, and essential minerals. The use of a green pressured liquid extraction technique allowed Perez-Vazquez et al., under specific experimental conditions of temperature, type of used solvent, extraction time, and pressure, to recover a high yield of active biomolecules to exploit on both a pharmaceutical and food industrial scale [The presence of bioactive compounds has also been Identified in marine environments. For example, seaweeds, such as red  and phycoerythrin (PE), two molecules naturally produced by the red marine microalga  closure . AltogetThe identification of phytomolecules with potential tailored applications represents a significant goal in the phytochemistry field. Particularly significant is the research discussed by Notaro et al. exploring the biochemical action of methyl gallate (MG), a gallotannin widely used in traditional Chinese phytotherapy to alleviate several cancer symptoms . The finActinidia chinensis) counteracted both H2O2-induced oxidative damage as well as malondialdehyde increase [However, beyond these effects, bioactive compounds present in plants have also been demonstrated to play a protective role against oxidative injury, an aspect recently studied by Lv et al. in Caco-2 cells. The use of proanthocyanidins purified from kiwi leaves  used alone and in different combination mixtures provided evidence of the high antioxidant activity of gallic acid by a ferric reducing antioxidant power (FRAP) technique and a good oxygen radical absorbance capacity of rosmarinic acid by ORAC assays . A relevWe would like to share our gratitude to all authors who submitted their outstanding research to this Special Issue. Their manuscripts highlighted the role of natural-derived compounds with antioxidant potential action to apply as preventative or adjuvant molecules in the treatment of some chronic human diseases. Additionally, the identification of extraction techniques and solvents that can maximize the extraction of bioactive compounds is of great importance.


Outcomes for patients with acute promyelocytic leukemia (APL) have improved over the past 40 years due to the development of targeted treatment regimens and an expanded understanding of the unique complications arising from both the disease and its treatments. Despite these advances, clinically significant bleeding and/or thrombosis in the first 30 days of treatment remain the most common causes of morbidity and mortality. Moreover, there is a lack of large clinical trials investigating approaches for the prevention and treatment of these complications. This review aims to provide a comprehensive overview and update on the coagulopathy associated with APL, compile current best practices for optimal management, and establish areas of potential future investigation to further improve outcomes for patients with APL.Acute promyelocytic leukemia (APL) has a well-established mechanism and a long-term prognosis that exceeds that of any other acute leukemia. These improving outcomes are due, in part, to all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), two targeted and highly active agents in this disease. However, there remains a considerable morbidity and mortality risk in APL secondary to clinically significant hemorrhagic and/or thrombotic events. Prevention and treatment of these coagulopathic complications remain significant impediments to further progress in optimizing outcomes for patients with APL. Moreover, the relative rarity of APL hinders adequately powered randomized controlled trials for evaluating APL coagulopathy management strategies. This review draws from peer-reviewed works falling between initial descriptions of APL in 1957 and work published prior to January 2023 and provides an updated overview of the pathophysiology of hemorrhagic and thrombotic complications in APL, outlines risk stratification parameters, and compiles current clinical best practices. An improved understanding of the pathophysiologic mechanisms driving hemorrhage and thrombosis along with the completion of well-designed trials of management strategies will assist clinicians in developing interventions that mitigate these devastating complications in an otherwise largely curable disease. Acute myeloid leukemia (AML) is a hematologic malignancy accounting for approximately 12,000 cases and 9000 deaths annually in the United States . The mosMucocutaneous and/or gastrointestinal bleeding is the most common presenting symptom of APL with up to 89% of patients presenting with these complaints . The utiThrombosis is also a significant contributor to morbidity in APL, with up to 20% of patients with APL experiencing a venous or arterial thrombotic event . These tAt physiologic baseline, a delicate balance between hemostatic mechanisms preventing blood loss and antithrombotic mechanisms preventing blood flow obstruction maintains blood flow homeostasis . Figure For the general treatment of bleeding diatheses, several agents are approved for clinical practice that act to stimulate or supplement components of the clotting cascade: tranexamic acid, recombinant FVIIa, prothrombin complex concentrate, and ε-aminocaproic acid ,27,28. SDespite the body of research investigating the pathophysiology driving the coagulopathy of APL, adequate clinical data to inform the treatment and prevention of these complications are insufficient. Prior studies have attributed this to APL’s rare disease status, a lack of commercial support for clinical research in this disease, and/or the relative success of ATRA overshadowing the need for further improvements in care ,30. ThisAPL has a characteristic presentation with 80–90% of patients exhibiting various forms of severe mucocutaneous, intracerebral, or intrapulmonary hemorrhage and/or a thromboembolic event ,32,33,34Similar to DIC, a component of the coagulopathy of APL is driven by a clotting factor consumptive state that contributes to both hemorrhagic and thrombotic outcomes . The traOne of the key differing mechanisms driving hemorrhage in APL is hyperfibrinolysis. Physiologic lysis of fibrin and fibrin-derived clots occurs via two major pathways. Primary fibrinolysis involves cleavage of plasminogen into its active form plasmin by tissue plasminogen activator (tPA) and urokinase (uPA) A. SecondThe physiologic pathway counteracting excess plasmin-mediated fibrinolysis involves regulatory compounds—such as plasminogen activation inhibitor 1 (PAI-1), alpha2-antiplasmin, and thrombin activatable fibrinolysis inhibitor (TAFI)—all acting to either reduce plasminogen activation or to directly inhibit plasmin-mediated fibrinogen and fibrin breakdown A. HoweveWhile a TF-driven consumptive coagulopathy and a hyperfibrinolytic state are likely the primary drivers of hemorrhage in APL, there is growing