Split application of polymer-coated urea combined with common urea improved nitrogen efficiency without sacrificing wheat yield and benefits while saving 20% nitrogen input.

Controlled-release nitrogen fertilizer (CRNF) has been expected to save labor input, reduce environmental pollution, and increase yield in crop production. However, the economic feasibility is still controversial due to its high cost. To clarify the suitable application strategy of CRNF in promoting the yield, nitrogen use efficiency and income on wheat grown in paddy soil, four equal N patterns were designed in 2017-2021 with polymer-coated urea (PCU) and common urea as material, including PCU applied once pre-sowing (M1), PCU applied 60% at pre-sowing and 40% at re-greening (M2), 30% PCU and 30% urea applied at pre-sowing, 20% PCU and 20% urea applied at re-greening (M3), and urea applied at four stage (CK, Basal:tillering:jointing:booting=50%:10%:20%:20%). In addition, M4-M6, which reduced N by 10%, 20% and 30% respectively based on M3, were designed in 2019-2021 to explore their potential for N-saving and efficiency-improving. The results showed that, compared with CK, M1 did not significantly reduce yield, but decreased the average N recovery efficiency (NRE) and benefits by 1.63% and 357.71 CNY ha-1 in the four years, respectively. M2 and M3 promoted tiller-earing, delayed the decrease of leaf area index (LAI) at milk-ripening stage, and increased dry matter accumulation post-anthesis, thereby jointly increasing spike number and grain weight of wheat, which significantly increased yield and NRE compared with CK in 2017-2021. Due to the savings in N fertilizer costs, M3 achieved the highest economic benefits. With the 20% N reduction, M5 increased NRE by 16.95% on average while decreasing yield and net benefit by only 6.39% and 7.40% respectively, compared with M3. Although NRE could continue to increase, but the yield and benefits rapidly decreased after N reduction exceeds 20%. These results demonstrate that twice-split application of PCU combined with urea is conducive to achieving a joint increase in yield, NRE, and benefits. More importantly, it can also significantly improve the NRE without losing yield and benefits while saving 20% N input.

TNEA therapy promotes the autophagic degradation of NLRP3 inflammasome in a transgenic mouse model of Alzheimer's disease via TFEB/TFE3 activation.

BACKGROUND: The impairment in the autophagy-lysosomal pathway (ALP) and the activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome represent two molecular events leading to neurodegeneration and neuroinflammation in Alzheimer's disease (AD), a devastating neurodegenerative disorder without a cure. Previously we demonstrated the cognitive-enhancing effect of a combined electroacupuncture (EA) therapy termed TNEA in a transgenic mouse model of AD, involving activation of transcription factor EB (TFEB), a master regulator of ALP. However, whether and how TNEA inhibits NLRP3 inflammasome via TFEB-mediated ALP in AD remains to be investigated. METHODS: 5xFAD mice overexpressing amyloid-ß (Aß) were treated with TNEA or EA on its composing acupoints (GB13 and GV24). The changes in the signaling pathways regulating NLRP3 inflammasome, the association of NLRP3 inflammasome with ALP, and the roles of TFEB/TFE3 in mice brains were determined by immunoblots, immunohistochemistry and AAV-mediated knockdown assays. RESULTS: TNEA inhibits the activation of NLRP3 inflammasome and the release of active interleukin 1ß (IL1B) in the hippocampi of 5xFAD mice. Mechanistically, TNEA promoted the autophagic degradation of inflammasome components via activating both TFEB and TFE3 by modulating kinases including AMPK and AKT. The composing acupoints in TNEA showed synergistic effects on regulating these molecular events and memory improvement. CONCLUSION: Our findings suggest that TNEA attenuates AD-associated memory impairment via promoting TFEB/TFE3-mediated autophagic clearance of Aß and NLRP3 inflammasome, and partially reveal the molecular basis of combined acupoints therapy originated from ancient wisdom.

Comparative transcriptomic and metabolomic analysis revealed molecular mechanism of two wheat near-isogenic lines response to nitrogen application.

