Study on the Mechanism of Lipid Peroxidation Induced by Carbonate Radicals.

Based on the reported research, hydroxyl radicals can be rapidly transformed into carbonate radicals in the carbonate-bicarbonate buffering system in vivo. Many of the processes considered to be initiated by hydroxyl radicals may be caused by carbonate radicals, which indicates that lipid peroxidation initiated by hydroxyl radicals can also be caused by carbonate radicals. To date, theoretical research on reactions of hydrogen abstraction from and radical addition to polyunsaturated fatty acids (PUFAs) of carbonate radicals has not been carried out systematically. This paper employs (3Z,6Z)-nona-3,6-diene (NDE) as a model for polyunsaturated fatty acids (PUFAs). Density functional theory (DFT) with the CAM-B3LYP method at the 6-311+g(d,p) level was used to calculate the differences in reactivity of carbonate radicals abstracting hydrogen from different positions of NDE and their addition to the double bonds of NDE under lipid solvent conditions with a dielectric constant of 4.0 (CPCM model). Grimme's empirical dispersion correction was taken into account through the D3 scheme. The energy barrier, reaction rate constants, internal energy, enthalpy and Gibbs free energy changes in these reactions were calculated With zero-point vibrational energy (ZPVE) corrections. The results indicated that carbonate radicals initiate lipid peroxidation primarily through hydrogen abstraction from diallyl carbon atoms. The reaction of hydrogen abstraction from diallyl carbon atoms exhibits the highest reaction rate, with a reaction rate constant approximately 43-fold greater than the second-ranked hydrogen abstraction from allyl carbon atoms. This process has the lowest energy barrier, internal energy, enthalpy, and Gibbs free energy changes, indicating that it is also the most spontaneous process.

Twisty C-TiO2 /PCN S-Scheme Heterojunction with Enhanced n→π * Electronic Excitation for Promoted Piezo-Photocatalytic Effect.

Herein, a twisty C-TiO2 /PCN (CNT) Step-scheme (S-scheme) heterojunction is fabricated and applied to degrade ciprofloxacin (CIP) with the assistance of ultrasonic vibration and visible light irradiation. The nitrogen-rich twisty polymeric carbon nitride (PCN) can not only induce a non-centrosymmetric structure with enhanced polarity for a better piezoelectric effect but also provide abundant lone pair electrons to promote n→π* transition during photocatalysis. Its hybridization with C-TiO2 particles can construct S-scheme heterojunction in CNT. During the piezo-photocatalysis, the strain-induced polarization electric field in the heterojunction can regulate the electron migration between the two components, resulting in a more effective CIP degradation. With the synergistic effect of ultrasonic vibration and visible light irradiation, the reaction rate constant of CIP degradation by CNT increases to 0.0517 min-1 , which is 1.86 times that of photocatalysis and 6.46 times that of ultrasound. This system exhibits a stable CIP decomposition efficiency under the interference of various environmental factors. In addition, the in-depth investigation found that three pathways and 12 major intermediates with reduced toxicity are produced after the reaction. Hopefully, the construction of this twisty CNT S-scheme heterojunction with enhanced piezo-photocatalytic effect offers inspiration for the design of environmentally functional materials.

Identification of Human Body Fluid Stains and Non-Biological Stains by Three-Dimensional Fluorescence Spectroscopy.

OBJECTIVES: To establish a rapid and nondestructive identification method for human body fluid stains and non-biological stains using three-dimensional fluorescence spectroscopy. METHODS: The collected three-dimensional fluorescence spectrum data of human saliva, 3% blood, coffee and Fanta® stains were processed with dimensionality reduction. After wavelet transform, spectral denoising and feature extraction, the classification formula was established. The Fisher discriminant was used for spectrum matching and recognition to establish the analysis method to distinguish stain types. RESULTS: According to the results of data training and comparison, all the recognition accuracies of Fanta®, coffee, saliva and blood were more than 91.39%. Among them, saliva reached 100% recognition accuracy. CONCLUSIONS: Three-dimensional fluorescence spectroscopy is a potential method for rapid and nondestructive identification of biological and non-biological stains.

αvß3-targeted sEVs for efficient intracellular delivery of proteins using MFG-E8.

