Molybdenum-containing nitrite reductases: Spectroscopic characterization and redox mechanism

Objectives: This review summarizes the spectroscopic results, which will provide useful suggestions for future research. In addition, the fields that urgently need more information are also advised.

Background: Nitrite-NO-cGMP has been considered as an important signaling pathway of NO in human cells. To date, all the four known human molybdenum-containing enzymes, xanthine oxidase, aldehyde oxidase, sulfite oxidase, and mitochondrial amidoxime-reducing component, have been shown to function as nitrite reductases under hypoxia by biochemical, cellular, or animal studies. Various spectroscopic techniques have been applied to investigate the structure and catalytic mechanism of these enzymes for more than 20 years.

Methods: We summarize the published data on the applications of UV-vis and EPR spectroscopies, and X-ray crystallography in studying nitrite reductase activity of the four human molybdenum-containing enzymes.

Results: UV-vis has provided useful information on the redox active centers of these enzymes. The utilization of EPR spectroscopy has been critical in determining the coordination and redox status of the Mo center during catalysis. Despite the lack of substrate-bound crystal structures of these nitrite reductases, valuable structural information has been obtained by X-ray crystallography.

Conclusions: To fully understand the catalytic mechanisms of these physiologically/pathologically important nitrite reductases, structural studies on substrate-redox center interaction are needed.     

Jones oxidation and high performance liquid chromatographic analysis of cholesterol in biological samples

A simple pre-column derivatization procedure for HPLC analysis of cholesterol in biological samples was developed. Cholesterol was treated with chromic acid and sulfuric acid in acetone (the Jones oxidation) and cholest-4-en-3,6-dione was formed. The reaction was finished in 5 min at room temperature and the product showed a strong UV absorbance at 250 nm that enabled an HPLC detection limit of 0.2 pmol. With stigmasterol as an internal standard, the reaction was applied to the analysis of total and free cholesterol in serum and high-density lipoproteins and the analysis showed a within-run and total coefficient of variation of about 0.2% and 0.5%, respectively.     

Chinese expert consensus statement on the diagnosis and treatment of fulminant myocarditis

Fulminant myocarditis is primarily caused by infection with any number of a variety of viruses. It arises quickly, progresses rapidly, and may lead to severe heart failure or circulatory failure presenting as rapid-onset hypotension and cardiogenic shock,with mortality rates as high as 50%–70%. Most importantly, there are no treatment options, guidelines or an expert consensus statement. Here, we provide the first expert consensus, the Chinese Society of Cardiology Expert Consensus Statement on the Diagnosis and Treatment of Fulminant Myocarditis, based on data from our recent clinical trial(NCT03268642). In this statement, we describe the clinical features and diagnostic criteria of fulminant myocarditis, and importantly, for the first time,we describe a new treatment regimen termed life support-based comprehensive treatment regimen. The core content of this treatment regimen includes(i) mechanical life support(applications of mechanical respirators and circulatory support systems,including intraaortic balloon pump and extracorporeal membrane oxygenation),(ii) immunological modulation by using sufficient doses of glucocorticoid, immunoglobulin and(iii) antiviral reagents using neuraminidase inhibitor. The proper application of this treatment regimen may and has helped to save the lives of many patients with fulminant myocarditis.     

Effects of cpxR on the growth characteristics and antibiotic production of Xenorhabdus nematophila