Nitrogen (N) is an essential nutrient element required for plant growth, and the development of wheat varieties with high nitrogen use efficiency (NUE) is an urgent need for sustainable crop production. However, the molecular mechanism of NUE between diverse wheat varieties in response to N application remains unclear. To reveal the possible molecular mechanisms underlying this complex phenomenon, we investigated the transcriptional and metabolic changes of flag leaves of two wheat near-isogenic lines (NILs) differing in NUE under two N fertilizer treatments. Comparative transcriptome analysis indicated that the expression levels of the genes responsible for carbon and nitrogen metabolism were significantly higher in high-NUE wheat. The metabolome comparison revealed that the activity of the tricarboxylic acid (TCA) cycle was enhanced in high-NUE wheat, while reduced in low-NUE wheat after the N fertilizer application. Additionally, amino acid metabolism increased in both wheat NILs but more increased in high-NUE wheat. In summary, more upregulated transcripts and metabolites were identified in high-NUE wheat, and this study provides valuable new insights for improving NUE in wheat.

Electroacupuncture ameliorates cerebrovascular impairment in Alzheimer's disease mice via melatonin signaling.

AIMS: Cerebrovascular impairment contributes to the pathogenesis of Alzheimer's disease (AD). However, it still lacks effective intervention in clinical practice. Here, we investigated the efficacy of electroacupuncture (EA) in cerebrovascular repair in 3xTg-AD mice and its mechanism. METHODS: 3xTg-AD mice were employed to evaluate the protective effect of EA at ST36 acupoint (EAST36). Behavioral tests were performed to assess neurological disorders. Laser speckle contrast imaging, immunostaining, and Western blot were applied to determine EAST36-boosted cerebrovascular repair. The mechanism was explored in 3xTg mice and endothelial cell cultures by melatonin signaling modulation. RESULTS: EAST36 at 20/100 Hz effectively alleviated the olfactory impairment and anxiety behavior and boosted cerebrovascular repair in AD mice. EAST36 attenuated cerebral microvascular degeneration in AD mice by modulating endothelial cell viability and injury. Consequently, the Aß deposits and neural damage in AD mice were reversed after EAST36. Mechanistically, we revealed that EAST36 restored melatonin levels in AD mice. Melatonin supplement mimicked the EAST36 effect on cerebrovascular protection in AD mice and endothelial cell cultures. Importantly, blockage of melatonin signaling by antagonist blunted EAST36-induced cerebrovascular recovery and subsequent neurological improvement. CONCLUSIONS: These findings provided strong evidence to support EAST36 as a potential nonpharmacological therapy against cerebrovascular impairment in AD. Further study is necessary to better understand how EAST36 treatment drives melatonin signaling.

[Effect of electroacupuncture on renal vascular microcirculation in diabetic mice based on in vivo two-photon microscopy imaging].

OBJECTIVE: To investigate the protective effect of electroacupuncture (EA) at "Zusanli"(ST36)and "Weiwanxiashu"(EX-B3) on capillary function around the renal tubule and renal tubule structure in diabetic mice based on two-photon microscopy (TPM) imaging, so as to providing visualizable evidence for the regulatory effect of EA on diabetic renal vascular microcirculation. METHODS: Spontaneous type Ⅱ diabetes mellitus mice (db/db) were employed for this study. Twenty db/db mice were randomly divided into model group (n=10) and EA group (n=10), and 10 db/m mice used as the control group. EA was applied to bilateral ST36 and EX-B3 for 20 min/time, 6 times a week for 6 weeks. The body weight was recorded and the fasting blood glucose measured before and after the intervention. The urine production and water consumption of mice in each cage were recorded after EA. The renal in vivo imaging method based on TPM was established to display the morphological structure of renal tubules, and the mouse renal blood flow velocity was detected by injecting 500 kDa dextran-fluorescein into femoral vein after the intervention. RESULTS: Compared with the control group, the proportion of mice with decreased body mass in the model group was increased, accounting for 40%, while that in the control group was 0%; and fasting blood glucose, urine production and water consumption were significantly increased in the model group (P<0.001, P<0.000 1). A renal in vivo imaging method based on TPM was successfully established, which can be applied to quantitatively analyze the renal blood flow and renal tubular diameter of mice. Based on this method, the results showed that compared with the control group, the blood flow velocity of peritubular capillary in the model group was significantly decreased (P<0.000 1, P<0.001), renal tubular cells were slightly exfoliated and the diameter of renal tubular was significantly increased (P<0.000 1). Compared with the model group, EA reduced the body weight loss ratio from 40% to 0%, and significantly decreased the fasting blood glucose, urine production and water consumption (P<0.01, P<0.000 1, P<0.001), and the blood flow velocity of peritubular capillary in the EA group was significantly increased (P<0.001, P<0.05) and tubule dilatation significantly alleviated (P<0.0 1). CONCLUSION: EA at ST36 and EX-B3 can ameliorate renal vascular microcirculation disorder to relieve the renal structure damage and improve renal function in diabetes mice.

