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AP-1 and AP-2 adaptor protein (AP) complexes mediate clathrin-dependent trafficking at the trans-Golgi network (TGN) and the plasma membrane, respectively. Whereas AP-1 is required for trafficking to plasma membrane and vacuoles, AP-2 mediates endocytosis. These AP complexes consist of four subunits (adaptins): two large subunits (β1 and γ for AP-1 and β2 and α for AP-2), a medium subunit μ, and a small subunit σ. In general, adaptins are unique to each AP complex, with the exception of β subunits that are shared by AP-1 and AP-2 in some invertebrates. Here, we show that the two putative Arabidopsis thaliana AP1/2β adaptins co-assemble with both AP-1 and AP-2 subunits and regulate exocytosis and endocytosis in root cells, consistent with their dual localization at the TGN and plasma membrane. Deletion of both β adaptins is lethal in plants. We identified a critical role of β adaptins in pollen wall formation and reproduction, involving the regulation of membrane trafficking in the tapetum and pollen germination. In tapetal cells, β adaptins localize almost exclusively to the TGN and mediate exocytosis of the plasma membrane transporters such as ATP-binding cassette (ABC)G9 and ABCG16. This study highlights the essential role of AP1/2β adaptins in plants and their specialized roles in specific cell types.

Clathrin plays a critical role in clathrin-mediated endocytosis (CME) in plants, and it is required for autophagy in mammals. However, the functional interconnection of clathrin with autophagy has not been firmly established in plants. Here, we demonstrate that loss of function of clathrin light chain (CLC) subunit 2 and 3 results in salicylic acid (SA)- and H2O2-dependent accelerated senescence and activated defense responses in Arabidopsis, which are hallmarks of the autophagy-related gene (ATG) mutants. Similar toatgmutants, theclc2-1clc3-1double mutant has enhanced sensitivity to both carbon and nitrogen starvation and enhanced resistance to biotrophic bacterial and fungal pathogens. In addition, the autophagy flux was significantly reduced in the roots ofclc2-1clc3-1mutant plants relative to Col-0 plants under carbon starvation conditions. Furthermore, our Yeast-2-hybrid (Y2H) and Luciferase complementation assays showed that CLC2 directly interacted with ATG8h and ATG8i. Mutations within the unique ATG8-interacting motif (AIM) of CLC2 as well as at the LIR/AIM-docking site (LDS) of ATG8h abolished the interaction between CLC2 and ATG8h. As anticipated, both GFP-ATG8h/GFP-ATG8i and CLC2 were subjected to autophagic degradation in the vacuoles. Together, our data revealed that the accelerated senescence and activated immune responses observed in Arabidopsisclc2-1clc3-1mutant plants result from impaired autophagy, and CLC2 participates in autophagy through direct interactions with ATG8h and ATG8i in an AIM1- and LDS-dependent manner. Our results unveil a previously unidentified link between the function of CLCs and autophagy.

Primexine deposition is essential for the formation of pollen wall patterns and is precisely regulated by the tapetum and microspores. While tapetum- and/or microspore-localized proteins are required for primexine biosynthesis, how their trafficking is established and controlled is poorly understood. In Arabidopsis thaliana, AP1σ1 and AP1σ2, two genes encoding the σ subunit of the trans-Golgi network/early endosome (TGN/EE)-localized ADAPTOR PROTEIN-1 complex (AP-1), are partially redundant for plant viability, and the loss of AP1σ1 function reduces male fertility due to defective primexine formation. Here, we investigated the role of AP-1 in pollen wall formation. The deposition of Acyl-CoA SYNTHETASE5 (ACOS5) and type III LIPID TRANSFER PROTEINs (LTPs) secreted from the anther tapetum, which are involved in exine formation, were impaired in ap1σ1 mutants. In addition, the microspore plasma membrane (PM) protein RUPTURED POLLEN GRAIN1 (RPG1), which regulates primexine deposition, accumulated abnormally at the TGN/EE in ap1σ1 mutants. We show that AP-1μ recognizes the YXXΦ motif of RPG1, thereby regulating its PM abundance through endocytic trafficking, and that loss of AP1σ1 decreases the levels of other AP-1 subunits at the TGN/EE. Our observations show that AP-1-mediated post-Golgi trafficking plays a vital role in pollen wall development by regulating protein transport in tapetal cells and microspores.

