Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death.

The perpetuation of inflammation is an important pathophysiological contributor to the global medical burden. Chronic inflammation is promoted by non-programmed cell death1,2; however, how inflammation is instigated, its cellular and molecular mediators, and its therapeutic value are poorly defined. Here we use mouse models of atherosclerosis-a major underlying cause of mortality worldwide-to demonstrate that extracellular histone H4-mediated membrane lysis of smooth muscle cells (SMCs) triggers arterial tissue damage and inflammation. We show that activated lesional SMCs attract neutrophils, triggering the ejection of neutrophil extracellular traps that contain nuclear proteins. Among them, histone H4 binds to and lyses SMCs, leading to the destabilization of plaques; conversely, the neutralization of histone H4 prevents cell death of SMCs and stabilizes atherosclerotic lesions. Our data identify a form of cell death found at the core of chronic vascular disease that is instigated by leukocytes and can be targeted therapeutically.

The effects of 17α-methyltestosterone on gonadal histology and gene expression along hypothalamic-pituitary-gonadal axis, germ cells, sex determination, and hypothalamus-pituitary-thyroid axis in zebrafish (Danio rerio).

As a synthetic androgen, 17α-methyltestosterone (MT) is widely used in aquaculture to induce sex reversal and may pose a potential risk to aquatic organisms. This ecological risk has attracted the attention of many scholars, but it is not comprehensive enough. Thus, the adverse effects of MT on zebrafish (Danio rerio) were comprehensively evaluated from gonadal histology, as well as the mRNA expression levels of 47 genes related to hypothalamic-pituitary-gonadal (HPG) axis, germ cell differentiation, sex determination, and hypothalamus-pituitary-thyroid (HPT) axis. Adult zebrafish with a female/male ratio of 5:7 were exposed to a solvent control (0.001% dimethyl sulfoxide) and three measured concentrations of MT (5, 51 and 583 ng/L) for 50 days. The results showed that MT had no significant histological effects on the ovaries of females, but the frequency of late-mature oocytes (LMO) showed a downward trend, indicating that MT could induce ovarian suppression to a certain extent. The transcriptional expression of activating transcription factor 4b1 (atf4b1), activating transcription factor 4b2 (atf4b2), calcium/calmodulin-dependent protein kinase II delta 1 (camk2d1), calcium/calmodulin-dependent protein kinase II delta 2 (camk2d2) and calcium/calmodulin-dependent protein kinase II inhibitor 2 (camk2n2) genes in the brain of females increased significantly at all treatment groups of MT, and the mRNA expression of forkhead box L2a (foxl2) and ovarian cytochrome P450 aromatase (cyp19a1a) genes in the ovaries were down-regulated by 5 and 583 ng/L group, which would translate into inhibition of oocyte development. As compared to females, MT had relatively little effects on the reproductive system of males, and only the transcriptional alterations of synaptonemal complex protein 3 (sycp3) and 17-alpha-hydroxylase/17,20-lyase (cyp17) genes were observed in the testes, not enough to affect testicular histology. In addition, MT at all treatments strongly increased corticotropin-releasing hormone (crh) transcript in the brain of females, as well as deiodinase 2 (dio2) transcript in the brain of males. The paired box protein 8 (pax8) gene was significantly decreased at 51 or 583 ng/L of MT in both female and male brains. The above results suggest that MT can pose potential adverse effects on the reproductive and thyroid endocrine system of fish.

Hybrid waveguide based augmented reality display system with extra large field of view and 2D exit pupil expansion.

For a waveguide display device, the field of view (FOV) is a key parameter for evaluating its optical performance. To address this issue, we propose a hybrid waveguide system, which is composed of two projectors, two in-couplers, two half-mirror arrays and an out-coupler. We use two projectors to generate the left and right parts of the output image separately, which can increase the upper limit of the FOV significantly. Unlike conventional waveguide-based system, we use half-mirror arrays instead of folding gratings to realize 2D exit pupil expansion. By doing so, the total internal reflection condition can always be met during the pupil expansion process. To solve the difficulty in designing collimating optical system with large FOV, we propose a method of tilting the projection system. The hybrid waveguide system can realize a FOV of 88°(H) × 53°(V).