High-fidelity imaging of amyloid-beta deposits with an ultrasensitive fluorescent probe facilitates the early diagnosis and treatment of Alzheimer's Disease.

Background: Imaging amyloid-beta (Aß) deposits with high fidelity in naturally aging brains is crucial for the early diagnosis of Alzheimer's disease (AD). However, this is impeded by the lack of highly sensitive probes. Methods: By conducting computational modelling to quantitatively fine-tune the twisted intramolecular charge transfer (TICT) tendency of Thioflavin T (ThT) analogues, we developed an ultrasensitive probe AH-2. AH-2 retained the binding affinity and binding mode of ThT towards Aß deposits, and exhibited ca 10-fold less background fluorescence and 5-10 folds of improved signal-to-background contrast upon binding Aß deposits. These desirable features endowed AH-2 the sensitivity to detect Aß deposition in naturally aging wild-type mice. Results: AH-2 imaging revealed that Aß puncta signals appeared near the nuclei in young mice and spread through the intracellular and extracellular compartments in older mice. Moreover, Aß deposits were observed to emerge earlier in mice cerebral cortex than in the hippocampus region. Given this desirable sensitivity and good spatiotemporal resolution, AH-2 was successfully applied in the preclinical evaluation of Aß-targeted treatment by melatonin. Conclusions: We expect that AH-2 is promising for early diagnosis of AD and will serve as a sensitive tool for studying Aß-related AD pathology.

In Situ Imaging of Formaldehyde in Live Mice with High Spatiotemporal Resolution Reveals Aldehyde Dehydrogenase-2 as a Potential Target for Alzheimer's Disease Treatment.

Alterations in formaldehyde (FA) homeostasis are associated with the pathology of Alzheimer's disease (AD). In vivo tracking of FA flux is important for understanding the underlying molecular mechanisms, but is challenging due to the lack of sensitive probes favoring a selective, rapid, and reversible response toward FA. In this study, we re-engineered the promiscuous and irreversible phenylhydrazines to make them selective and reversible toward FA by tuning their nucleophilicity. This effort resulted in PFM309, a selective (selectivity coefficient KFA,methylglyoxal = 0.06), rapid (t1/2 = 32 s at [FA] = 200 µM), and reversible fluorogenic probe (K = 6.24 mM-1) that tracks the FA flux in both live cells and live mice. In vivo tracking of the FA flux was realized by PFM309 imaging, which revealed the gradual accumulation of FA in the live mice brain during normal aging and its further increase in AD mice. We further identified the age-dependent loss of catabolism enzymes ALDH2 and ADH5 as the primary mechanism responsible for formaldehyde excess. Activating ALDH2 with the small molecular activator Alda1 significantly protected neurovascular cells from formaldehyde overload and consequently from impairment during AD progress both in vitro and in vivo. These findings revealed PFM309 as a robust tool to study AD pathology and highlight ALDH2 as a potential target for AD drug development.

Evaluating and Screening of Agro-Physiological Indices for Salinity Stress Tolerance in Wheat at the Seedling Stage.

