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.

     

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     

Reprogramming of ovine adult fibroblasts to pluripotency via drug-inducible expression of defined factors

Reprogramming of somatic cells in the enucleated egg made Dolly, the sheep, the first successfully cloned mammal in 1996. However, the mechanism of sheep somatic cell reprogramming has not yet been addressed. Moreover, sheep embryonic stem (ES) cells are still not available, which limits the generation of precise gene-modified sheep. In this study, we report that sheep somatic cells can be directly reprogrammed to induced pluripotent stem (iPS) cells using defined factors (Oct4, Sox2, c-Myc, Klf4, Nanog, Lin28, SV40 large T and hTERT). Our observations indicated that somatic cells from sheep are more difficult to reprogram than somatic cells from other species, in which iPS cells have been reported. We demonstrated that sheep iPS cells express ES cell markers, including alkaline phosphatase, Oct4, Nanog, Sox2, Rex1, stage-specific embryonic antigen-1, TRA-1-60, TRA-1-81 and E-cadherin. Sheep iPS cells exhibited normal karyotypes and were able to differentiate into all three germ layers both in vitro and in teratomas. Our study may help to reveal the mechanism of somatic cell reprogramming in sheep and provide a platform to explore the culture conditions for sheep ES cells. Moreover, sheep iPS cells may be directly used to generate precise gene-modified sheep.     

The Molecular Mechanism of Long Non-Coding RNA MALAT1-Mediated Regulation of Chondrocyte Pyroptosis in Ankylosing Spondylitis

Long non-coding RNAs (lncRNAs) may be important regulators in the progression of ankylosing spondylitis (AS). The competing endogenous RNA (ceRNA) activity of lncRNAs plays crucial roles in osteogenesis. We identified the mechanism of the differentially expressed lncRNA MALAT1 in AS using bioinformatic analysis and its ceRNA mechanism. The interaction of MALAT1, microRNA-558, and GSDMD was identified using integrated bioinformatics analysis and validated. Loss- and gain-of-function assays evaluated their effects on the viability, apoptosis, pyroptosis and inflammation of chondrocytes in AS. We found elevated MALAT1 and GSDMD but reduced miR-558 in AS cartilage tissues and chondrocytes. MALAT1 contributed to the suppression of cell viability and facilitated apoptosis and pyroptosis in AS chondrocytes. GSDMD was a potential target gene of miR-558. Depletion of MALAT1 expression elevated miR-558 by inhibiting GSDMD to enhance cell viability and inhibit inflammation, apoptosis and pyroptosis of chondrocytes in AS. In summary, our key findings demonstrated that knockdown of MALAT1 served as a potential suppressor of AS by upregulating miR-558 via the downregulation of GSDMD expression.