Nitrogen addition promotes terrestrial plants to allocate more biomass to aboveground organs: A global meta-analysis.

A significant increase in reactive nitrogen (N) added to terrestrial ecosystems through agricultural fertilization or atmospheric deposition is considered to be one of the most widespread drivers of global change. Modifying biomass allocation is one primary strategy for maximizing plant growth rate, survival, and adaptability to various biotic and abiotic stresses. However, there is much uncertainty as to whether and how plant biomass allocation strategies change in response to increased N inputs in terrestrial ecosystems. Here, we synthesized 3516 paired observations of plant biomass and their components related to N additions across terrestrial ecosystems worldwide. Our meta-analysis reveals that N addition (ranging from 1.08 to 113.81 g m-2 year-1 ) increased terrestrial plant biomass by 55.6% on average. N addition has increased plant stem mass fraction, shoot mass fraction, and leaf mass fraction by 13.8%, 12.9%, and 13.4%, respectively, but with an associated decrease in plant reproductive mass (including flower and fruit biomass) fraction by 3.4%. We further documented a reduction in plant root-shoot ratio and root mass fraction by 27% (21.8%-32.1%) and 14.7% (11.6%-17.8%), respectively, in response to N addition. Meta-regression results showed that N addition effects on plant biomass were positively correlated with mean annual temperature, soil available phosphorus, soil total potassium, specific leaf area, and leaf area per plant. Nevertheless, they were negatively correlated with soil total N, leaf carbon/N ratio, leaf carbon and N content per leaf area, as well as the amount and duration of N addition. In summary, our meta-analysis suggests that N addition may alter terrestrial plant biomass allocation strategies, leading to more biomass being allocated to aboveground organs than belowground organs and growth versus reproductive trade-offs. At the global scale, leaf functional traits may dictate how plant species change their biomass allocation pattern in response to N addition.

Macrophage ALDH2 (Aldehyde Dehydrogenase 2) Stabilizing Rac2 Is Required for Efferocytosis Internalization and Reduction of Atherosclerosis Development.

BACKGROUND: Clinical studies show that the most common single-point mutation in humans, ALDH2 (aldehyde dehydrogenase 2) rs671 mutation, is a risk factor for the development and poor prognosis of atherosclerotic cardiovascular diseases, but the underlying mechanism remains unclear. Apoptotic cells are phagocytosed and eliminated by macrophage efferocytosis during atherosclerosis, and enhancement of arterial macrophage efferocytosis reduces atherosclerosis development. METHODS: Plaque areas, necrotic core size, apoptosis, and efferocytosis in aortic lesions were investigated in APOE-/- mice with bone marrow transplanted from APOE-/-ALDH2-/- and APOE-/- mice. RNA-seq, proteomics, and immunoprecipitation experiments were used to screen and validate signaling pathways affected by ALDH2. Efferocytosis and protein levels were verified in human macrophages from wild-type and rs671 mutation populations. RESULTS: We found that transplanting bone marrow from APOE-/-ALDH2-/- to APOE-/- mice significantly increased atherosclerosis plaques compared with transplanting bone marrow from APOE-/- to APOE-/- mice. In addition to defective efferocytosis in plaques of APOE-/- mice bone marrow transplanted from APOE-/-ALDH2-/- mice in vivo, macrophages from ALDH2-/- mice also showed significantly impaired efferocytotic activity in vitro. Subsequent RNA-seq, proteomics, and immunoprecipitation experiments showed that wild-type ALDH2 directly interacted with Rac2 and attenuated its degradation due to decreasing the K48-linked polyubiquitination of lysine 123 in Rac2, whereas the rs671 mutant markedly destabilized Rac2. Furthermore, Rac2 played a more crucial role than other Rho GTPases in the internalization process in which Rac2 was up-regulated, activated, and clustered into dots. Overexpression of wild-type ALDH2 in ALDH2-/- macrophages, rather than the rs671 mutant, rescued Rac2 degradation and defective efferocytosis. More importantly, ALDH2 rs671 in human macrophages dampened the apoptotic cells induced upregulation of Rac2 and subsequent efferocytosis. CONCLUSIONS: Our study has uncovered a pivotal role of the ALDH2-Rac2 axis in mediating efferocytosis during atherosclerosis, highlighting a potential therapeutic strategy in cardiovascular diseases, especially for ALDH2 rs671 mutation carriers.

