Phosphorylation and O-GlcNAcylation at the same α-synuclein site generate distinct fibril structures.

α-Synuclein forms amyloid fibrils that are critical in the progression of Parkinson's disease and serves as the pathological hallmark of this condition. Different posttranslational modifications have been identified at multiple sites of α-synuclein, influencing its conformation, aggregation and function. Here, we investigate how disease-related phosphorylation and O-GlcNAcylation at the same α-synuclein site (S87) affect fibril structure and neuropathology. Using semi-synthesis, we obtained homogenous α-synuclein monomer with site-specific phosphorylation (pS87) and O-GlcNAcylation (gS87) at S87, respectively. Cryo-EM revealed that pS87 and gS87 α-synuclein form two distinct fibril structures. The GlcNAc situated at S87 establishes interactions with K80 and E61, inducing a unique iron-like fold with the GlcNAc molecule on the iron handle. Phosphorylation at the same site prevents a lengthy C-terminal region including residues 73 to 140 from incorporating into the fibril core due to electrostatic repulsion. Instead, the N-terminal half of the fibril (1-72) takes on an arch-like fibril structure. We further show that both pS87 and gS87 α-synuclein fibrils display reduced neurotoxicity and propagation activity compared with unmodified α-synuclein fibrils. Our findings demonstrate that different posttranslational modifications at the same site can produce distinct fibril structures, which emphasizes link between posttranslational modifications and amyloid fibril formation and pathology.

Specific binding of Hsp27 and phosphorylated Tau mitigates abnormal Tau aggregation-induced pathology.

Amyloid aggregation of phosphorylated Tau (pTau) into neurofibrillary tangles is closely associated with Alzheimer's disease (AD). Several molecular chaperones have been reported to bind Tau and impede its pathological aggregation. Recent findings of elevated levels of Hsp27 in the brains of patients with AD suggested its important role in pTau pathology. However, the molecular mechanism of Hsp27 in pTau aggregation remains poorly understood. Here, we show that Hsp27 partially co-localizes with pTau tangles in the brains of patients with AD. Notably, phosphorylation of Tau by microtubule affinity regulating kinase 2 (MARK2), dramatically enhances the binding affinity of Hsp27 to Tau. Moreover, Hsp27 efficiently prevents pTau fibrillation in vitro and mitigates neuropathology of pTau aggregation in a Drosophila tauopathy model. Further mechanistic study reveals that Hsp27 employs its N-terminal domain to directly interact with multiple phosphorylation sites of pTau for specific binding. Our work provides the structural basis for the specific recognition of Hsp27 to pathogenic pTau, and highlights the important role of Hsp27 in preventing abnormal aggregation and pathology of pTau in AD.

Interaction of RAGE with α-synuclein fibrils mediates inflammatory response of microglia.

Microglia-mediated neuroinflammation and α-synuclein (α-syn) aggregation, both as pathological hallmarks of Parkinson's disease (PD), crosstalk to exacerbate degeneration of dopaminergic neurons and PD progression. However, the mechanism underlying their interaction is poorly understood, which obstructs effective therapeutic inhibition of α-syn-induced neuroinflammation. Here, we initiate from structure-based interaction predictions and find that receptor for advanced glycation end products (RAGE) serves as a receptor of α-syn fibrils on microglia. Results of nuclear magnetic resonance (NMR) spectroscopy and mutagenesis validate that the V domain of RAGE that contains an alkaline surface can bind with acidic C-terminal residues of α-syn. Furthermore, the binding of α-syn fibrils with RAGE induces neuroinflammation, which is blocked by both genetic depletion of RAGE and inhibitor FPS-ZM1. Our work shows the important role, as well as the structural mechanism, of RAGE in mediating the inflammatory response of microglia to α-syn fibrils, which may help to establish effective therapeutic strategies to alleviate α-syn-induced neuroinflammation and neuronal damage.

Febuxostat Protects Human Aortic Valve Endothelial Cells From Oxidized Low-density Lipoprotein-Induced Injury and Monocyte Attachment.

