马铃薯晚疫病菌侵染的Solophenyl Flavine荧光染色方法研究

在植物病害研究中,观察寄主植物被病原菌入侵的过程非常重要。Solophenyl Flavine 7GFE染料可附着于菌丝,在波长为330~380 nm的激发光下被激发出蓝色荧光。为了更好的观察寄主植物中病原真菌的侵染情况,本实验以Solophenyl Flavine 7GFE为染剂,对寄主植物中病原真菌的侵染情况进行了观察研究。结果显示,用0.002%（M/V）Solophenyl Flavine 7GFE溶于0.1 mol/L Tris-HCl（p H 8.5）配制的染色液染色5 min的效果最佳;使用95%乙醇溶液替代0.15%三氯乙酸（W/V）酒精溶液∶氯仿（V/V）（4∶1）对寄主植物叶片脱色的方法操作简便、毒害较低;染色时省略了番红预染步骤。将改良的染色方法用于晚疫病菌入侵的马铃薯叶片观察取得了良好的效果。该技术是一种改良的、快速有效、安全无毒的观察真菌菌丝入侵植物的荧光染色方法,也适用于观察其他真菌入侵寄主植物组织的过程。

Recent Advances in Photoenzymatic Catalysis

Photoenzymatic catalysis has become an emerging field in organic synthetic chemistry that provides eco-friendly alternatives to traditional methods. This comprehensive review examines the developing field of photoenzymatic catalysis, categorized by reaction types and focusing on its application in organic synthesis. This article highlights recent advances in the use of photoenzymatic reactions in carbon-carbon cross-coupling, ketone and alkene reduction, hydroamination, and hydrosulfonylation, mostly by flavin-dependent “ene”-reductases and nitroreductases. In each case, we exemplified the substrate scope that produces products with high yield and enantioselectivity. Additionally, the emerging trends in developing new enzymatic variants and novel reaction pathways that broaden the scope and enhance yield of these reactions were discussed.

MITOCHONDRIAL REDOX IMAGING FOR CANCER DIAGNOSTIC AND THERAPEUTIC STUDIES

Mitochondrial redox states provide important information about energy-linked biological processes and signaling events in tissues for various disease phenotypes including cancer.The redox scanning method developed at the Chance laboratory about 30 years ago has allowed 3D highresolution(∼50×50×10µm^(3))imaging of mitochondrial redox state in tissue on the basis of the fluorescence of NADH(reduced nicotinamide adenine dinucleotide)and Fp(oxidized flavoproteins including flavin adenine dinucleotide,i.e.,FAD).In this review,we illustrate its basic principles,recent technical developments,and biomedical applications to cancer diagnostic and therapeutic studies in small animal models.Recently developed calibration procedures for the redox imaging using reference standards allow quantification of nominal NADH and Fp concentrations,and the concentration-based redox ratios,e.g.,Fp/(Fp+NADH)and NADH/(Fp+NADH)in tissues.This calibration facilitates the comparison of redox imaging results acquired for different metabolic states at different times and/or with different instrumental settings.A redox imager using a CCD detector has been developed to acquire 3D images faster and with a higher in-plane resolution down to 10µm.Ex vivo imaging and in vivo imaging of tissue mitochondrial redox status have been demonstrated with the CCD imager.Applications of tissue redox imaging in small animal cancer models include metabolic imaging of glioma and myc-induced mouse mammary tumors,predicting the metastatic potentials of human melanoma and breast cancer mouse xenografts,differentiating precancerous and normal tissues,and monitoring the tumor treatment response to photodynamic therapy.Possible future directions for the development of redox imaging are also discussed.

Mechanism of Cerium Ions Scavenging Superoxide Radical

The mechanism of cerium ions scavenging superoxide radical(O÷2) has been studied. The results show that (1) Ce^3+ can reduce O÷2 to H_2O_2 while it is oxidized to Ce^4+; (2) Ce^4+ can oxidize O÷2 to O_2 while it is reduced to Ce^3+.

Rapid and Non-Invasive Screening System for Early Detection of Diabetic Retinopathy via Auto Fluorescence of <i>Falvin</i>Proteins

Diabetic retinopathy is one of the most serious complications of diabetes, which is also one of the most important causes of blindness around the world. Autofluorescence of flavin protein in retinal pigment epithelial cells is considered as a marker of early tissue damage. This study was designed to image spontaneous fluorescence of falvin proteins in fundus macular center area. The average intensity of the fluorescence signal and the characteristics of the histogram distribution were obtained and the significant differences in the fluorescence signals between the diabetic and the normal people were also compared with statistical methods, and then a rapid and non-invasive screening method and equipment for early detection of diabetic retinopathy were developed.