evidence to support other prohemorrhagic mechanisms. The first involves promyeloblast CD44 surface expression contributing to fibrin abnormalities . A recenA second suggested mechanism contributing to hyperfibrinolysis in APL involves abnormal protease activity. Sepsis-driven DIC involves leukocyte elastase degradation of cross-linked fibrin outside of the physiologic plasmin-driven fibrin degradation pathway . MoreoveA final potential mechanism promoting hemorrhage in APL is a deficit in primary hemostasis. Most patients with APL are thrombocytopenic at the time of diagnosis, which may significantly contribute to early bleeding events. A study of platelet defects in APL demonstrated that while hemostatic variables improved during induction therapy, platelet counts remained significantly diminished until after four weeks of therapy . On a plEarly death in APL induction is commonly defined as death within 30 days of treatment induction ,65. PrioHemorrhagic death rates reported in the literature may be an underestimate of the true overall rate, as many patients not included in these studies present to hospitals with less capacity for the supportive care and therapy needed to achieve longer-term survival and confirmation of diagnosis. Of note, in one report of 963 APL patients treated at hospitals in California only 13.2% were treated at an NCI-CC, and treatment at a non-NCI-CC was associated with increased 30-day mortality . MoreoveAlthough the Sanz score and other risk stratification parameters have been used for assessing overall APL disease risk, there is not a standardized means of hemorrhagic risk stratification in APL. Naymagon and Mascarenhas recently conducted a review of parameters as predictors of hemorrhage in APL . Table 29/L at presentation to risk-stratify adult and pediatric patients [The most universally suggested and supported parameter for determining risk of hemorrhage, overall disease severity, and overall risk of early death in APL is leukocytosis. Clinicians and clinical trials commonly employ a WBC cutoff of >10 × 10patients ,79. Alonpatients ,85. LeukPlatelet concentration, fibrinogen, and other hemostatic parameters have been proposed as adjunctive predictors of hemorrhagic risk, but the literature supporting these factors is less consistent overall than for total WBC count. Low fibrinogen levels correlate with significant risk of hemorrhage, coinciding with the hyperfibrinolytic mechanism underlying the hemorrhagic diathesis of APL ,76,80,81Other non-hemostatic laboratory values are also associated with risk of bleeding. Multiple studies have shown lactate dehydrogenase (LDH) strongly correlates with hemorrhagic risk, likely secondary to lysis of leukemic cells, contributing both to elevated LDH and to release of factors altering physiologic hemostasis in APL ,62,78,84Thrombotic events in APL are less common than hemorrhage, but they are still a major contributor to morbidity and a minor contributor to mortality. Virchow’s triad  drives pathophysiologic thrombosis. Accumulating evidence suggests that APL causes thrombotic events via vascular inflammation that leads to endothelial damage and induces a systemic hypercoagulable state via multiple mechanisms outlined below.The primary mechanism driving thrombosis in APL is a consumptive coagulopathy that also contributes to the prohemorrhagic state outlined previously. APL promyeloblasts contain large amounts of tissue factor (TF) driven by aberrant RARa activation of the TF promotor ,37,38. MIn multiple cancer types, intravascular microparticles derived from cancer cell and endothelial cell membrane components, platelets, and monocyte remnants cause a hypercoagulable state via surface expression of tissue factor  ,89. MultAnother contributing driver of hypercoagulability in APL involves cancer procoagulant, a cysteine proteinase that activates factor X without needing factor VII . As showA final etiology contributing to the prothrombotic state of APL is the cancer-driven release of inflammatory cytokines that cause vascular damage and upregulation of multiple components of the coagulation cascade. The cytokines IL-1B, IL-6, and TNF-α are overexpressed in patients with AML and APL ,94,95. TDespite improvements in APL outcomes, early thrombotic events still contribute to APL-associated morbidity . Deep ve9/L) demonstrated that elevated WBC/D-dimer ratio and reduced D-dimer/fibrinogen ratio were independent predictors of thrombosis [Evidenced-based risk factors are less well defined for thrombosis compared with hemorrhage. A recent study of 84 patients with high-risk APL  in lieu of cytotoxic chemotherapy in high-risk APL . Three sConsensus guidelines for the management of APL were first published in 2009 by an expert panel on behalf of the European LeukemiaNet . The relThe mainstay of addressing the coagulopathy of APL involves the immediate initiation of ATRA along with supportive transfusion of fibrinogen concentrate/cryoprecipitate, platelets, and/or FFP as soon as APL is suspected. To prevent delays in treatment, the bedside clinical team and blood bank should be alerted that an intensive supportive transfusion regimen may be needed multiple times per day for the first 3–5 days of treatment. Moreover, invasive procedures such as central venous catheter placement or lumbar puncture should be avoided early in the disease course to avoid hemorrhagic complications of these procedures . The proHeparin has been utilized to mitigate the consumptive coagulopathy of DIC in sepsis and has been reported in patients with APL. Heparin inhibits fibrinogenesis via activation of antithrombin III, leading to inhibition of factors II and X . HoweverThrombomodulin, an anticoagulant normally expressed by endothelial cells that binds thrombin—leading to protein C activation and cleavage of factors V and VIII—has shown promise in stabilizing the coagulopathy of APL in limited retrospective studies . RecombiThe use of antifibrinolytic therapy is not recommended outside of clinical trials due to a lack of adequately powered, randomized-controlled clinical trials . A retroAnother agent that has been reported, mostly in the setting of APL patients with severe intracerebral hemorrhage, is recombinant Factor VIIa (rFVIIa). Multiple case reports/case series document the use of rFVIIa in treating life-threatening bleeding, mainly ICH, in patients with APL ,136,137.While less common than hemorrhage, thrombotic events still contribute to early morbidity and mortality in APL. Many targeted antithrombotic agents are utilized in other scenarios that warrant consideration for use in APL. The first is recombinant antithrombin III (rAT-III). Antithrombin–thrombin complexes are associated with active intravascular coagulation, and levels are