CpxR is a global response regulator that negatively influences the antimicrobial activities of Xenorhabdus nematophila. Herein, the wildtype and ΔcpxR mutant of X. nematophila were cultured in a 5-l and 70-l bioreactor. The kinetic analysis showed that ΔcpxR significantly increased the cell biomass and antibiotic activity. The maximum dry cell weight (DCW) and antibiotic activity of ΔcpxR were 20.77 ± 1.56 g L⁻¹ and 492.0 ± 31.2 U ml⁻¹ and increased by 17.28 and 97.33% compared to the wildtype respectively. Xenocoumacin 1 (Xcn1), a major antimicrobial compound, was increased 3.07-fold, but nematophin was decreased by 48.7%. In 70-l bioreactor, DCW was increased by 18.97%, while antibiotic activity and Xcn1 were decreased by 27.71% and 11.0% compared to that in 5-l bioreactor respectively. Notably, pH had remarkable effects on the cell biomass and antibiotic activity of ΔcpxR, where ΔcpxR was sensitive to alkaline pH conditions. The optimal cell growth and antibiotic activity of ΔcpxR occurred at pH 7.0, while Xcn1 was increased 5.45- and 3.87-fold relative to that at pH 5.5 and 8.5 respectively. These findings confirmed that ΔcpxR considerably increased the biomass of X. nematophila at a late stage of fermentation. In addition, ΔcpxR significantly promoted the biosynthesis of Xcns but decreased the production of nematophin.     

Effect of Ibuprofen on Autophagy of Astrocytes During Pentylenetetrazol-Induced Epilepsy and its Significance: An Experimental Study

Epilepsy is a chronic neurological disease. Astrogliosis is an important pathological change in epileptic lesions. Studies have reported that ibuprofen can affect autophagy and/or inhibit cell proliferation in many diseases. This study investigated the effect and significance of ibuprofen on autophagy of astrocytes during pentylenetetrazol (PTZ) induced epilepsy. 60 male Sprague–Dawley (SD) rats were randomly divided into five groups: control group (received normal saline), PTZ group, 3-methyladenine (3-MA)?+?PTZ group, ibuprofen?+?PTZ group and 3-MA?+?ibuprofen?+?PTZ group. Dose of each agent was 35 mg/kg (PTZ), 10 mg/kg (3-MA) and 30 mg/kg (ibuprofen) and all drugs were administered intraperitoneally 15 times on alternate days (29 days). Human astrocytes were cultured in vitro. Behavioral performance (i.e., latency, grade and duration of seizures) and EEG of rats were observed and recorded. Proliferation of astrocytes was detected by CCK-8 method. Immunofluorescence and Western blot test were used to detect the expression of LC3 and GFAP. Mean number, grade and duration of seizures were markedly reduced in ibuprofen?+?PTZ group and 3-MA?+?ibuprofen?+?PTZ group (P?<?0.05). Similarly, peak of EEG waves were markedly reduced in ibuprofen?+?PTZ group and 3-MA?+?ibuprofen?+?PTZ group (P?<?0.05). Compared to the control group, the level of LC3 in ibuprofen group was significantly increased in vitro (P?<?0.05). While, levels of LC3 were significantly higher and that of GFAP were significantly lower in ibuprofen?+?PTZ group (P?<?0.05) compared to PTZ group in vivo. Ibuprofen reduces the proliferation of astrocytes by increasing autophagy, thus affecting the development of epilepsy. Therefore, ibuprofen may be used as an adjuvant to improve efficacy of treatment in epilepsy.

     

土地管理对植被变绿的潜在贡献——以中国东北农业区为例

植被变绿现象一般指绿度指标统计上呈现年际增加趋势。大尺度植被变绿现象及其归因与影响研究广泛开展中。其中,量化各种驱动因素的贡献仍然十分困难,特别是土地管理影响的研究尚显不足。选择了中国农业分布最广泛的东北农业区,尝试从植被变绿的年际变化和季节特征对土地管理因素的潜在贡献进行了推断分析,并选择增强植被指数EVI作为指标。在2000—2019年期间,EVI在农田和自然植被(林地和草地)的变化趋势分别为2.19×10^-3/a(P<0.05)和1.86×10^-3/a(P<0.05)(1.83×10^-3/a(P<0.05)和1.94×10^-3/a(P<0.05)),即处于相同量级。但是,农田EVI的增长主要集中在6—9月,增长率为4.99×10^-3/a(P<0.05),而同期自然植被的增长速率仅为2.30×10^-3/a(P<0.05)(林地和草地分别为1.79×10^-3/a(P<0.05)和3.71×1...     

