Assembly and comparative analysis of the complete multichromosomal mitochondrial genome of Cymbidium ensifolium, an orchid of high economic and ornamental value.

BACKGROUND: Orchidaceae is one of the largest groups of angiosperms, and most species have high economic value and scientific research value due to their ornamental and medicinal properties. In China, Chinese Cymbidium is a popular ornamental orchid with high economic value and a long history. However, to date, no detailed information on the mitochondrial genome of any species of Chinese Cymbidium has been published. RESULTS: Here, we present the complete assembly and annotation of the mitochondrial genome of Cymbidium ensifolium (L.) Sw. The mitogenome of C. ensifolium was 560,647 bp in length and consisted of 19 circular subgenomes ranging in size from 21,995 bp to 48,212 bp. The genome encoded 35 protein-coding genes, 36 tRNAs, 3 rRNAs, and 3405 ORFs. Repeat sequence analysis and prediction of RNA editing sites revealed a total of 915 dispersed repeats, 162 simple repeats, 45 tandem repeats, and 530 RNA editing sites. Analysis of codon usage showed a preference for codons ending in A/T. Interorganellar DNA transfer was identified in 13 of the 19 chromosomes, with plastid-derived DNA fragments representing 6.81% of the C. ensifolium mitochondrial genome. The homologous fragments of the mitochondrial genome and nuclear genome were also analysed. Comparative analysis showed that the GC content was conserved, but the size, structure, and gene content of the mitogenomes varied greatly among plants with multichromosomal mitogenome structure. Phylogenetic analysis based on the mitogenomes reflected the evolutionary and taxonomic statuses of C. ensifolium. Interestingly, compared with the mitogenomes of Cymbidium lancifolium Hook. and Cymbidium macrorhizon Lindl., the mitogenome of C. ensifolium lost 8 ribosomal protein-coding genes. CONCLUSION: In this study, we assembled and annotated the mitogenome of C. ensifolium and compared it with the mitogenomes of other Liliidae and plants with multichromosomal mitogenome structures. Our findings enrich the mitochondrial genome database of orchid plants and reveal the rapid structural evolution of Cymbidium mitochondrial genomes, highlighting the potential for mitochondrial genes to help decipher plant evolutionary history.

Diversity and Network Relationship Construction of Soil Fungal Communities in Lactarius hatsudake Tanaka Orchard during Harvest.

Lactarius hatsudake Tanaka is a mycorrhizal edible mushroom with rich economic and nutritional value. Although it is artificially planted, its yield is unstable. Soil fungi, including L. hatsudake, coexist with many other microorganisms and plants. Therefore, complex microbial communities have an influence on the fruiting body formation of L. hatsudake. L. hatsudake and its interactions with the rest of the fungal community over time are not completely understood. In this study, we performed high-throughput sequencing of microorganisms in the basal soil of the fruiting body (JT), mycorrhizosphere soil (JG), and non-mushroom-producing soil (CK) in a 6-year-old L. hatsudake plantation at harvest. The results showed that the soil of the L. hatsudake plantation was rich in fungal communities and a total of 10 phyla, 19 classes, 53 orders, 90 families, 139 genera, and 149 species of fungi were detected. At the phylum level, the major groups were Basidiomycota and Ascomycota. At the genus level, the dominant groups were Lactarius, Trichoderma, Suillus, and Penicillium. Among them, L. hatsudake had an absolute dominant position in the soil fungal community of the plantation, and was the only group of Lactarius in the plantation soil. Penicillium cryptum and Penicillium adametzii were unique to the JT soil sample. Chaetopsphaeria, Myxocephala, Devriesia, and Psathyrella were positively correlated with L. hatsudake. In the constructed fungal network, the total number of nodes were ranked in descending order as JG (441) > CK (405) > JT (399), while the total number of edges were ranked in descending order as CK (1360) > JG (647) > JT (586). Analysis of the fungal assembly process revealed that groups CK and JG have determinative processes that dominated community building, while the JT group exhibited a dominant random process with a 0.60 probability. The results indicated that L. hatsudake was successfully colonized in the plantation soil. During harvest, the CK group exhibited the largest network size and the most complex fungal interactions, while the fungal community structure in the mushroom cultivation zone (JT and JG) was stable and less susceptible to external environmental interference. L. hatsudake affects the fungal community in the soil surrounding its fruiting body.

Mycena subpiligera sp. nov., a Symbiotic Species from China Associated with the Seed Germination of Gastrodia elata.