significantly increased in APL ,88. rAT-Free tissue factor pathway inhibitor (TFPI) is elevated in patients with APL, and free TFPI inhibits activated factor X and factor VIIa-TF complexes . The useA final potential avenue for addressing the prothrombotic diathesis of APL is recombinant human protein C . Physiologically, protein C regulates excess thrombosis, reduces inflammation, and promotes fibrinolysis . ProteinDespite advancements in treatment, APL remains a disease with high risk of early mortality due to a characteristic coagulopathy. In today’s clinical landscape, a final major hurdle for minimizing APL morbidity and mortality is its associated coagulopathy. Research efforts continue to elucidate the underlying mechanisms of this coagulopathy, identify predictive markers for patient risk stratification, and determine optimal preventative and therapeutic approaches.It is now clear that the coagulopathy of APL is distinct from sepsis-induced DIC. Beyond a consumptive coagulopathy, hyperfibrinolysis and defects in primary hemostasis are important components of the predisposition towards hemorrhage. Thrombosis in APL is driven by aberrant activation of tissue factor along with cancer procoagulant and prothrombotic inflammatory cytokine expression.Many studies have attempted to identify markers and/or scoring parameters to predict hemorrhagic and/or thrombotic risk, with the aim of improving patient monitoring throughout the pre- and post-induction periods. The aim is to promote optimal triaging of patients in need of intensive monitoring, expedited allocation of transfusion products, and earlier identification of patients who may require novel approaches to mitigate severe coagulopathic events. There is a need for a consensus multicenter retrospective approach to identify consensus markers for predicting hemorrhagic and/or thrombotic risk in APL.Improve the early recognition of APL across all health care systems based on clinical symptoms and laboratory abnormalities, and, when possible, transfer patients to a center with experience in the care of individuals with acute leukemiaRisk-stratify patients utilizing common laboratory parameter cutoffsMinimize the time to induction treatment, including ATRA at first suspicion of diagnosis of APL and supportive blood product transfusionThe delicate balance between a prothrombotic and prohemorrhagic state in APL has made it challenging to combat complications of one without tipping the balance in favor of the other. Beyond the addition of ATRA and ATO, the only supported interventions for reducing the risk of coagulopathy include avoiding high-risk interventions/medications and aggressive blood product support . Some agFuture studies should focus on retrospective and/or prospective multicenter collaborations aimed at achieving adequate power to ensure relevant findings will have both statistical and clinical significance. At this time, the most critical areas of focus include the following: (1) developing a unified set of markers predicting hemorrhagic and thrombotic risk to be incorporated into current risk stratification tools and used for future clinical trials; (2) expanding the preventative treatment armamentarium to include modalities outside of blood product transfusion; and (3) identifying effective treatments for life-threatening bleeding events such as intracranial hemorrhage. With these initiatives, clinicians and researchers will be able to further improve long-term survival in this disease.


To the best of our knowledge, neurophysiological markers indicating changes induced by non-invasive brain stimulation (NIBS) on cognitive performance, especially one of the most investigated under these procedures, working memory (WM), are little known. Here, we will briefly introduce frontal midline theta (FM-theta) oscillation (4–8 Hz) as a possible indicator for NIBS effects on WM processing. Electrophysiological recordings of FM-theta oscillation seem to originate in the medial frontal cortex and the anterior cingulate cortex, but they may be driven more subcortically. FM-theta has been acknowledged to occur during memory and emotion processing, and it has been related to WM and sustained attention. It mainly occurs in the frontal region during a delay period, in which specific information previously shown is no longer perceived and must be manipulated to allow a later (delayed) response and observed in posterior regions during information maintenance. Most NIBS studies investigating effects on cognitive performance have used n-back tasks that mix manipulation and maintenance processes. Thus, if considering FM-theta as a potential neurophysiological indicator for NIBS effects on different WM components, adequate cognitive tasks should be considered to better address the complexity of WM processing. Future research should also evaluate the potential use of FM-theta as an index of the therapeutic effects of NIBS intervention on neuropsychiatric disorders, especially those involving the ventral medial prefrontal cortex and cognitive dysfunctions. Few years ago, when investigating clinical effects of non-invasive brain stimulation (NIBS) technique applied over the dorsolateral prefrontal cortex (dlPFC) in substance use disorders, we looked for biological evidence. Using low-resolution brain electromagnetic tomography (LORETA) focused on event related potential (ERP) P3 segment, we observed that a region located more ventrally in the medial prefrontal cortex  showed the highest electrophysiological change under drug-related cues in patients with alcohol or crack-cocaine use disorders maintaining abstinence after multiple sessions of the bilateral transcranial direct current stimulation (tDCS) over the dlPFC . With thMost neuropsychiatric disorders are associated with important dysfunction in frontal regions, more specifically the vmPFC . This brBrain oscillations have been suggested as biological markers in neuropsychiatric disorders , althougHowever, frontal midline theta oscillations seems to be related to anxiety conditions and have been suggested as an instrument to evaluate symptoms of anxiety in generalized anxiety disorder as its appearance seems to be closely related to improved anxiety symptoms under treatment with anti-anxiety drugs . It alsoThus, we wondered if NIBS induced changes in vmPFC activity, and probably a subsequent frontal theta synchronicity within cognitive networks could indicate therapeutic effects of these interventions, especially when aiming to improve cognition in neuropsychiatric disorders .Indeed, one major goal of NIBS, such as TMS, tDCS, or transcranial alternating current stimulation (tACS), is to promote neuroenhancement and neurorehabilitation using the potential of protocols to promote changes in cortical excitability and generate neuroplasticity in circuits underlying working memory (WM) and executive functions (EFs) . HoweverConsidering the evidence in the literature showing that theta waves consistently occur in the frontal midline during the processing of memory and emotion related to WM and EFs, we started to explore whether this electrophysiological rhythm could indicate the effects of NIBS on these cognitive processes. We first searched for articles published in the last 10 years (2012–2022) on the MEDLINE/PUBMED database, using the terms : “Theta Rhythm” combined (using the Boolean operator AND) with “Transcranial Direct Current Stimulation,” “Transcranial Alternating Current Stimulation,” “Transcranial Magnetic Stimulation,” “Medial Prefrontal Cortex,” resulting in 478 articles. 