Structurally and mechanically tuned macroporous hydrogels for scalable mesenchymal stem cell-extracellular matrix spheroid production.

Mesenchymal stem cells (MSCs) are essential in regenerative medicine. However, conventional expansion and harvesting methods often fail to maintain the essential extracellular matrix (ECM) components, which are crucial for their functionality and efficacy in therapeutic applications. Here, we introduce a bone marrow-inspired macroporous hydrogel designed for the large-scale production of MSC-ECM spheroids. Through a soft-templating approach leveraging liquid-liquid phase separation, we engineer macroporous hydrogels with customizable features, including pore size, stiffness, bioactive ligand distribution, and enzyme-responsive degradability. These tailored environments are conducive to optimal MSC proliferation and ease of harvesting. We find that soft hydrogels enhance mechanotransduction in MSCs, establishing a standard for hydrogel-based 3D cell culture. Within these hydrogels, MSCs exist as both cohesive spheroids, preserving their innate vitality, and as migrating entities that actively secrete functional ECM proteins. Additionally, we also introduce a gentle, enzymatic harvesting method that breaks down the hydrogels, allowing MSCs and secreted ECM to naturally form MSC-ECM spheroids. These spheroids display heightened stemness and differentiation capacity, mirroring the benefits of a native ECM milieu. Our research underscores the significance of sophisticated materials design in nurturing distinct MSC subpopulations, facilitating the generation of MSC-ECM spheroids with enhanced therapeutic potential.

Zero Voltage-Degradation of Li2MnO3 with Ultrathin Amorphous Li─Mn─O Coating.

Manganese-based lithium-rich layered oxides (Mn-LLOs) are promising candidate cathode materials for lithium-ion batteries, however, the severe voltage decay during cycling is the most concern for their practical applications. Herein, an Mn-based composite nanostructure constructed Li2MnO3 (LMO@Li2MnO3) is developed via an ultrathin amorphous functional oxide LixMnOy coating at the grain surface. Due to the thin and universal LMO amorphous surface layer etched from the lithiation process by the high-concentration alkaline solution, the structural and interfacial stability of Li2MnO3 are enhanced apparently, showing the significantly improved voltage maintenance, cycle stability, and energy density. In particular, the LMO@Li2MnO3 cathode exhibits zero voltage decay over 200 cycles. Combining with ex situ spectroscopic and microscopic techniques, the Mn2+/4+ coexisted behavior of the amorphous LMO is revealed, which enables the stable electrochemistry of Li2MnO3. This work provides new possible routes for suppressing the voltage decay of Mn-LLOs by modifying with the composite functional unit construction.

Role of the PARP1/NF-κB Pathway in DNA Damage and Apoptosis of TK6 Cells Induced by Hydroquinone.

Hydroquinone(HQ) is a widely used industrial raw material and is a topical lightening product found in over-the-counter products. However, inappropriate exposure to HQ can pose certain health hazards. This study aims to explore the mechanisms of DNA damage and cell apoptosis caused by HQ, with a focus on whether HQ activates the nuclear factor-κB (NF-κB) pathway to participate in this process and to investigate the correlation between the NF-κB pathway activation and poly(ADP-ribose) polymerase 1(PARP1). Through various experimental techniques, such as DNA damage detection, cell apoptosis assessment, cell survival rate analysis, immunofluorescence, and nuclear-cytoplasmic separation, the cytotoxic effects of HQ were verified, and the activation of the NF-κB pathway was observed. Simultaneously, the relationship between the NF-κB pathway and PARP1 was verified by shRNA interference experiments. The results showed that HQ could significantly activate the NF-κB pathway, leading to a decreased cell survival rate, increased DNA damage, and cell apoptosis. Inhibiting the NF-κB pathway could significantly reduce HQ-induced DNA damage and cell apoptosis and restore cell proliferation and survival rate. shRNA interference experiments further indicated that the activation of the NF-κB pathway was regulated by PARP1. This study confirmed the important role of the NF-κB pathway in HQ-induced DNA damage and cell apoptosis and revealed that the activation of the NF-κB pathway was mediated by PARP1. This research provides important clues for a deeper understanding of the toxic mechanism of HQ.