ABSTRACT: Atherosclerosis (AS) is a common cardiovascular disease with high morbidity and mortality. The pathogenesis of AS is closely related to endothelial dysfunction, which is mainly induced by oxidative stress, inflammation, and enhanced adhesion of monocytes to endothelial cells on the vessel wall. Febuxostat is a novel antigout agent recently reported to exert protective effects on endothelial dysfunction. This study aims to investigate the protective capacity of febuxostat against oxidized low-density lipoprotein (ox-LDL)-induced injury and monocyte attachment to endothelial cells. Human aortic valve endothelial cells (HAVECs) were stimulated with ox-LDL in the presence or absence of febuxostat (5 and 10 µM) for 6 hours. Mitochondrial reactive oxygen species were measured using MitoSox red staining, and the level of protein carbonyl was detected using enzyme-linked immunosorbent assay (ELISA). The expressions of IL-6, TNF-α, tissue factor (TF), VCAM-1, and ICAM-1 were evaluated with qRT-PCR assay and ELISA. Calcein-AM staining was used to determine the attachment of U937 monocytes to HAVECs. quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blot were used to measure the expression level of early growth response 1 (Egr-1) in HAVECs. First, the elevated expression of LOX-1, activated oxidative stress, excessive secreted inflammatory factors, and promoted expression of TF induced by stimulation with ox-LDL were significantly reversed by febuxostat, indicating a protective effect of febuxostat against endothelial dysfunction. Second, the upregulated VCAM-1 and ICAM-1, as well as the increased proportion of adhered monocytes to HAVECs induced by ox-LDL, were significantly alleviated by febuxostat. Finally, the promoted expression level of Egr-1 induced by ox-LDL was pronouncedly suppressed by febuxostat. We conclude that febuxostat protected HAVECs from ox-LDL-induced injury and monocyte attachment.

Biochemical and biophysical characterization of pathological aggregation of amyloid proteins.

Protein amyloid fibrillation, a process of liquid to solid phase transition, is involved in the pathogenesis of a variety of human diseases. Several amyloid proteins including α-synuclein (α-syn), Tau, amyloid ß (Aß) protein, and TAR DNA-binding protein 43 kDa (TDP-43) form pathological fibrils and deposit in patient brains of different neurodegenerative diseases (NDs) such as Parkinson's disease (PD), Alzheimer's disease (AD) and Amyotrophic lateral sclerosis (ALS). Preparation and characterization of amyloid fibrils in vitro are essential for studying the molecular mechanism underlying the dynamic amyloid aggregation and its pathogenesis in diseases. In this protocol, we take PD-associated α-syn as an example, and describe amyloid protein purification and fibrillation approaches. We then introduce biochemical and biophysical characterization of amyloid fibrils by Thioflavin-T (ThT) fluorescence kinetics assay, transmission electron microscopy (TEM), atomic force microscopy (AFM) and multiple fibril stability measurement assays. The approaches described here are applicable to different amyloid proteins, and are of importance for further study on the structure determination of amyloid fibrils and their pathological function in cells and animal models.

Cellular and animal models to investigate pathogenesis of amyloid aggregation in neurodegenerative diseases.

Abnormal aggregation of amyloid proteins, e.g. amyloid ß (Aß), Tau and α-synuclein (α-syn), is closely associated with a variety of neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Cellular and animal models are useful to explore the neuropathology of amyloid aggregates in disease initiation and progression. In this protocol, we describe detailed procedures for how to establish neuronal and PD mouse models to evaluate amyloid pathologies including self-propagation, cell-to-cell transmission, neurotoxicity, and impact on mouse motor and cognitive functions. We use α-syn, a key pathogenic protein in PD, as an example to demonstrate the application of the protocol, while it can be used to investigate the pathologies of other amyloid proteins as well. The established disease models are also useful to assess the activities of drug candidates for therapeutics of neurodegenerative diseases.

Optimizing the Phase-Separated Domain Size of the Active Layer via Sequential Crystallization in All-Polymer Solar Cells.

The proper domain size of the active layer plays a key role in determining the exciton dissociation and charge transport in all-polymer solar cells (all-PSCs). However, fine-tuning the domain size remains challenging due to low glass transition temperature and negligible mixing entropy in polymer blends. Herein, we systematically studied the influence of "crystallization kinetics" on the domain size and proposed that if the donor and acceptor crystallize simultaneously, they are prone to form a large domain, while if sequential crystallization of the donor and acceptor occurs, a fine phase separation structure with the proper domain size can be obtained. Taking PBDB-T/PNDI blends for instance, the domain size was decreased by using sequential crystallization; meanwhile, the crystallinity and molecular orientation were also optimized, boosting the power conversion efficiency from 6.55% to 7.78%. This work provides a novel way to finely tune the heterojunction phase separation structures.