365 were excluded initially because of title inadequacy followed by abstracts that did not fit within the scope of our goal. The remaining 113 publications underwent full reading, of which 49 were considered relevant to be included in this brief review. Additionally, 14 articles were subsequently included to support the context.Frontal midline theta oscillations were first named FM-theta (or Fmθ) by Frontal midline-theta has been associated with broad cognitive functions  and it hThe characteristics of FM-theta were detailed by It is also essential to consider a few limitations. For instance, it seems that not all individuals display theta oscillations on EEGs signals during cognitive task performance, being of low occurrence, especially in old adults . Thus, iHere we will bring evidence proving the relationship between FM-theta and WM. Let us start by defining WM. It can be defined as a system or a process constituted by multiple components or a working system assembling distinct cognitive processes . It contThus, WM processing requires the storage buffer and central EFs to be coordinated with the contribution of different brain regions with diverse functional roles. The neural mechanisms governing these processes seem to be signaled by theta and alpha waves detected in EEG recordings . The relBehavioral tasks that seem to better examine the neural mechanisms underlying WM functions are those with delayed response . In thesPatterns of enhanced FM-theta coherence, meaning increased synchronization between two cortical sites, seem specific to the delay period in delayed response tasks . MoreoveIndeed, FM-theta power seems to rise in conditions with high WM load and task difficulty, and the increase in theta activity during WM also appears to predict later long-term memory retrieval for correct responses as it has not been observed when the responses are incorrect .However, according to In this section we will introduce NIBS effects on WM followed by electrophysiological evidence under these conditions. Although FM-theta has been consistently observed during many cognitive tasks, here we will focus on WM. This is because this cognitive function is one of the most studied under NIBS. Most of these studies have investigated the effects of the stimulation of the left dorsolateral prefrontal cortex (dlPFC) on the performance of n-back tasks, but, unfortunately, showing mixed results.N-back tasks involve the continuous presentation of items, in which the participant must indicate whether an actual item presented is the same as presented one-, two-, three-, or n-times back, usually with no delay introduced, so items are presented consecutively. A clear relationship exists between this task and FM-theta spectral activity, attributed to the allocation of attentional resources related to WM . HoweverIn their meta-analysis, However, Using a delayed WM task, Differently from tDCS, a frequency-modulated brain stimulation can be implemented with TMS and tACS . These tStudies investigating the tACS, especially in theta and gamma frequencies, most over the left dlPFC, with some including parietal cortex, show inconsistent results on WM, especially in young, healthy participants or in a single session. Yet, it seems more favorable, either in one or in multiple sessions, in low performers such as old adults and patients with neurodegenerative diseases .When applied bilaterally over the dlPFC, tACS improved verbal WM accuracy in young subjects , and wheUsing TMS alone delivered in theta or alpha frequencies over the left frontal or left parietal region, matching or not to the task-driven theta or alpha oscillations, seen by neuroimaging analysis during a retro-cued delayed WM task, Employing TMS at high frequency to frontal (Fz), temporal (TP7) or parietal (Pz) areas paired with EEG during an auditory delayed WM task, So far, evidence is growing that NIBS may induce FM-theta oscillations related to WM processing. To that end, they likely share biochemical and molecular mechanisms.Few neurochemistry studies have associated the variation in FM-theta with variation in monoamine  systems. It seems that not only their levels but the balance between them is important in the control of this frontal rhythm .Anodal tDCS and TMS enhance excitatory synaptic transmissions possibly by facilitating cortical glutamate transmission and suppressing gamma-aminobutyric acid transmission. They also modulate positively or negatively the activities of dopamine, serotonin, and acetylcholine transmissions, events that may change the balance between excitatory and inhibitory inputs .The molecular basis of the synchronicity of theta oscillation between the medial prefrontal cortex and amygdala and hippocampus in conditioning responses associated with fear and reward in rodents, seems to be associated with the expression of receptors and proteins fundamental to the process of brain plasticity, such as NMDA (N-methyl D-Aspartate) receptors, dopamine receptors, c-FOS and CREB (cyclic-AMP response element binding protein) .These molecular mechanisms also mediate cortical changes induced by NIBS . Animal Bearing in mind experimental limitations and limited evidence, FM-theta could be a potential electrophysiological indicator for NIBS effects on distinct WM components. Furthermore, FM-theta oscillations could also indicate the potential therapeutical effects of NIBS on neuropsychiatric disorders associated with vmPFC and cognitive dysfunctions. Evidently, this hypothesis needs to be specifically investigated in the future.EN-P conceived the original idea, directed the discussion of references included in the text, assembled the parts worked by the other authors, and wrote the main text. AF schematized the search keys in the literature with all the other authors and drafted the introduction. QA, LP, MZ, CR, and PL worked and drafted parts of the text included in the main text. All authors discussed the content and contributed to the final manuscript.


