Overexpression of cat2 restores antioxidant properties and production traits in degenerated strains of Volvariella volvacea.

Strain degeneration is an important factor hindering the development of the edible fungus industry. Strain degeneration is associated with the excessive accumulation of reactive oxygen species (ROS) in vivo. Catalase (CAT), an important antioxidant enzyme, can promote the clearance of ROS. In this study, the cat2 gene of Volvariella volvacea was first cloned into an overexpression plasmid via homologous recombination. Finally, through Agrobacterium-mediated transformation, this plasmid was inserted into degenerated strains of V. volvacea T19. The physiological properties, antioxidant properties, ROS content, matrix degradation activity, and cultivation properties of the transformants were tested. The results showed that the cloned cat2 gene was 99.94% similar to the reference sequence. Screening revealed that six positive transformants were successfully obtained. After the overexpression of cat2, the growth rate and biomass of the mycelium increased significantly in the transformant strains (versus the V. volvacea T19 degenerated strains). Moreover, the accumulation of superoxide radical (O2•-) and hydrogen peroxide (H2O2) was significantly reduced, and the activity of the enzymes CAT, superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPX) was significantly increased. Meanwhile, the expression of cat2, Mnsod1, Mnsod2, gpx, and gr was significantly upregulated, and the activity of eight matrix degradation-related enzymes was increased to varying degrees. More importantly, the overexpression of the cat2 gene promoted the regrowth of fruiting bodies in degenerated strains of V. volvacea T19. This study provides a new biotechnological strategy to control the degeneration of V. volvacea and other edible fungi.

MethylRAD Sequencing Technology Reveals DNA Methylation Characteristics of Apostichopus japonicus of Different Ages.

The A. japonicus industry has expanded significantly, but no research has focused on determining the age of A. japonicus during farming. Correctly estimating the age of A. japonicus can provide a decision-making basis for the breeding process and data for the protection of A. japonicus aquatic germplasm resources. DNA methylation levels in the body wall of Apostichopus japonicus at 4 months, 1 year, 2 years, and 3 years old were determined using MethylRAD-Seq, and differentially methylated genes were screened. A total of 441 and 966 differentially methylated genes were detected at the CCGG and CCWGG sites, respectively. Aspartate aminotransferase, succinate semialdehyde dehydrogenase, isocitrate dehydrogenase, the histone H2AX, heat shock protein Hsp90, aminopeptidase N, cell division cycle CDC6, Ras GTPase activating protein (RasGAP), slit guidance ligand slit1, integrin-linked kinase ILK, mechanistic target of rapamycin kinase Mtor, protein kinase A Pka, and autophagy-related 3 atg3 genes may play key roles in the growth and aging process of A. japonicus. This study provides valuable information regarding age-related genes for future research, and these candidate genes can be used to create an "epigenetic clock".

Molecular mechanisms that regulate the heat stress response in sea urchins (Strongylocentrotus intermedius) by comparative heat tolerance performance and whole-transcriptome RNA sequencing.

In the context of climate change and extreme high temperature, the commercially important sea urchin Strongylocentrotus intermedius suffers high mortality during summer in Northern China. How sea urchins respond to high temperatures is of great concern to academia and industry. How to understand the heat tolerance of sea urchin from the whole transcriptome level. In this study, the heat-resistant S. intermedius bred by our team and its control group were used as the research objects, then we applied whole-transcriptome RNA sequencing to detect differentially expressed mRNAs, microRNAs, long noncoding RNAs that respond to heat stress in the heat-resistant and control S. intermedius. A competitive endogenous RNA (ceRNA) regulatory network was constructed with predicted pairs of differentially expressed mRNAs and noncoding RNAs and revealed the molecular regulatory mechanisms in S. intermedius responding to heat stress. A functional analysis suggested that the ceRNAs were involved in basal metabolism, calcium ion transport, endoplasmic reticulum stress, and apoptosis. This is the whole-transcriptomic analysis of S. intermedius under heat stress to propose ceRNA networks that will provide a basis for studying the potential functions of long noncoding RNAs and miRNAs in the heat stress response in S. intermedius and provide a theoretical basis for the study of the molecular mechanism of sea urchins in response to environmental changes.

Differences in body wall metabolites of Apostichopus japonicus of different ages in liquid chromatography-tandem mass spectrometry.

Apostichopus japonicus is a marine invertebrate with high economic value. Analysis of the effects of age on the structure of this species is important for understanding the status of the population and conservation. In this study, metabolite analysis of the body walls of A. japonicus of three different ages (1, 2, and 3 years) was performed using LC-MS/MS. Metabolite analysis was performed for the KEGG metabolic pathway. The partial least squares-discriminant analysis is clearly distinguished metabolites in the three groups of A. japonicus. A total of 59, 222, and 179 different metabolites were detected in the age 3 vs age 2, age 3 vs age 1, and age 2 vs age 1 controls, respectively (P < 0.05). These metabolic differences involved several metabolic pathways, including phenylalanine metabolism, taurine and hypotaurine metabolism, sulfur metabolism et al. Thiamin, riboflavin, lipoic acid, acetyl l-carnitine levels increase with age, trans-10-heptadecenoic acid, stearic acid, pentadecanoic acid, dibutyl sebacate, and 8,15-diHETE levels decrease with age, which these metabolites can be used as potential markers to determine the age of A. japonicus. Our results provide the foundation for determining the age of A. japonicus and are important for the conservation of germplasm resources of A. japonicus.