BACKGROUND: Small extracellular vesicles (sEVs) are nanometer-sized membranous particles shed by many types of cells and can transfer a multitude of cargos between cells. Recent studies of sEVs have been focusing on their potential to be novel drug carriers due to natural composition and other promising characteristics. However, there are challenges in sEVs-based drug delivery, one of which is the inefficient loading of drugs into sEVs, especially for large biomolecules. RESULTS: In this study, we proposed a membrane-associated protein, milk fat globule-epidermal growth factor 8 protein (MFG-E8), to produce αvß3-targeted sEVs with high delivery efficiency of interested protein. MFG-E8 is a secreted protein with NH2-terminal epidermal growth factor (EGF)-like domains, containing an Arg-Gly-Asp(RGD) sequence that binds αvß3 and αvß5 integrins, and COOH terminal domains C1 and C2, which can bind to lipid membrane with strong affinity. Firstly, we transiently expressed MFG-E8 in HEK293F cells and found that this protein could be secreted and adhere to the cell membrane. The recombinant MFG-E8 is also found to locate at the outer membrane of sEVs. Then we generated engineered sEVs by expressing high levels of the EGFP fused to MFG-E8 in HEK293F cells and showed that MFG-E8 could increase the delivery efficiency of EGFP into sEVs. Further delivery of Gaussia luciferase (GL) by fusion expression with MFG-E8 in donor cells demonstrated that target proteins fused with MFG-E8 still kept their activity. Finally, we identified the sEVs' target to integrin αvß3 by comparing the transfection efficiency with MFG-E8 loaded sEVs (MFG-E8-sEVs) in αvß3 positive cells and αvß3 negative cells. Analysis showed higher target protein could transfect into αvß3 positive cells with MFG-E8-sEVs than with EGFP loaded sEVs (EGFP-sEVs), meaning the engineered sEVs with MFG-E8 not only could increase the delivery of target protein into sEVs, but also could target the αvß3 positive cells. CONCLUSION: This study suggests that recombinant MFG-E8 is an ideal protein to increasingly deliver the drug into sEVs and give sEVs the ability to target the αvß3 positive cells.

Soluble expression of bioactive recombinant porcine-human chimeric uricase mutant employing MBP-SUMO fusion system.

Urate oxidase is a promising biological medicine for hyperuricemia treatment, but immunogenicity obstructs the development of its clinical application. The recombinant porcine-human chimeric uricase mutant named dHU-wPU is a humanized chimeric uricase based on wild porcine uricase (wPU), which can effectively reduce the limitation of potential immunogenicity with a high homology (92.76%) to deduced human uricase (dHU). Unfortunately, the insoluble expression form of dHU-wPU in E. coli increases the difficulty of production. In this study, we described a more convenient method to efficiently obtain recombinant dHU-wPU protein from E. coli. Combination small ubiquitin-related modifier protein (SUMO) and maltose-binding protein (MBP) was employed to achieve the soluble expression of dHU-wPU. MBP-SUMO-dHU-wPU fusion protein was not only overexpressed in a soluble form, but also showed high purification and cleavage efficiency. Subsequently, we optimized the culture conditions of shake flasks and expanded the production of MBP-SUMO-dHU-wPU fusion protein in a 5 L bioreactor. Finally, about 15 mg of recombinant dHU-wPU was obtained from 1 L M9 fermentation culture by using two-step affinity chromatography, with a SDS-PAGE purity over 90%. In vitro activity analysis showed that dHU-wPU had better ability to catalyze uric acid than wPU.

Aponeurosis discission, a low-detergent method for tissue-engineered acellular ligament scaffolds.

Detergent treatment is the most commonly used method for the decellularization of ligaments and tendon grafts. However, it is well recognized that detergent treatment can also adversely affect the extracellular matrix. This study found that discission into the aponeurosis layer of the patellar tendon (PT) before decellularization is conducive to extracting cells from the PT using a low quantity of detergent in a short time period. The acellular aponeurosis discission ligament (AADL) retains its native collagen fibril structure and mechanical properties. Moreover, the PT retained cell and tissue compatibility in vitro and in vivo. After implantation into a defective allogeneic PT, we found that the AADL healed well in the host, and its collagen structure exhibited gradual improvement 12 months after implantation with satisfactory reconstruction. IMPACT: The aponeurosis of tendons/ligaments is the main barrier to achieving complete decellularization, and it thus prevents complete recellularization for applications in tissue engineering. Aponeurosis can obstruct the removal of cell components. We found that excising the aponeurosis before decellularization allows for the removal of cellular components with a reduced amount of detergent, thus improving the biological properties of the acellular ligament. To the best of our knowledge, no similar studies have been performed. Graphical abstract.

