Comparative analysis of proteomes and transcriptomes revealed the molecular mechanism of development and nutrition of Pleurotus giganteus at different fruiting body development stages.

Pleurotus giganteus is a commercially cultivated high-temperature mushroom. Investigating the molecular mechanism of fruiting body development will help us to better understand the regulation of substrates and energy in this process. However, little information has been reported on the development and nutrients of the P. giganteus fruiting body. In the present study, P. giganteus is cultivated in a climate chamber, and comparative transcriptome, proteome, and nutritional analysis of P. giganteus fruiting bodies were performed. Our results revealed that Cytochrome P450 monooxygenases and hydrophobin proteins play important roles during the differentiation in the elongation stage. Later, carbon metabolism dominate the fruiting body metabolism and genes related to the carbohydrate metabolic process, glycolytic process, and gluconeogenesis were up-regulated in the mature fruiting bodies. The up-regulation of carbohydrate substrates utilization CAZymes genes and inconsistent protein expression in pileus indicated a reverse transportation of mRNA from the fruiting body to vegetative mycelia. In addition, protein concentration in the pileus is higher than that in the stem, while the stem is the major nitrogen metabolic and amino acid synthetic location. The integrated transcriptomic, proteomic, and nutritional analysis indicated a two-way transportation of substrates and mRNAs in P. giganteus. Stem synthesizes amino acids and transported them to pileus with reducing sugars, while pileus induces the expression of substrate degradation mRNA according to the needs of growth and development and transports them in the other direction.

Multi-stage nuclear transcriptomic insights of morphogenesis and biparental role changes in Lentinula edodes.

Based on six offspring with different mitochondrial (M) and parental nuclear (N) genotypes, the multi-stage morphological characteristics and nuclear transcriptomes of Lentinula edodes were compared to investigate morphogenesis mechanisms during cultivation, the key reason for cultivar resistance to genotype changes, and regulation related to biparental role changes. Six offspring had specific transcriptomic data and morphological characteristics that were mainly regulated by the two parental nuclei, followed by the cytoplasm, at different growth stages. Importing a wild N genotype easily leads to failure or instability of fruiting; however, importing wild M genotypes may improve cultivars. Major facilitator superfamily (MFS) transporter genes encoding specific metabolites in spawns may play crucial roles in fruiting body formation. Pellets from submerged cultivation and spawns from sawdust substrate cultivation showed different carbon metabolic pathways, especially in secondary metabolism, degradation of lignin, cellulose and hemicellulose, and plasma membrane transport (mainly MFS). When the stage of small young pileus (SYP) was formed on the surface of the bag, the spawns inside were mainly involved in nutrient accumulation. Just broken pileus (JBP) showed a different expression of plasma membrane transporter genes related to intracellular material transport compared to SYP and showed different ribosomal proteins and cytochrome P450 functioning in protein biosynthesis and metabolism than near spreading pileus (NSP). Biparental roles mainly regulate offspring metabolism, growth, and morphogenesis by differentially expressing specific genes during different vegetative growth stages. Additionally, some genes encoding glycine-rich RNA-binding proteins, F-box, and folliculin-interacting protein repeat-containing proteins may be related to multi-stage morphogenesis. KEY POINTS: • Replacement of nuclear genotype is not suitable for cultivar breeding of L. edodes. • Some genes show a biparental role-divergent expression at mycelial growth stage. • Transcriptomic changes of some sawdust substrate cultivation stages have been elucidated.

Corrigendum: An initial prediction and fine-tuning model based on improving GCN for 3D human motion prediction.

[This corrects the article DOI: 10.3389/fncom.2023.1145209.].

An initial prediction and fine-tuning model based on improving GCN for 3D human motion prediction.

Human motion prediction is one of the fundamental studies of computer vision. Much work based on deep learning has shown impressive performance for it in recent years. However, long-term prediction and human skeletal deformation are still challenging tasks for human motion prediction. For accurate prediction, this paper proposes a GCN-based two-stage prediction method. We train a prediction model in the first stage. Using multiple cascaded spatial attention graph convolution layers (SAGCL) to extract features, the prediction model generates an initial motion sequence of future actions based on the observed pose. Since the initial pose generated in the first stage often deviates from natural human body motion, such as a motion sequence in which the length of a bone is changed. So the task of the second stage is to fine-tune the predicted pose and make it closer to natural motion. We present a fine-tuning model including multiple cascaded causally temporal-graph convolution layers (CT-GCL). We apply the spatial coordinate error of joints and bone length error as loss functions to train the fine-tuning model. We validate our model on Human3.6m and CMU-MoCap datasets. Extensive experiments show that the two-stage prediction method outperforms state-of-the-art methods. The limitations of proposed methods are discussed as well, hoping to make a breakthrough in future exploration.