Toward systematic understanding of adsorptive removal of legacy and emerging per-and polyfluoroalkyl substances (PFASs) by various activated carbons (ACs).

Per-and polyfluoroalkyl substances (PFASs) have received great attention due to their persistence, bioaccumulation and toxicity. Various activated carbons (ACs) exhibit wide variability in adsorptive performance towards PFASs. In order to gain a systematic understanding of adsorptive removal of legacy and emerging PFASs by ACs, the adsorption of ten PFASs on various ACs was comprehensively investigated. Results showed that granular activated carbon-1 (GAC-1) and powdered activated carbon-1 (PAC-1) removed more than 90% of all target PFASs. Particle size, surface charge, and micropores quantity of ACs were closely related to their performance for PFASs removal. Electrostatic interaction, hydrophobic interaction, surface complexation and hydrogen bonding were the adsorption mechanisms, with hydrophobic interaction being the predominant adsorptive force. Physical and chemical adsorption were both involved in PFAS adsorption. The removal rates of PFASs by GAC-1 decreased from 93%-100% to 15%-66% in the presence of 5 mg/L fulvic acid (FA). GAC was able to remove more PFASs under acidic medium, whereas PAC removed hydrophobic PFASs better under the neutral medium. The removal rates of PFASs by GAC-3 increased significantly from 0%-21% to 52%-97% after being impregnated with benzalkonium chlorides (BACs), demonstrating the superiority of this modification method. Overall, this study provided theoretical support for removing PFASs from water phase with ACs.

Endotoxinemia Accelerates Atherosclerosis Through Electrostatic Charge-Mediated Monocyte Adhesion.

BACKGROUND: Acute infection is a well-established risk factor of cardiovascular inflammation increasing the risk for a cardiovascular complication within the first weeks after infection. However, the nature of the processes underlying such aggravation remains unclear. Lipopolysaccharide derived from Gram-negative bacteria is a potent activator of circulating immune cells including neutrophils, which foster inflammation through discharge of neutrophil extracellular traps (NETs). Here, we use a model of endotoxinemia to link acute infection and subsequent neutrophil activation with acceleration of vascular inflammation Methods: Acute infection was mimicked by injection of a single dose of lipopolysaccharide into hypercholesterolemic mice. Atherosclerosis burden was studied by histomorphometric analysis of the aortic root. Arterial myeloid cell adhesion was quantified by intravital microscopy. RESULTS: Lipopolysaccharide treatment rapidly enhanced atherosclerotic lesion size by expansion of the lesional myeloid cell accumulation. Lipopolysaccharide treatment led to the deposition of NETs along the arterial lumen, and inhibition of NET release annulled lesion expansion during endotoxinemia, thus suggesting that NETs regulate myeloid cell recruitment. To study the mechanism of monocyte adhesion to NETs, we used in vitro adhesion assays and biophysical approaches. In these experiments, NET-resident histone H2a attracted monocytes in a receptor-independent, surface charge-dependent fashion. Therapeutic neutralization of histone H2a by antibodies or by in silico designed cyclic peptides enables us to reduce luminal monocyte adhesion and lesion expansion during endotoxinemia. CONCLUSIONS: Our study shows that NET-associated histone H2a mediates charge-dependent monocyte adhesion to NETs and accelerates atherosclerosis during endotoxinemia.

