A dual channel optical sensing composite for the detection of picric acid (PA) was constructed, using luminescent lanthanide MOF as supporting substrate and a rhodamine-based dye as sensing probe, ...respectively. These rod-liked composite nanocrystals had dual sensing channels towards PA, including colorimetric sensing and ratiometric fluorescent sensing. A linear working curve with limit of detection as low as 10μM was obtained. Good selectivity towards PA was confirmed.
Display omitted
•A dual channel optical sensing composite for the detection of picric acid was constructed.•Colorimetric sensing and ratiometric fluorescent sensing were realized.•A linear working curve with limit of detection as low as 10μM was obtained.•Sensing mechanism was the combination of rhodamine emission increase and nitrophenyl-triggered Eu emission quenching.
For the present work, a dual channel optical sensing composite for the detection of picric acid (PA) was constructed, using luminescent lanthanide MOF as supporting substrate and a rhodamine-based dye as sensing probe, respectively. This composite material was discussed in detail by means of electron microscopy images, XRD, IR, TGA, absorption and emission spectra. These rod-liked composite nanocrystals had dual sensing channels towards PA, including colorimetric sensing and ratiometric fluorescent sensing. A linear working curve with limit of detection as low as 10μM was obtained. Good selectivity towards PA was confirmed. Their sensing mechanism was discussed and found as the combination of proton-triggered rhodamine structural transformation and nitrophenyl-triggered Eu emission quenching. This composite material showed a superior sensing performance over literature ones owing to its dual sensing channels and the possibility of naked eye detection.
Cultivating oyster mushrooms (
Pleurotus ostreatus
), a typical primary decomposer of lignocellulose, on a short composting substrate is a novel procedure which possesses energy conserves, reduced ...the chance of infection by competitive species, shorter production duration and achieved high production efficiency. However, the microbiome and microbial metabolic functions in the composting substrate during the mushroom cropping is unknown. In the present study, the contents of hemicellulose, cellulose and lignin and the activities of protease, laccase and cellulase were evaluated in the corncob short composting substrate from before oyster mushroom spawning to first flush fructification; meanwhile the changes in the microbiome and microbial metabolic functions were surveyed by using metagenomic sequencing. Results showed that the hemicellulose, cellulose and lignin in the short composting substrate were decomposed of 42.76, 34.01, and 30.18%, respectively, during the oyster mushroom cropping process. In addition, the contents of hemicellulose, cellulose and lignin in the composting substrate were reduced rapidly and negatively correlated with the abundance of the Actinobacteria phylum. The activities of protease, laccase and cellulase fastly increased in the period of before oyster mushroom spawning to full colonization and were positively correlated to the abundance of Actinobacteria phylum. The total abundance of bacteria domain gradually decreased by only approximately 15%, while the abundance of Actinobacteria phylum increased by 68% and was positively correlated with that of oyster mushroom. The abundance of oyster mushroom increased by 50 times from spawning to first flush fructification. The dominant genera, all in the order of Actinomycetales, were
Cellulosimicrobium
,
Mycobacterium
,
Streptomyces
and
Saccharomonospora
. The total abundance of genes with functions annotated in the Clusters of Orthologous Groups of proteins (COG) for Bacteria and Archaea and Kyoto Encyclopedia of Genes and Genomes (KEGG) database for all three life domains was positively correlated.
The three metabolic pathways for carbohydrates, amino acids and energy were the primary enrichment pathways in KEGG pathway, accounting for more than 30% of all pathways, during the mushroom cropping in which the glycine metabolic pathway, carbon fixation pathways in prokaryotes and methane metabolism were all dominated by bacteria. The genes of cellulolytic enzymes, hemicellulolytic enzymes, laccase, chitinolytic enzymes, peptidoglycanlytic enzymes and ammonia assimilation enzymes with abundances from 0.28 to 0.24%, 0.05 to 0.02%, 0.02 to 0.01%, 0.14 to 0.08%, 0.39 to 0.16%, and 0.13 to 0.12% during the mushroom cropping identified in the Evolutionary Genealogy of Genes: Non-supervised Orthologous Groups (eggNOG) database for all three life domains were all aligned to COG database. These results indicated that bacteria, especially Actinomycetales, were the main metabolism participants in the short composting substrate during the oyster mushroom cropping. The relationship between oyster mushrooms and bacteria was cooperative, Actinomycetales were oyster mushroom growth promoting bacteria (OMGPB).