Mycena subpiligera, a new taxon in sect. Fragilipedes that can strongly enhance the germination efficiency of Gastrodia elata seeds, was discovered in subtropical areas of China. As revealed by a morphological comparison with related Mycena species as well as maximum likelihood (ML) and Bayesian phylogenetic analyses based on sequences of the internal transcribed spacer (ITS) and the large subunit (LSU) regions of nuclear ribosomal RNA, the new taxon can be distinguished from phenotypically similar and phylogenetically related species. Optimal cultural conditions for M. subpiligera basidiomata are reported, and the germination rate of the new species is compared with that of M. citrinomarginata.

A high-quality genome assembly of Lactarius hatsudake strain JH5.

Lactarius hatsudake is a species of Lactarius commonly found in pine forests, is edible with a delicious and nutritious fruiting body, and exhibits medicinal properties. It is an ideal natural multifunctional food with bioactive components including fungal polysaccharides, crude fiber, unsaturated fatty acids, nucleic acid derivatives, various amino acids, and vitamins. However, biological and genomic analyses of this mycorrhizal mushroom are sparse, thereby hindering large-scale cultivation. Previously, we isolated and screened L. hatsudake JH5 strains and have applied our garnered knowledge to the large-scale cultivation of mycorrhizal seedlings. In this study, we produced a high-quality genome assembly of L. hatsudake JH5 by combining Illumina paired-end and PacBio single molecule real-time sequencing, resulting in PacBio single molecule real-time reads of 7.67 Gb and Illumina Pair-End reads of 1,560 Mb. Based on the distribution of k-mer frequencies, the genome size of this strain was estimated to be 63.84 Mb (1.14% heterozygosity). Based on de novo genome assembly, the final genome size was determined to be 76.7 Mb, with scaffold N50 of 223.2 kb and N90 of 54.5 kb, and a GC content of 54.38%. BUSCO assessment showed that genome completeness was 89.0%. The N50 length of the JH5 genome was 43.6% longer than that of the previously published L. hatsudake MG20 genome. This high-quality L. hatsudake genome assembly will facilitate research on the functional genome, molecular breeding, yield enhancement, and sustainability of L. hatsudake cultivation.

Association of non-alcoholic fatty liver disease with diabetic retinopathy in type 2 diabetic patients: A meta-analysis of observational studies.

AIMS/INTRODUCTION: Non-alcoholic fatty liver disease (NAFLD) is becoming more and more prevalent in type 2 diabetes mellitus. Evidence connecting NAFLD to diabetic retinopathy (DR) is increasing, but the results vary. Thus, we undertook a meta-analysis to explore the effect of NAFLD on diabetic retinopathy in patients with type 2 diabetes mellitus. MATERIALS AND METHODS: PubMed, Embase, Cochrane and Scopus database were searched for until September 30, 2019. Original studies analyzing the association between NAFLD and diabetic retinopathy in the type 2 diabetic population were included. This meta-analysis was processed by RevMan 5.3 software. Subgroup analyses based on countries were carried out. The pooled odds ratios and 95% confidence intervals were used to evaluate the association between NAFLD and diabetic retinopathy incidence. The I2 test was used to assess heterogeneity of studies. RESULTS: We retrieved 414 articles, and nine studies involving 7,170 patients were included in the final analysis. The pooled effects estimate suggested that NAFLD was not associated with the risk of diabetic retinopathy in patients with type 2 diabetes mellitus. Subgroup analysis suggested that in China, Korea and Iran, patients with type 2 diabetes mellitus with NAFLD had a decreased risk for diabetic retinopathy compared with the non-NAFLD individuals. However, in Italy and India, patients with type 2 diabetes mellitus with NAFLD had an increased risk for diabetic retinopathy compared with the non-NAFLD individuals. In addition, no relevance between NAFLD and diabetic retinopathy was found in America. CONCLUSIONS: On the whole, there was no association between NAFLD and diabetic retinopathy in individuals with type 2 diabetes mellitus. However, subgroup analysis showed that a difference of country may have an influence on the result.

The effects and safety of activators of glucokinase versus placebo in patients with type 2 diabetes mellitus: a systematic review and meta-analysis.