Soil salinity is a worldwide issue that affects wheat production. A comprehensive understanding of salt-tolerance mechanisms and the selection of reliable screening indices are crucial for breeding salt-tolerant wheat cultivars. In this study, 30 wheat genotypes (obtained from a rapid selection of 96 original varieties) were chosen to investigate the existing screening methods and clarify the salinity tolerance mechanisms in wheat. Ten-day-old seedlings were treated with 150 mM NaCl. Eighteen agronomic and physiological parameters were measured. The results indicated that the effects of salinity on the agronomic and physiological traits were significant. Salinity stress significantly decreased K+ content and K+/Na+ ratio in the whole plant, while the leaf K+/Na+ ratio was the strongest determinant of salinity tolerance and had a significantly positive correlation with salt tolerance. In contrast, salinity stress significantly increased Na+ concentration and relative gene expression (TaHKT1;5, TaSOS1, and TaAKT1-like). The Na+ transporter gene (TaHKT1;5) showed a significantly greater increase in expression than the K+ transporter gene (TaAKT1-like). We concluded that Na+ exclusion rather than K+ retention contributed to an optimal leaf K+/Na+ ratio. Furthermore, the present exploration revealed that, under salt stress, tolerant accessions had higher shoot water content, shoot dry weight and lower stomatal density, leaf sap osmolality, and a significantly negative correlation was observed between salt tolerance and stomatal density. This indicated that changes in stomata density may represent a fundamental mechanism by which a plant may optimize water productivity and maintain growth under saline conditions. Taken together, the leaf K+/Na+ ratio and stomatal density can be used as reliable screening indices for salt tolerance in wheat at the seedling stage.

Immobilization of Fibronectin-Loaded Polyelectrolyte Nanoparticles on Cardiovascular Material Surface to Improve the Biocompatibility.

Vascular stent interventional therapy is the main method for clinical treatment of coronary artery diseases. However, due to the insufficient biocompatibility of cardiovascular materials, the implantation of stents often leads to serious adverse cardiac events. Surface biofunctional modification to improve the biocompatibility of vascular stents has been the focus of current research. In this study, based on the structure and function of extracellular matrix on vascular injury healing, a novel fibronectin-loaded poly-l-lysine/heparin nanoparticles was constructed for stent surface modification. In vitro blood compatibility evaluation results showed that the nanoparticles-modified surface could effectively reduce platelet adhesion and activation. In vitro cellular compatibility evaluation results indicated that the nanocoating may provide adequate efficacy in promoting the adhesion and proliferation of endothelial cells and thereby accelerate endothelialization. This study provides a new approach for the surface biological function modification of vascular stents.

Visualizing Autophagic Flux during Endothelial Injury with a Pathway-Inspired Tandem-Reaction Based Fluorogenic Probe.

Autophagy is a dynamic and complicated catabolic process. Imaging autophagic flux can clearly advance knowledge of its pathophysiology significance. While the most common way autophagy is imaged relies on fluorescent protein-based probes, this method requires substantial genetic manipulation that severely restricts the application. Small fluorescent probes capable of tracking autophagic flux with good spatiotemporal resolution are highly demanable. Methods: In this study, we developed a small-molecule fluorogenic probe (AFG-1) that facilitates real-time imaging of autophagic flux in both intact cells and live mice. AFG-1 is inspired by the cascading nitrosative and acidic microenvironments evolving during autophagy. It operates over two sequential steps. In the first step, AFG-1 responds to the up-regulated peroxynitrite at the initiation of autophagy by its diphenylamino group being oxidatively dearylated to yield a daughter probe. In the second step, the daughter probe responds to the acidic autolysosomes at the late stage of autophagy by being protonated. Results: This pathway-dependent mechanism has been confirmed first by sequentially sensing ONOO- and acid in aqueous solution, and then by imaging autophagic flux in live cells. Furthermore, AFG-1 has been successfully applied to visualize autophagic flux in real-time in live mice following brain ischemic injury, justifying its robustness. Conclusion: Due to the specificity, easy operation, and the dynamic information yielded, AFG-1 should serve as a potential tool to explore the roles of autophagy under various pathological settings.