Blocking cholesterol efflux mechanism is a potential target for antilymphoma therapy.

Cholesterol is an essential plasma membrane lipid for the maintenance of cellular homeostasis and cancer cell proliferation. Free cholesterol is harmful to cells; therefore, excessive free cholesterol must be quickly esterified by acetyl-coenzyme A:cholesterol acetyltransferase (ACAT) and exported by scavenger receptor class B member I (SR-BI) or ATP-binding cassette protein A1 from specific cells such as macrophage foam cells, which contain cholesteryl ester-derived vacuoles. Many vacuoles are present in the cytoplasm of Burkitt lymphoma cells. In this study, we observed that these vacuoles are often seen in high-grade lymphomas. Cell culture study using lymphoma cell lines found that esterified cholesterol is the main component of these vacuoles and the expression of cholesterol metabolism-related molecules was significantly upregulated in lymphoma cell lines, with SR-BI and ACAT inhibitors (BLT-1 and CI-976, respectively) impeding lymphoma cell proliferation. Cytoplasmic free cholesterol was increased by ACAT and SR-BI inhibitors, and the accumulation of free cholesterol induced lymphoma cell apoptosis by inducing endoplasmic reticulum stress. Furthermore, synergistic effects of SR-BI and ACAT inhibitors were observed in a preclinical study. Treatment with SR-BI inhibitor suppressed lymphoma progression in a tumor-bearing mouse model, whereas ACAT inhibitor did not. Therefore, SR-BI inhibitors are potential new antilymphoma therapeutics that target cholesterol metabolism.

A Standardized Step-by-Step Approach for the Diagnosis and Treatment of Sepsis.

Sepsis is the major culprit of death among critically ill patients who are hospitalized in intensive care units (ICUs). Although sepsis-related mortality is steadily declining year-by-year due to the continuous understanding of the pathophysiological mechanism on sepsis and improvement of the bundle treatment, sepsis-associated hospitalization is rising worldwide. Surviving Sepsis Campaign (SSC) guidelines are continuously updating, while their content is extremely complex and comprehensive for a precisely implementation in clinical practice. As a consequence, a standardized step-by-step approach for the diagnosis and treatment of sepsis is particularly important. In the present study, we proposed a standardized step-by-step approach for the diagnosis and treatment of sepsis using our daily clinical experience and the latest researches, which is close to clinical practice and is easy to implement. The proposed approach may assist clinicians to more effectively diagnose and treat septic patients and avoid the emergence of adverse clinical outcomes.

Exploring value of CT coronary imaging combined with machine-learning methods to predict myocardial ischemia.

PURPOSE: To establish a machine-learning (ML) model based on coronary computed tomography angiography (CTA) images for evaluating myocardial ischemia in patients diagnosed with coronary atherosclerosis. METHODS: This retrospective analysis includes CTA images acquired from 110 patients. Among them, 58 have myocardial ischemia and 52 have normal myocardial blood supply. The patients are divided into training and test datasets with a ratio 7 : 3. Deep learning model-based CQK software is used to automatically segment myocardium on CTA images and extract texture features. Then, seven ML models are constructed to classify between myocardial ischemia and normal myocardial blood supply cases. Predictive performance and stability of the classifiers are determined by receiver operating characteristic curve with cross validation. The optimal ML model is then validated using an independent test dataset. RESULTS: Accuracy and areas under ROC curves (AUC) obtained from the support vector machine with extreme gradient boosting linear method are 0.821 and 0.777, respectively, while accuracy and AUC achieved by the neural network (NN) method are 0.818 and 0.757, respectively. The naive Bayes model yields the highest sensitivity (0.942), and the random forest model yields the highest specificity (0.85). The k-nearest neighbors model yields the lowest accuracy (0.74). Additionally, NN model demonstrates the lowest relative standard deviations (0.16 for accuracy and 0.08 for AUC) indicating the high stability of this model, and its AUC applying to the independent test dataset is 0.72. CONCLUSION: The NN model demonstrates the best performance in predicting myocardial ischemia using radiomics features computed from CTA images, which suggests that this ML model has promising potential in guiding clinical decision-making.