Mutations in FMN binding pocket diminish chromate reduction rates for Gh-ChrR isolated from <i>Gluconacetobacter hansenii</i>

A putative chromate ion binding site was identified proximal to a rigidly bound FMN from electron densities in the crystal structure of the quinone reductase from Gluconacetobacter hansenii (Gh-ChrR) (3s2y.pdb). To clarify the location of the chromate binding site, and to understand the role of FMN in the NADPH-dependent reduction of chromate, we have expressed and purified four mutant enzymes involving the site-specific substitution of individual side chains within the FMN binding pocket that form non-covalent bonds with the ribityl phosphate (i.e., S15A and R17A in loop 1 between β1 sheet and α1 helix) or the isoalloxanzine ring (E83A or Y84A in loop 4 between the β3 sheet and α4 helix). Mutations that selectively disrupt hydrogen bonds between either the N3 nitrogen on the isoalloxanzine ring (i.e., E83) or the ribitylphos- phoate (i.e., S15) respectively result in 50% or 70% reductions in catalytic rates of chromate reduction. In comparison, mutations that disrupt π-π ring stacking interactions with the isoal-loxanzine ring (i.e., Y84) or a salt bridge with the ribityl phosphate result in 87% and 97% inhibittion. In all cases there are minimal alterations in chromate binding affinities. Collectively, these results support the hypothesis that chromate binds proximal to FMN, and implicate a structural role for FMN positioning for optimal chromate reduction rates. As side chains proximal to the β3/α4 FMN binding loop 4 contribute to both NADH and metal ion binding, we propose a model in which structural changes around the FMN binding pocket couples to both chromate and NADH binding sites.

中国可再生能源的未来来;自世界观察所的观察——专访世界观察研究所主席Christopher·Flavin

北京,五月下旬,清晨,风依然有些冷.我应邀专访前来中国考察的世界观察研究所主席Christopher·Flavin先生.短暂的寒暄后,我们开始了这次专访的主题;中国可再生能源的未来.

Effect of exogenous flavins on the microbial corrosion by Geobacter sulfurreducens via iron-to-microbe electron transfer

Microbes can cause or accelerate metal corrosion,leading to huge losses in corrosion damages each year.Geobacter sulfurreducens is a representative electroactive bacterium in many soils,sediments,and wastew-ater systems.It has been confirmed to directly extract electrons from elemental metals.However,little is known about the effect of electron shuttles in G.sulfurreducens corrosion on stainless steel.In this study,we report that exogenous flavins promote iron-to-microbe electron transfer,accelerating micro-bial corrosion.G.sulfurreducens caused 1.3 times deeper pits and increased electron uptake(with 2 times increase of i_(corr))from stainless steel when riboflavin was added to the culture medium.OmcS-deficient mutant data suggest that G.sulfurreducens utilizes riboflavin as a bound-cofactor in outer membrane c-type cytochromes.The finding that,in the presence of microbes,riboflavin can substantially accelerate corrosion highlights the role of flavin redox cycling for enhanced iron-to-microbe electron transfer by G.sulfurreducens and provides new insights in microbial corrosion.

Biomimetic asymmetric catalysis

Enzymes are the core for biological transformations in nature.Their structures and functions have drawn enormous attention from biologists as well as chemists since last century.The large demand of bioactive molecules and the pursuit of efficiency and greenness of synthesis have spurred the rapid development of biomimetic chemistry in the past several decades.Biomimetic asymmetric catalysis,mimicking the structures and functions of enzymes,has been recognized as one of the most promising synthetic strategies for the synthesis of valuable chiral compounds.This review summarizes the evolution of asymmetric catalysis inspired by aldolases,vitamin B_(1)/B_(6)-dependent enzymes,NAD(P)H,flavin,hydrogenases,heme oxygenases,nonheme oxygenases,and dinuclear/multinuclear metalloenzymes in aspects of biomimetic design,catalyst development and related catalytic transformations.Those well-established synthetic approaches originating from biological reactions have demonstrated the unique prowess of biomimetic asymmetric catalysis in bridging the gap between bio-catalysis and chemical synthesis.