Recent advances in the chemistry of aryltriazene.

Triazene is one of the most versatile building blocks in organic synthesis. Generally, it is viewed as a safe equivalent of diazonium salt, thus immediately finding numerous applications in preparative chemistry and medicinal chemistry. Besides, it can be used as a removable directing group in C-H functionalization or play a smart role as a precursor for aryl cation/radical generation. In this review, we will highlight recent noteworthy developments in this field.

Long non-coding RNA LINC01480 is activated by Foxo3a and promotes hydroquinone-induced TK6 cell apoptosis by inhibiting the PI3K/AKT pathway.

Long non-coding RNAs (lncRNAs) have been shown to play a critical role in the damage caused to the body by environmental exogenous chemicals; however, few studies have explored their effects during exposure to benzene and its metabolite, hydroquinone (HQ). An emerging lncRNA, LINC01480, was found to be associated with the immune microenvironment of some cancers, but its specific function remains unknown. Therefore, this study aimed to investigate the role of LINC01480 in HQ-induced apoptosis. The biological function of LINC01480 was investigated through gain-of-function and loss-of-function experiments. Mechanically, nuclear-cytoplasmic fractionation experiment, chromatin immunoprecipitation (ChIP), dual-luciferase reporter assay, and rescue experiments were performed. In this study, when TK6 cells were treated with HQ (0, 5, 10, and 20 µM) for 12, 24, 48, and 72 h, the expression of LINC01480 was increased in a dose-dependent manner. Meanwhile, the phosphorylation levels of PI3K and AKT decreased, and apoptosis increased. As compared to the control group, HQ-induced apoptosis was significantly reduced, and the relative survival rate of TK6 cells increased after silencing LINC01480, while overexpression of LINC01480 further sensitized TK6 cells to HQ-induced apoptotic cell death. LINC01480 negatively regulated the PI3K/AKT pathway in TK6 cells, and the apoptosis-inhibiting effect of LINC01480 silencing was reversed after inhibition of the PI3K/AKT pathway. In addition, ChIP and the dual-luciferase reporter assays showed that the transcription factor Foxo3a promoted LINC01480 transcription by directly binding to the promoter regions - 149 to - 138 of LINC01480. Moreover, short-term HQ exposure promoted the expression of Foxo3a. From these findings, we can conclude that LINC01480 is activated by Foxo3a, and promotes HQ-induced apoptosis by inhibiting the PI3K/AKT pathway, suggesting that LINC01480 might become a possible target for therapeutic intervention of HQ-induced toxicity.

Hydroquinone-induced endoplasmic reticulum stress affects TK6 cell autophagy and apoptosis via ATF6-mTOR.

Hydroquinone (HQ), one of the main active metabolites of benzene in vivo, 7is commonly used as a surrogate for benzene in in vitro studies and has been shown to be cytotoxic. The aim of this study was to investigate the role of endoplasmic reticulum stress (ERS) in HQ-induced autophagy and apoptosis in human lymphoblastoid cells (TK6) and how activating transcription factor 6 (ATF-6) is involved. We treated TK6 cells with HQ to establish a cytotoxicity model and found that HQ induced cellular ERS, autophagy and apoptosis by Western blot, flow cytometry and transmission electron microscopy. In addition, inhibition of both reactive oxygen species (ROS) and ERS inhibited cellular autophagy and apoptosis, suggesting that ERS may be induced by ROS, which in turn affects autophagy and apoptosis. Our study also found that HQ could inhibit ATF6 expression and mTOR activation. Knockdown of ATF6 enhanced autophagy and apoptosis levels and further inhibited mTOR activation; activation of ATF6 by AA147 enhanced cellular activity, suggesting that ATF6 may affect cellular autophagy and apoptosis through mTOR. In conclusion, our data suggest that ROS mediated ERS may promote autophagy and apoptosis by inhibiting ATF6-mTOR pathway after HQ treatment of TK6 cells.