Integrative mRNA-miRNA interaction analysis associated with the immune response of Strongylocentrotus intermedius to Vibrio harveyi infection.

Strongylocentrotus intermedius is one of the most economically valuable sea urchin species in China and has experienced mass mortality owing to outbreaks of bacterial diseases such as black mouth disease. This has caused serious economic losses to the sea urchin farming industry. To investigate the immune response mechanism of S. intermedius with different tube feet colors in response to Vibrio harveyi infection, we examined the different tube feet-colored S. intermedius under V. harveyi challenge and compared their transcriptome and microRNA (miRNA) profiles using RNA-Seq. We obtained 1813 differentially expressed genes (DEGs), 28 DE miRNAs, and 303 DE miRNA-DEG pairs in different tube feet-colored S. intermedius under V. harveyi challenge. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the most significant DEGs were associated with the Notch signaling and phagosome pathways. The target genes of immune-related miRNAs (miR-71, miR-184, miR-193) and genes (CALM1, SPSB4, DMBT, CSRP1) in S. intermedius were predicted and validated. This study provides insight into the molecular mechanisms that regulate genes involved in the immune response of S. intermedius infected with V. harveyi.

Whole-genome resequencing reveals genetic differences and the genetic basis of parapodium number in Russian and Chinese Apostichopus japonicus.

BACKGROUND: Apostichopus japonicus is an economically important species in the global aquaculture industry. Russian A. japonicus, mainly harvested in the Vladivostok region, exhibits significant phenotypic differentiation, including in many economically important traits, compared with Chinese A. japonicus owing to differences in their habitat. However, both the genetic basis for the phenotypic divergence and the population genetic structure of Russian and Chinese A. japonicus are unknown. RESULT: In this study, 210 individuals from seven Russian and Chinese A. japonicus populations were sampled for whole-genome resequencing. The genetic structure analysis differentiated the Russian and Chinese A. japonicus into two groups. Population genetic analyses indicated that the Russian population showed a high degree of allelic linkage and had undergone stronger positive selection compared with the Chinese populations. Gene ontology terms enriched among candidate genes with group selection analysis were mainly involved in immunity, such as inflammatory response, antimicrobial peptides, humoral immunity, and apoptosis. Genome-wide association analysis yielded eight single-nucleotide polymorphism loci significantly associated with parapodium number, and these loci are located in regions with a high degree of genomic differentiation between the Chinese and Russia populations. These SNPs were associated with five genes. Gene expression validation revealed that three of these genes were significantly differentially expressed in individuals differing in parapodium number. AJAP08772 and AJAP08773 may directly affect parapodium production by promoting endothelial cell proliferation and metabolism, whereas AJAP07248 indirectly affects parapodium production by participating in immune responses. CONCLUSIONS: This study, we performed population genetic structure and GWAS analysis on Chinese and Russian A. japonicus, and found three candidate genes related to the number of parapodium. The results provide an in-depth understanding of the differences in the genetic structure of A. japonicus populations in China and Russia, and provide important information for subsequent genetic analysis and breeding of this species.

Successive mycelial subculturing decreased lignocellulase activity and increased ROS accumulation in Volvariella volvacea.

Strain degradation is a common problem in many artificially-cultivated edible mushrooms. As a fungus with poor tolerance to low-temperature, Volvariella volvacea cannot delay its degradation by long-term low temperature storage like other fungi, so its degradation is particularly severe, which hinders industrial applications. Periodic mycelial subculture is a common storage method for V. volvacea, but excessive subculturing can also lead to strain degeneration. After 20 months of continuous subculturing every 3 days, V. volvacea strains S1-S20 were obtained, and their characteristics throughout the subculture process were analyzed. With increasing number of subculture, the growth rate, mycelial biomass, the number of fruiting bodies and biological efficiency gradually decreased while the production cycle and the time to primordium formation was lengthened. Strains S13-S20, obtained after 13-20 months of mycelial subculturing, also lacked the ability to produce fruiting bodies during cultivation experiments. Determination of reactive oxygen species (ROS) content as well as enzyme activity showed that decreased lignocellulase activity, along with excessive accumulation of ROS, was concomitant with the subculture-associated degeneration of V. volvacea. Reverse transcription polymerase chain reaction (RT-PCR) was eventually used to analyze the gene expression for lignocellulase and antioxidant enzymes in subcultured V. volvacea strains, with the results found to be consistent with prior observations regarding enzyme activities. These findings could form the basis of further studies on the degeneration mechanism of V. volvacea and other fungi.