PIN-FORMED (PIN)-dependent directional auxin transport is crucial for plant development. Although the redistribution of auxin mediated by the polarization of PIN3 plays key roles in modulating hypocotyl cell expansion, how PIN3 becomes repolarized to the proper sites within hypocotyl cells is poorly understood. We previously generated the clathrin light chain clc2-1 clc3-1 double mutant in Arabidopsis thaliana and found that it has an elongated hypocotyl phenotype compared to the wild type. Here, we performed genetic, cell biology, and pharmacological analyses combined with live-cell imaging to elucidate the molecular mechanism underlying the role of clathrin light chains in hypocotyl elongation. Our analyses indicated that the defects of the double mutant enhanced auxin maxima in epidermal cells, thus, promoting hypocotyl elongation. PIN3 relocated to the lateral sides of hypocotyl endodermal cells in clc2-1 clc3-1 mutants to redirect auxin toward the epidermal cell layers. Moreover, the loss of function of PIN3 largely suppressed the long hypocotyl phenotype of the clc2-1 clc3-1 double mutant, as did treatment with auxin transport inhibitors. Based on these data, we propose that clathrin modulates PIN3 abundance and polarity to direct auxin flux and inhibit cell elongation in the hypocotyl, providing novel insights into the regulation of hypocotyl elongation. This article is protected by copyright. All rights reserved.

Clathrin-mediated endocytosis (CME) is the major endocytic pathway in eukaryotic cells that directly regulates abundance of plasma membrane (PM) proteins. Clathrin triskelia are composed of clathrin heavy chains (CHCs) and light chains (CLCs), and the phytohormone auxin differentially regulates membrane-associated CLCs and CHCs, modulating the endocytosis and therefore the distribution of auxin efflux transporter PIN-FORMED2 (PIN2). However, the molecular mechanisms by which auxin regulates clathrin are still poorly understood. Transmembrane kinase family proteins (TMKs) are considered to contribute to auxin signaling and plant development, it remains unclear whether they are involved in PIN transport by CME. We assessed TMKs involvement in the regulation of clathrin by auxin, using genetic, pharmacological, and cytological approaches including live-cell imaging and immunofluorescence. In tmk1 mutant seedlings, auxin failed to rapidly regulate abundance of both CHC and CLC and to inhibit PIN2 endocytosis, leading to an impaired asymmetric distribution of PIN2 and therefore auxin. Furthermore, TMK3 and TMK4 were shown not involved in regulation of clathrin by auxin. In summary, TMK1 is essential for auxin-regulated clathrin recruitment and CME. TMK1 therefore plays a critical role in the establishment of an asymmetric distribution of PIN2 and an auxin gradient during root gravitropism. This article is protected by copyright. All rights reserved.

Clathrin-dependent endocytosis and exocytosis are coordinated to maintain cell homeostasis in Arabidopsis roots., Coupling of post-Golgi and endocytic membrane transport ensures that the flow of materials to/from the plasma membrane (PM) is properly balanced. The mechanisms underlying the coordinated trafficking of PM proteins in plants, however, are not well understood. In plant cells, clathrin and its adaptor protein complexes, AP-2 and the TPLATE complex (TPC) at the PM, and AP-1 at the trans-Golgi network/early endosome (TGN/EE), function in clathrin-mediated endocytosis (CME) and post-Golgi trafficking. Here, we utilized mutants with defects in clathrin-dependent post-Golgi trafficking and CME, in combination with other cytological and pharmacological approaches, to further investigate the machinery behind the coordination of protein delivery and recycling to/from the TGN/EE and PM in Arabidopsis (Arabidopsis thaliana) root cells. In mutants with defective AP-2-/TPC-dependent CME, we determined that clathrin and AP-1 recruitment to the TGN/EE as well as exocytosis are significantly impaired. Likewise, defects in AP-1-dependent post-Golgi trafficking and pharmacological inhibition of exocytosis resulted in the reduced association of clathrin and AP-2/TPC subunits with the PM and a reduction in the internalization of cargoes via CME. Together, these results suggest that post-Golgi trafficking and CME are coupled via modulation of clathrin and adaptor protein complex recruitment to the TGN/EE and PM.