We undertook a systematic review and meta-analysis to assess the effects and safety of activators of glucokinase (GKAs) in patients with type 2 diabetes mellitus (T2DM). 11 RCTs, including 2,429 participants, are enrolled in our study. According to different doses, we divided the studies into 3 groups: low-dose group, medium-dose group and high-dose group for subgroup analysis. There were decreases of HbA1c in all dose group (WMD = -0.27, 95%CI (-0.51~ -0.03), Z = 2.17, p = 0.03; WMD = -0.37, 95%CI (-0.58~ -0.16), Z = 3.41, p = 0.0006; WMD = -0.60, 95%CI (-0.86~ -0.33), Z = 4.43, p < 0.00001). Though the total risk of hypoglycemia is absolutely low, in the high-dose group higher hypoglycemia than the placebo can be observed (RR = 0.03, 95%CI (0.00~0.06), Z = 2.27, p = 0.02). In addition, the study found that the drug was less likely to have adverse reactions such as diarrhea, headache and dizziness, nasopharyngitis and upper respiratory tract infection (RR = 0.76, 95%CI (0.36~1.60), Z = 0.73, p = 0.47; RR = 1.26, 95%CI (0.73~2.17), Z = 0.83, p = 0.41; RR = 0.71, 95%CI (0.41~1.22), Z = 1.25, p = 0.21; RR = 1.61, 95%CI (0.77~3.36), Z = 1.26, p = 0.21). It concludes that GKAs are relatively effective and safe in the treatment of patients with T2DM, but in consideration of the potential risk of hypoglycemia in the high-dose group, the low-dose and medium-dose group, in the clinical practice, can be an excellent choice.

Genome and secretome analysis of Pochonia chlamydosporia provide new insight into egg-parasitic mechanisms.

Pochonia chlamydosporia infects eggs and females of economically important plant-parasitic nematodes. The fungal isolates parasitizing different nematodes are genetically distinct. To understand their intraspecific genetic differentiation, parasitic mechanisms, and adaptive evolution, we assembled seven putative chromosomes of P. chlamydosporia strain 170 isolated from root-knot nematode eggs (~44 Mb, including 7.19% of transposable elements) and compared them with the genome of the strain 123 (~41 Mb) isolated from cereal cyst nematode. We focus on secretomes of the fungus, which play important roles in pathogenicity and fungus-host/environment interactions, and identified 1,750 secreted proteins, with a high proportion of carboxypeptidases, subtilisins, and chitinases. We analyzed the phylogenies of these genes and predicted new pathogenic molecules. By comparative transcriptome analysis, we found that secreted proteins involved in responses to nutrient stress are mainly comprised of proteases and glycoside hydrolases. Moreover, 32 secreted proteins undergoing positive selection and 71 duplicated gene pairs encoding secreted proteins are identified. Two duplicated pairs encoding secreted glycosyl hydrolases (GH30), which may be related to fungal endophytic process and lost in many insect-pathogenic fungi but exist in nematophagous fungi, are putatively acquired from bacteria by horizontal gene transfer. The results help understanding genetic origins and evolution of parasitism-related genes.

The complete mitochondrial genome of the nematophagous fungus Lecanicillium saksenae.

Lecanicillium saksenae CGMCC5329 is a useful biological control agent against plant-parasitic nematodes. The complete mitogenome sequence of L. saksenae is reported for the first time. The mitochondrial genome is 25 919 bp long with 14 typical protein-coding genes, an intronic ORF coding for a putative ribosomal protein (rps3), 2 ribosomal RNA genes and a set of 26 transfer RNA genes. The phylogeny based on 12 protein-coding genes (except the loss of other two genes in Acremonium implicatum) suggests the close phylogenetic relationship between L. saksenae and L. muscarium. Comparative analysis reveals that mitogenome of L. saksenae is 1420 bp larger than L. muscarium, mainly due to the intergenic region between cox2 and trnR. The trnC between cob and cox1 is conserved in the mitogenomes of three nematophagous fungus of Pochonia chlamydosporia, A. implicatum and L. saksenae, but absent in L. muscarium. This study may provide valuable information for further research on mitochondrial evolution of nematophagous fungus and Lecanicillium species.

The complete mitochondrial genome of the nematophagous fungus Acremonium implicatum.

The complete mitochondrial genome of the nematophagous fungus Acremonium implicatum is reported for the first time. The genome is concatenated with 22,367 bp in length, encoding 13 protein-coding genes, 2 ribosomal RNA genes and a set of 17 transfer RNA genes. The synteny analysis reveals that 50.35% of A. implicatum mitochondrial sequences matched to 48.21% of Acremonium chrysogenum mitochondrial sequences with 85.68% identity. Two proteins of cox3 and nad6, as well as seven tRNAs are lost in A. implicatum mitogenome compared to A. chrysogenum mitogenome. The gene orders in A. implicatum and A. chrysogenum mitogenome is different, which is mainly due to the location of nad4 and cox2. In addition, one transposition event related to tRNAs is identified in these two mitogenomes. This study may provide valuable mitochondrial information for research on A. implicatum and facilitate the study of mitochondrial evolution.