Di-(2-ethylhexyl) phthalate impairs angiogenesis and hematopoiesis via suppressing VEGF signaling in zebrafish.

Di-(2-ethylhexyl) phthalate (DEHP) is among the most widely used plasticizers in plastic production, which has been detected in various environments. However, DEHP safety remains poorly known. Using zebrafish models, the effects of DEHP on the angiogenesis and hematopoiesis, and the underlying mechanism, were studied. Transgenic zebrafish embryos with specific fluorescence of vascular endothelial cells, myeloid cells, or hematopoietic stem cells were exposed to 0, 100, 150, 200, or 250 nM of DEHP for 22, 46 or 70 h, followed by fluorescence observation, endogenous alkaline phosphatase activity measurement, erythrocyte staining, and gene expression analysis by quantitative PCR and whole mount in situ hybridization. High DEHP concentrations decreased the sprouting rate, average diameter, and length, and the expansion area of the vessels lowered the EAP activity and suppressed the vascular endothelial growth factor (vegf) and hematopoietic marker genes, including c-myb, hbae1, hbbe1, and lyz expressions. DEHP treatment also decreased the number of hematopoietic stem cells, erythrocytes, and myeloid cells at 24 and 72 hpf. These DEHP-induced angiogenetic and hematopoietic defects might be alleviated by vegf overexpression. Our results reveal a plausible mechanistic link between DEHP exposure-induced embryonic angiogenetic defect and hematopoietic impairment.

Anti-CXCR2 antibody-coated nanoparticles with an erythrocyte-platelet hybrid membrane layer for atherosclerosis therapy.

Atherosclerosis is the leading cause of mortality globally. RBC-platelet hybrid membrane-coated nanoparticles ([RBC-P]NPs), which biologically mimic platelets in vivo, display evidence of anti-atherosclerotic activity. The efficacy of a targeted RBC-platelet hybrid membrane-coated nanoparticles ([RBC-P]NP)-based approach was investigated as a primary preventive measure against atherosclerosis. A ligand-receptor interactome analysis conducted with circulating platelets and monocytes derived from CAD patients and healthy controls identified CXCL8-CXCR2 as a key platelet ligand-monocyte receptor dyad in CAD patients. Based on this analysis, a novel anti-CXCR2 [RBC-P]NP that specifically binds to CXCR2 and blocks the interaction between CXCL8 and CXCR2 was engineered and characterized. Administering anti-CXCR2 [RBC-P]NPs to Western diet-fed Ldlr-/- mice led to diminished plaque size, necrosis, and intraplaque macrophage accumulation relative to control [RBC-P]NPs or vehicle. Importantly, anti-CXCR2 [RBC-P]NPs demonstrated no adverse bleeding/hemorrhagic effects. A series of in vitro experiments was conducted to characterize anti-CXCR2 [RBC-P]NP's mechanism of action in plaque macrophages. Mechanistically, anti-CXCR2 [RBC-P]NPs inhibited p38α (Mapk14)-mediated, pro-inflammatory M1 skewing and corrected efferocytosis in plaque macrophages. This targeted [RBC-P]NP-based approach, in which the cardioprotective effects of anti-CXCR2 [RBC-P]NP therapy overweighs its bleeding/hemorrhagic risks, could potentially be used to proactively manage atherosclerotic progression in at-risk populations.

Stroke-induced hexokinase 2 in circulating monocytes exacerbates vascular inflammation and atheroprogression.