Manipulating Adenovirus Hexon Hypervariable Loops Dictates Immune Neutralisation and Coagulation Factor X-dependent Cell Interaction In Vitro and In Vivo

Adenoviruses are common pathogens, mostly targeting ocular, gastrointestinal and respiratory cells, but in some cases infection disseminates, presenting in severe clinical outcomes. Upon dissemination and contact with blood, coagulation factor X (FX) interacts directly with the adenovirus type 5 (Ad5) hexon. FX can act as a bridge to bind heparan sulphate proteoglycans, leading to substantial Ad5 hepatocyte uptake. FX “coating” also protects the virus from host IgM and complement-mediated neutralisation. However, the contribution of FX in determining Ad liver transduction whilst simultaneously shielding the virus from immune attack remains unclear. In this study, we demonstrate that the FX protection mechanism is not conserved amongst Ad types, and identify the hexon hypervariable regions (HVR) of Ad5 as the capsid proteins targeted by this host defense pathway. Using genetic and pharmacological approaches, we manipulate Ad5 HVR interactions to interrogate the interplay between viral cell transduction and immune neutralisation. We show that FX and inhibitory serum components can co-compete and virus neutralisation is influenced by both the location and extent of modifications to the Ad5 HVRs. We engineered Ad5-derived HVRs into the rare, native non FX-binding Ad26 to create Ad26.HVR5C. This enabled the virus to interact with FX at high affinity, as quantified by surface plasmon resonance, FX-mediated cell binding and transduction assays. Concomitantly, Ad26.HVR5C was also sensitised to immune attack in the absence of FX, a direct consequence of the engineered HVRs from Ad5. In both immune competent and deficient animals, Ad26.HVR5C hepatic gene transfer was mediated by FX following intravenous delivery. This study gives mechanistic insight into the pivotal role of the Ad5 HVRs in conferring sensitivity to virus neutralisation by IgM and classical complement-mediated attack. Furthermore, through this gain-of-function approach we demonstrate the dual functionality of FX in protecting Ad26.HVR5C against innate immune factors whilst determining liver targeting.     

Molecular basis for ligand activation of the human KCNQ2 channel

The voltage-gated potassium channel KCNQ2 is responsible for M-current in neurons and is an important drug target to treat epilepsy, pain and several other diseases related to neuronal hyper-excitability. A list of synthetic compounds have been developed to directly activate KCNQ2, yet our knowledge of their activation mechanism is limited, due to lack of high-resolution structures. Here, we report cryo-electron microscopy (cryo-EM) structures of the human KCNQ2 determined in apo state and in complex with two activators, ztz240 or retigabine, which activate KCNQ2 through different mechanisms. The activator-bound structures, along with electrophysiology analysis, reveal that ztz240 binds at the voltage-sensing domain and directly stabilizes it at the activated state, whereas retigabine binds at the pore domain and activates the channel by an allosteric modulation. By accurately defining ligand-binding sites, these KCNQ2 structures not only reveal different ligand recognition and activation mechanisms, but also provide a structural basis for drug optimization and design.Subject terms: Cryoelectron microscopy, Mechanisms of disease     

东海原甲藻与中肋骨条藻的种间竞争特征

对东海原甲藻和中肋骨条藻按照起始Chl-a比1∶5、1∶1和5∶1进行了f/2条件下的共培养实验,以探讨这两种藻的种间竞争特征。实验结果表明在共培养体系中,中肋骨条藻完全占优势,而东海原甲藻的生长受到明显的抑制。应用Lotka-Volterra种间竞争模型对共培养实验进行模拟的结果表明,东海原甲藻与中肋骨条藻的种间竞争结果与初始密度配比无关,中肋骨条藻总会竞争胜过东海原甲藻。为了探讨他感作用对东海原甲藻和中肋骨条藻种间竞争的影响,采用了中肋骨条藻的无藻细胞滤液来进行培养实验。实验结果显示,中肋骨条藻滤液对东海原甲藻及其本身的生长均无明显影响,这表明他感作用并非中肋骨条藻获得优势的主要竞争方式。