Coagulopathy and associated bleeding and venous thromboembolism (VTE) are major causes of morbidity and mortality in patients with acute leukemia. The underlying mechanisms of these complications have not been fully elucidated.To evaluate the associations between biomarker levels and bleeding and VTE in acute leukemia patients.We examined plasma levels of activators, inhibitors and biomarkers of the coagulation and fibrinolytic pathways in patients ≥18 years with newly diagnosed acute leukemia compared to healthy controls. Multivariable regression models were used to examine the association of biomarkers with bleeding and VTE in acute leukemia patients. The study included 358 patients with acute leukemia , and 30 healthy controls.Patients with acute leukemia had higher levels of extracellular vesicle (EV) tissue factor (TF) activity, phosphatidylserine-positive EVs, plasminogen activator inhibitor-1 (PAI-1), plasmin-antiplasmin complexes, cell-free DNA and lower levels of citrullinated histone H3-DNA complexes compared to healthy controls. APL patients had the highest levels of EVTF activity and the lowest levels of tissue plasminogen activator among the acute leukemia patients. There were 41 bleeding and 37 VTE events in acute leukemia patients. High EVTF activity was associated with increased risk of bleeding  whereas high PAI-1 was associated with increased risk of VTE  in these patients.Our study shows alterations in several biomarkers in acute leukemia and identifies biomarkers associated with risk of bleeding and VTE. Acute leukemia is a rapidly progressing hematologic malignancy and includes two distinct major morphologic types, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). While the 5-year survival rates are improving for acute leukemia, early morbidity and mortality from disseminated intravascular coagulation (DIC) and associated complications, such as bleeding and venous thromboembolism (VTE) remain high.–12 AML aTissue factor (TF) is a transmembrane receptor for factor (F) VII/VIIa. The TF/FVIIa complex is the major physiological initiator of the coagulation protease cascade. LeukemiaMany cells, including cancer cells and activated monocytes, release submicron vesicles called extracellular vesicles (EVs) that express TF and expose procoagulant phospholipids, such as phosphatidylserine (PS), on their surface., 39 NB4 Leukemic blasts release cell-free (cf) DNA into the circulation. Indeed, The current study had two goals. The first was to compare levels of activators, inhibitors and biomarkers of coagulation and fibrinolysis in patients with acute leukemia with healthy controls. The second was to determine the association between these biomarkers with bleeding and VTE in acute leukemia patients.This prospective cohort study included adult patients (≥18 years at diagnosis) with newly diagnosed acute leukemia who provided blood samples for the University of Alabama at Birmingham (UAB) biorepository between May 2016 and April 2021. Of the 826 subjects enrolled in the study, 570 had a diagnosis of acute leukemia. Of these, patients with relapsed/refractory disease at the time of sample collection, insufficient clinical data, and those who did not have adequate plasma sample were excluded, yielding a final sample size of 358 . Blood sDemographic information, clinical data regarding leukemia diagnosis, comorbidities, chemotherapy, and outcomes (VTE and bleeding) were abstracted from the medical records until blood or marrow transplantation, last contact with patient or death, whichever occurred first. Laboratory values as part of routine clinical care included WBC count, hemoglobin, platelet count, prothrombin time (PT), partial thromboplastin time (PTT), D-dimer, fibrinogen, creatinine, lactate dehydrogenase (LDH) and peripheral blood blast cell percentage at leukemia diagnosis. This study was conducted with the approval of the institutional review board at UAB and informed consent was obtained according to the Declaration of Helsinki.Plasma samples were obtained from 30 healthy individuals from whole blood drawn into EDTA tubes from Innovative Research  using the same methodology as leukemia patients.™ MP-ACTIVITY assay kit . H3Cit-DNA complexes were measured using an in-house ELISA as described previously.[™ PicoGreen™ dsDNA Assay Kit . PAP , tissue plasminogen activator  and active PAI-1  were measured using commercial assays. EVTF activity data from 6 patients were used in a publication[The transfer to the University of North Carolina at Chapel Hill and measurement of biomarkers was approved by the Institutional Review Board of the University of North Carolina at Chapel Hill (21–0554). Biomarkers in the coagulation and fibrinolytic pathways, and NETs were measured at the University of North Carolina at Chapel Hill. EVTF activity were measured using an in-house 2-stage FXa generation assay as described previously. PS+ EVs eviously. cfDNA weblication for compOutcomes and characteristics of interest for this study included overt DIC at the time of acute leukemia diagnosis, bleeding and VTE. Overt DIC was defined based on the scoring system developed by the ISTH, using levels of D-dimer, fibrinogen, PT, and platelet count at the time of initial diagnosis., 61 Bleet test or the Mann-Whitney U test depending on normality. The Kruskal-Wallis test with the Dunn’s multiple comparisons test was used to compare biomarker levels in multiple groups. These analyses were performed with PRISM version 7.03 . We used SAS software v9.4  to analyze biomarker associations with bleeding and VTE outcomes. Age at leukemia diagnosis and body mass index (BMI) were treated as continuous variables; the remaining variables were considered categorical. We calculated cumulative incidence of bleeding or VTE, using Fine and Gray methods, treating death as a competing risk.[t test or Wilcoxon rank-sum test (for continuous variables) and chi-square test  to compare differences between groups. Using proportional sub-distribution hazards multivariable regression analysis,[p <0.05 were considered statistically significant. Statistical analyses are described in the The Shapiro-Wilk test was used for normality testing. For biomarker comparisons between leukemia patients and controls, we used the unpaired 2-tailed Student ing risk. We used analysis, we examiyohei_hisada@med.unc.eduFor original data, please contact Demographic and clinical characteristics of acute leukemia patients and controls as well as routine laboratory parameters at leukemia diagnosis are shown in EVTF activity was higher in leukemia patients compared to healthy controls . APL patLevels of PAP were significantly higher in the leukemia patients compared to healthy controls . tPA levNext, we determined if EVTF activity is associated with leukocytosis, high percentage of blast cells, D-dimer and platelet count in acute leukemia patients. We compared leukocyte count, peripheral blood blast cell percentage, D-dimer levels and platelet count between acute leukemia patients with the top 10% of EVTF activity  and the rest of acute leukemia patients . We found that both WBC count and peripheral blood