BACKGROUND: Stroke accelerates inflammatory monocyte recruitment to the endothelium and consequent atheroprogression via high-mobility group box 1-receptor for advanced glycation end products signaling. Notably, Hmgb1 interacts with multiple toll-like receptors (TLRs) and promotes TLR4-mediated proinflammatory myeloid cell activation. Therefore, TLR-associated mechanism(s) within monocytes may play a role in Hmgb1-driven poststroke atheroprogression. OBJECTIVES: We aimed to elucidate the TLR-associated mechanism(s) within monocytes that contribute to stroke-induced exacerbation of atherosclerotic disease. METHODS: A weighted gene coexpression network analysis on the whole blood transcriptomes of stroke model mice identified hexokinase 2 (HK2) as a key gene associated with TLR signaling in ischemic stroke. We conducted a cross-sectional analysis of monocyte HK2 levels in patients with ischemic stroke patients. We performed in vitro and in vivo studies using high-cholesterol diet-fed myeloid-specific Hk2-null ApoE-/- (ApoE-/-;Hk2ΔMφ) mice and ApoE-/-;Hk2fl/fl controls. RESULTS: We found markedly higher monocyte HK2 levels in patients with ischemic stroke patients during the acute and subacute phases poststroke. Similarly, stroke model mice displayed a profound increase in monocyte Hk2 levels. Using aortas and aortic valve samples collected from high-cholesterol diet-fed ApoE-/-;Hk2ΔMφ mice and ApoE-/-;Hk2fl/fl controls, we found that stroke-induced monocyte Hk2 upregulation enhanced poststroke atheroprogression and inflammatory monocyte recruitment to the endothelium. Stroke-induced monocyte Hk2 upregulation induced inflammatory monocyte activation, systemic inflammation, and atheroprogression via Il-1ß. Mechanistically, we demonstrated that stroke-induced monocyte Hk2 upregulation was dependent upon Hmgb1-driven p38-dependent hypoxia-inducible factor-1α stabilization. CONCLUSION: Stroke-induced monocyte Hk2 upregulation is a key mechanism underlying poststroke vascular inflammation and atheroprogression.

New preservation and detection technologies for edible mushrooms: A review.

Edible mushrooms are nutritious, tasty, and have medicinal value, which makes them very popular. Fresh mushrooms have a high water content and a crisp texture. They demonstrate strong metabolic activity after harvesting. However, they are prone to textural changes, microbial infestation, and nutritional and flavor loss, and they therefore require appropriate post-harvest processing and preservation. Important factors affecting safety and quality during their processing and storage include their quality, source, microbial contamination, physical damage, and chemical residues. Thus, these aspects should be tested carefully to ensure safety. In recent years, many new techniques have been used to preserve mushrooms, including electrofluidic drying and cold plasma treatment, as well as new packaging and coating technologies. In terms of detection, many new detection techniques, such as nuclear magnetic resonance (NMR), imaging technology, and spectroscopy can be used as rapid and effective means of detection. This paper reviews the new technological methods for processing and detecting the quality of mainstream edible mushrooms. It mainly introduces their working principles and application, and highlights the future direction of preservation, processing, and quality detection technologies for edible mushrooms. Adopting appropriate post-harvest processing and preservation techniques can maintain the organoleptic properties, nutrition, and flavor of mushrooms effectively. The use of rapid, accurate, and non-destructive testing methods can provide a strong assurance of food safety. At present, these new processing, preservation and testing methods have achieved good results but at the same time there are certain shortcomings. So it is recommended that they also be continuously researched and improved, for example through the use of new technologies and combinations of different technologies. © 2023 Society of Chemical Industry.

Salt reducing and saltiness perception enhancing strategy for shiitake (Lentinus edodes) bud using novel combined treatment of yeast extract and radio frequency.

This study investigated the potential of yeast extract and radio frequency (RF) treatment as a strategy of reducing salt and enhancing saltiness perception for Lentinus edodes bud. The results of E-nose demonstrated yeast extract and RF treatment improved the saltiness of Lentinus edodes bud. Meanwhile, yeast extract and RF treatment significantly decreased the addition of salt (P < 0.05), and led to the formation of special flavor substances, whereas amino acid nitrogen content decreased. On the other hand, sensory attribute, hardness, total flavonoid and phenolic content, antioxidant capacity of L. edodes buds significantly (P < 0.05) increased after the yeast extract combined with RF treatment. In addition, the modification of water distribution, the formation of dense structure, uniform microstructure and Na+ distribution were observed in treated sample, causing the enhancement of saltiness perception. Accordingly, the alteration of properties contributed to higher sensory properties of texture, taste, flavor, and overall acceptability.

Improved understanding on biochar effect in electron supplied anaerobic soil as evidenced by dechlorination and methanogenesis processes.