Transformation of the endophytic fungus Acremonium implicatum with GFP and evaluation of its biocontrol effect against Meloidogyne incognita.

Acremonium implicatum is an endophytic fungus with biocontrol potential against Meloidogyne incognita based on its opportunistic egg-parasitic, hatching inhibition, and toxic properties. To understand its mode of plant endophytism and opportunistic egg parasitism, GFP-tagged A. implicatum was constructed by PEG-mediated protoplast transformation. By laser scanning confocal microscopy (LSCM), we evaluated the endophytism and opportunistic egg parasitism of a stable gfp transformant (Acr-1). Acr-1 could colonize epidermal tissue, cortical tissue, and xylem of roots and form a mutualistic symbiosis with tomato host plants. LSCM of Acr-1 infecting M. incognita eggs revealed that hyphae penetrated the shell and grew inside eggs to form trophic hyphae. A large number of hyphae enveloped parasitized eggs. In addition, the egg shell integrity was destroyed by fungal penetration. The percentage of egg parasitism was 33.8 %. There were no marked differences between the wild type and mutant in nematode second-stage juvenile mortality and egg hatching and in fungal control efficiency in a pot experiment. In conclusion, gfp-transformation did not change the nematicidal activity of A. implicatum and is a tool to examine the mode of plant endophytism and opportunistic egg parasitism of A. implicatum.

Analysis of the complete mitochondrial genome of Pochonia chlamydosporia suggests a close relationship to the invertebrate-pathogenic fungi in Hypocreales.

BACKGROUND: The fungus Pochonia chlamydosporia parasitizes nematode eggs and has become one of the most promising biological control agents (BCAs) for plant-parasitic nematodes, which are major agricultural pests that cause tremendous economic losses worldwide. The complete mitochondrial (mt) genome is expected to open new avenues for understanding the phylogenetic relationships and evolution of the invertebrate-pathogenic fungi in Hypocreales. RESULTS: The complete mitogenome sequence of P. chlamydosporia is 25,615 bp in size, containing the 14 typical protein-coding genes, two ribosomal RNA genes, an intronic ORF coding for a putative ribosomal protein (rps3) and a set of 23 transfer RNA genes (trn) which recognize codons for all amino acids. Sequence similarity studies and syntenic gene analyses show that 87.02% and 58.72% of P. chlamydosporia mitogenome sequences match 90.50% of Metarhizium anisopliae sequences and 61.33% of Lecanicillium muscarium sequences with 92.38% and 86.04% identities, respectively. A phylogenetic tree inferred from 14 mt proteins in Pezizomycotina fungi supports that P. chlamydosporia is most closely related to the entomopathogenic fungus M. anisopliae. The invertebrate-pathogenic fungi in Hypocreales cluster together and clearly separate from a cluster comprising plant-pathogenic fungi (Fusarium spp.) and Hypocrea jecorina. A comparison of mitogenome sizes shows that the length of the intergenic regions or the intronic regions is the major size contributor in most of mitogenomes in Sordariomycetes. Evolutionary analysis shows that rps3 is under positive selection, leading to the display of unique evolutionary characteristics in Hypocreales. Moreover, the variability of trn distribution has a clear impact on gene order in mitogenomes. Gene rearrangement analysis shows that operation of transposition drives the rearrangement events in Pezizomycotina, and most events involve in trn position changes, but no rearrangement was found in Clavicipitaceae. CONCLUSIONS: We present the complete annotated mitogenome sequence of P. chlamydosporia. Based on evolutionary and phylogenetic analyses, we have determined the relationships between the invertebrate-pathogenic fungi in Hypocreales. The invertebrate-pathogenic fungi in Hypocreales referred to in this paper form a monophyletic group sharing a most recent common ancestor. Our rps3 and trn gene order results also establish a foundation for further exploration of the evolutionary trajectory of the fungi in Hypocreales.

Determination of total tritium in urine from residents living in the vicinity of nuclear power plants in Qinshan, China.