Effect of mir-92a-3p on hydroquinone induced changes in human lymphoblastoid cell cycle and apoptosis.

Hydroquinone (HQ), one of the metabolites of benzene in humans, has significant hepatotoxic properties. Chronic exposure to HQ can lead to leukemia. In a previous study by this group, we constructed a model of malignant transformation of human lymphoblastoid cells (TK6) induced by chronic exposure to HQ with significant subcutaneous tumorigenic capacity in nude mice. miR-92a-3p is a tumor factor whose role in HQ-induced malignant transformation is not yet clear. In the present study, raw signal analysis and dual-luciferase reporter gene results suggested that miR-92a-3p could target and regulate TOB1, and the expression level of miR-92a-3p was significantly upregulated in the long-term HQ-induced TK6 malignant transformation model, while the anti-proliferative factor TOB1 was significantly downregulated. To investigate the mechanism behind this, we inhibited miR-92a-3p in a malignant transformation model and found a decrease in cell viability, a decrease in MMP-9 protein levels, a G2/M phase block in the cell cycle, and an upregulation of the expression of G2/M phase-related proteins cyclinB1 and CDK1. Inhibition of miR-92a-3p in combination with si-TOB1 restored cell viability, inhibited cyclin B1 and CDK1 protein levels, and attenuated the G2/M phase block. Taken together, miR-92a-3p reduced the cell proliferation rate of HQ19 and caused cell cycle arrest by targeting TOB1, which in turn contributed to the altered malignant phenotype of the cells. This study suggests that miR-92a-3p is likely to be a biomarker for long-term HQ-induced malignant transformation of TK6 and could be a potential therapeutic target for leukemia caused by long-term exposure to HQ.

Insights into a novel nitrogen removal process based on simultaneous anammox and denitrification (SAD) following nitritation with in-situ NOB elimination.

Simultaneous anammox and denitrification (SAD) is an efficient approach to treat wastewater having a low C/N ratio; however, few studies have investigated a combination of SAD and partial nitritation (PN). In this study, a lab-scale up-flow blanket filter (UBF) and zeolite sequence batch reactor (ZSBR) were continuously operated to implement SAD and PN advantages, respectively. The UBF achieved a high total nitrogen (TN) removal efficiency of over 70% during the start-up stage (days 1-50), and reached a TN removal efficiency of 96% in the following 90 days (days 51-140) at COD/NH4+-N ratio of 2.5. The absolute abundance of anammox bateria increased to the highest value of 1.58 × 107 copies/µL DNA; Comamonadaceae was predominant in the UBF at the optimal ratio. Meanwhile, ZSBR was initiated on day 115 as fast nitritation process to satisfy the influent requirement for the UBF. The combined process was started on day 140 and then lasted for 30 days, during the combined process, between the two reactors, the UBF was the main contributor for TN (66.5% ± 4.5%) and COD (71.8% ± 4.9%) removal. These results demonstrated that strong SAD occurred in the UBF when following a ZSBR with in-situ NOB elimination. This research presents insights into a novel biological nitrogen removal process for low C/N ratio wastewater treatment.

A Trinuclear Zinc Coordination Cluster Exhibiting Fluorescence, Colorimetric Sensitivity, and Recycling of Silver Ion and Detection of Cupric Ion.