Research interest in biochar as an environmental remediation material has rapidly increased over the past few years. However, the effect of biochar on typical environmental processes in anaerobic soil environment has been insufficiently discussed. By regulating the electron donors with sodium acetate or pyruvate, the effects and underpinning chemical-microbiological coupling mechanisms of biochar under anaerobic conditions were disclosed. Unlike the electron limited condition, the addition of electron donors alleviated the competition for electrons among various reduction processes in the soil. The effect of biochar in regulating the electron transfer processes was lessened. But more than doubled methane emissions were resulted by the exogenous substances, especially with the synergic effect of biochar. Biochar addition increased soil environmental heterogeneity. It might indirectly affect the reductive transformation of γ-HCH via increasing the bioavailability of pollutants through adsorption and promoting the metabolism of some rare microorganisms. Anaerolineaceae, Peptococcaceae and Methanosarcina had coherent phylogenetic patterns and were likely to be the enablers for the reductive dechlorination process in flooded soil. ENVIRONMENTAL IMPLICATION: Previous studies have widely reported the performance characteristics of biochar, but its effects under anaerobic environments are not systematically understood. By regulating the electron donors, the competition for electrons among various reduction processes in the soil might be alleviated, resulting in a lessened effect of biochar in regulating the electron transfer processes. The findings presented in this study highlight the role of biochar to the dynamic changes of reduction processes under anaerobic environments. The relevant soil conditions such as the electron donors and the functional microbial groups should be adequately considered for maximizing the all-around beneficial efficiency of biochar amendments.

Facile synthesis of isoquinolines and isoquinoline N-oxides via a copper-catalyzed intramolecular cyclization in water.

A highly efficient method for the facile access of isoquinolines and isoquinoline N-oxides via a Cu(i)-catalyzed intramolecular cyclization of (E)-2-alkynylaryl oxime derivatives in water has been developed. This protocol was performed under simple and mild conditions without organic solvent, additives or ligands. By switching on/off a hydroxyl protecting group of oximes, the selective N-O/O-H cleavage could be triggered, delivering a series of isoquinolines and isoquinoline N-oxides, respectively, in moderate to high yields with good functional group tolerance and high atom economy. Moreover, the practicality of this method was further demonstrated by the total synthesis of moxaverine in five steps.

Whole-genome sequence of a high-temperature edible mushroom Pleurotus giganteus (zhudugu).

Most of the sequenced wood-rotting edible mushroom produce fruiting body at relatively low temperatures. Little information has been known about the high-temperature wood-rotting mushroom. Here, we performed de novo sequencing and assembly of the genome of a high-temperature edible mushroom Pleurotus giganteus from a monokaryotic strain zhudugu2 using the Illumina and Pac-Bio CLR sequencing technologies. P. giganteus, also known as Zhudugu in China, is a well-known culinary edible mushroom that has been widely distributed and cultivated in China, Southeast Asia, and South Asia. The genome consists of 40.00 Mb in 27 contigs with a contig N50 of 4.384 Mb. Phylogenetic analysis reveals that P. giganteus and other strains in Pleurotus clustered in one clade. Phylogenetic analysis and average nucleotide identity analysis indicated that the P. giganteus genome showed a closer relationship with other Pleurotus species. Chromosome collinearity analysis revealed a high level of collinearity between P. ostreatus and P. giganteus. There are 12,628 protein-coding genes annotated in this monoploid genome. A total of 481 enzymes accounting for 514 carbohydrate-active enzymes (CAZymes) terms were identified in the P. giganteus genome, including 15 laccases and 10 class II peroxidases predicted in the genome, which revealed the robustness of lignocellulose degradation capacity of P. giganteus. The mating-A type locus of P. giganteus consisted of a pair of homeodomain mating-type genes HD1 and HD2. The mating-B type locus of P. giganteus consisted of at least four pheromone receptor genes and three pheromone genes. The genome is not only beneficial for the genome-assisted breeding of this mushroom but also helps us to understand the high-temperature tolerance of the edible mushroom.

Responses of the Mushroom Pleurotus ostreatus under Different CO2 Concentration by Comparative Proteomic Analyses.

