Isolation and Evaluation of Erinacine A Contents in Mycelia of Hericium erinaceus Strains.

Hericium erinaceus has long been favored for its remarkable nutritional and health-promoting benefits, and erinacine A is the key component responsible for the neuroprotective properties of H. erinaceus. Establishing an efficient method for separating erinacine A from H. erinaceus and screening the erinacine A-enriched strains is crucial to maximizing its benefits. Herein, we first reported that high-speed counter current chromatography (HSCCC) is an effective method for separating high-purity erinacine A. Using a two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water (4.5:5:4.5:5, v/v/v/v), erinacine A with a purity of over 95% was separated. Then, we evaluated the content and yield of erinacine A in the liquid-fermented mycelia of Hericium germplasms. Both the content and yield of erinacine A varied greatly among the surveyed strains. The significant effect of the strain on the erinacine A content and yield was revealed by an analysis of variance. The highest erinacine A content and yield were observed in the mycelia of a wild strain HeG, reaching 42.16 mg/g and 358.78 mg/L, which is superior to the current highest outcomes achieved using submerged cultivation. The isolation method established and the strains screened in this study can be beneficial for the scaling up of erinacine A extraction and nutraceutical development to industrial levels.

The complete mitochondrial genome of Hericium erinaceus (Bull.:Fr.) Pers., 1797 (Russulales, Basidiomycota): an edible and medicinal fungus.

Hericium erinaceus (Bull.:Fr.) Pers., 1797, is an edible and medicinal fungus found in China. In this study, specimens of H. erinaceus HE0021 were collected from southeastern China (Yunhe County, Lishui City, Zhejiang Province, 28°7'12″N, 119°34'12″E). The whole mitochondrial genome of H. erinaceus HE0021 was sequenced using next-generation sequencing (NGS) technology, which comprised 15 protein-coding genes (PCGs), 27 transfer RNAs (tRNAs), two ribosomal RNAs, with a total length of 83,518 base pairs (bp). The results of the phylogenetic analysis show that H. erinaceus and H. coralloides were clustered in the same clade. The complete mitogenome sequence provides essential data for the subsequent investigation of Hericium and Russulales.

Imprinting but not cytonuclear interactions determines seed size heterosis in Arabidopsis hybrids.

The parent-of-origin effect on seeds can result from imprinting (unequal expression of paternal and maternal alleles) or combinational effects between cytoplasmic and nuclear genomes, but their relative contributions remain unknown. To discern these confounding factors, we produced cytoplasmic-nuclear substitution (CNS) lines using recurrent backcrossing in Arabidopsis (Arabidopsis thaliana) ecotypes Col-0 and C24. These CNS lines differed only in the nuclear genome (imprinting) or cytoplasm. The CNS reciprocal hybrids with the same cytoplasm displayed ∼20% seed size difference, whereas the seed size was similar between the reciprocal hybrids with fixed imprinting. Transcriptome analyses in the endosperm of CNS hybrids using laser-capture microdissection identified 104 maternally expressed genes (MEGs) and 90 paternally expressed genes (PEGs). These imprinted genes were involved in pectin catabolism and cell wall modification in the endosperm. Homeodomain Glabrous9 (HDG9), an epiallele and one of 11 cross-specific imprinted genes, affected seed size. In the embryo, there were a handful of imprinted genes in the CNS hybrids but only 1 was expressed at higher levels than in the endosperm. AT4G13495 was found to encode a long-noncoding RNA (lncRNA), but no obvious seed phenotype was observed in lncRNA knockout lines. Nuclear RNA Polymerase D1 (NRPD1), encoding the largest subunit of RNA Pol IV, was involved in the biogenesis of small interfering RNAs. Seed size and embryos were larger in the cross using nrpd1 as the maternal parent than in the reciprocal cross, supporting a role of the maternal NRPD1 allele in seed development. Although limited ecotypes were tested, these results suggest that imprinting and the maternal NRPD1-mediated small RNA pathway play roles in seed size heterosis in plant hybrids.