Background Nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) plays an important role in the formation of oxidative stress in brain tissues. We intended to investigate relationship between serum NOX4 concentrations and severity, delayed cerebral ischemia (DCI) plus prognosis after aneurysmal subarachnoid hemorrhage (aSAH). Methods Serum NOX4 concentrations were gauged in a total of 165 aSAH patients. The World Federation of Neurological Surgeons (WFNS) scale and modified Fisher grading scale were recorded for assessing hemorrhagic severity. Relations of serum NOX4 concentrations to DCI and 90-day poor outcome (Glasgow outcome scale score of 1–3) were determined using multivariate analysis. Results Serum NOX4 concentrations were substantially higher in patients with 90-day poor outcome or DCI than in other remainders. Serum NOX4 concentrations of patients were intimately correlated with WFNS scores and modified Fisher scores. Serum NOX4 appeared as an independent predictor for DCI and 90-day poor outcome after aSAH. Under ROC curve analysis, serum NOX4 concentrations possessed significantly high predictive capability for DCI and 90-day poor outcome following hemorrhagic stroke. Conclusions Serum NOX4 concentrations, in close correlation with hemorrhagic severity, were independently associated with DCI and poor clinical outcome after hemorrhagic stroke, substantializing serum NOX4 as a promising prognostic biomarker for aSAH.

The blood‐brain barrier (BBB) damage is a momentous pathological process of ischaemic stroke. NADPH oxidases 4 (NOX4) boosts BBB damage after ischaemic stroke and its expression can be influenced by microRNAs. This study aimed to probe into whether miR‐92b influenced the BBB damage after ischaemic stroke by regulating NOX4 expression. Here, miR‐92b expression was lessened in the ischaemic brains of rats and oxygen‐glucose deprivation (OGD)‐induced brain microvascular endothelial cells (BMECs). In middle cerebral artery occlusion (MCAo) rats, miR‐92b overexpression relieved the ameliorated neurological function and protected the BBB integrity. In vitro model, miR‐92b overexpression raised the viability and lessened the permeability of OGD‐induced BMECs. miR‐92b targeted NOX4 and regulated the viability and permeability of OGD‐induced BMECs by negatively modulating NOX4 expression. The transcription factor Foxo1 bound to the miR‐92b promoter and restrained its expression. Foxo1 expression was induced by OGD‐induction and its knockdown abolished the effects of OGD on miR‐92b and NOX4 expressions, cell viability and permeability of BMECs. In general, our findings expounded that Foxo1‐induced lessening miR‐92b boosted BBB damage after ischaemic stroke by raising NOX4 expression.

Cerebral ischemia/reperfusion (IR) after ischemic stroke causes deleterious microglial activation. Protein tyrosine phosphatase 1B (PTP1B) exacerbates neuroinflammation, yet the effect of the inhibition on microglial activation and cerebral IR injury is unknown. A cerebral IR rat model was induced by middle cerebral artery occlusion (MCAO) and reperfusion. The PTP1B inhibitor, sc-222227, was administered intracerebroventricularly. Neurologic deficits, infarct volume, and brain water content were examined. An in vitro oxygen glucose deprivation/reoxygenation (OGD/R) model was established in primary microglia and BV-2 cells. Microglial activation/polarization, endoplasmic reticulum (ER) stress, autophagy, and apoptosis were detected using western blot, immunohistology, ELISA, and real-time PCR. Protein interaction was assessed by a proximity ligation assay. The results showed a significant increase in microglial PTP1B expression after IR injury. Sc-222227 attenuated IR-induced microglial activation, ER stress, and autophagy and promoted M2 polarization. Upon OGD/R, sc-222227 mitigated microglial activation by inhibiting ER stress-dependent autophagy, the effect of which was abolished by PERK activation, and PERK inhibition attenuated microglial activation. The PTP1B-phosphorylated PERK protein interaction was significantly increased after OGD/R, but decreased upon sc-222227 treatment. Finally, sc-222227 mitigated neuronal damage and neurologic deficits after IR injury. Treatment targeting microglial PTP1B might be a potential therapeutic strategy for ischemic stroke treatment.