Lyophilized bovine acellular tendon linear fiber material for the reconstruction of attachment structure of paraspinous muscles: an animal in vivo study.

Low back pain is common after lumbar spine surgery and the injury from extensive detachment of paraspinal muscles during the surgery may play a vital role. Previously, we prepared a bovine acellular tendon fiber (ATF) material through lyophilization and proved that it could retain its original fibrillar structure and mechanical properties. The objective of this study is to evaluate the effectiveness of this new fiber material used for attachment structure reconstruction of paraspinal muscle. Defect of spinous process, interspinous and supraspinous ligament was established on lumbar spine in rabbit and rat and ATF linear material was implanted to reconstruct the attachment structure. Ultrasound showed the cross-sectional area of the paraspinal muscle in ATF group was larger than that of control group in rats. MRI showed the irregular shape and high signal changes in control group, but regular shape and uniform signal in the ATF group in rabbit. For Electromyogram, the frequency of evoked potential in control group was lower than ATF group and normal rats. HE and Masson staining showed good tissue healing, and immunohistochemical results showed the immune rejection of ATF is significantly lower than that of suture. Reconstruction of the attachment structure of paraspinous muscles with ATF linear material could maintain the morphology, volume and function of paraspinal muscle. ATF material has the potential to be used to manufacture personalized ligaments and other tissue engineering scaffolds. Graphical abstract.

KdPT alleviates imiquimod-induced psoriasis-like skin lesion in mice via inhibiting proliferation and inflammation response.

Psoriasis is a complex chronic skin inflammatory disease characterized by abnormal proliferation, differentiation of keratinocytes and infiltration of lymphocytes and neutrophils. The tripeptide KdPT, structurally derived from the C-terminal amino acid of alpha-melanocyte-stimulating hormone, has shown a significant anti-inflammatory effect on mild-to-moderate active ulcerative colitis in previous reports. In this research, we investigated whether KdPT could consistently ameliorate disease in a mouse model of imiquimod (IMQ)-induced psoriasis by inhibiting proliferation and inflammation response. We demonstrated that KdPT in vitro significantly inhibited the proliferation of human keratinocytes and endothelial cells, and also downgraded the expression of inflammatory factors in LPS-induced RAW264.7, including IL-6, TNF-α and NO. In vivo, KdPT attenuates the severity of IMQ-induced psoriasis-like phenotype in mice. Such an effect was achieved by downregulating the expression of the inflammatory cytokines interleukin (IL)-6, TNF-α, and the proliferation marker Ki67. These results suggested that KdPT might be useful in the treatment for psoriasis.

SCF-FBXO24 regulates cell proliferation by mediating ubiquitination and degradation of PRMT6.

The protein arginine methyltransferase 6 (PRMT6) is a coregulator of gene expression by methylation of the histone H3 on arginine 2 (H3R2), H4R3 and H2AR3 [1,2]. PRMT6 is aberrantly expressed in various types of human cancer, and abnormal methylation in cancers caused by overexpression of PRMT6 is considered to correlate with poor recovery prognosis [3,4]. However, mechanisms that regulate PRMT6 protein stability in cells remain largely unknown. Here we identified that an orphan F-box protein, FBXO24, that binds to 270 to 275 amino acid residues of PRMT6 to cause polyubiquitination of lysine at position 369 of PRMT6, which mediates its degradation via the ubiquitin-proteasome pathway. Overexpression of FBXO24 or knockout of PRMT6 was found to inhibit cell proliferation, migration, and invasion in H1299 cells. PRMT6 K369R mutant became resistant to degradation. Overexpression of PRMT6 K369R caused cell cycle progression, resulting in cell proliferation. Thus, our data confirm that FBXO24 regulates cell proliferation by mediating ubiquitin-dependent proteasomal degradation of PRMT6.

Preparation of a monoPEGylated derivative of cyanovirin-N and its virucidal effect on acyclovir-resistant strains of herpes simplex virus type 1.