Imprinting but not cytonuclear interactions affects parent-of-origin effect on seed size in Arabidopsis hybrids.

The parent-of-origin effect on seed size can result from imprinting or a combinational effect between cytoplasmic and nuclear genomes, but their relative contributions remain unknown. To discern these confounding effects, we generated cytoplasmic-nuclear substitution (CNS) lines using recurrent backcrossing in the Arabidopsis thaliana ecotypes Col-0 and C24. These CNS lines differ only in the nuclear genome (imprinting) or in the cytoplasm. The CNS reciprocal hybrids with the same cytoplasm display a ~20% seed size difference as observed in the conventional hybrids. However, seed size is similar between the reciprocal cybrids with fixed imprinting. Transcriptome analyses in the endosperm of CNS hybrids using laser-capture microdissection have identified 104 maternally expressed genes (MEGs) and 90 paternally-expressed genes (PEGs). These imprinted genes are involved in pectin catabolism and cell wall modification in the endosperm. HDG9, an epiallele and one of 11 cross-specific imprinted genes, controls seed size. In the embryo, a handful of imprinted genes is found in the CNS hybrids but only one is expressed higher in the embryo than endosperm. AT4G13495 encodes a long-noncoding RNA (lncRNA), but no obvious seed phenotype is observed in the lncRNA knockout lines. NRPD1, encoding the largest subunit of RNA Pol IV, is involved in the biogenesis of small interfering RNAs. Seed size and embryo is larger in the cross using nrpd1 as the maternal parent than in the reciprocal cross. In spite of limited ecotypes tested, these results suggest potential roles of imprinting and NRPD1-mediated small RNA pathway in seed size variation in hybrids.

Quantification of macromolecule crowding at single-molecule level.

Macromolecule crowding has a prominent impact on a series of biochemical processes in the cell. It is also expected to promote macromolecular complexation induced by excluded volume effects, which conflicts with recent advances in the thermodynamic interaction between inert, synthetic polymers, and nucleic acids. Along this line, a method combining high-resolution magnetic tweezers and extended crowder-oxDNA model was applied to resolve these discrepancies by systematically studying the kinetics and thermodynamics of the folding-unfolding transition for an individual DNA hairpin in a crowded environment. More specifically, from the magnetic tweezers-based experiments, the linear dependence of the critical force of the DNA hairpin on the polyethylene glycol (PEG) concentration was demonstrated, which is consistent with the results based on the crowder-oxDNA model in which the Lennard-Jones potential was adopted to express the interaction between the crowders and the DNA hairpin. These consistencies highlight that the excluded volume effects are mainly responsible for the interaction between PEG and the DNA hairpin, which is different from the interaction between dextran and the DNA hairpin. In the meantime, the dependence of the folding rate on the molecule weight of PEG, which was different from fluorescence resonance energy transfer-based results, was identified. The proposed method opens a path to detect the interaction between an inert, synthetic molecule, and the DNA hairpin, which is important to accurately mimic the cytosolic environments using mixtures of different inert molecules.

Stability of DNA and RNA hairpins: a comparative study based on ox-DNA.

Advances in single-molecule experiments on macromolecular crowding urgently need an efficient simulation method to resolve their discrepancies quantitatively. Ox-DNA model has been since reworked to treat the thermodynamics and mechanical properties of DNA/RNA hairpin at a stretching force. In hopping experiments, the critical forces of RNA hairpins at different temperatures are greater than those of DNA hairpins, in addition, the Gibbs free energy at a fixed temperature required to convert an RNA hairpin into a single-stranded molecule at zero force is obviously greater than that of DNA hairpin and gradually decreases by increasing the temperature. As far as force-ramping experiments are concerned, the first-rupture forces of RNA/DNA hairpins corresponding to the maximum probability density linearly pertain to the force-loading rate, with those of RNA hairpins being greater. The extended ox-DNA model could potentially identify the interaction between biologically inert polymer and RNA/DNA hairpins in crowded environments.