blast cell percentage were significantly higher in the high EVTF activity group compared to the mid to low EVTF activity group . We alsoWe calculated the ISTH overt DIC score and found that 90% of APL patients, 34% of non-APL AML patients, and 45% of ALL patients had overt DIC at diagnosis. EVTF activity and WBC count were significantly higher in patients with APL with overt DIC compared to patients without DIC (referred to as no-DIC) . In patiThe characteristics of acute leukemia patients with and without bleeding are shown in When examining the associations between biomarkers and bleeding, we combined all leukemia types to ensure sufficient statistical power. Multivariable sub-distribution hazards model with death as competing risk and adjusting for age, sex, race/ethnicity showed that EVTF activity , PT , WBC count , and LDH  were associated with increased bleeding risk . After fThe characteristics of acute leukemia patients with and without VTE are shown in In a sub-analysis of patients with non-APL AML, EVTF activity , WBC count  and LDH  were associated with bleeding risk. Low levels of tPA  and high levels of PAI-1  were associated with VTE risk .Although the study was not designed to perform sub-analysis for intracranial hemorrhage and this sub-analysis is underpowered, we found that low levels of tPA and fibrinogen were associated with intracranial hemorrhage, and high EVTF activity was approaching statistical significance. .In this large cohort study, we found high rates of ISTH overt DIC score in APL, non-APL AML and ALL patients. We found alterations in levels of several activators, inhibitors of the coagulation and fibrinolytic pathways along with NET markers in patients with acute leukemia compared to healthy controls. EVTF activity was associated with a high risk of bleeding and PAI-1 was associated with a high risk of VTE in acute leukemia patients.55 This complex functions as a receptor for both tPA and plasminogen to effectively generate plasmin in a fibrin-free mechanism on the cell surface.[In a previous study, PBMCs (including blast cells) from AML patients had higher levels of TF procoagulant activity compared to PBMCs from ALL patients. In addit surface. This may surface., 70. AssWe found high levels of cfDNA in acute leukemia patients consistent with previous studies., 47 We aWe also found that acute leukemia patients with high EVTF activity had higher WBC count and peripheral blood blast percentage compared to patients with mid to low EVTF activity. These data suggest that WBC and blast cells are the sources of TF+EVs in these patients. Similarly, acute leukemia patients with high EVTF activity had higher D-dimer levels compared to patients with mid to low EVTF activity. This data suggests that EVTF activity is associated with activation of coagulation and fibrinolysis. In contrast, there is no difference of platelet counts between acute leukemia patients with high EVTF activity and mid to low EVTF activity. This data suggests that EVTF activity is not associated with platelet count.In addition to the known high risk of DIC in APL, we also found high rates of DIC in non-APL AML and ALL patients. While the majority of bleeding and VTE complications occurred early in the clinical course of APL, we found a continued risk of these complications in non-APL AML beyond the first month. We also compared levels of biomarkers between patients with overt DIC and those without DIC. In APL and non-APL AML patients, EVTF activity was significantly higher in patients with overt DIC compared to patients without DIC. There are two possible interpretations of these data. The first is that TF+EVs are the major driver of overt DIC in these patients. The second is that EVTF activity may reflect more severe disease. In APL and non-APL AML patients, WBC count was significantly higher in overt DIC patients compared to patients without DIC. This data suggests that leukocytosis is associated with overt DIC in APL and non-APL AML patients. In addition, peripheral blood blast cell percentage and LDH level were significantly higher in non-APL AML patients with overt DIC compared to patients without DIC. Collectively, these data suggest that leukemia cell burden and disease severity are associated with overt DIC in these patients. Interestingly, levels of cfDNA and PTT were significantly higher in non-APL AML patients with overt DIC compared to patients without DIC. These data suggest that the intrinsic coagulation pathway is possibly activated by cfDNA that results in consumption of the intrinsic coagulation factors in non-APL AML patients with overt DIC leading to prolonged PTT. This needs to be further investigated. Importantly, the incidence of bleeding was significantly higher in non-APL AML patients with overt DIC compared to non-APL AML patients without DIC. We also found lower levels of PS+ EVs in ALL patients with overt DIC compared to ALL patients without DIC. In this regard, it is notable that WBC counts were lower, and platelet counts were significantly lower in ALL patients with overt DIC compared to ALL patients without DIC. Altogether, we believe that the total number of PS+ EVs are low in ALL patients with overt DIC because the sources of PS+ EVs, such as WBC and platelets are low. PAP and D-dimer levels were significantly higher in ALL patients with overt DIC compared to ALL patients without DIC supporting the conclusion that ALL patients with overt DIC have greater activation of fibrinolysis. In addition, we found that hemoglobin levels were significantly lower in ALL patients with overt DIC compared to ALL patients without DIC.Libourel and colleagues showed that the ISTH overt DIC score and D-dimer significantly predicted venous and arterial thrombosis in non-APL AML patients. In our sTo our knowledge, this is the largest cohort study evaluating the association between different biomarkers of activation and inhibition of the coagulation and fibrinolytic pathways with bleeding and VTE in acute leukemia patients. We found an association between levels of EVTF activity and risk of bleeding, and between PAI-1 levels with VTE in acute leukemia patients. We hypothesize that TF+EVs contribute to over-activation of coagulation that can ultimately result in bleeding due to consumption of coagulation factors in acute leukemia patients. In contrast, PAI-1 inhibits fibrinolysis which may contribute to VTE. Our data suggests that acute leukemia patients may have a sensitive balance between bleeding and VTE that is driven by EVTF activity and PAI-1, respectively. It is also notable that in our study acute leukemia patients showed much higher EVTF activity than levels reported in previous smaller studies., 42 One There are some limitations in the current study. First, there are only 30 healthy control plasma although we have a total of 358 acute leukemia patients’ plasma. The COVID-19 pandemic made it difficult to collect enough number of healthy control plasma. Second, VTE and bleeding are dynamic events in the clinical course of leukemia patients, and this risk is determined by several patient and treatment related factors. Our study did not examine the role of chemotherapy, central venous catheters, and other complications such as infection that might contribute to VTE and/or bleeding risk. We were also not able to capture laboratory parameters at the time of VTE and bleeding. Smaller event rates also preclude examining the associations between biomarkers with outcomes in sub-types of leukemia. Third, 27 out of 29 APL patients received at least one dose of ATRA treatment before sample collection as ATRA is usually administered immediately upon suspicion of APL, before these subjects were enrolled in the study. Previous studies have shown that ATRA reduces TF protein levels in APL cells.