The surface nuclear magnetic resonance (SNMR) method is widely used in groundwater detection because of its sensitivity to hydrogen in water and direct water detection. However, low signal-to-noise ratios (SNRs) restrict the development of this technique. An optimum pulse sequence is designed according to correspondence between the pulse moment strength and its best detection depth. Because only selection of the pulse intensity distribution according to the target aquifer depth is required and the "on-resonance" pulse pattern is still employed, this pulse sequence emission can be easily achieved using existing SNMR instrumentation. Numerical simulation results and field experiments show that, compared with traditional exponential growth pulses, the optimum pulse sequence effectively improves the SNR of the SNMR method. The aquifer boundary, water content, and pore characteristics of the inversion result are thus more consistent with characteristics of underground structures. Additionally, because the optimum pulse sequence focuses most of the pulse moments in the target depth range, in situations where two aquifers are separated by a relatively narrow aquitard, it is better able to resolve the individual aquifers than the traditional pulses. Optimum pulse moments improve the SNR by enhancing the signal amplitude, compared with various filtering methods, and obtain a better detection effect. This kind of pulse sequence can be used as an alternative pulse sequence form of the SNMR method.

The JinDeng Temple grottoes are large-scale grottoes that were dug in natural cliffs more than 500 years ago. The grotto art provides an important legacy and is of high historical, cultural, and artistic value. Over the course of more than 500 years, some areas of the grottoes have been seriously affected by seepage damage. Non-destructive detection methods (surface nuclear magnetic resonance and spontaneous potential) were used to investigate the cause of the seepage. The surface nuclear magnetic resonance method was successfully used to detect the spatial occurrence of groundwater and the water content per unit volume in the mountain above the grottoes. The results of spontaneous potential revealed the recharge and flow directions in the upper part of the mountain. The two methods used were complementary and provided mutually consistent results regarding the cause of water seepage in the grottoes, and the relationship between the positions of the water-bearing areas in the upper part of the mountain and the seepage. In addition, the two methods provided important basic data for the formulation of a work plan to protect and preserve the cultural relics.

Background. Moyamoya disease (MMD) is a progressive occlusive cerebrovascular disease that is characterized by abnormal angiogenesis at the base of the brain. This pathological abnormal angiogenesis is susceptible to disturbances, including spontaneous hemorrhage and vasogenic edema. However, the underlying mechanisms of pathological angiogenesis and occurrence of hemorrhage are unclear. Angiopoietins play a fundamental role in the pathophysiology of central nervous system disorders in angiogenesis. This study was aimed at examining whether angiopoietins are associated with formation of abnormal collateral vessels and the occurrence of hemorrhage in adult-onset moyamoya disease (HMMD). Methods. A total of 27 consecutive adult patients with HMMD were enrolled from June 2011 to May 2017. Serum levels of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) were examined by enzyme-linked immunosorbent assay. Patients with HMMD were compared with those with spontaneous hemorrhage (controls) and nonhemorrhagic-onset MMD (NHMMD). Results. Serum Ang-2 levels were significantly higher in patients with adult HMMD than in those with spontaneous hemorrhage and NHMMD. The ROC curve identified that a baseline serum Ang-2 level>1230 ng/ml may be associated with adult HMMD with 88.39% sensitivity and 70.37% specificity (area under the curve (AUC), 0.89; 95% CI, 0.808-0.973; P<0.001). Moreover, serum Ang-2 levels were significantly elevated in stages II, III, and IV. In subgroup analysis of a high and low degree of moyamoya vessels, serum Ang-2 levels were significantly higher in the high moyamoya vessel group than in the low moyamoya vessel group. Serum Ang-2 levels were also significantly higher in the low moyamoya vessel group compared with the control group. Serum Ang-1 levels were not significantly different among the groups. Conclusion. Increased serum Ang-2 levels may contribute to pathological abnormal angiogenesis and/or to the instability of vascular structure and function, thus causing brain hemorrhage in adult HMMD.