BACKGROUND: Pleurotus ostreatus is a popular edible mushroom in East Asian markets. Research on the responses of P. ostreatus under different carbon dioxide concentrations is limited. METHODS: Label-free LC-MS/MS quantitative proteomics analysis technique was adopted to obtain the protein expression profiles of P. ostreatus fruiting body pileus collected under different carbon dioxide concentrations. The Pearson correlation coefficient analysis and principal component analysis were performed to reveal the correlation among samples. The differentially expressed proteins (DEPs) were organized. Gene ontology analysis was performed to divide the DEPs into different metabolic processes and pathways. RESULTS: The expansion of stipes was inhibited in the high CO2 group compared with that in the low CO2 group. There were 415 DEPs (131 up- and 284 down-regulated) in P. ostreatus PH11 treated with 1% CO2 concentration compared with P. ostreatus under atmospheric conditions. Proteins related to hydrolase activity, including several amidohydrolases and cell wall synthesis proteins, were highly expressed under high CO2 concentration. Most of the kinases and elongation factors were significantly down-regulated under high CO2 concentration. The results suggest that the metabolic regulation and development processes were inhibited under high CO2 concentrations. In addition, the sexual differentiation process protein Isp4 was inhibited under high CO2 concentrations, indicating that the sexual reproductive process was also inhibited under high CO2 concentrations, which is inconsistent with the small fruiting body pileus under high CO2 concentrations. CONCLUSIONS: This research reports the proteome analysis of commercially relevant edible fungi P. ostreatus under different carbon dioxide concentrations. This study deepens our understanding of the mechanism for CO2-induced morphological change in the P. ostreatus fruiting body, which will facilitate the artificial cultivation of edible mushrooms.

Immunogenomic landscape analyses of immune molecule signature-based risk panel for patients with triple-negative breast cancer.

Triple-negative breast cancer (TNBC) presented as high heterogeneous immunogenicity that lacks useful clinical signatures to risk-stratify immune-benefit subtypes. We hypothesized that molecular-based phenotypic characterization of TNBC tumors and their immunity may overcome these challenges. We enrolled 1,145 patients with TNBC for analysis. Through combining algorithm integration analysis and TNBC datasets, a tumor immune risk score (TIRS) panel consisting of 8 potential biomarkers was identified. The TIRS panel represented excellent effectiveness as an independent predictor. High- and low risk stratification of patients was further achieved by TIRS, and significant survival and immune-infiltration pattern differences were found in each cohort, both at the transcriptome and protein levels. Non-negative matrix factorization clustering further identified four different tumor immune microenvironment types (TIMTs), among which TIMT-II was associated with the best prognosis and immune status, whereas TIMT-IV had the opposite effect, TIMT-III was associated with highly unstable genomes, and TIMT-I displayed stem-cell-related characteristics along with high stromal scores and may have extensive enrichment of tumor-associated fibroblasts and vascular cells. In conclusion, our TIRS panel could serve as a robust prognostic signature and provide therapeutic benefits for immunotherapy. Additionally, coordinating four TIMTs may be helpful for clinical decision-making in TNBC patients.

Proangiogenesis effects of compound danshen dripping pills in zebrafish.

BACKGROUND: The compound Danshen Dripping Pill (CDDP), which is a mixture of extracts from Radix Salviae and Panax notoginseng, is a patented traditional Chinese medicine that is widely used in multiple countries for relieving coronary heart disease (CHD), but its pharmacological mechanism has not been fully elucidated. In this study, we screened the key pharmacological pathways and targets of CDDP that act on CHD using a network pharmacology-based strategy, and the angiogenic activity of CDDP was directly visually investigated in zebrafish embryos in vivo. METHODS: The potential therapeutic targets and pathways were predicted through a bioinformatics analysis. The proangiogenic effects of CDDP were examined using vascular sprouting assays on subintestinal vessels (SIVs) and optic arteries (OAs) as well as injury assays on intersegmental vessels (ISVs). Pharmacological experiments were applied to confirm the pathway involved. RESULTS: Sixty-five potential therapeutic targets of CDDP on CHD were identified and enriched in the PI3K/AKT and VEGF/VEGFR pathways. An in vivo study revealed that CDDP promoted angiogenesis in SIVs and OAs in a dose-dependent manner and relieved the impairments in ISVs induced by lenvatinib, a VEGF receptor kinase inhibitor (VRI). In addition, Vegfaa and Kdrl expression were significantly upregulated after CDDP treatment. Furthermore, the proangiogenic effect of CDDP could be abolished by PI3K/AKT pathway inhibitors. CONCLUSIONS: CDDP has a proangiogenic effect, the mechanism of which involves the VEGF/VEGFR and PI3K/AKT signaling pathways. These results suggest a new insight into the cardiovascular protective effect of CDDP.