Automated µFTIR Imaging Demonstrates Taxon-Specific and Selective Uptake of Microplastic by Freshwater Invertebrates.

Microplastic particles can be deposited to sediments and subsequently ingested by benthic organisms. It is unknown to what extent ingestion of microplastic is taxon-specific or whether taxa can be selective toward certain types of microplastics. Here, we used state-of-the-art automated micro-Fourier-transform infrared (µFTIR) imaging and attenuated total reflectance FTIR spectroscopy to determine small-size (20-500 µm) and large-size (500-5000 µm) microplastic particles in sediments and a range of benthic invertebrate species sampled simultaneously from the Dommel River in the Netherlands. Microplastic number concentrations differed across taxa at the same locations, demonstrating taxon-specific uptake, whereas size distributions were the same across sediments and taxa. At the site with the highest concentration, microplastic occupied up to 4.0% of the gut volume of Asellidae. Particle shape distributions were often not statistically different between sediments and taxa, except for Astacidea at one of the locations where the proportion of particles with a length to width ratio >3 (i.e., fibers) was twice as high in sediments than in Astacidea. Acrylates/polyurethane/varnish was predominately found in sediments, while soft and rubbery polymers ethylene propylene diene monomer and polyethylene-chlorinated were the dominant polymers found in invertebrates. Microplastic polymer composition and thus polymer density differed significantly between invertebrates and their host sediment. Trophic transfer at the base of the food web appears to have a filter function with respect to microplastic particle types and shapes. Together with the very high ingestion rates, this has clear implications for ecological and human health risks, where uptake concerns edible species (e.g., Astacidea).

Distribution, partitioning behavior and potential source of legacy and alternative per- and polyfluoroalkyl substances (PFASs) in water and sediments from a subtropical Gulf, South China Sea.

Legacy per- and polyfluoroalkyl acids (PFASs) have received global concern over the scientific and public community since this century. However, the information on alternative PFASs pollution in the marine environment, especially in the subtropical marine environment is extremely limited. This study investigated the occurrence, partitioning, potential sources, and ecological risks of PFASs, including perfluoroalkane sulfonic acids (PFSAs), perfluoroalkyl carboxylic acids (PFCAs), and alternative PFASs, in surface water and sediments from the subtropical Beibu Gulf, South China. Concentrations of total PFASs (∑PFASs) were in the range of 0.98-2.64 ng/L in water and 0.19-0.66 ng/g (dry weight, dw) in sediment, respectively. Perfluorooctanoic acid (PFOA) was the most abundant PFAS in water, while PFASs in sediment were dominated by perfluorooctanesulfonic acid (PFOS) and PFOA. Among investigated environmental parameters (total organic carbon (TOC), grain size, water pH, sediment pH, and salinity), TOC and salinity were the dominant factors influencing the sediment-water distribution coefficient (Kd) of PFOA, perfluorodecanoic acid (PFDA), and perfluorononanoic acid (PFNA). Log Kd and log soil organic carbon-water distribution coefficient (Koc) both increase with increasing carbon chain length of PFASs. Significantly positive correlations between PFOS and perfluorohexanoic acid (PFHxA) (p < 0.05), PFOA and perfluoro-1-butane-sulfonamide (FBSA) were observed, suggesting that these PFASs might have similar sources and transport routes. Preliminary environmental risk assessment showed that PFOA and PFOS would not pose risks to the marine aquatic environment. This is the first comprehensive survey of legacy and alternative PFASs in a subtropical area of the Beibu Gulf, which provides significant data and scientific basis to better understand the fate of PFASs and pollution control management.

Response of periphytic biofilm in water to estrone exposure: Phenomenon and mechanism.