Rationale: Maintaining iron homeostasis is essential for proper cardiac function. Both iron deficiency and iron overload are associated with cardiomyopathy and heart failure via complex mechanisms. Although ferritin plays a central role in iron metabolism by storing excess cellular iron, the molecular function of ferritin in cardiomyocytes remains unknown. Objective: To characterize the functional role of Fth (ferritin H) in mediating cardiac iron homeostasis and heart disease. Methods and Results: Mice expressing a conditional Fth knockout allele were crossed with 2 distinct Cre recombinase-expressing mouse lines, resulting in offspring that lack Fth expression specifically in myocytes (MCK-Cre) or cardiomyocytes (Myh6-Cre). Mice lacking Fth in cardiomyocytes had decreased cardiac iron levels and increased oxidative stress, resulting in mild cardiac injury upon aging. However, feeding these mice a high-iron diet caused severe cardiac injury and hypertrophic cardiomyopathy, with molecular features typical of ferroptosis, including reduced glutathione (GSH) levels and increased lipid peroxidation. Ferrostatin-1, a specific inhibitor of ferroptosis, rescued this phenotype, supporting the notion that ferroptosis plays a pathophysiological role in the heart. Finally, we found that Fth-deficient cardiomyocytes have reduced expression of the ferroptosis regulator Slc7a11, and overexpressing Slc7a11 selectively in cardiomyocytes increased GSH levels and prevented cardiac ferroptosis. Conclusions: Our findings provide compelling evidence that ferritin plays a major role in protecting against cardiac ferroptosis and subsequent heart failure, thereby providing a possible new therapeutic target for patients at risk of developing cardiomyopathy.

Background. Buyang Huanwu decoction (BYHWD), an important traditional Chinese medicine (TCM), has been used clinically for centuries for the treatment of various diseases. The study aims to explore the BYHWD effects on angiogenesis and neuroprotection after cerebral ischemia/reperfusion (CI/R) injury in rats and to explore the underlying angiogenic roles and mechanisms of BYHWD in hydrogen peroxide (H2O2) induced oxidative stress in human umbilical vein endothelial cells (HUVECs) model. Methods. The effects of BYHWD on neurological function were screened by measuring neurological deficits, spatial memory function, and angiogenesis (by microvascular density (MVD) and cerebral blood flow (CBF)) after CI/R injury in middle cerebral artery occlusion (MCAO) in vivo in rats. In vitro, we examined the angiogenic roles and mechanisms of action of BYHWD in an H2O2-induced oxidative stress HUVECs model by measuring cell viability, apoptosis, vascular tube formation, intracellular ROS generation, NADPH oxidase (Nox) activity, and Nox4 protein expression. Results. BYHWD significantly improved neurological function, including neurological deficits and spatial learning and memory, and significantly increased MVD and CBF in the ischemic penumbra after CI/R injury in rats. BYHWD significantly increased cell viability, inhibited apoptosis, induced vascular tube formation, decreased intracellular ROS generation, and reduced Nox activity and Nox4 protein expression in H2O2-treated HUVECs in a dose-dependent manner. Conclusions. Our study demonstrates that BYHWD promotes neurological function recovery and increases angiogenesis. BYHWD exerts angiogenic effects against cerebral ischemic injury through the downregulation of Nox4, which results in the reduction of ROS generation.

Mass spectrometry analyses of Arabidopsis suspension-cultured cell clathrin-coated vesicles delineate the plant clathrin-coated vesicle (CCV) proteome and identify evolutionarily conserved and plant-specific CCV-associated factors. In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor-protein assembly polypeptide-1 (AP-1) complex operates as part of the secretory pathway at the trans-Golgi network (TGN), while the AP-2 complex and the TPLATE complex jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant-specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched TGN/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis (Arabidopsis thaliana) cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data.