The detection and reusage of transition-metal ions play a crucial role in human health and environmental protection. Recently, various analytical methods and substances have been successfully applied to probe or sense silver ions; however, rare representative examples have been presented regarding the simultaneous detection of silver and silver recycling with the elemental silver powder form. Herein, an unparalleled sensing mechanism for silver ions and recycling silver in its elemental form is exemplified by a fluorescent trinuclear zinc coordination cluster possessing the dual function of colorimetric sensing of silver and responding cupric ions. A Schiff-base-based trinuclear zinc coordination cluster, 1, with formula Zn3(L1)2(CH3COO)2(H2O)2, has been successfully synthesized by the initial exploration of multidentate ligand H2L1-((E)-2,4-di-tert-butyl-6-((2-hydroxy-3-methoxybenzy-lidene)amino)phenol) with various metal ions under self-assembly reactions. Complex 1 is highly fluorescent in solution and as a solid, in addition to acting as a fluorescence sensor toward AgI in ethanol media. Compound 1 displays distinctive sensing of AgI through the fluorescence quenching effect at 576 nm and signal augment at 446 nm over 11 kinds of cations in the absence of interference. The proposed sensing mechanism is attributed to the ligands in 1 which interact with AgI; the ligands undergo oxidation cyclization reaction, leading to the formation of 2 with the formula Zn3(L2)4(CH3COO)2·2CH3CH2OH·H2O, and AgI reduction to elemental Ag powder. Compound 1 presents specific selectivity and sensitivity for AgI in ethanolic solution with a detection limit of 0.1722 µM. The orange color of 1 changes to colorless during the mixing of a small amount of AgI, revealing its potential practical application in naked-eye detection of AgI. Furthermore, 2 exhibits obvious fluorescence emission at 448 nm (λex = 380 nm) and selectively responds to CuII over 11 kinds of metal ions with the fluorescence "turn-off" owing to the formation of 3 in ethanolic solution; it also has a detection limit of 0.0226 µM.

An Inconspicuous Six-Coordinate Neutral DyIII Single-Ion Magnet with Remarkable Magnetic Anisotropy and Stability.

It is a crucial challenge to address both magnetic anisotropy and stability for single-molecule magnets (SMMs) used in next-generation nanodevices. Highly axial lanthanide SMMs with neutral charge and moderate coordination numbers represent promising magnetic materials. Here, using iodide ions with large volume and low surface charge density as weak donors, we report a six-coordinate neutral dysprosium SMM [Dy(Cy3PO)2I3(CH3CN)] with a certain degree of stability exhibiting a huge thermal barrier of 1062 K and hysteresis loops open up to 9 K. Through the elaborate reduction of ligand field strength, an apparent strongly axial crystal field is provided which elicits prominent crystal-field splitting and high axiality with the thermally activated relaxation via the third-excited Kramers' doublet. Moreover, the profound influence of strong equatorial ligand substitution on the electronic structure and relaxation pathway is clearly explored in DyIII analogues. The result suggests the great potential of the reducing the transverse ligand field in the improvement of SMMs performance.

Probabilistic sensitivity analysis on Markov models with uncertain transition probabilities: an application in evaluating treatment decisions for type 2 diabetes.

Markov models are commonly used for decision-making studies in many application domains; however, there are no widely adopted methods for performing sensitivity analysis on such models with uncertain transition probability matrices (TPMs). This article describes two simulation-based approaches for conducting probabilistic sensitivity analysis on a given discrete-time, finite-horizon, finite-state Markov model using TPMs that are sampled over a specified uncertainty set according to a relevant probability distribution. The first approach assumes no prior knowledge of the probability distribution, and each row of a TPM is independently sampled from the uniform distribution on the row's uncertainty set. The second approach involves random sampling from the (truncated) multivariate normal distribution of the TPM's maximum likelihood estimators for its rows subject to the condition that each row has nonnegative elements and sums to one. The two sampling methods are easily implemented and have reasonable computation times. A case study illustrates the application of these methods to a medical decision-making problem involving the evaluation of treatment guidelines for glycemic control of patients with type 2 diabetes, where natural variation in a patient's glycated hemoglobin (HbA1c) is modeled as a Markov chain, and the associated TPMs are subject to uncertainty.