A switchable [2]rotaxane with two active alkenyl groups.

A novel functional [2]rotaxane containing two alkenyl bonds was designed, synthesized and characterized by 1H, 13C NMR spectroscopy and HRESI mass spectrometry. The introduction of alkenyl bonds endowed the [2]rotaxane a fascinating ability to react with versatile functional groups such as alkenyl and thiol functional groups. The reversible shuttling movement of the macrocycle between two different recognition sites on the molecular thread can be driven by external acid and base. This kind of rotaxane bearing functional groups provides a powerful platform for preparing stimuli-responsive polymers.

Myt1l induced direct reprogramming of pericytes into cholinergic neurons.

OBJECTIVE: The cholinergic deficit is thought to underlie progressed cognitive decline in Alzheimer Disease. The lineage reprogramming of somatic cells into cholinergic neurons may provide strategies toward cell-based therapy of neurodegenerative diseases. METHODS AND RESULTS: Here, we found that a combination of neuronal transcription factors, including Ascl1, Myt1l, Brn2, Tlx3, and miR124 (5Fs) were capable of directly converting human brain vascular pericytes (HBVPs) into cholinergic neuronal cells. Intriguingly, the inducible effect screening of reprogramming factors showed that a single reprogramming factor, Myt1l, induced cells to exhibit similarly positive staining for Tuj1, MAP2, ChAT, and VAChT upon lentivirus infection with the 5Fs after 30 days. HBVP-converted neurons were rarely labeled even after long-term incubation with BrdU staining, suggesting that induced neurons were directly converted from HBVPs rather than passing through a proliferative state. In addition, the overexpression of Myt1l induced the elevation of Ascl1, Brn2, and Ngn2 levels that contributed to reprogramming. CONCLUSIONS: Our findings provided proof of the principle that cholinergic neurons could be produced from HBVPs by reprogramming factor-mediated fate instruction. Myt1l was a critical mediator of induced neuron cell reprogramming. HBVPs represent another excellent alternative cell resource for cell-based therapy to treat neurodegenerative disease.

Changes in mineral elements and starch quality of grains during the improvement of japonica rice cultivars.

BACKGROUND: The improvement of rice cultivars plays an important role in yield increase. However, little is known about the changes in starch quality and mineral elements during the improvement of rice cultivars. This study was conducted to investigate the changes in starch quality and mineral elements in japonica rice cultivars. RESULTS: Twelve typical rice cultivars, applied in the production in Jiangsu province during the last 60 years, were grown in the paddy fields. These cultivars were classified into six types according to their application times, plant types and genotypes. The nitrogen (N), phosphorus (P) and, and potassium (K) were mainly distributed in endosperm, bran and bran, respectively. Secondary and micromineral nutrients were distributed throughout grains. With the improvement of cultivars, total N contents gradually decreased, while total P, K and magnesium contents increased in grains. Total copper and zinc contents in type 80'S in grains were highest. The improvement of cultivars enhanced palatability (better gelatinisation enthalpy and amylose content), taste (better protein content) and protein quality (better protein components and essential amino acids). Correlation analysis indicated the close relationship between mineral elements and starch quality. CONCLUSION: The mineral elements and starch quality of grains during the improvement of japonica rice cultivars are improved. © 2017 Society of Chemical Industry.

Cholinergic Grb2-Associated-Binding Protein 1 Regulates Cognitive Function.

Grb2-associated-binding protein 1 (Gab1) is a docking/scaffolding molecule known to play an important role in cell growth and survival. Here, we report that Gab1 is decreased in cholinergic neurons in Alzheimer's disease (AD) patients and in a mouse model of AD. In mice, selective ablation of Gab1 in cholinergic neurons in the medial septum impaired learning and memory and hippocampal long-term potentiation. Gab1 ablation also inhibited SK channels, leading to an increase in firing in septal cholinergic neurons. Gab1 overexpression, on the other hand, improved cognitive function and restored hippocampal CaMKII autorphosphorylation in AD mice. These results suggest that Gab1 plays an important role in the pathophysiology of AD and may represent a novel therapeutic target for diseases involving cholinergic dysfunction.