In vivo evaluation of riboflavin receptor targeted fluorescent USPIO in mice with prostate cancer xenografts

Riboflavin （Rf） receptors bind and translocate Rf and its phosphorylated forms （e.g. flavin mononucleotide, FMN） into cells where they mediate various cellular metabolic pathways. Previously, we showed that FMN-coated ultrasmall superparamagnetic iron oxide （FLUSPIO） nanoparticles are suitable for labeling metabolically active cancer and endothelial cells in vitro. In this study, we focused on the in vivo application of FLUSPIO using prostate cancer xenografts. Size, charge, and chemical composition of FLUSPIO were evaluated. We explored the in vitro specificity of FLUSPIO for its cellular receptors using magnetic resonance imaging （MRI） and Prussian blue staining. Competitive binding experiments were performed in vivo by injecting free FMN in excess. Bio-distribution of FLUSPIO was determined by estimating iron content in organs and tumors using a colorimetric assay. AFM analysis and zeta potential measurements revealed a particulate morphology approximately 20-40 nm in size and a negative zeta potential （-24.23±0.15 mV） in water. X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry data confirmed FMN present on the USPIO nanoparticle surface. FLUSPIO uptake in prostate cancer cells and human umbilical vein endothelial cells was significantly higher than that of control USPIO, while addition of excess of free FMN reduced accumulation. Similarly, in vivo MRI and histology showed specific FLUSPIO uptake by prostate cancer cells, tumor endothelial cells, and tumor-associated macrophages. Besides prominent tumor accumulation, FLUSPIO accumulated in the liver, spleen, lung, and skin. Hence, our data strengthen our hypothesis that targeting riboflavin receptors is an efficient approach to accumulate nanomedicines in tumors opening perspectives for the development of diagnostic and therapeutic systems.

LOV to BLUF： Flavoprotein Contributions to the Optogenetic Toolkit

Optogenetics is an emerging field that combines optical and genetic approaches to non-invasively interfere with cellular events with exquisite spatiotemporal control. Although it arose originally from neuroscience, optogenetics is widely applicable to the study of many different biological systems and the range of applications arising from this tech- nology continues to increase. Moreover, the repertoire of light-sensitive proteins used for devising new optogenetic tools is rapidly expanding. Light, Oxygen, or Voltage sensing （LOV） and Blue-Light-Utilizing flavin adenine dinucleotide （FAD） （BLUF） domains represent new contributors to the optogenetic toolkit. These small （100-140-amino acids） flavoprotein modules are derived from plant and bacterial photoreceptors that respond to UV-A/blue light. In recent years, considerable progress has been made in uncovering the photoactivation mechanisms of both LOV and BLUF domains. This knowledge has been applied in the design of synthetic photoswitches and fluorescent reporters with applications in cell biology and biotechnology. In this review, we summarize the photochemical properties of LOV and BLUF photosensors and highlight some of the recent advances in how these flavoproteins are being employed to artificially regulate and image a variety of biological processes.

FMO3-TMAO axis modulates the clinical outcome in chronic heart-failure patients with reduced ejection fraction:evidence from an Asian population

The association among plasma trimethylamine-N-oxide(TMAO),FMO3 polymorphisms,and chronic heart failure(CHF)remains to be elucidated.TMAO is a microbiota-dependent metabolite from dietary choline and carnitine.A prospective study was performed including 955 consecutively diagnosed CHF patients with reduced ejection fraction,with the longest follow-up of 7 years.The concentrations of plasma TMAO and its precursors,namely,choline and carnitine,were determined by liquid chromatography-mass spectrometry,and the FMO3 E158K polymorphisms(rs2266782)were genotyped.The top tertile of plasma TMAO was associated with a significant increment in hazard ratio(HR)for the composite outcome of cardiovascular death or heart transplantation(HR=1.47,95%CI=1.13-1.91,P=0.004)compared with the lowest tertile.After adjustments of the potential confounders,higher TMAO could still be used to predict the risk of the primary endpoint(adjusted HR=1.33,95%CI=1.01-1.74,P=0.039).This result was also obtained after further adjustment for carnitine(adjusted HR=1.33,95%CI=1.01-1.74,P=0.039).The FM03 rs2266782 polymorphism was associated with the plasma TMAO concentrations in our cohort,and lower TMAO levels were found in the AA-genotype.Thus,higher plasma TMAO levels indicated increased risk of the composite outcome of cardiovascular death or heart transplantation independent of potential confounders,and the FMO3 AA-genotype in rs2266782 was related to lower plasma TMAO levels.