Biological technologies for the remediation of co-contaminated soil.

Compound contamination in soil, caused by unreasonable waste disposal, has attracted increasing attention on a global scale, particularly since multiple heavy metals and/or organic pollutants are entering natural ecosystem through human activities, causing an enormous threat. The remediation of co-contaminated soil is more complicated and difficult than that of single contamination, due to the disparate remediation pathways utilized for different types of pollutants. Several modern remediation technologies have been developed for the treatment of co-contaminated soil. Biological remediation technologies, as the eco-friendly methods, have received widespread concern due to soil improvement besides remediation. This review summarizes the application of biological technologies, which contains microbial technologies (function microbial remediation and composting or compost addition), biochar, phytoremediation technologies, genetic engineering technologies and biochemical technologies, for the remediation of co-contaminated soil with heavy metals and organic pollutants. Mechanisms of these technologies and their remediation efficiencies are also reviewed. Based on this study, this review also identifies the future research required in this field.

The interactions of composting and biochar and their implications for soil amendment and pollution remediation: a review.

Compost and biochar, used for the remediation of soil, are seen as attractive waste management options for the increasing volume of organic wastes being produced. This paper reviews the interaction of biochar and composting and its implication for soil amendment and pollution remediation. The interaction of biochar and composting affect each other's properties. Biochar could change the physico-chemical properties, microorganisms, degradation, humification and gas emission of composting, such as the increase of nutrients, cation exchange capacity (CEC), organic matter and microbial activities. The composting could also change the physico-chemical properties and facial functional groups of biochar, such as the improvement of nutrients, CEC, functional groups and organic matter. These changes would potentially improve the efficiency of the biochar and composting for soil amendment and pollution remediation. Based on the above review, this paper also discusses the future research required in this field.

Experimental and Theoretical Interpretation on the Magnetic Behavior in a Series of Pentagonal-Bipyramidal DyIII Single-Ion Magnets.

Taking advantage of the steric hindrance and charge-driving effects, four air-stable pentagonal bipyramidal mononuclear DyIII compounds were hydrothermally synthesized. With a tetradentate ligand, N,N'-bis(2-methylenepyridinyl)ethylenediamine (Bpen), invariably coordinates to DyIII in an equatorial plan, 1-3 achieve an orderly transformation of the ligand field by sequentially replacing the remaining sites of the DyIII ion. Compound 4 possesses the same coordination atoms but a different peripheral coordination sphere with 3. Magnetic characterizations display that the compounds are field-induced single-ion magnets (SIM) with actually low barriers, even though 2 has both the same atoms and a similar geometry of the first sphere compared with [Dy(bbpen)Cl] (2', H2 bbpen=N,N'-bis(2-hydroxybenzyl)-N,N'-bis(2-methylpyridyl)ethylenediamin), a high-performance SIM previously reported. Detailed ab initio calculations have been employed to further elucidate the electronic and magnetic structure of the low-lying energy levels of compounds 1-4 and 2'. The theoretical results indicate there is an apparent difference in the electronic structure for these compounds. The analysis on the electrostatic potential further demonstrates that although the pentagonal bipyramidal D5h is one of the ideal configurations expected, the electron density of the donor atoms from the different hybridizations and other functional groups, outside the first sphere, should also be considered in the rational design of promising molecular magnets.

Magnetic Interaction Affecting the Zero-Field Single-Molecule Magnet Behaviors in Isomorphic {NiII2DyIII2} and {CoII2DyIII2} Tetranuclear Complexes.