Intracranial atherosclerotic disease (ICAD) is a common cause of stroke with high rates of ischemic recurrence. We aimed to investigate the role of circulating exosomal microRNAs (e-miRNAs) in recurrent ischemic events in ICAD. Consecutive patients with severe ICAD undergoing intensive medical management (IMM) were prospectively enrolled. Those with recurrent ischemic events despite IMM during 6-month follow up were algorithmically matched to IMM responders. Baseline blood e-miRNA expression levels of the matched patients were measured using next generation sequencing. A total of 122 e-miRNAs were isolated from blood samples of 10 non-responders and 11 responders. Thirteen e-miRNAs predicted IMM failure with 90% sensitivity and 100% specificity. Ingenuity pathway analysis (IPA) determined 10 of the 13 e-miRNAs were significantly associated with angiogenesis-related biological functions (p

ObjectiveTo investigate the correlation between gene mutations and glucocorticoid resistance in pediatric acute lymphoblastic leukemia (ALL).MethodsA total of 71 children with ALL admitted to our center between September 2019 and September 2021 were enrolled. DNA obtained from bone marrow or peripheral blood samples at initial diagnosis was used for genetic testing via whole exome sequencing. Meanwhile, patient clinical information was collected. Subsequently, the correlations of gene mutations with clinical features and glucocorticoid resistance were analyzed.ResultsOf the 71 children enrolled, 61 (85.9%) had B-cell ALL (B-ALL) and 10 (14.1%) had T-cell ALL (T-ALL). The five genes with the highest mutation frequency in B-ALL were TTN (24.4%), FLT3 (14.6%), TP53 (14.6%), MUC16 (9.8%), and EPPK1 (9.8%). In contrast, those with the highest frequency in T-ALL were NOTCH1 (54.5%), FBXW7 (27.3%), TTN (27.3%), MUC16 (27.3%), and PHF6 (18.2%). Upon statistical analysis, TTN and NOTCH1 mutations were found to be associated with prednisone resistance. Further, TTN and MUC16 mutations were associated with a lower age at diagnosis, and NOTCH1 mutations were associated with T-ALL in female patients. Leukocyte counts and LDH levels did not differ based on the presence of any common gene mutation, and no association between these gene mutations and overall survival was observed.ConclusionsOur study is the first to demonstrate the association between TTN mutation and glucocorticoid resistance in ALL. Our findings could guide strategies for overcoming drug resistance and aid in the development of drug targets.

Objective: We aimed to investigate the distribution of [68Ga]Ga-p14-032, a novel PET ligand that binds to vascular amyloid, in patients diagnosed clinically with probable cerebral amyloid angiopathy (CAA) compared with patients with Alzheimer's disease (AD) and normal controls (NC).Methods: This longitudinal cohort study was composed of 10 subjects (three probable CAA patients, two AD patients, five NC subjects), recruited from a clinic in China. CAA patients had a history of lobar intracerebral hemorrhage (ICH) and met modified Boston criteria for probable CAA. All participants were aged at least 55 years and underwent [68Ga] Ga-p14-032 PET/CT or/and PET/MRI, and the Montreal Cognitive Assessment on initial assessment. Demographics were measured at baseline (diabetes, hypertension, hypercholesterolemia, ischemic stroke, and ICH). Two PET imaging experts reviewed the PET images with cortical standardized uptake value ratio (SUVr) displayed on a color scale and visually classified the images as positive or negative. The mean of SUVr was calculated using the pons as reference.Results: In CAA patients, PET scans were positive in regions with higher numbers of CMBs. No significant signal was seen in AD subjects or controls. The relative [68Ga]Ga-p14-032 retention in the cortex was stronger in patients with CAA than AD and NC (median SUVr 2.68 ± 1.53 vs. 1.77 ± 0.08 and 0.83 ± 0.24).Conclusions: Our results provide early evidence that the [68Ga] Ga-p14-032 PET probe binds preferentially to vascular amyloid and may be a useful tracer for diagnosing CAA.

In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor- protein AP-1 complex operates as part of the secretory pathway at the trans-Golgi network, while the AP-2 complex and the TPLATE complex (TPC) jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant- specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched trans-Golgi network/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data.