In this study, Taisui TS-2007S, a previously unidentified biological object discovered in soil in China, was identified. TS-2007S was shown to contain abundant carbohydrates but a scarcity of ...protein, fat, and minerals. The exopolymers of TS-2007S showed FT-IR spectra that were similar to those of xanthan gum (XG) but that were dissimilar to those of polyvinyl alcohol (PVA). The NMR spectra of TS-2007S exopolymers in D
2
O were similar to those of PVA but differed from those of xanthan gum. Unlike PVA, TS-2007S exopolymers and xanthan gum were not soluble in dimethyl sulfoxide (DMSO). Furthermore, the exopolymers contained many monosaccharide components, including fucose, rhamnose, mannose, and glucuronic acid in a molar ratio of 87.90:7.49:4.45:0.15. The exopolymers also included traces of glucuronic acid, galactose, and xylose. Taken together, these results suggest that the exopolymers are microbial extracellular polymeric substances (EPSs). The microbial community structure in TS-2007S showed that the predominant bacterial, archaeal, and fungal phyla were Proteobacteria, Euryarchaeota, and Ascomycota at high relative abundances of 90.77, 97.15, and 87.43%, respectively, different from those observed in water and soil environments. Based on these results, we strongly propose that TS-2007S should be defined as a microbial mat formed in soil.
Dominant Vγ2Vδ2 T-cell subset exist only in primates, and recognize phosphoantigen from selected pathogens including M. tuberculosis(Mtb). In vivo function of Vγ2Vδ2 T cells in tuberculosis remains ...unknown. We conducted mechanistic studies to determine whether earlier expansion/differentiation of Vγ2Vδ2 T cells during Mtb infection could increase immune resistance to tuberculosis in macaques. Phosphoantigen/IL-2 administration specifically induced major expansion and pulmonary trafficking/accumulation of phosphoantigen-specific Vγ2Vδ2 T cells, significantly reduced Mtb burdens and attenuated tuberculosis lesions in lung tissues compared to saline/BSA or IL-2 controls. Expanded Vγ2Vδ2 T cells differentiated into multifunctional effector subpopulations capable of producing anti-TB cytokines IFNγ, perforin and granulysin, and co-producing perforin/granulysin in lung tissue. Mechanistically, perforin/granulysin-producing Vγ2Vδ2 T cells limited intracellular Mtb growth, and macaque granulysin had Mtb-bactericidal effect, and inhibited intracellular Mtb in presence of perforin. Furthermore, phosphoantigen/IL2-expanded Vγ2Vδ2 T effector cells produced IL-12, and their expansion/differentiation led to enhanced pulmonary responses of peptide-specific CD4+/CD8+ Th1-like cells. These results provide first in vivo evidence implicating that early expansion/differentiation of Vγ2Vδ2 T effector cells during Mtb infection increases resistance to tuberculosis. Thus, data support a rationale for conducting further studies of the γδ T-cell-targeted treatment of established TB, which might ultimately help explore single or adjunctive phosphoantigen expansion of Vγ2Vδ2 T-cell subset as intervention of MDR-tuberculosis or HIV-related tuberculosis.
Mushroom β-glucans are potent immunological stimulators in medicine, but their productivities are very low. In this study, we successfully improved its production by promoter engineering in Pleurotus ...ostreatus. The promoter for β-1,3-glucan synthase gene (GLS) was replaced by the promoter of glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans. The homologous recombination fragment for swapping GLS promoter comprised five segments, which were fused by two rounds of combined touchdown PCR and overlap extension PCR (TD-OE PCR), and was introduced into P. ostreatus through PEG/CaCl2-mediated protoplast transformation. The transformants exhibited one to three fold higher transcription of GLS gene and produced 32% to 131% higher yield of β-glucans than the wild type. The polysaccharide yields had a significant positive correlation to the GLS gene expression. The infrared spectra of the polysaccharides all displayed the typical absorption peaks of β-glucans. This is the first report of successful swapping of promoters in filamentous fungi.