Great interest is being shown in investigating magnetic interactions that efficiently influence lanthanide single-molecule magnet behavior. A series of heterometallic complexes [M2Ln2(Hhms)2(CH3COO)6(CH3OH)2(H2O)2]·(NO3)2 (M = NiII, Ln = DyIII (1), GdIII (2), and YIII (3); M = CoII, Ln = DyIII (4), GdIII (5), and YIII (6)) have been prepared with a compartmental Schiff-base ligand, 1-(2-hydroxy-3-methoxybenzylidene)-semicarbazide (H2hms), featuring a zigzag-shaped MII-LnIII-LnIII-MII metallic core arrangement. In complexes 1-6, a unique monophenoxo/diacetate asymmetric bridging connects MII ion with LnIII ion, and four acetates bridge two LnIII ions where acetates play essential roles as coligand in generating the tetranuclear units. Magnetic studies reveal the presence of predominant ferromagnetic coupling in DyIII and GdIII derivatives, and slow relaxation of magnetization is observed for {Ni2IIDy2III} and {CoII2DyIII2} with an energy barrier of 16.0 K for {Ni2IIDy2III} and 6.7 K for {CoII2DyIII2} under zero static field. Compared with the analogue {CoII2DyIII2}, the {Ni2IIDy2III} shows longer relaxation time and an absence of the quantum tunnelling of the magnetization (QTM) at low temperatures. Ab initio calculations suggest that the zero-field QTM of {Ni2IIDy2III} is effectively interrupted thanks to the ferromagnetic exchange coupling generated between NiII and DyIII ions. The presence of ferromagnetic exchange between NiII and DyIII ions is more conducive to zero-field single-molecule magnet behaviors than in isomorphic {CoII2DyIII2} where the exchange is antiferromagnetic.

Responses of bacterial community and functional marker genes of nitrogen cycling to biochar, compost and combined amendments in soil.

Biochar and compost are seen as two attractive waste management options and are used for soil amendment and pollution remediation. The interaction between biochar and composting may improve the potential benefits of biochar and compost. We investigated soil physicochemical properties, bacterial community, bacterial 16S rRNA, and functional marker genes of nitrogen cycling of the soil remedied with nothing (S), compost (SC), biochar (SB), a mixture of compost and biochar (SBC), composted biochar (SBced), and a composted mixture of biochar and biomass (SBCing). The results were that all amendments (1) increased the bacterial community richness (except SB) and SBCing showed the greatest efficiency; (2) increased the bacterial community diversity (SBCing > SBC > SC > SBced > SB > S); and (3) changed the gene copy numbers of 16S rRNA, nirK, nirS, and nosZ genes of bacteria, ammonia-oxidizing archaea (AOA), and ammonia-oxidizing bacteria (AOB). All amendments (except SB) could increase the gene copy number of 16S rRNA, and SBCing had the greatest efficiency. The changes of soil bacterial community richness and diversity and the gene copy numbers of 16S rRNA, nirK, nirS, nosZ, AOA, and AOB would affect carbon and nitrogen cycling of the ecosystem and also implied that BCing had the greatest efficiency on soil amendment.

Response of rhizosphere microbial community structure and diversity to heavy metal co-pollution in arable soil.

Due to the emerging environmental issues related to heavy metals, concern about the soil quality of farming lands near manufacturing district is increasing. Investigating the function of soil microorganisms exposed to long-term heavy metal contamination is meaningful and important for agricultural soil utilization. This article studied the potential influence of several heavy metals on microbial biomass, activity, abundance, and community composition in arable soil near industrial estate in Zhuzhou, Hunan province, China. The results showed that soil organic contents (SOC) were significantly positive correlated with heavy metals, whereas dehydrogenase activity (DHA) was greatly depressed by the heavy metal stress. Negative correlation was found between heavy metals and basal soil respiration (BSR), and no correlation was found between heavy metals and microbial biomass content (MBC). The quantitative PCR (QPCR) and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis could suggest that heavy metal pollution has significantly decreased abundance of bacteria and fungi and also changed their community structure. The results could contribute to evaluate heavy metal pollution level in soil. By combining different environmental parameters, it would promote the better understanding of heavy metal effect on the size, structure, and activity of microbial community in arable soil.