The treatment for patients with poor-grade aneurysms defined as World Federation of Neurosurgical Societies (WFNS) grade IV-V is still unclear and controversial. In this research, we compared the clinical efficacy and safety between clipping and coiling in the treatment of ruptured poor-grade anterior circulation aneurysms.We conducted a retrospective analysis of a hospital database. From January 2013 to May 2018, 94 patients who presented with poor-grade anterior circulation aneurysms were included. Preoperative baseline, postprocedure complications, and outcome (3-month and 6-month modified Rankin Scale scores) were analyzed. Multivariate logistic regression analysis was conducted to identify risk factors of short-term (in-hospital, 30-day) mortality.A total of 21 (22%) patients died during short-term follow-up; there was a greater short-term mortality in coiling group (38% vs. 15%, P = 0.015). The incidence of delayed cerebral ischemia and intracranial infection in the clipping group was significantly greater than the coiling group: (33% vs. 14%, P = 0.045) and (68% vs. 41%, P = 0.016). However, coiling group had a greater rate of shunt-dependent hydrocephalus (21% vs. 6%, P = 0.035). Multivariate logistic regression analysis revealed cerebral vasospasm (odds ratio [OR], 9.22; P0.01), admission WFNS grade V (OR, 15.43; P0.01), coiling (OR, 5.92; P = 0.013), and postoperative aneurysm rebleeding (OR, 40.04; P = 0.01) would influence the mortality.Patients with ruptured poor-grade anterior circulation aneurysms who undergo microsurgical clipping seem to have a lower short-term mortality. Cerebral vasospasm, WFNS grade V, and postoperative aneurysm rebleeding are associated with short-term mortality.

Background Vascular endothelial growth factor-A165 (VEGF-A165) has been identified as a combination of 2 alternative splice variants: proangiogenic VEGF-A165a and antiangiogenic VEGF-A165b. Intracranial atherosclerotic disease (ICAD) and moyamoya disease (MMD) are 2 main types of intracranial arterial steno-occlusive disorders with distinct capacities for collateral formation. Recent studies indicate that VEGF-A165 regulates collateral growth in ischemia. Therefore, we investigated if there is a distinctive composition of VEGF-A165 isoforms in ICAD and MMD. Methods Sixty-six ICAD patients, 6 MMD patients, and 5 controls were enrolled in this prospective study. ICAD and MMD patients received intensive medical management upon enrollment. Surgery was offered to 9 ICAD patients who had recurrent ischemic events, 6 MMD patients, and 5 surgical controls without ICAD. VEGF-A165a and VEGF-A165b plasma levels were measured at baseline, within 1 week after patients having surgery, and at 1, 3, and 6 months after treatment. Results A significantly higher baseline VEGF-A165a/b ratio was observed in MMD compared to ICAD (P = .016). The VEGF-A165a/b ratio increased significantly and rapidly after surgical treatment in ICAD (P = .026) more so than in MMD and surgical controls. In patients with ICAD receiving intensive medical management, there was also an elevation of the VEGF-A165a/b ratio, but at a slower rate, reaching the peak at 3 months after initiation of treatment (baseline versus 3 months VEGF-A165a/b ratio, P = .028). Conclusions Our study shows an increased VEGF-A165a/b ratio in MMD compared to ICAD, and suggests that both intensive medical management and surgical revascularization elevate the VEGF-A165a/b ratio in ICAD patients.

In plants, calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) regulate Ca2+ signalling and so responses to biotic and abiotic stresses. However, the details of specific CIPKs functions in various stress responses are poorly understood. Here, we report roles of dicot and monocot CIPK2 genes in response to salinity and heavy metals. Arabidopsis thaliana AtCIPK2 was found to be universally expressed in different tissues and organs and furthermore induced by salinity. Overexpression of AtCIPK2 or Tibetan Plateau wild barley (Hordeum spontaneum) HsCIPK2 in Arabidopsis alleviated toxic effects of NaCl and mercury on seed germination and root growth. Similarly, reduced toxic effects of copper and cadmium on seed germination, but not on root growth, were observed in these transgenic lines. Live-cell fluorescence imaging analysis revealed that HsCIPK2 was predominantly distributed in the cytoplasm and nucleus and weakly localized at the plasma membrane (PM), but its PM association was rapidly enhanced upon exposure to high salinity and mercury. These results suggest an involvement of CIPK2 in plant tolerance to salinity and mercury and provide a new insight into physiological functions of CIPKs in plant response to heavy metals.

A molecular probe with aggregation-induced phosphorescence (AIP) properties for calcium ion-specific detection and imaging in vivo was designed. The long-lived phosphorescence caused by calcium-triggered aggregation enables establishment of a first time-gated detection method for calcium ions and long-term imaging in Arabidopsis thaliana.