The responses of pure strains to contaminant (i.e., estrone, E1) exposure have been widely studied. However, few studies about the responses of multispecies microbial aggregates (e.g., periphytic biofilm) to E1 exposure are available. In this study, the changes in physiological activity and community composition of periphytic biofilms before and after E1 exposure were investigated. The results showed that periphytic biofilms exhibited high adaptability to E1 exposure at a concentration of 0.5 mg L-1 based on physiological results. The increase in productivity of extracellular polymeric substances (EPS) after exposure to E1 was the main factor preventing association between E1 and microbial cells. The increase in the activity of superoxide dismutase (SOD) and ATP enzyme activity and the change in the co-occurrence pattern of microbial communities (increasing the relative abundance of Xanthomonadaceae and Cryomorphacea) also protected biofilms from E1 exposure. However, exposure to a high concentration of E1 (>10 mg L-1) significantly decreased EPS productivity and metabolic activity due to the excessive accumulation of reactive oxygen species. In addition, the abundance of some sensitive species, such as Pseudanabaenaceae, decreased sharply at this concentration. Overall, this study highlighted the feasibility of periphytic biofilms to adapt to E1 exposure at low concentrations in aquatic environments.

Histological and transcriptional effects of androstenedione in adult zebrafish (Danio rerio).

As a natural androgen, androstenedione (AED) may pose potential risks to aquatic organisms due to its ubiquitousness in aquatic environments. Here we assessed the adverse effects of AED on histology of gonads, as well as mRNA expression levels of 34 genes concerned with hypothalamic-pituitary-gonadal (HPG) axis, germ-cell differentiation and sex differentiation in zebrafish (Danio rerio). Adult zebrafish were exposed to solvent control and three measured concentrations of 0.2, 2.3 and 23.7 µg/L AED for 60 days. The results showed that AED did not induce any obvious histological effects in the ovaries and testes. Of the investigated genes, transcriptional expression levels of amh and cyp11c1 genes in the ovaries of females were significantly increased by AED at 2.3 or 23.7 µg/L. However, different exposure concentrations of AED significantly inhibited mRNA expression of gnrh3, atf4b1 and cyp19a1b in the brain of males. In the testes of males, AED at 2.3 µg/L led to a significant induction of sox9b gene, but it at 23.7 µg/L down-regulated nr5a1b gene. These observed transcriptional changes indicated that AED could pose potential androgenic effects in zebrafish.

Enhanced computed tomography radiomics-based machine-learning methods for predicting the Fuhrman grades of renal clear cell carcinoma.

OBJECTIVE: To develop and test an optimal machine learning model based on the enhanced computed tomography (CT) to preoperatively predict pathological grade of clear cell renal cell carcinoma (ccRCC). METHODS: A retrospective analysis of 53 pathologically confirmed cases of ccRCC was performed and 25 consecutive ccRCC cases were selected as a prospective testing set. All patients underwent routine preoperative abdominal CT plain and enhanced scans. Renal tumor lesions were segmented on arterial phase images and 396 radiomics features were extracted. In the training set, seven discrimination classifiers for high- and low-grade ccRCCs were constructed based on seven different machine learning models, respectively, and their performance and stability for predicting ccRCC grades were evaluated through receiver operating characteristic (ROC) analysis and cross-validation. Prediction accuracy and area under ROC curve were used as evaluation indices. Finally, the diagnostic efficacy of the optimal model was verified in the testing set. RESULTS: The accuracies and AUC values achieved by support vector machine with radial basis function kernel (svmRadial), random forest and naïve Bayesian models were 0.860±0.158 and 0.919±0.118, 0.840±0.160 and 0.915±0.138, 0.839±0.147 and 0.921±0.133, respectively, which showed high predictive performance, whereas K-nearest neighborhood model yielded lower accuracy of 0.720±0.188 and lower AUC value of 0.810±0.150. Additionally, svmRadial had smallest relative standard deviation (RSD, 0.13 for AUC, 0.17 for accuracy), which indicates higher stability. CONCLUSION: svmRadial performs best in predicting pathological grades of ccRCC using radiomics features computed from the preoperative CT images, and thus may have high clinical potential in guiding preoperative decision.