Ethylene regulates mycelial growth, primordium formation, and postharvest mushroom maturation and senescence in the white button mushroom, Agaricus bisporus. However, it remains unknown how ethylene ...is detected by the mushroom. In this study, we found that two hybrid histidine kinases in the mushroom, designated
ETR1 and
ETR2, showed domain structures similar to those of plant ethylene receptors. The transmembrane helices of
ETR1 and
ETR2 were expressed in yeast cells and showed ethylene-binding activities. Mushroom strains with downregulated expressions of
ETR1 and
ETR2 showed reduced sensitivity to the ethylene inhibition of mycelial growth, ethylene regulation of their own synthesis, postharvest mushroom maturation, and senescence and expression of maturation- and senescence-related genes. Therefore,
ETR1 and
ETR2 are expected to be biologically functional ethylene receptors and exhibit a different mode of action from that of the receptors of plants. Here, we fill gaps in the knowledge pertaining to higher fungus ethylene receptors, discover a novel mode of action of ethylene receptors, confirm ethylene as a novel fungal hormone, and provide a facilitated approach for preventing the maturation and senescence of postharvest button mushrooms.
Ethylene regulates diverse physiological activities in bacteria, cyanobacteria, fungi, and plants, but how to perceive ethylene by fungi only remains unknown. In this study, we identify two biologically functional ethylene receptors in the basidiomycete fungus Agaricus bisporus, which fills the gaps of deficient fungal ethylene receptors. Furthermore, we found that decreased expression of the ethylene receptors facilitates preventing the maturation and senescence of postharvest button mushrooms, indicating that the two fungal ethylene receptors positively regulate the ethylene response, in contrast to that in plants.
The precise editing of genes mediated by CRISPR-Cas9 necessitates the application of donor DNA with appropriate lengths of homologous arms and fragment sizes. Our previous development, ...SSB/CRISPR-Cas9, has demonstrated high efficiency in homologous recombination and non-homologous end joining gene editing within bacteria. In this study, we optimized the lengths and sizes of homologous arms of the donor DNA within this system. Two sets of donor DNA constructs were generated: one set comprised donors with only 10–100 bp homologous arms, while the other set included donors with homologous arms ranging from 10–100 bp, between which was a tetracycline resistance expression cassette (1439 bp). These donor constructs were transformed into Escherichia coli MG1655 cells alongside pCas-SSB/pTargetF-lacZ. Notably, when the homologous arms ranged from 10 to 70 bp, the transformation efficiency of non-selectable donors was significantly higher than that of selectable donors. However, within the range of 10–100 bp homologous arm lengths, the homologous recombination rate of selectable donors was significantly higher than that of non-selectable donors, with the gap narrowing as the homologous arm length increased. For selectable donor DNA with homologous arm lengths of 10–60 bp, the homologous recombination rate increased linearly, reaching a plateau when the homologous arm length was between 60–100 bp. Conversely, for non-selectable donor DNA, the homologous recombination rate increased linearly with homologous arm lengths of 10–90 bp, plateauing at 90–100 bp. Editing two loci simultaneously with 100 bp homologous arms, whether selectable or non-selectable, showed no difference in transformation or homologous recombination rates. Editing three loci simultaneously with 100 bp non-selectable homologous arms resulted in a 45% homologous recombination rate. These results suggest that efficient homologous recombination gene editing mediated by SSB/CRISPR-Cas9 can be achieved using donor DNA with 90–100 bp non-selectable homologous arms or 60–100 bp selectable homologous arms.