Endothelial ErbB4 deficit induces alterations in exploratory behavior and brain energy metabolism in mice.

AIMS: The receptor tyrosine kinase ErbB4 is present throughout the primate brain and has a distinct functional profile. In this study, we investigate the potential role of endothelial ErbB4 receptor signaling in the brain. RESULTS: Here, we show that the endothelial cell-specific deletion of ErbB4 induces decreased exploratory behavior in adult mice. However, the water maze task for spatial memory and the memory reconsolidation test reveal no changes; additionally, we observe no impairment in CaMKII phosphorylation in Cdh5Cre;ErbB4f/f mice, which indicates that the endothelial ErbB4 deficit leads to decreased exploratory activity rather than direct memory deficits. Furthermore, decreased brain metabolism, which was measured using micro-positron emission tomography, is observed in the Cdh5Cre;ErbB4f/f mice. Consistently, the immunoblot data demonstrate the downregulation of brain Glut1, phospho-ULK1 (Ser555), and TIGAR in the endothelial ErbB4 conditional knockout mice. Collectively, our findings suggest that endothelial ErbB4 plays a critical role in regulating brain function, at least in part, through maintaining normal brain energy homeostasis. CONCLUSIONS: Targeting ErbB4 or the modulation of endothelial ErbB4 signaling may represent a rational pharmacological approach to treat neurological disorders.

Nitration of TRPM2 as a Molecular Switch Induces Autophagy During Brain Pericyte Injury.

AIMS: Dysfunction of neurovascular pericytes underlies breakdown of the blood-brain barrier, but the molecular mechanisms are largely unknown. In this study, we evaluated the role of the transient receptor potential melastatin-related 2 (TRPM2) channel and autophagy during brain pericyte injury both in vitro and in vivo. RESULTS: A rapid induction in autophagy in human brain vascular pericytes, in the zinc oxide nanoparticles (ZnO-NP)-induced cell stress model, was paralleled with an increase in the expression of the TRPM2-S truncated isoform, which was abolished by treatment with a nitric oxide synthase inhibitor and a peroxynitrite scavenger. Furthermore, Y1485 in the C-terminus of the TRPM2 protein was identified as the tyrosine nitration substrate by mass spectrometry. Overexpression of the Y1485S TRPM2 mutant reduced LC3-II accumulation and pericyte injury induced by ZnO-NP. Consistently, LC3-II accumulation was reduced and pericytes were better preserved in intact brain microvessels of the TRPM2 knockout mice after ZnO-NP-induced vascular injury. Innovation and Conclusions: Our present study has revealed a novel mechanism of autophagy disturbance secondary to nitrosative stress-induced tyrosine nitration of TRPM2 during pericyte injury. Antioxid. Redox Signal. 27, 1297-1316.

Functional Genomic Analyses Identify Pathways Dysregulated in Animal Model of Autism.

BACKGROUND: Autism spectrum disorders (ASDs) are a heterogeneous group of neurodevelopmental disorders that display complicated behavioral symptoms. METHODS: Using gene expressing profiling and the weighted gene co-expression network analysis (WGCNA), we studied genes coregulated by similar factors such as genetic variants or environmental effects in the hippocampus in an animal model of autism. RESULTS: From microarray data, we identified 21,388 robustly expressed genes of which 721 genes were found to be differently expressed in the valproic acid-treated group compared to the control group. WGCNA identified multiple co-expression modules known to associate with cognitive function, inflammation, synaptic, and positive regulation of protein kinase activating. Many of these modules, however, have not been previously linked to autism spectrum disorders which included G-protein signaling, immunity, and neuroactive ligand-receptor interaction pathway. The downregulation of the highly connected (hub) genes Taar7h and Taar7b in neuroactive ligand-receptor interaction pathway was validated by qRT-PCR. Immunoblotting and immunohistochemistry further showed that TAAR7 expression was downregulated not only in valproic acid-treated animals, but also BTBR T+tf/J mice. CONCLUSIONS: This study highlights the advantages of gene microarrays to uncover co-expression modules associated with autism and suggests that Taars and related gene regulation networks may play a significant role in autism.