The long-term administration of acyclovir (ACV) for therapy against herpes simplex virus type 1 (HSV-1) infections can result in the emergence of ACV-resistant HSV strains. It is therefore urgent to develop new anti-herpetic compounds with mechanisms that differ from that of ACV. Cyanovirin-N (CV-N) is an antiviral agent that has an inhibitory effect on HSV-1 infections, and PEGylation of CV-N is potentially useful for pharmaceutical applications. Here, a (Gly4Ser)3 linker molecule was attached to the N-terminus of CV-N, and the resulting compound, linker-CV-N (LCV-N), was produced on a pilot scale with purity up to 95%. Then, PEG10k-LCV-N was synthesized by modifying at the α-amine group of the N-terminus of LCV-N with 10-kDa polyethylene glycol propionaldehyde (mPEG-ALD). CV-N, LCV-N and PEG10k-LCV-N were all found to have potent inhibitory activity against ACV-resistant HSV strains with IC50 values in the nM range. LCV-N was the most potent of these three compounds against both normal and ACV-resistant HSV strains. Although PEG10k-LCV-N showed less antiviral activity than CV-N and LCV-N, it still exhibited significant and universal virucidal activity against drug-resistant viruses. The toxicity and immunogenicity of PEG10k-LCV-N were dramatically lower than those of CV-N and LCV-N. In conclusion, we suggest that LCV-N and PEG10k-LCV-N are promising and safe microbicides for the control and/or treatment of ACV-resistant HSV infection.

Study of the mechanism for increased protein expression via transcription potency reduction of the selection marker.

Stable transfection of mammalian cells using various expression cassettes for exogenous gene expression has been well established. The impact of critical factors in these cassettes, such as promoter and enhancer elements, on recombinant protein production in mammalian cells has been studied extensively to optimize the expression efficiency. However, few studies on the correlation between the strength of selection marker and the expression of gene of interest (GOI) have been reported. Here we investigated the correlation between the strength of a widely used selection marker, glutamine synthetase (GS) gene, and gene of interest in which the expression of GOI is driven by mouse cytomegalovirus (mCMV) major immediate early (MIE) promoter whereas the expression of GS is controlled by SV40E (Simian vacuolating virus 40E) promoter. We used a green fluorescent protein and the adalimumab antibody (heavy and light chain) as two distinct examples for the gene of interest. We then decreased the expression of GS gene by engineering a specific region of its SV40E promoter in these expression cassettes. By comparing the expression of GS and GOI at transcription and translation level before and after the SV40E promoter was weakened, we found that lower GS expression due to weaker SV40E transcription correlated well with the higher expression of recombinant proteins, mainly by increasing the copy number of GS and GOI integration into host cell genome.

LPS impairs oxygen utilization in epithelia by triggering degradation of the mitochondrial enzyme Alcat1.

Cardiolipin (also known as PDL6) is an indispensable lipid required for mitochondrial respiration that is generated through de novo synthesis and remodeling. Here, the cardiolipin remodeling enzyme, acyl-CoA:lysocardiolipin-acyltransferase-1 (Alcat1; SwissProt ID, Q6UWP7) is destabilized in epithelia by lipopolysaccharide (LPS) impairing mitochondrial function. Exposure to LPS selectively decreased levels of carbon 20 (C20)-containing cardiolipin molecular species, whereas the content of C18 or C16 species was not significantly altered, consistent with decreased levels of Alcat1. Alcat1 is a labile protein that is lysosomally degraded by the ubiquitin E3 ligase Skp-Cullin-F-box containing the Fbxo28 subunit (SCF-Fbxo28) that targets Alcat1 for monoubiquitylation at residue K183. Interestingly, K183 is also an acetylation-acceptor site, and acetylation conferred stability to the enzyme. Histone deacetylase 2 (HDAC2) interacted with Alcat1, and expression of a plasmid encoding HDAC2 or treatment of cells with LPS deacetylated and destabilized Alcat1, whereas treatment of cells with a pan-HDAC inhibitor increased Alcat1 levels. Alcat1 degradation was partially abrogated in LPS-treated cells that had been silenced for HDAC2 or treated with MLN4924, an inhibitor of Cullin-RING E3 ubiquitin ligases. Thus, LPS increases HDAC2-mediated Alcat1 deacetylation and facilitates SCF-Fbxo28-mediated disposal of Alcat1, thus impairing mitochondrial integrity.

Steady-state structural fluctuation is a predictor of the necessity of pausing-mediated co-translational folding for small proteins.

Translational pausing coordinates protein synthesis and co-translational folding. It is a common factor that facilitates the correct folding of large, multi-domain proteins. For small proteins, pausing sites rarely occurs in the gene body, and the 3'-end pausing sites are only essential for the folding of a fraction of proteins. The determinant of the necessity of the pausings remains obscure. In this study, we demonstrated that the steady-state structural fluctuation is a predictor of the necessity of pausing-mediated co-translational folding for small proteins. Validated by experiments with 5 model proteins, we found that the rigid protein structures do not, while the flexible structures do need 3'-end pausings to fold correctly. Therefore, rational optimization of translational pausing can improve soluble expression of small proteins with flexible structures, but not the rigid ones. The rigidity of the structure can be quantitatively estimated in silico using molecular dynamic simulation. Nevertheless, we also found that the translational pausing optimization increases the fitness of the expression host, and thus benefits the recombinant protein production, independent from the soluble expression. These results shed light on the structural basis of the translational pausing and provided a practical tool for industrial protein fermentation.