–36 ThereIn summary, we have shown that APL patients had the highest levels of EVTF activity and PAI-1, and lowest levels of tPA among different types of acute leukemia. ALL patients had the highest levels of cfDNA. Our data suggests that high levels of EVTF activity, WBC, PT and LDH were associated with increased risk of bleeding in acute leukemia patients. High levels of PAI-1 were associated with increased risk of VTE in acute leukemia patients. Future studies investigating these biomarkers in serial plasma samples and the impact of chemotherapy on these biomarkers will help in understanding the mechanisms of coagulopathy in acute leukemia.Supplement 1


Consumers who are environmentally and health conscious are increasingly looking for plant-based alternatives to replace animal-based products in their daily diets. Among these alternatives, there is a growing demand for meat analogues that closely resemble the taste and texture of meat. As a result, significant efforts have been dedicated to developing meat analogues with a desirable meat-like structure. Currently, soy protein and wheat gluten are the main ingredients used for producing these meat analogues due to their availability and unique functionalities. This study observed that high moisture extrusion at moisture levels of 50–80% has become a common approach for creating fibrous structures, with soy protein and wheat gluten being considered incompatible proteins. After the structuring process, they form two-phase filled gels, with wheat gluten acting as the continuous phase and soy protein serving as a filler material. Moreover, the formation of soy protein and wheat gluten networks relies on a combination of covalent and non-covalent interaction bonds, including hydrogen bonds that stabilize the protein networks, hydrophobic interactions governing protein chain associations during thermo-mechanical processes, and disulfide bonds that potentially contribute to fibrous structure formation. This review provides case studies and examples that demonstrate how specific processing conditions can improve the overall structure, aiming to serve as a valuable reference for further research and the advancement of fibrous structures. Plant-based diets have gained global attention for their positive impact on both environmental sustainability and health benefits, resulting in a growing trend of replacing animal-based products with plant-based products ,2. A sigThe soy protein industry has undergone significant development, with substantial investments made throughout its value chain from farm to fork, ensuring a cost-effective price for soy protein ingredients . AlthougThe fibrous structure is a crucial element in ensuring consumer acceptance of meat analogue products ,15. ThisIn this overview, we provide an initial introduction to soy protein and wheat gluten, which act as the main ingredients in plant-based meat analogues. Then, we delve into the discussion of advanced technologies used for creating the desired structure. Emphasis is placed on protein interactions and the potential underlying mechanisms behind fibrous structure formation. Furthermore, we elaborate on different formulations for the development of meat analogues and present case studies and examples that highlight how specific processing conditions can enhance the overall structure.Soybeans contain both water-soluble and insoluble proteins, which can be separated into storage globulin and whey fractions by acidification to an approximate pH of 4.5 . The whoSoy protein ingredients, such as soy protein concentrate (SPC) and soy protein isolate (SPI), are broadly used in various industries. SPC typically contains around 65% protein, while SPI contains at least 85% protein. There are two common pathways for fractionating SPC and SPI . To prodSoy protein ingredients were developed to enhance the economic feasibility of soy refining . They haThe approach of separating wheat flour into starch and gluten has been understood for nearly 300 years. However, it was only during the second half of the 20th century that wheat gluten began to be traded as a commodity, eventually gaining recognition as a valuable vegetable protein  32]. Cu. Cu32]. Wheat gluten is a cohesive and viscoelastic proteinaceous substance obtained as the by-product of wheat starch production. Gluten protein constitutes approximately 85% of all wheat proteins that are deposited in the wheat endosperm cells, and consists of a diverse mixture of proteins. About half of these proteins are monomeric gliadins, while the remaining portion forms the polymeric glutenin fraction through disulfide cross-linking . GliadinGluten has an extensible nature and can be stretched and pulled, which corresponds with its unique film-forming properties. The elastic properties of gluten can be attributed to glutenin, while gliadin contributes to the viscosity of the protein network . GluteniSoy protein and wheat gluten are plant proteins with distinct fractionation approaches, protein profiles, properties, and functionalities. However, they fulfill the requirements for nutritional and functional characteristics, and act as bulk ingredients in the development of meat alternatives. In order to achieve the desired texture and appearance in current meat analogues, the selection of appropriate protein-rich ingredients is important; furthermore, the utilization of established texturization methods also play a crucial role in creating meat-like structures.Various technologies have been studied and developed for creating a meat-like fibrous structure using soy protein and wheat gluten, including low/high moisture extrusion (LME/HME), spinning, shear cell, and 3D printing. HME is the most commonly used technology in the industry, while the others are still in the developing stage at laboratories. Extrusion is a popular method used in the production of protein-based foods, and it was first introduced in the 1970s to create soy-based meat substitutes; the obtained products appear as a relatively flat, elongated, and longitudinally aligned fibrous mass that closely resembles meat in terms of compactness and chewiness . CurrentThe process of creating meat analogues through extrusion Involves three main steps: preconditioning, mixing/cooking, and cooling. This process typically requires a setup that includes a preconditioning system, a feeding system, a screw, a heated barrel equipped with a single screw or twin screw, and a cooling die a 61]. I. I61]. IThe