Ultra-stretchable chitin-based branched elastomers with enhanced mechanical properties via RAFT polymerization.

In this work, a chitin-based macromolecular chain transfer agent (Chitin-CTA) was designed to graft polymers from chitin at the molecular level. Homogeneous reversible addition-fragmentation chain transfer (RAFT) polymerization was performed to prepare branched MA elastomers, chitin-graft-poly(methyl acrylate) (Chitin-g-PMA) copolymers, which were thermally stable and showed tunable glass transition temperatures. These ultra-stretchable branched MA elastomers exhibit unique strain-hardening behavior and significantly enhanced mechanical properties. Mechanical tests indicate that the chitin backbones in branched MA elastomers can act as cross-linking points to improve the tensile strength, toughness, and elasticity simultaneously. The macroscopic performance of branched MA elastomers c be further promoted by introducing hydrogen bonding as non-covalent interaction to form an additional reversible physical network. This robust and versatile grafting strategy can provide new opportunities to prepare chitin-based branched MA elastomers with extraordinary mechanical properties.

Chromosomal genome and population genetic analyses to reveal genetic architecture, breeding history and genes related to cadmium accumulation in Lentinula edodes.

BACKGROUND: Lentinula edodes (Berk.) is the second most productive mushroom in the world. It contains compounds effective for antiviral, antitumor, antioxidant and immune regulation. Although genomes have previously been reported for this species, a high-quality chromosome-level reference for L. edodes is unavailable. This hinders detailed investigation of population genetics, breeding history of strains and genes related to environmental stress responses. RESULTS: A high-quality chromosome-level genome was constructed. We separated a monokaryon from protoplasts of the commercial L. edodes strain L808 and assembled the genome of L. edodes using PacBio long-read and Illumina short-read sequencing, along with the high-throughput chromatin conformation capture (Hi-C) technique. We assembled a 45.87 Mb genome, and 99% of the sequences were anchored onto 10 chromosomes. The contig and scaffold N50 length were 2.17 and 4.94 Mb, respectively. Over 96% of the complete Benchmarking Universal Single-Copy Orthologs (BUSCO) were identified, and 9853 protein-coding genes were predicted. We performed population genome resequencing using 34 wild strains and 65 commercial cultivars of L. edodes originating from China, Japan, the United States and Australia. Based on whole-genome variants, we showed substantial differences in the Chinese wild population, which divided into different branches according to the main areas of their geographical distribution. We also determined the breeding history of L. edodes at the molecular level, and demonstrated that the cultivated strains in China mainly originated from wild strains from China and Northeast Asia. Phenotypic analysis showed that 99 strains exhibited differences on the Cd accumulation. Three significant loci in the of L. edodes genome were identified using the genome-wide association study (GWAS) of Cd accumulation traits. Functional genes associated with Cd accumulation traits were related to DNA ligase and aminoacyl tRNA synthetase, indicating that DNA damage repair and in vivo protein translation may be responses to Cd stress. CONCLUSIONS: A high-quality chromosome-level genome and population genetic data of L. edodes provide genetic resources for functional genomic, evolutionary and artificial breeding studies for L. edodes.

Cultivating Lentinula edodes on Substrate Containing Composted Sawdust Affects the Expression of Carbohydrate and Aromatic Amino Acid Metabolism-Related Genes.

In mushroom cultivation, composting the substrate can make the nutrients more easily absorbed by hyphae due to the degradation of lignin, cellulose, and other organic matter. However, the effects of cultivating Lentinula edodes on composted substrate and the related molecular mechanisms have not been studied systemically. We applied transcriptomics, qRT-PCR, and proteomics to study L. edodes cultivated on substrates with fresh (CK) and composted (ND) sawdust, focusing on the brown film formation stage. The time of brown film formation was shorter and the mycelium growth rate and crude polysaccharide content of the brown film were higher in ND than in CK. The faster growth rate in ND may have been due to the higher nitrogen content in ND than in CK. Among the 9,455 genes annotated using transcriptomics, 96 were upregulated and 139 downregulated in ND compared with CK. Among the 2,509 proteins identified using proteomics sequencing, 74 were upregulated and 113 downregulated. In the KEGG pathway analyses, both differentially expressed genes and proteins were detected in cyanoamino acid metabolism, inositol phosphate metabolism, pentose and glucuronate interconversions, phosphatidylinositol signaling system, RNA polymerase, starch and sucrose metabolism, and tyrosine metabolism pathways. A large number of differentially expressed genes (DEGs) related to aromatic amino acid metabolic and biosynthetic process were upregulated in ND. Most of the DEGs annotated to carbohydrate active enzymes were downregulated in L. edodes growing on composted sawdust containing substrate, possibly due to the lower hemicellulose and cellulose contents in the composted sawdust. The results suggested that using composted substrate may decrease the cultivation time and improve the quality of L. edodes and revealed the underlying molecular mechanisms. IMPORTANCE Composted substrates are not commonly used in the cultivation of Lentinula edodes, thus the effects of cultivating L. edodes on composted substrate and the related molecular mechanisms have not been studied systemically. We studied L. edodes cultivated on substrates with fresh (CK) and composted (ND) sawdust, focusing on the brown film formation stage, and determined the composting related differences in the substrate and in the growth and gene expression of L. edodes. Cultivation on composted substrate was beneficial and showed potential for decreasing the cultivation time and improving the quality of L. edodes. Analyzing the expression levels of genes and proteins in brown film revealed gene and metabolism pathway level changes that accompanied the cultivation on composted substrate.