Landscape of meiotic crossovers in Hericium erinaceus.

Meiotic crossover shows marked interspecific and intraspecific variation, and knowledge about the molecular mechanism of crossover variation remains limited. Herein, we described the genome-wide scanning of crossover in one mushroom-forming fungus Hericium erinaceus. Utilizing the whole-genome single-nucleotide polymorphism (SNP) data-sets of a 127 F1 haploid progeny, we localized a total of 1316 crossover events and found that they were more likely to occur in the genic than intergenic regions. More than 30 % of the crossovers were concentrated in 59 crossover hotspots that were preferentially located close to chromosome ends. We then examined the genomic features around crossover hotspots. Results showed that the crossover hotspots were associated with increased gene density and guanine-cytosine (GC) content. An 8-bp GC-rich motif (GCGTCAGC) was found to be significantly enriched in these hotspots. The presence of mating-type loci affected the crossover at local scale rather than the overall crossover number. In order to dissect the genetic mechanisms shaping crossover variation, we then conducted quantitative trait locus (QTL) mapping for the total crossovers (TCO) and the crossover events that solely occurred within hotspots (HCO). Genome-wide QTL scanning identified four TCO-QTLs and two HCO-QTLs, which all located within or next to the crossover-hotspots. Crossover variations were shaped by multiple small-effect loci, with individual QTL contributing 6.9 %-11.7 % of variation. A few recombination pathway genes, including Spo11, Msh5, and Smc5 were found to be co-localized with the mapped crossover QTLs. Taken together, findings of this study offer insights into the crossover distribution and genetic factors conferring crossover variation in H. erinaceus, and advance our understandings for meiotic recombination in mushroom-forming fungi.

Simultaneous measurement of temperature and strain based on SCF-based MZI cascaded with FBG.

A novel seven-core fiber (SCF)-based sensor capable of simultaneous measurement of temperature and strain was proposed and experimentally demonstrated. The sensor consists of a fiber Bragg grating (FBG) and a SCF-based Mach-Zehnder interferometer (MZI). The MZI is formed by splicing a short section of SCF between two single-mode fibers (SMFs), and in the meantime, two fiber tapers that act as light splitter and coupler are formed at both ends of the SCF by the fusion splicing method. In addition, the FBG with a central wavelength of 1547.98 nm is cascaded at the end of one fiber taper, and thus simultaneous measurement of temperature and strain can be realized. The experiment result shows that the SCF-based MZI is only sensitive to temperature and has the higher temperature sensitivity of 93.11 pm/°C, while the FBG is sensitive to temperature and strain with sensitivities of 11.46 pm/°C and 0.627pm/µÎµ, respectively. The corresponding temperature and strain resolutions of the sensor are 0.21°C and 27.95µÎµ, respectively. The proposed sensor has the advantages of simple fabrication and high repeatability and has potential applications in many fields such as engineering machinery and building structure health monitoring.

Ex-ante online risk assessment for building emergency evacuation through multimedia data.

Ex-ante online risk assessment for building emergency evacuation is essential to protect human life and property. Current risk assessment methods are limited by the tradeoff between accuracy and efficiency. In this paper, we propose an online method that overcomes this tradeoff based on multimedia data (e.g. videos data from surveillance cameras) and deep learning. The method consists of two parts. The first estimates the evacuee position as input for training the assessment model to then perform risk assessment in real scenarios. The second considers a social force model based on the evacuation simulation for the output of training model. We verify the proposed method in simulation and real scenarios. Model sensitivity analyses and large-scale tests demonstrate the usability and superiority of the proposed method. By the method, the computation time of risk assessment could be decreased from 10 minutes (by traditional simulation method) to 2.18 s.

Measuring spatio-temporal accessibility to emergency medical services through big GPS data.