The 1-aminocyclopropane-1-carboxylic acid (ACC) pathway that synthesizes ethylene is shared in seed plants, fungi and probably other organisms. However, the evolutionary relationship of the key ...enzyme ACC oxidase (ACO) in the pathway among organisms remains unknown. Herein, we cloned, expressed and characterized five ACOs from the straw mushroom (
) and the oyster mushroom (
): VvACO1-4 and PoACO. The five mushroom ACOs and the previously identified AbACO of the button mushroom contained all three conserved residues that bound to Fe(II) in plant ACOs. They also had variable residues that were conserved and bound to ascorbate and bicarbonate in plant ACOs and harbored only 1-2 of the five conserved ACO motifs in plant ACOs. Particularly, VvACO2 and AbACO had only one ACO motif 2. Additionally, VvACO4 shared 44.23% sequence identity with the cyanobacterium
putative functional ACO. Phylogenetic analysis showed that the functional ACOs of monocotyledonous and dicotyledonous plants co-occurred in Type I, Type II and Type III, while putative functional gymnosperm ACOs also appeared in Type III. The putative functional bacterial ACO, functional fungi and slime mold ACOs were clustered in ancestral Type IV. These results indicate that ACO motif 2, ACC and Fe(II) are essential for ACO activity. The ACOs of the other organisms may come from the horizontal transfer of fungal ACOs, which were found ordinarily in basidiomycetes. It is mostly the first case for the horizontal gene transfers from fungi to seed plants. The horizontal transfer of ACOs from fungi to plants probably facilitates the fungal-plant symbioses, plant-land colonization and further evolution to form seeds.
The biosynthetic pathway from linoleic acid to 1-octen-3-ol in Agaricus bisporus has long been established, in which linoleic acid is converted to 10-hydroperoxide (10-HPOD) by deoxygenation, and ...10-HPOD is subsequently cleaved to yield 1-octene-3-ol and 10-oxodecanoic acid. However, the corresponding enzymes have not been identified and cloned. In the present study, four putative genes involved in oxylipid biosynthesis, including one lipoxygenase gene named AbLOX, two linoleate diol synthase genes named AbLDS1 and AbLDS2, and one hydroperoxide lyase gene named AbHPL were retrieved from the A. bisporus genome by a homology search and cloned and expressed prokaryotically. AbLOX, AbLDS1, and AbLDS2 all exhibited fatty acid dioxygenase activity, catalyzing the conversion of linoleic acid to generate hydroperoxide, and AbHPL showed a cleaving hydroperoxide activity, as was determined by the KI-starch method. AbLOX and AbHPL catalyzed linoleic acid to 1-octen-3-ol with an optimum temperature of 35 °C and an optimum pH of 7.2, whereas AbLDS1, AbLDS2, and AbHPL catalyzed linoleic acid without 1-octen-3-ol. Reduced AbLOX expression in antisense AbLOX transformants was correlated with a decrease in the yield of 1-octen-3-ol. AbLOX and AbHPL were highly homologous to the sesquiterpene synthase Cop4 of Coprinus cinerea and the yeast sterol C-22 desaturase, respectively. These results reveal that the enzymes for the oxidative cleavage of linoleic acid to synthesize 1-octen-3-ol in A. bisporus are the multifunctional fatty acid dioxygenase AbLOX and hydroperoxide lyase AbHPL.
Single-stranded DNA-binding proteins (SSBs) are essential for all living organisms. Whether SSBs can repair DNA double-strand breaks (DSBs) and improve the efficiency of CRISPR/Cas9-mediated genome ...editing has not been determined. Here, based on a pCas/pTargetF system, we constructed pCas-SSB and pCas-T4L by replacing the λ-Red recombinases with
SSB and phage T4 DNA ligase in pCas, respectively. Inactivation of the
gene with homologous donor dsDNA increased the gene editing efficiency of pCas-SSB/pTargetF by 21.4% compared to pCas/pTargetF. Inactivation of the
gene via NHEJ increased the gene editing efficiency of pCas-SSB/pTargetF by 33.2% compared to pCas-T4L/pTargetF. Furthermore, the gene-editing efficiency of pCas-SSB/pTargetF in
(Δ
, Δ
, Δ
) with or without donor dsDNA did not differ. Additionally, pCas-SSB/pTargetF with donor dsDNA successfully deleted the
gene in
sp. UW4. These results demonstrate that
SSB repairs DSBs caused by CRISPR/Cas9 and effectively improves CRISPR/Cas9 genome editing in
and
.
PDF