Expression Regulation Mechanisms of Sea Urchin (Strongylocentrotus intermedius) Under the High Temperature: New Evidence for the miRNA-mRNA Interaction Involvement.

In the context of global warming and continuous high temperatures in the northern part of China during summer, the mortality rate of our main breeding species, Strongylocentrotus intermedius, reached 80% in 2020. How sea urchins respond to high temperatures is of great concern to academia and industry. In this study, we examined the antioxidant enzyme activities of different color tube-footed sea urchins under heat stress and compared their transcriptome and microRNA (miRNA) profiles using RNA-Seq. The results showed that the antioxidant enzyme activities of sea urchins were altered by thermal stress, and the changes in peroxidase activities of red tube-footed sea urchins were particularly significant. Investigations revealed that 1,079 differentially expressed genes (DEGs), 11 DE miRNAs, and 104 "DE miRNA-DEG" pairs in total were detected in sea urchins under high temperature stress. Several mRNA and miRNAs were significantly changed (e.g. HSP70, DnaJ11, HYAL, CALR, miR-184-p5, miR-92a, miR-92c, and miR-124-p5), suggesting these genes and miRNAs exerted important functions in response to high temperature. At the transcriptional level, red tube-footed sea urchins were found to be more sensitive to high temperature and could respond to high temperature rapidly. DE miRNA-mRNA network showed that miR-92b-3p and PC-5p-7420 were the most corresponding miRNAs. Five mRNAs (DnaJ11, SAR1B, CALR, HYOU1, TUBA) may be potential markers of sea urchin response to high temperature. Possible interaction between miRNA-mRNA could be linked to protein folding in the endoplasmic reticulum, Phagosomes, and calcium transport. This study provides a theoretical basis for the molecular mechanism of sea urchin heat tolerance and information that will aid in the selection and breeding of sea urchins with high temperature tolerance.

Gene expression patterns of sea urchins (Strongylocentrotus intermedius) exposed to different combinations of temperature and hypoxia.

Strongylocentrotus intermedius is one of the most economically valuable sea urchin species in China, and its growth and survival are severely constrained by ocean warming and the hypoxia that often accompanies high water temperatures. To elucidate the molecular mechanisms of S. intermedius that regulate gene expression in response to multi-causal environmental stresses. We performed a de novo transcriptome analysis of coelomocyte from S. intermedius to heat (25 °C), hypoxia (2 mg/L), and the combined stress. We identified 35,635, 29,107, and 29,440 differentially expressed genes (DEGs) in S. intermedius cultured under high temperature, low oxygen, and combined stress, respectively. Further Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analyses revealed that these DEGs mainly enriched the functional categories of "Protein processing in endoplasmic reticulum," and "Glutathione metabolism" by heat stress, such as HSP70, GSTO1, PDIA4. After hypoxic stress, "Notch signaling pathway" and metabolism-related pathways such as "Glycerolipid metabolism", "Pyruvate metabolism" were significantly enriched. Exposure to combined stress resulted in a two-factor additive effect at the transcriptome level and have a more extensive impact on the immune correlated pathways in S. intermedius than single stress, the expression of related immune genes (C3, C5, and AIFM2) were up-regulated. Quantitative real-time PCR (qRT-PCR) analysis of the expression of 18 DEGs confirmed the RNA-Seq results. Observations in the present study will improve the understanding of the molecular mechanism of S. intermedius in response to multi-causal environmental stress.

Characterization of the complete chloroplast genome of Syringa wolfii.

Syringa wolfii (Syringa: Syringeae), an upright shrub, is ornamental species used in urban greenification. In this study, we determined the complete chloroplast (cp) genome of S. wolfii using next-generation sequencing (NGS). The size of the chloroplast genome is 156,571 bp in length, including a large single-copy region (LSC) of 86,684 bp, a small single-copy region (SSC) of 19,109 bp, and a pair of inverted repeat (IR) regions with 25,362 bp. The GC content of the chloroplast genome was 37.95%. Moreover, a total of 131 functional genes were annotated, including 87 protein-coding genes, 36 tRNA genes, and 8 rRNA genes. The neighbor-joining phylogenetic tree suggested that S. wolfii was closely related to S. yunnansis.

[Applying Swanson's Caring Theory to Manage Spiritual Distress in a Patient With Terminal Lung Cancer].

This case report shares a nursing experience that applied Swanson's Caring Theory as part of the care regimen for a terminal lung cancer patient suffering from spiritual distress. The nursing care was provided from March 20th to April 25th, 2012. Data were collected through observation and conversation and recorded using sketches and written notes. The nurse assessed the patient's relationships with the self, with others, with the environment, and with God in accordance with Stoll's spiritual interrelatedness hypothesis. Several spiritual distresses were reported. Interventions such as "knowing", "being with", "doing for", "enabling" and "maintaining belief" were applied to manage the patient's spiritual distress and address spiritual needs. This case report is intended to help increase the awareness and sensitivity of nurses to patients' spiritual needs and to help nurses provide effective spiritual care.