Calpain-Dependent ErbB4 Cleavage Is Involved in Brain Ischemia-Induced Neuronal Death.

Disturbance of neuregulin-1ß/ErbB4 signaling is considered to be associated with brain ischemia, but the mechanisms of this disruption are largely unknown. In the present study, we provide evidence that degradation of ErbB4 is involved in neuronal cell death in response to ischemia. Our data showed that the application of neuregulin-1ß provided significant protection against oxygen-glucose deprivation (OGD)-induced neuronal death as detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, annexin V/propidium iodide flow cytometry analysis and terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) staining. Furthermore, neuregulin-1ß treatment significantly reduced the infarct volume of ischemic mice, and this result was not seen in the ErbB4 knockout mice. We found that brain ischemia induced the breakdown of ErbB4 in a time-dependent manner in vivo, but not that of ErbB2. In vitro studies further indicated that recombinant calpain induced the cleavage of ErbB4 in a dose-dependent way, whereas the calpain inhibitor significantly reduced the OGD-induced ErbB4 breakdown. Additionally, OGD-induced apoptosis was partially abolished by transfection with the ErbB4E872K mutant. Taken together, neuregulin-1ß elicits its neuroprotective effect in an ErbB4-dependent manner, and the cleavage of ErbB4 by calpain contributes to a neuronal cell death cascade during brain ischemia.

Valproic Acid Influences MTNR1A Intracellular Trafficking and Signaling in a ß-Arrestin 2-Dependent Manner.

Valproate exposure is associated with increased risks of autism spectrum disorder. To date, the mechanistic details of disturbance of melatonin receptor subtype 1 (MTNR1A) internalization upon valproate exposure remain elusive. By expressing epitope-tagged receptors (MTNR1A-EGFP) in HEK-293 and Neuro-2a cells, we recorded the dynamic changes of MTNR1A intracellular trafficking after melatonin treatment. Using time-lapse confocal microscopy, we showed in living cells that valproic acid interfered with the internalization kinetics of MTNR1A in the presence of melatonin. This attenuating effect was associated with a decrease in the phosphorylation of PKA (Thr197) and ERK (Thr202/Tyr204). VPA treatment did not alter the whole-cell currents of cells with or without melatonin. Furthermore, fluorescence resonance energy transfer imaging data demonstrated that valproic acid reduced the melatonin-initiated association between YFP-labeled ß-arrestin 2 and CFP-labeled MTNR1A. Together, we suggest that valproic acid influences MTNR1A intracellular trafficking and signaling in a ß-arrestin 2-dependent manner.

Visualizing peroxynitrite fluxes in endothelial cells reveals the dynamic progression of brain vascular injury.

Accumulating evidence suggests that formation of peroxynitrite (ONOO(-)) in the cerebral vasculature contributes to the progression of ischemic damage, while the underlying molecular mechanisms remain elusive. To fully understand ONOO(-) biology, efficient tools that can realize the real-time tracing of endogenous ONOO(-) fluxes are indispensable. While a few ONOO(-) fluorescent probes have been reported, direct visualization of ONOO(-) fluxes in the cerebral vasculature of live mice remains a challenge. Herein, we present a fluorescent switch-on probe (NP3) for ONOO(-) imaging. NP3 exhibits good specificity, fast response, and high sensitivity toward ONOO(-) both in vitro and in vivo. Moreover, NP3 is two-photon excitable and readily blood-brain barrier penetrable. These desired photophysical and pharmacokinetic properties endow NP3 with the capability to monitor brain vascular ONOO(-) generation after injury with excellent temporal and spatial resolution. As a proof of concept, NP3 has enabled the direct visualization of neurovascular ONOO(-) formation in ischemia progression in live mouse brain by use of two-photon laser scanning microscopy. Due to these favorable properties, NP3 holds great promise for visualizing endogenous peroxynitrite fluxes in a variety of pathophysiological progressions in vitro and in vivo.