Medical accessibility is an important indicator for evaluating the effectiveness of public health services. However, the previous medical accessibility studies mainly focus on spatial accessibility without considering temporal variation in population distribution which is significant for evaluating access to emergency medical service (EMS). This paper proposes a model of spatio-temporal accessibility to EMS called ST-E2SFCA based on adapting the enhanced two-step floating catchment area (E2SFCA) method. We apply our method to the greater Tokyo area for a large volume of GPS dataset with millions of users and compare the accessibility difference over space and time. To evaluate our model, we also analyze the distinction of our model over different weight sets and compare the performance of ST-E2SFCA with the traditional E2SFCA. The result shows that our method can illustrate the temporal difference and is suitable for measuring the spatio-temporal accessibility to EMS, thus can guide the hospital location selection and urban planning.

The gymnosperm ortholog of the angiosperm central cell-specification gene CKI1 provides an essential clue to endosperm origin.

A defining feature of angiosperms is double fertilization involving the female gametophyte central cell and formation of a nutrient-storing tissue called endosperm. The route for the evolutionary origin of endosperm from a gymnosperm ancestor, particularly the molecular steps involved, has remained elusive. Recently, the histidine kinase gene Cytokinin-Independent 1 (CKI1), an activator of cytokinin signaling, was described as a key to specification of the endosperm precursor central cell in Arabidopsis. Here, we have investigated the function and expression of a putative ortholog of CKI1 in the gymnosperm Ginkgo biloba. We demonstrate that Ginkgo CKI1 can partially rescue an Arabidopsis cki1 mutant and promote weak activation of the cytokinin signaling pathway in the Arabidopsis embryo sac, but does not confer central cell specification. Ginkgo CKI1 is expressed in both male and female gametophytes of Ginkgo. In the latter, it is expressed in the ventral canal cell, which is sister to the egg cell in the archegonium. As in Arabidopsis, Ginkgo CKI1 is not expressed in the egg cell. The similarities in expression patterns of CKI1 in Ginkgo and Arabidopsis female gametophytes suggest that extant gymnosperms possess an essential component of the molecular machinery required for angiosperm endosperm development, and provide new insights into endosperm origin from a gymnospermous ancestor.

ARF2-ARF4 and ARF5 are Essential for Female and Male Gametophyte Development in Arabidopsis.

The plant hormone auxin plays critical roles in plant growth and development. Auxin response factors (ARFs) are a class of transcription factors which regulate auxin-mediated gene expression. While the functions of ARFs in sporophytic development have been well characterized, their functions specific to gametophytic development have not been studied extensively. In this study, Arabidopsis ARF genes were selectively down-regulated in gametophytes by misexpression of targeted microRNAs (amiRARF234, amiRARFMP and MIR167a) to silence AtARF2-AtAEF4, AtARF5, AtARF6 and AtARF8. Embryo sacs in amiRARF234- and amiRARFMP-expressing plants exhibited identity defects in cells at the micropylar pole, such as formation of two cells with egg cell-like morphology, concomitant with loss of synergid marker expression and seed abortion. The pollen grains of the transgenic plants were morphologically aberrant and unviable, and the inclusions and nuclei were lost in the abnormal pollen grains. However, plants misexpressing MIR167a showed no obvious abnormal phenotypes in the embryo sacs and pollen grains. Overall, these results provide evidence that AtARF2-AtARF4 and AtARF5 play significant roles in regulating both female and male gametophyte development in Arabidopsis.

AHP2, AHP3, and AHP5 act downstream of CKI1 in Arabidopsis female gametophyte development.