Effect of animal protein diet on the prognosis of children with Henoch-Schönlein purpura. / 动物蛋白饮食对过敏性紫癜患儿预后的影响分析.

OBJECTIVES: To study the association of animal protein diet with the recurrence of Henoch-Schönlein purpura (HSP)/skin rash and the risk factors for recurrence of HSP. METHODS: A prospective analysis was performed for 121 children with HSP who were admitted to the Beijing Children's Hospital from October to December 2020. The children were given the doctor's advice of the same diet (animal protein diet could be added after 1 week without new-onset skin rash). Follow-up was performed at the outpatient service for half a year. According to the presence or absence of animal protein intake, the children were divided into an observation group with 65 children and a control group with 56 children. The times of skin rash recurrence, the incidence of HSP recurrence, and the incidence of kidney injury were compared between the two groups. According to the presence or absence of recurrence, the children were divided into a recurrence group with 32 children and a non-recurrence group with 89 children. A questionnaire on food frequency was used to record the daily intake of animal protein in the two groups. A multivariate logistic regression analysis was used to identify the risk factors for recurrence of HSP in children. RESULTS: There was no significant difference between the observation and control groups in the times of skin rash recurrence, the incidence rate of HSP recurrence, and the incidence rate of kidney injury (P>0.05). There was no significant difference in the daily intake of animal protein between the recurrence and non-recurrence groups (P>0.05). The multivariate logistic regression analysis showed that presence of kidney injury at initial onset, respiratory infection after cure for the first time, and lack of exercise control after cure for the first time were independent risk factors for the recurrence of HSP in children (P<0.05). CONCLUSIONS: There is no significant association between animal protein diet and the recurrence of HSP or skin rash. Timely treatment of kidney injury, avoidance of infection after cure, and limitation of strenuous exercise may help to reduce the recurrence rate of HSP in children. Citation.

ALDH2 (Aldehyde Dehydrogenase 2) Protects Against Hypoxia-Induced Pulmonary Hypertension.

OBJECTIVE: Hypoxia-induced pulmonary hypertension (HPH) increases lipid peroxidation with generation of toxic aldehydes that are metabolized by detoxifying enzymes, including ALDH2 (aldehyde dehydrogenase 2). However, the role of lipid peroxidation and ALDH2 in HPH pathogenesis remain undefined. Approach and Results: To determine the role of lipid peroxidation and ALDH2 in HPH, C57BL/6 mice, ALDH2 transgenic mice, and ALDH2 knockout (ALDH2-/-) mice were exposed to chronic hypoxia, and recombinant tissue-specific ALDH2 overexpression adeno-associated viruses were introduced into pulmonary arteries via tail vein injection for ALDH2 overexpression. Human pulmonary artery smooth muscle cells were used to elucidate underlying mechanisms in vitro. Chronic hypoxia promoted lipid peroxidation due to the excessive production of reactive oxygen species and increased expression of lipoxygenases in lung tissues. 4-hydroxynonenal but not malondialdehyde level was increased in hypoxic lung tissues which might reflect differences in detoxifying enzymes. ALDH2 overexpression attenuated the development of HPH, whereas ALDH2 knockout aggravated it. Specific overexpression of ALDH2 using AAV1 (adeno-associated virus)-ICAM (intercellular adhesion molecule) 2p-ALDH2 and AAV2-SM22αp (smooth muscle 22 alpha)-ALDH2 viral vectors in pulmonary artery smooth muscle cells, but not endothelial cells, prevented the development of HPH. Hypoxia or 4-hydroxynonenal increased stabilization of HIF (hypoxia-inducible factor)-1α, phosphorylation of Drp1 (dynamin-related protein 1) at serine 616, mitochondrial fission, and pulmonary artery smooth muscle cells proliferation, whereas ALDH2 activation suppressed the latter 3. CONCLUSIONS: Increased 4-hydroxynonenal level plays a critical role in the development of HPH. ALDH2 attenuates the development of HPH by regulating mitochondrial fission and smooth muscle cell proliferation suggesting ALDH2 as a potential new therapeutic target for pulmonary hypertension.