To estimate the tritium doses of the residents living in the vicinity of a nuclear power plant, urine samples of 34 adults were collected from residents living near the Qinshan nuclear power plant. The tritium-in-urine (HTO plus OBT) was measured by liquid scintillation counting. The doses of tritium-in-urine from participants living at 2, 10 and 22 km were in a range of 1.26-6.73 Bq/L, 1.31-3.09 Bq/L and 2.21-3.81 Bq/L, respectively, while the average activity concentrations of participants from the three groups were 3.53 ± 1.62, 2.09 ± 0.62 and 2.97 ± 0.78 Bq/L, respectively. The personal committed effective doses for males were 2.5 ± 1.7 nSv and for females they were 2.9 ± 1.3 nSv. These results indicate that tritium concentrations in urine samples from residents living at 2 km from a nuclear power plant are significantly higher than those at 10 km. It may be the downwind direction that caused a higher dose in participants living at 22 km. All the measured doses of tritium-in-urine are in a background level range.

Development of a high-efficiency gene knockout system for Pochonia chlamydosporia.

The nematophagous fungus Pochonia chlamydosporia, which belongs to the family Clavicipitaceae (Ascomycota: Pezizomycotina: Sordariomycetes: Hypocreales), is a promising biological control agent for root-knot and cyst nematodes. Its biocontrol effect has been confirmed by pot and field trials. The genome sequence of the fungus was completed recently; therefore, genome-wide functional analyses will identify its infection-associated genes. Gene knockout techniques are useful molecular tools to study gene functions. However, cultures of P. chlamydosporia are resistant to high levels of a range of fungal inhibitors, which makes the gene knockout technique difficult in this fungus. Fortunately, we found that the wild P. chlamydosporia strain PC-170 could not grow on medium containing 150µgml(-1) G418 sulfate, representing a new selectable marker for P. chlamydosporia. The neomycin-resistance gene (neo), which was amplified from the plasmid pKOV21, conferred G418-resistance on the fungus; therefore, it was chosen as the marker gene. We subsequently developed a gene knockout system for P. chlamydosporia using split-marker homologous recombination cassettes with resistance selection and protoplast transformation. The split-marker cassettes were developed using fusion PCR, and involved only two rounds of PCR. The final products comprised two linear constructs. Each construct contained a flanking region of the target gene and two thirds of the neo gene. Alkaline serine protease and chitinase were confirmed to be produced by P. chlamydosporia during infection of nematode eggs and could participate in lysis of the eggshell of nematode eggs. Here, we knocked out one chitinase gene, VFPPC_01099, and two protease genes (VFPPC_10088, VFPPC_06535). We obtained approximately 100 suspected mutants after each transformation. After screening by PCR, the average rate of gene knockout was 13%: 11% (VFPPC_01099), 13% (VFPPC_10088) and 15% (VFPPC_06535). This efficient and convenient technique will accelerate functional genomic studies in P. chlamydosporia.

Expression of IL-27, Th1 and Th17 in patients with aplastic anemia.

BACKGROUND: Aplastic anemia (AA) is an autoimmune disease and interleukin-27 (IL-27) is an important cytokine involved in the pathogenesis of autoimmune diseases. To date there have been no reports concerning the intrinsic association among IL-27 and Thelper (Th) 1 and Th17 cells in AA. MATERIALS AND METHODS: Enzyme-linked immunosorbent assay (ELISA) to assay IL-27, interferon gamma (IFN-γ) and IL-17 levels, flow cytometry to measure the percentages of Th1 and Th17 cells among peripheral blood mononuclear cells (PBMCs), real-time reverse transcriptase polymerase chain reaction (PCR) for the mRNA levels of IL-27, IFN-γ, T-bet and IL-17 and retinoid related orphan receptor gamma (RORγt) in PBMCs were performed. In addition, the effect of exogenous rhIL-27 on the differentiation of T cells into Th1 and Th17 cells was investigated in vitro. RESULTS: Plasma and mRNA levels of IL-27 in PBMCs from AA patients were significantly higher than those in healthy controls. A positive correlation was found between plasma levels of IL27 and IFN-γ. The proportions of Th1 and Th17 cells accompanied by the mRNA expression of RORγt and T-bet were significantly higher in AA patients than in healthy controls. Plasma levels of IL-27 correlated positively with frequencies of Th1 cells in AA patients. Exogenous rhIL-27 could significantly upregulate the frequency of Th1 cells and the mRNA levels of T-bet and IFN-γ and the application of rhIL-27 in vitro could inhibit the expression of RORγt mRNA. CONCLUSION: The upregulation of IL-27 might cause Th1 differentiation and immune disorders in AA patients. Blocking the expression of IL-27 could therefore be a reasonable therapeutic strategy for AA.