successful production of meat analogue products with a fibrous structure is influenced by various processing conditions, such as the screw diameter, rotor speed, barrel temperatures, and the specific plant proteins and formulations used . SeveralIn the early 2000s, shear cell devices were developed for studying the effects of simple shear on the overall properties of biopolymers like starch or proteins ,72. Two The implementation of the shearing process to induce fibrous hierarchical structures in dense calcium caseinate has led to the recognition of shear cell technology as a potential tool for creating fibrous anisotropic structures for meat analogue development . EvaluatApart from soy protein and wheat gluten systems, shear cell technology has successfully created a meat-like fibrous structure using a greater variety of plant-based materials ,76,77,783D printing, initially developed in the 1980s for material science applications, is a revolutionary digital process that allows for the creation of intricate solid forms  Figure c. The laDepositing liquid-based materials can be achieved through extrusion and inkjet printing, with extrusion printing being the most commonly used method . Coaxial3D printing technology offers the advantage of creating intricate and visually appealing structures in edible products, thus enhancing consumer interest and appetite . In theoSpinning, a well-established technique, was first observed in the early 1900s and patented in the 1930s . Wet spiThe wet spinning process of proteins presents a challenge as most proteins cannot be spun under food-grade conditions. In the case of the electrospinning process, it has been investigated to structure proteins on a nanometer-length scale, and it is able to act as a means to produce thin fibrils that can serve as textural building blocks for meat analogue products . Due to Despite significant advancements in the technology used to structure protein-rich ingredients, the impact of structuring processes on the physical and covalent interactions between proteins remains a topic of extensive research. This complexity arises from the intricate chemical bonding responsible for protein structure. By investigating the interactions between proteins during the structuring process, we can gain valuable insights into the nature of protein–protein bonding.Gaining a comprehensive understanding of the interactions between soy protein and wheat gluten, as well as their interactions with other components in the system, could greatly contribute to the comprehension of the underlying mechanisms involved in the formation of fibrous structures during the development of meat analogue products. This knowledge has the potential to resolve discrepancies found in existing literature, enhance production techniques, and ultimately elevate the quality of the end products.Various factors influence the structural formation of multi-component gels through the interaction of two or more proteins. These factors include the proteins’ thermodynamic compatibility, their potential for interaction, and their individual mechanisms of gelation. When different types of protein are mixed, they can be classified as incompatible, semi-compatible, or compatible, depending on whether they form two or more immiscible phases, partially mix at the molecular level, or form a single thermodynamically stable phase . The claWheat gluten has the ability to form fibrous structures independently after undergoing thermo-mechanical processes such as extrusion and shear cell, and fibrous formations were still observed with the addition of up to 50% soy protein ,75. HencThe heat-induced gelation of soy protein occurs in four steps: dissociation of small aggregates, association into dense spherical particles, formation of self-similar aggregates, and ultimately, network formation . HydrophThe heating and structuring process of soy protein and wheat gluten involves the application of heat and shear, impacting various bonds that stabilize protein structure. As temperature increases, hydrogen bonds are weakened, and hydrophobic interactions can increase ,107. ReaEnvironmentally and health conscious consumers are increasingly seeking plant-based alternatives to replace animal-based products in their diets, particularly in relation to dairy. The market is seeing a growing demand for meat analogue products that mimic the taste and texture of meat. Currently, the most commonly used plant protein resources for developing these products are soy protein and wheat gluten, mainly due to their availability and specific functionalities. Extensive research has focused on understanding how these proteins interact and form a fibrous structure through various structuring processes. Our findings indicate that both HME and shear cell processes share similar basic processing steps, while 3D-printing and spinning processes exhibit some similarities to a certain extent. Among all of the techniques, extrusion has become the industrially used technique for fibrous structure formation, while other advanced technologies are actively being developed and tested at both the lab-scale and pilot-scale levels. In addition to processing techniques, our research also summarizes formulations and processing conditions and introduces hypotheses regarding the protein–protein interactions during the process. According to our hypothesis, soy protein and wheat gluten generally form two-phase filled gels, and a combination of covalent and non-covalent interaction bonds plays a crucial role in forming soy protein and wheat gluten networks. These bonds include hydrogen bonds, which help to stabilize the networks of proteins, hydrophobic interactions that govern protein chain associations during thermo-mechanical processes, and disulfide bonds that potentially contribute to the formation of fibrous structures.In light of the increasing demand for plant-based meat analogue products and the advancements made in understanding protein interactions and structuring processes, future research should focus on several key areas. Firstly, it is necessary to explore different fractionation pathways to produce a wider range of soy protein and wheat gluten ingredients. Currently, their existing fractionation processes are not fully optimized for meat analogue development. By doing so, the applicability and sustainability of plant protein ingredients can be further enhanced. Moreover, more in-depth studies are required to understand the network-forming behavior of soy protein and wheat gluten. The mechanisms behind their formation have not been fully elucidated yet. Gaining a better understanding of these mechanisms would enable precise control over the texture and structure of meat analogue products, ultimately improving consumer acceptance. Furthermore, additional research is needed to optimize the production processes of meat analogues, such as 3D-printing and spinning techniques. Taking a comprehensive approach that incorporates multiple developed technologies will ensure that future developments in meat analogues meet the evolving needs of environmentally and health conscious consumers.