Aryl C–H iodination: are there actual flavin-dependent iodinases in nature?

Flavin-dependent halogenases (FDHs) are well known to introduce carbon halide bonds (mainly C–Cl and C–Br) into natural products with the assistance of a partner protein flavin reductase to generate reduced flavin (FADH_(2)or FMNH_(2)).Compared with the common chloride-and bromide-containing natural products (approximately 5,000 compounds),iodinated natural products(approximately 100 compounds) are very limited.Specific iodinases have also rarely been identified in nature to date.This study discovered a novel relationship between iodination and flavin reductases for the first time.Through mechanistic studies,it was identified that peroxide (H_(2)O_(2)) was released from the uncoupling reaction of flavin reductases and then reacted with iodide ions(I^(-)) to produce hypoiodous acid (IOH) for the final iodination.Furthermore,this study also unintentionally verified that the recently reported flavin-dependent iodinase Vir X1 from the marine virus and its two homologs (MBG and NCV) did not catalyze iodination in the in vitro biochemical system but likely belonged to a new phylogenetic clade in the tryptophan halogenase superfamily.As a consequence,actual flavin-dependent iodinases in nature remain to be discovered by the scientific community in the future.

Kinetics and mechanism of electron-induced splitting of a cyclobutane pyrimidine dimer with or without an electron acceptor

Utilizing a pulse radiolysis equipment with time-resolved optical detector, kinetic processes of electron-induced splitting of cis-syn 1,3-dimethyluracil cyclobutane dimer (DMUD) in aqueous solution were investigated in the presence or absence of riboflavin (RF) or flavin adenine dinucleotide (FAD). It has been observed that the cyclobutane pyrimidine dimer reacting with hydrated electron splits spontaneously to give a monomer and a monomer radical anion, and the anion transfers one electron to RF or FAD. From the buildup kinetics of radical species, the rate constants of electron transfer from the monomer radical anion to RF and FAD have been determined. On the basis of comparison of the interactions between DMUD and hydrated electron in the presence and absence of RF or FAD, a chain reaction process in the absence of RF or FAD has been demonstrated.

Mechanistic Study of Oxidoreductase AprQ Involved in Biosynthesis of Aminoglycoside Antibiotic Apramycin

Main observation and conclusion The aminoglycoside antibiotic apramycin contains a unique bicyclic octose moiety,and biosynthesis of this moiety involves an oxidoreductase AprQ.Unlike other known“Q”series proteins involved in aminoglycosides biosynthesis,AprQ does not work with an aminotransferase partner,and performs a four-electron oxidation that converts a CH2OH moiety to a carboxylate group.

SPECTRA PROPERTIES OF RAT LIVER MITOCHONDRIAL CHOLINE DEHYDROGENASE

Choline dehydrogenase contains the prosthetic group FAD, non-haem iron and acid labile sulfur. However, the absorption spectra of the purified enzyme do not change after adding substrate. The reduced absorption spectra of choline dehydrogenase can only be determined after the addition of dithionite. Those choline dehydrogenases situated in the mitochondrial inner membrane can be reduced by substrate and exist in the reduced state. When cholate was used to solubilize the substrate-reduced choline dehydrogenase, the reduced spectra will gradually disappear. However, if solubilization is carried out under anaerobic conditions, the reduced spectra can be retained, suggesting that the solubilized choline dehydrogenase can use oxygen as an acceptor.

Exploration of 5-cyano-6-phenylpyrimidin derivatives containing an 1,2,3-triazole moiety as potent FAD-based LSD1 inhibitors

Histone lysine specific demethylase 1(LSD1)has become a potential therapeutic target for the treatment of cancer.Discovery and develop novel and potent LSD1 inhibitors is a challenge,although several of them have already entered into clinical trials.Herein,for the first time,we reported the discovery of a series of 5-cyano-6-phenylpyrimidine derivatives as LSD1 inhibitors using flavin adenine dinucleotide(FAD)similarity-based designing strategy,of which compound 14 q was finally identified to repress LSD1 with IC50=183 nmol/L.Docking analysis suggested that compound 14 q fitted well into the FAD-binding pocket.Further mechanism studies showed that compound 14 q may inhibit LSD1 activity competitively by occupying the FAD binding sites of LSD1 and inhibit cell migration and invasion by reversing epithelial to mesenchymal transition(EMT).Overall,these findings showed that compound14 q is a suitable candidate for further development of novel FAD similarity-based LSD1 inhibitors.