Relationship between Cefquinome PK/PD Parameters and Emergence of Resistance of Staphylococcus aureus in Rabbit Tissue-Cage Infection Model.

In order to explore the relationship between different antibiotic dosing regimens and selective enrichment of resistant strains, tissue-cage infection model was established in rabbits to study relationship between cefquinome pharmacokinetic/pharmacodynamic parameters and the change of susceptibility of Staphylococcus aureus (S. aureus). In this model, above 10(8) CFU/mL of S. aureus culture were exposed to cefquinome concentrations below the MIC99 (the minimal concentration that inhibits colony formation by 99% in vitro, 0.3 µg/mL), between the MIC99 and the MPC (the mutant prevent concentration in vitro, 1.6 µg/mL), and above the MPC after intramuscular injection with cefquinome at doses of 4, 8, 16, and 32 mg/kg of body weight (bw) once daily for 5 days or 4, 8, 16, and 24 mg/kg of bw twice daily for 2.5 days. Samples of tissue-cage fluid were collected from the tissue-cage at 2, 4, 6, 8, 10, 12, 24 h after each dosing (one dosing daily) or at 2, 4, 6, 8, 10, and 12 h (two dosing daily). Cefquinome concentration, susceptibility of S. aureus to cefquinome, and bacterial numbers at the infected site were monitored. The MICs of S. aureus and the fraction of resistant bacteria both increased when cefquinome concentrations fluctuated between the MIC99 and MPC. Resistant bacteria were selected in vivo when %T > MPC was < 58% of administration interval or %T > MIC99 was ≥70% of administration interval. These findings demonstrate that low-level, cefquinome-resistant S. aureus were selected predominantly when drug concentrations fell inside a concentration window in in vivo model, which was evidenced by pulsed-field gel electrophoresis. The selection of resistant bacteria arose from both susceptible bacteria being killed and resistant bacteria re-growth. Keeping drug concentrations above the MPC for ≥58% of administration interval provides a strategy to achieve effective antibacterial activity and minimize the emergence of resistance to cefquinome.

Prevalence of extended-spectrum cephalosporin-resistant Escherichia coli in a farrowing farm: ST1121 clone harboring IncHI2 plasmid contributes to the dissemination of bla CMY-2.

During a regular monitoring of antimicrobial resistance in a farrowing farm in Southern China, 117 Escherichia coli isolates were obtained from sows and piglets. Compared with the isolates from piglets, the isolates from sows exhibited higher resistance rates to the tested cephalosporins. Correspondingly, the total detection rate of the bla CMY-2/bla CTX-M genes in the sow isolates (34.2%) was also significantly higher than that of the piglet isolates (13.6%; p < 0.05). The bla CMY-2 gene had a relatively high prevalence (11.1%) in the E. coli isolates. MLST and PFGE analysis revealed the clonal spread of ST1121 E. coli in most (7/13) of the bla CMY-2-positive isolates. An indistinguishable IncHI2 plasmid harboring bla CMY-2 was also identified in each of the seven ST1121 E. coli isolates. Complete sequence analysis of this IncHI2 plasmid (pEC5207) revealed that pEC5207 may have originated through recombination of an IncHI2 plasmid with a bla CMY-2-carrying IncA/C plasmid like pCFSAN007427_01. In addition to bla CMY-2, pEC5207 also carried other resistance determinants for aminoglycosides (aacA7), sulfonamides (sul1), as well as heavy metals ions, such as Cu and Ag. The susceptibility testing showed that the pEC5207 can mediate both antibiotic and heavy metal resistance. This highlights the role of pEC5207 in co-selection of bla CMY-2-positive isolates under the selective pressure of heavy metals, cephalosporins, and other antimicrobials. In conclusion, clonal spread of an ST1121 type E. coli strain harboring an IncHI2 plasmid contributed to the dissemination of bla CMY-2 in a farrowing farm in Southern China. We also have determined the first complete sequence analysis of a bla CMY-2-carrying IncHI2 plasmid.