Environmental factors shaping the abundance and distribution of laccase-encoding bacterial community with potential phenolic oxidase capacity during composting.

Increasing molecular evidence points to a wide occurrence of laccase-like multicopper oxidase (LMCO)-encoding genes in bacteria. Most researches mainly focused on the bacterial LMCO diversity, whereas the processes and the environmental factors responsible for structuring bacterial LMCO communities remain relatively unknown in a composting system. Six gene libraries were constructed from samples in representative stages during composting. A total of 185 sequences obtained from sample DNA extracts were classified to 59 operational taxonomic units (OTUs) based on 10 % cutoff. The distribution profile of bacterial LMCO genes showed that proteobacterial- and actinobacterial-associated species were the dominant communities during composting. Pearson correlation analysis indicated that the pile temperature and water-soluble carbon (WSC) content were significantly positively correlated with bacterial LMCO gene OTU numbers, Chao1 and Shannon index, whereas the humic acid (HA)-like carbon content had the most significant effect on the distribution of the bacterial LMCO genes during composting by redundancy analysis. These findings will improve the understanding of the mutual relationship between environmental factors and bacterial LMCO community compositions in composting.

Spatial and temporal variation of heavy metal risk and source in sediments of Dongting Lake wetland, mid-south China.

Surface sediments of Dongting Lake wetland were collected from ten sites to investigate variation trend, risk and sources of heavy metal distribution in dry seasons of 2011∼2013. The three-year mean concentrations (mg/kg) of Cr, Cu, Pb, Cd, Hg and As were 91.33, 36.27, 54.82, 4.39, 0.19 and 25.67, respectively, which were all higher than the corresponding background values. Sediment quality guidelines (SQGs) and Geo-accumulation index (Igeo) were used for the assessment of pollution level of heavy metals. The pollution risk of Cd, Hg and As were great and that of Cr needed urgent attention because of its obvious increase. Pollution load index (PLI) and geographic information system (GIS) methods were conducted to assess spatial and temporal variation of heavy metal contamination. Results confirmed an increased contamination contribution inflow from Xiang River. Multivariate statistical analyses were applied to identify contribution sources of heavy metal, which showed anthropogenic origin mainly from mining, smelting, chemical industry and agricultural activity.

Diversity of two-domain laccase-like multicopper oxidase genes in Streptomyces spp.: identification of genes potentially involved in extracellular activities and lignocellulose degradation during composting of agricultural waste.

Traditional three-domain fungal and bacterial laccases have been extensively studied for their significance in various biotechnological applications. Growing molecular evidence points to a wide occurrence of more recently recognized two-domain laccase-like multicopper oxidase (LMCO) genes in Streptomyces spp. However, the current knowledge about their ecological role and distribution in natural or artificial ecosystems is insufficient. The aim of this study was to investigate the diversity and composition of Streptomyces two-domain LMCO genes in agricultural waste composting, which will contribute to the understanding of the ecological function of Streptomyces two-domain LMCOs with potential extracellular activity and ligninolytic capacity. A new specific PCR primer pair was designed to target the two conserved copper binding regions of Streptomyces two-domain LMCO genes. The obtained sequences mainly clustered with Streptomyces coelicolor, Streptomyces violaceusniger, and Streptomyces griseus. Gene libraries retrieved from six composting samples revealed high diversity and a rapid succession of Streptomyces two-domain LMCO genes during composting. The obtained sequence types cluster in 8 distinct clades, most of which are homologous with Streptomyces two-domain LMCO genes, but the sequences of clades III and VIII do not match with any reference sequence of known streptomycetes. Both lignocellulose degradation rates and phenol oxidase activity at pH 8.0 in the composting process were found to be positively associated with the abundance of Streptomyces two-domain LMCO genes. These observations provide important clues that Streptomyces two-domain LMCOs are potentially involved in bacterial extracellular phenol oxidase activities and lignocellulose breakdown during agricultural waste composting.