Single-Cell Transcriptome Analysis Reveals Embryonic Endothelial Heterogeneity at Spatiotemporal Level and Multifunctions of MicroRNA-126 in Mice.

BACKGROUND: Endothelial cells (ECs) play a critical role in angiogenesis and vascular remodeling. The heterogeneity of ECs has been reported at adult stages, yet it has not been fully investigated. This study aims to assess the transcriptional heterogeneity of developmental ECs at spatiotemporal level and to reveal the changes of embryonic ECs clustering when endothelium-enriched microRNA-126 (miR-126) was specifically knocked out. METHODS: C57BL/6J mice embryos at day 14.5 were harvested and digested, followed by fluorescence-activated cell sorting to enrich ECs. Then, single-cell RNA sequencing was applied to enriched embryonic ECs. Tie2 (Tek receptor tyrosine kinase)-cre-mediated ECs-specific miR-126 knockout mice were constructed, and ECs from Tie2-cre-mediated ECs-specific miR-126 knockout embryos were subjected to single-cell RNA sequencing. RESULTS: Embryonic ECs were clustered into 11 groups corresponding to anatomic characteristics. The vascular bed (arteries, capillaries, veins, lymphatics) exhibited transcriptomic similarity across the developmental stage. Embryonic ECs had higher proliferative potential than adult ECs. Integrating analysis showed that 3 ECs populations (hepatic, mesenchymal transition, and pulmonary ECs) were apparently disorganized after miR-126 being knocked out. Gene ontology analysis revealed that disrupted ECs were mainly related to hypoxia, glycometabolism, and vascular calcification. Additionally, in vivo experiment showed that Tie2-cre-mediated ECs-specific miR-126 knockout mice exhibited excessive intussusceptive angiogenesis; reductive glucose and pyruvate tolerance; and excessive accumulation of calcium. Agonist miR-126-3p agomir significantly rescued the phenotype of glucose metabolic dysfunction in Tie2-cre-mediated ECs-specific miR-126 knockout mice. CONCLUSIONS: The heterogeneity of ECs is established as early as the embryonic stage. The deficiency of miR-126 disrupts the differentiation and diversification of embryonic ECs, suggesting that miR-126 plays an essential role in the maintenance of ECs heterogeneity.

The study of early screening technique for fruiting ability of Lentinula edodes hybrid progenies.

Crossbreeding is the most commonly used method in breeding of Lentinula edodes, however low fruiting rate of the hybrids has always caused troubles and barriers for breeders. An early screening method of the fruiting ability could make the breeding work more efficient. In this paper, a rapid and high-throughput laccase activity detection method based on agar diffusion principle was developed. In this way, we investigated the constitutive and inducible extracellular laccase activity of 36 strains in a breeding population of L. edodes on different media and performed a correlation analysis with fruiting ability of these strains. The results showed the laccase activity of mycelium cultured in non-induced medium for 8 d could be used as an early screening index to judge whether it had fruiting ability at the later stage. Early rapid and simple screening method for hybrid populations was established based on laccase activity characteristics of mycelia. 127 strains from another 5 different hybrid populations were used to verify the early screening method. From the validation results, the early screening method was effective, but the appropriate screening threshold was needed to select according to the cross population, which would greatly to improve the breeding efficiency of L. edodes.