Features and variations of a radial artery approach in southern Chinese populations and their clinical significance in percutaneous coronary intervention.

BACKGROUND: More and more percutaneous coronary intervention were done from radial artery approach. But the great limitation of radial artery approach and main failure cause of transradial coronary intervention is smaller size and more variations of a radial artery approach. The aim of the study is to explore the features and variations of a radial artery approach in southern Chinese populations and their clinical significance in percutaneous coronary intervention. METHODS: A total of 1400 patients who underwent scheduled first time transradial coronary angiography between July 2007 and September 2010 were enrolled. Radial arteriography was performed in all patients to detect the anatomical variations of this vessel. All patients' radial and ulnar artery inner diameters were measured using a computer assisted quantification method. A detailed patient history was recorded. Multivariate Logistic regression analysis was performed to evaluate the predictive value of variables (including age, gender, ethnicity, height, weight, body mass index, smoking, diabetes, hypertension and dyslipidemia) in arterial tortuosities and variations of this vessel. RESULTS: In southern Chinese populations, there were no significant differences in the diameters of the forearm arteries: the mean radial artery inner diameter was (3.04 ± 0.43) mm in ethnic Han Chinese and (3.05 ± 0.42) mm in ethnic Zhuang Chinese, P > 0.05), the mean ulnar artery inner diameter was (3.03 ± 0.38) mm in Han Chinese and (3.05 ± 0.36) mm in Zhuang Chinese, P > 0.05). It was estimated that the inner diameter of the radial artery was not smaller than a 6F Cordis sheath in 86.1% of male patients and in 57.0% of female patients, and not smaller than a 7F Cordis sheath in 59.3% of male patients and 24.9% of female patients. The factors found to positively affect the size of the radial artery were sex (bj = 0.309, P < 0.01), weight (bj = 0.103, P < 0.01), and diabetes mellitus (bj = -0.088, P < 0.01) was found to negatively affect radial artery size. Arterial tortuosities occurred in 12.1% of patients and arterial variations in 4.1%. The incidence of tortuosities and variations included radial artery tortuosity (3.6%), high origin of radial artery (1.7%), radial artery loop (0.6%), double radial artery (0.1%), brachial artery tortuosity (0.4%), double brachial artery (0.1%), subclavian artery tortuosity (5.4%), small subclavian artery (0.4%), right retro-esophageal subclavian artery (0.6%), brachiocephalic trunk tortuosity (2.8%), small brachiocephalic artery (0.1%), and brachiocephalic artery anomaly (0.4%). For people in Guangxi province, tortuosities of the subclavian artery and radial artery are the most common among the vascular tortuosities of the radial artery approach. The overall rate of transradial procedural success was 96.1%. Procedural failure was more common in patients with anomalous radial artery approach than in patients with normal radial artery approach (22.8% vs. 1.8%, P = 0.000). According to multivariate Logistic regression analysis, age (OR = 2.695, 95%CI 2.232 - 3.253, P = 0.000), female gender (OR = 5.127, 95%CI 3.000 - 8.762, P = 0.000), height (OR = 0.612, 95%CI 0.465 - 0.807, P = 0.000), body mass index (OR = 2.377, 95%CI 1.834 - 3.082, P = 0.000), hypertension (OR = 1.668, 95%CI 1.132 - 2.458, P = 0.010), hyperlipidemia (OR = 1.273, 95%CI 1.425 - 2.049, P = 0.034) and smoking (OR = 5.750, 95%CI 3.636 - 9.093, P = 0.000), were independently associated with arterial tortuosities of the radial artery approach. Female gender was independently associated with arterial variations of the radial artery approach (OR = 3.613, 95%CI 3.208 - 7.826, P = 0.000). CONCLUSIONS: The diameters of the radial and ulnar arteries between the Han people and the Zhuang people in southern Chinese populations are similar. In a transradial operation, the most southern Chinese populations, the use of a 6F sheath and guiding catheter is safe, and using a 7F sheath and guiding catheter is feasible in some selected patients. Radial arterial tortuosities and variations in southern Chinese populations are relatively common and are a significant cause of the failure of transradial coronary procedure. Old age, female gender, short stature, high body mass index, hypertension, hyperlipidemia and smoking, were independently associated with an increased risk of arterial tortuosity. In addition, female gender was an independent predictor of arterial variations.