Antineoplastic activity of linear leucine homodipeptides and their potential mechanisms of action.

Galaxamide is a rare cyclic homopentapeptide composed of three leucines and two N-methyl leucines isolated from marine algae Galaxaura filamentosa. The strong antitumor activity of this compound makes it a promising candidate for tumor therapy. The synthesis of galaxamide, however, is a complex process, and it has poor water solubility. On the basis of its special chemical composition, we designed a series of linear leucine homopeptides. Among seven dipeptide derivatives, five compounds with terminal protection groups and methyl substitution of the hydrogen in the amido group showed remarkable inhibitory effects against various cancer cells. N-tertbutyl-D-leucine-N-methyl-D-leucinebenzyl (A7), the only stereomer condensed by two D-leucines, showed the highest antineoplastic activity. A7-treated cells showed cell cycle arrest and morphological changes typical of cells undergoing apoptosis. The population of Annexin-V positive/propidium iodide-negative cells also increased, indicating the induction of early apoptosis. A7 promoted the cleavage of caspase-9 and caspase-3, as well as increased intracellular Ca levels and decreased the mitochondrial membrane potential. Collectively, certain linear leucine dipeptides derived from cyclic pentapeptide are able to inhibit tumor cell proliferation through cell cycle arrest and apoptosis induction. The N-methyl group in the side chain and the D/L conformation of the amino-acid residue are critical for their activity.

Histone deacetylase 2 (HDAC2) protein-dependent deacetylation of mortality factor 4-like 1 (MORF4L1) protein enhances its homodimerization.

Histone acetyltransferase mortality factor 4-like 1 (MORF4L1) is a relatively new histone acetyltransferase component that exists as a homodimer to exert its epigenetic function. The mechanism of MORF4L1 self-assembly is unknown. Here we report that Lys-148 deacetylation is indispensable for facilitating MORF4L1 self-assembly into a homodimeric unit. Among a stretch of ∼10 amino acids in the NH2 terminus between the chromodomain and MORF4-related gene (MRG) domain within MORF4L1, Lys-148 is normally acetylated. Substitution of Lys-148 with arginine augments MORF4L1 self-assembly. However, acetylation mimics of MORF4L1, including K148L and K148Q, abolished its self-assembly of the histone acetyltransferase component. HDAC2, a deacetylase, interacts with and keeps MORF4L1 in a deacetylation status at Lys(148) that triggers MORF4L1 self-assembly. Knockdown of HDAC2 reduces MORF4L1 self-assembly. HDAC2-dependent deacetylation of MORF4L1 enhances MORF4L1 homodimerization, thus facilitating the functionality of complex formation to repress cell proliferation.

Characterization and applications of Nanobodies against human procalcitonin selected from a novel naïve Nanobody phage display library.

BACKGROUND: Nanobodies (Nbs) are single-domain antigen-binding fragments derived from the camelids heavy-chain only antibodies (HCAbs). Their unique advantageous properties make Nbs highly attractive in various applications. The general approach to obtain Nbs is to isolate them from immune libraries by phage display technology. However, it is unfeasible when the antigens are toxic, lethal, transmissible or of low immunogenicity. Naïve libraries could be an alternative way to solve the above problems. RESULTS: We constructed a large camel naïve phage display Nanobody (Nb) library with great diversity. The generated library contains to 6.86 × 10(11) clones and to our best of knowledge, this is the biggest naïve phage display Nb library. Then Nbs against human procalcitonin (PCT) were isolated from this library. These Nbs showed comparable affinity and antigen-binding thermostability at 37°C and 60°C compared to the PCT Nbs from an immune phage-displayed library. Furthermore, two PCT Nbs that recognize unique epitopes on PCT have been successfully applied to develop a sandwich enzyme-linked immunosorbent assay (ELISA) to detect PCT, which showed a linear working range from 10-1000 ng/mL of PCT. CONCLUSION: We have constructed a large and diverse naïve phage display Nb library, which potentially functioning as a good resource for selecting antigen-binders with high quality. Moreover, functional Nbs against PCT were successfully characterized and applied, providing great values on medical application.