Poly (ADP-ribose) polymerase inhibition protects against myocardial ischaemia/reperfusion injury via suppressing mitophagy.

Myocardial ischaemia/reperfusion (I/R) injury attenuates the beneficial effects of reperfusion therapy. Poly(ADP-ribose) polymerase (PARP) is overactivated during myocardial I/R injury. Mitophagy plays a critical role in the development of myocardial I/R injury. However, the effect of PARP activation on mitophagy in cardiomyocytes is unknown. In this study, we found that I/R induced PARP activation and mitophagy in mouse hearts. Poly(ADP-ribose) polymerase inhibition reduced the infarct size and suppressed mitophagy after myocardial I/R injury. In vitro, hypoxia/reoxygenation (H/R) activated PARP, promoted mitophagy and induced cell apoptosis in cardiomyocytes. Poly(ADP-ribose) polymerase inhibition suppressed H/R-induced mitophagy and cell apoptosis. Parkin knockdown with lentivirus vectors inhibited mitophagy and prevented cell apoptosis in H/R-treated cells. Poly(ADP-ribose) polymerase inhibition prevented the loss of the mitochondrial membrane potential (ΔΨm). Cyclosporin A maintained ΔΨm and suppressed mitophagy but FCCP reduced the effect of PARP inhibition on ΔΨm and promoted mitophagy, indicating the critical role of ΔΨm in H/R-induced mitophagy. Furthermore, reactive oxygen species (ROS) and poly(ADP-ribosylation) of CypD and TSPO might contribute to the regulation of ΔΨm by PARP. Our findings thus suggest that PARP inhibition protects against I/R-induced cell apoptosis by suppressing excessive mitophagy via the ΔΨm/Parkin pathway.

Allosteric modulation of the catalytic VYD loop in Slingshot by its N-terminal domain underlies both Slingshot auto-inhibition and activation.

Slingshots are phosphatases that modulate cytoskeleton dynamics, and their activities are tightly regulated in different physiological contexts. Recently, abnormally elevated Slingshot activity has been implicated in many human diseases, such as cancer, Alzheimer's disease, and vascular diseases. Therefore, Slingshot-specific inhibitors have therapeutic potential. However, an enzymological understanding of the catalytic mechanism of Slingshots and of their activation by actin is lacking. Here, we report that the N-terminal region of human Slingshot2 auto-inhibits its phosphatase activity in a noncompetitive manner. pH-dependent phosphatase assays and leaving-group dependence studies suggested that the N-terminal domain of Slingshot2 regulates the stability of the leaving group of the product during catalysis by modulating the general acid Asp361 in the catalytic VYD loop. F-actin binding relieved this auto-inhibition and restored the function of the general acid. Limited tryptic digestion and biophysical studies identified large conformational changes in Slingshot2 after the F-actin binding. The dissociation of N-terminal structural elements, including Leu63, and the exposure of the loop between α-helix-2 and ß-sheet-3 of the phosphatase domain served as the structural basis for Slingshot activation via F-actin binding in vitro and via neuregulin stimulation in cells. Moreover, we designed a FlAsH-BRET-based Slingshot2 biosensor whose readout was highly correlated with the in vivo phosphatase activities of Slingshot2. Our results reveal the auto-inhibitory mechanism and allosteric activation mechanisms of a human Slingshot phosphatase. They also contribute to the design of new strategies to study Slingshot regulation in various cellular contexts and to screen for new activators/inhibitors of Slingshot activity.