Histidine phosphotransfer proteins (HPs) are key elements of the two-component signaling system, which act as a shuttle to transfer phosphorylation signals from histidine kinases (HKs) to response regulators (RRs). CYTOKININ INDEPENDENT 1 (CKI1), a key regulator of central cell specification in the Arabidopsis female gametophyte, activates the cytokinin signaling pathway through the Arabidopsis histidine phosphotransfer proteins (AHPs). There are five HP genes in Arabidopsis, AHP1-AHP5, but it remains unknown which AHP genes act downstream of CKI1 in Arabidopsis female gametophyte development. Promoter activity analysis of AHP1-AHP5 in embryo sacs revealed AHP1, AHP2, AHP3, and AHP5 expression in the central cell. Phenotypic studies of various combinations of ahp mutants showed that triple mutations in AHP2, AHP3, and AHP5 resulted in defective embryo sac development. Using cell-specific single and double markers in the female gametophyte, the ahp2-2 ahp3 ahp5-2/+ triple mutant ovules showed loss of central cell and antipodal cell fates and gain of egg cell or synergid cell attributes, resembling the cki1 mutant phenotypes. These data suggest that AHP2, AHP3, and AHP5 are the major factors acting downstream of CKI1 in the two-component cytokinin signaling pathway to promote Arabidopsis female gametophyte development.

The CKI1 Histidine Kinase Specifies the Female Gametic Precursor of the Endosperm.

Since the discovery of double fertilization, it has been recognized that flowering plants produce two highly dimorphic female gametes, the egg cell and central cell. These give rise, respectively, to the embryo and the endosperm, a nourishing tissue unique to flowering plants. Here we show that in Arabidopsis, endosperm formation requires the CYTOKININ INDEPENDENT 1 (CKI1) histidine kinase, an activator of the cytokinin signaling pathway, which specifies central cells and restricts egg cell fate. Dimorphism of the two adjacent gametes is mechanistically established in the syncytial embryo sac by spatially restricted CKI1 expression, followed by translocation of ER-localized CKI1 protein via nuclear migration. Cell specification by CKI1 likely involves activation of the cytokinin signaling pathway mediated by histidine phosphotransferases. Ectopic CKI1 expression generates non-propagating seeds with dual fertilized endosperms and no embryos. We conclude that CKI1-directed specification of the endosperm precursor central cell results in seeds containing an embryo and an endosperm.

Antipodal cells persist through fertilization in the female gametophyte of Arabidopsis.

The female gametophyte of most flowering plants forms four cell types after cellularization, namely synergid cell, egg cell, central cell and antipodal cell. Of these, only the antipodal cells have no established functions, and it has been proposed that in many plants including Arabidopsis, the antipodal cells undergo programmed cell death during embryo sac maturation and prior to fertilization. Here, we examined the expression of female gametophyte-specific fluorescent reporters in mature embryo sacs of Arabidopsis, and in developing seeds shortly after fertilization. We observed expression of the fluorescence from the reporter genes in the three antipodal cells in the mature stage embryo sac, and continuing through the early syncytial endosperm stages. These observations suggest that rather than undergoing programmed cell death and degenerating at the mature stage of female gametophyte as previously supposed, the antipodal cells in Arabidopsis persist beyond fertilization, even when the other cell types are no longer present. The results support the concept that the Arabidopsis female gametophyte at maturity should be considered to be composed of seven cells and four cell types, rather than the previously prevailing view of four cells and three cell types.

Molecular characterization of the glauce mutant: a central cell-specific function is required for double fertilization in Arabidopsis.

Double fertilization of the egg cell and the central cell by two sperm cells, resulting in the formation of the embryo and the endosperm, respectively, is a defining characteristic of flowering plants. The Arabidopsis thaliana female gametophytic mutant glauce (glc) can exhibit embryo development without any endosperm. Here, we show that in glc mutant embryo sacs one sperm cell successfully fuses with the egg cell but the second sperm cell fails to fuse with the central cell, resulting in single fertilization. Complementation studies using genes from the glc deletion interval identified an unusual genomic locus having homology to BAHD (for BEAT, AHCT, HCBT, and DAT) acyl-transferases with dual transcription units and alternative splicing that could rescue the sterility defect of glc. Expression of these transcripts appears restricted to the central cell, and expression within the central cell is sufficient to restore fertility. We conclude that the central cell actively promotes its own fertilization by the sperm cell through a signaling mechanism involving products of At1g65450. Successful fertilization of the egg cell is not blocked in the glc mutant, suggesting that evolution of double fertilization in flowering plants involved acquisition of specific functions by the central cell to enable its role as a second female gamete.