Image reconstruction method based on CCD calibration in frequency domain.

We demonstrate a method to reconstruct CCD images by calculating out the pixel response function accurately with laser interference patterns. This method is proven theoretically to have the ability to improve image quality greatly and thus may find great application in high-quality imaging fields.

High-density lipoprotein induces cyclooxygenase-2 expression and prostaglandin I-2 release in endothelial cells through sphingosine kinase-2.

High-density lipoprotein (HDL) has a significant cardioprotective effects. HDL induces cyclooxygenase-2 (COX-2) expression and prostacyclin I-2 (PGI-2) release in vascular endothelial cells, which contributes to its anti-atherogenic effects. However, the underlying mechanisms are not fully understood. In the present study, we observed that HDL-stimulated COX-2 expression and PGI-2 production in human umbilical vein endothelial cells (HUVECs) in a time- and dose-dependent manner. These effects triggered by HDL were inhibited by pertussis toxin (PTX), protein kinase C (PKC) inhibitor GF109203X, and ERK inhibitor PD98059, suggesting that Gαi/Gαo-coupled GPCR, PKC, and ERK pathways are involved in HDL-induced COX-2/PGI-2 activation. More importantly, we found that silencing of sphingosine kinase 2 (SphK-2) also blocked HDL-induced COX-2/PGI-2 activation. In addition, HDL-activated SphK-2 phosphorylation accompanied by increased S1P level in the nucleus. Our ChIP data demonstrated that SphK-2 is associated with CREB at the COX-2 promoter region. Collectively, these results indicate that HDL induces COX-2 expression and PGI-2 release in endothelial cells through activation of PKC, ERK1/2, and SphK-2 pathways. These findings implicate a novel mechanism underlying anti-atherothrombotic effects of HDL.

Disordered antireflective subwavelength structures using Ag nanoparticles on fused silica windows.

In this paper, we have demonstrated an effective method for fabricating disordered subwavelength structures (d-SWSs) on fused silica using thermal dewetted Ag nanoparticles at lower temperatures (<300°C) with a vacuum. Theoretically and experimentally, we investigate the effects of the film thickness, annealing temperature, and etching time on the antireflective properties of the d-SWS arrays. The measured data and calculated results obtained by rigorous coupled-wave analysis exhibit reasonably similar tendencies. For the sample with a 10-nm-thick Ag film, good optical transmission characteristics (on one side, T(ave)∼95.6%) over a wide wavelength region of 500-1300 nm were obtained, and a maximum value of ∼96% at a wavelength of 850 nm was also obtained. Furthermore, the d-SWSs exhibit excellent optical and thermal stability at high temperatures of 800°C and 1000°C compared to a conventional Ta2O5/SiO2 multilayer coating.

Evaluating oxidation-reduction properties of dissolved organic matter from Chinese milk vetch (Astragalus sinicus L.): a comprehensive multi-parametric study.

Green manuring is a common practice in replenishment of soil organic matter and nutrients in rice paddy field. Owing to the complex interplay of multiple factors, the oxidation--reduction (redox) properties of dissolved organic matter (DOM) from green manure crops are presently not fully understood. In this study, a variety of surrogate parameters were used to evaluate the redox capacity and redox state of DOM derived from Chinese milk vetch (CMV, Astragalus sinicus L.) via microbial decomposition under continuously flooded (CF) and non-flooded (NF) conditions. Additionally, the correlation between the surrogate parameters of CMV-DOM and the kinetic parameters of relevant redox reactions was evaluated in a soil-water system containing CMV-DOM. Results showed that the redox properties of CMV-DOM were substantially different between the fresh and decomposed CMV-DOM treatments. Determination of the surrogate parameters via ultraviolet-visible/Fourier transform infrared absorption spectroscopy and gel permeation chromatography generally provided high-quality data for predicting the redox capacity of CMV-DOM, while the surrogate parameters determined by elemental analysis were suitable for predicting the redox state of CMV-DOM. Depending on the redox capacity and redox state of various moieties/components, NF-decomposed CMV-DOM could easily accelerate soil reduction by shuttling electrons to iron oxides, because it contained more reversible redox-active functional groups (e.g. quinone and hydroquinone pairs) than CF-decomposed CMV-DOM. This work demonstrates that a single index cannot interpret complex changes in multiple factors that jointly determine the redox reactivity of CMV-DOM. Thus, a multi-parametric study is needed for providing comprehensive information on the redox properties of green manure DOM.