Reconstruction of gene innovation associated with major evolutionary transitions in the kingdom Fungi.

BACKGROUND: Fungi exhibit astonishing diversity with multiple major phenotypic transitions over the kingdom's evolutionary history. As part of this process, fungi developed hyphae, adapted to land environments (terrestrialization), and innovated their sexual structures. These changes also helped fungi establish ecological relationships with other organisms (animals and plants), but the genomic basis of these changes remains largely unknown. RESULTS: By systematically analyzing 304 genomes from all major fungal groups, together with a broad range of eukaryotic outgroups, we have identified 188 novel orthogroups associated with major changes during the evolution of fungi. Functional annotations suggest that many of these orthogroups were involved in the formation of key trait innovations in extant fungi and are functionally connected. These innovations include components for cell wall formation, functioning of the spindle pole body, polarisome formation, hyphal growth, and mating group signaling. Innovation of mitochondria-localized proteins occurred widely during fungal transitions, indicating their previously unrecognized importance. We also find that prokaryote-derived horizontal gene transfer provided a small source of evolutionary novelty with such genes involved in key metabolic pathways. CONCLUSIONS: The overall picture is one of a relatively small number of novel genes appearing at major evolutionary transitions in the phylogeny of fungi, with most arising de novo and horizontal gene transfer providing only a small additional source of evolutionary novelty. Our findings contribute to an increasingly detailed portrait of the gene families that define fungal phyla and underpin core features of extant fungi.

Variable Spontaneous Mutation and Loss of Heterozygosity among Heterozygous Genomes in Yeast.

Mutation and recombination are the primary sources of genetic variation. To better understand the evolution of genetic variation, it is crucial to comprehensively investigate the processes involving mutation accumulation and recombination. In this study, we performed mutation accumulation experiments on four heterozygous diploid yeast species in the Saccharomycodaceae family to determine spontaneous mutation rates, mutation spectra, and losses of heterozygosity (LOH). We observed substantial variation in mutation rates and mutation spectra. We also observed high LOH rates (1.65-11.07×10-6 events per heterozygous site per cell division). Biases in spontaneous mutation and LOH together with selection ultimately shape the variable genome-wide nucleotide landscape in yeast species.

Comparative genomics reveals a core gene toolbox for lifestyle transitions in Hypocreales fungi.

Fungi have evolved diverse lifestyles and adopted pivotal new roles in both natural ecosystems and human environments. However, the molecular mechanisms underlying their adaptation to new lifestyles are obscure. Here, we hypothesize that genes shared across all species with the same lifestyle, but absent in genera with alternative lifestyles, are crucial to that lifestyle. By analysing dozens of species within four genera in a fungal order, with each genus following a different lifestyle, we find that genus-specific genes are typically few in number. Notably, not all genus-specific genes appear to derive from de novo birth, with most instead reflecting recurrent loss across the fungi. Importantly, however, a subset of these genus-specific genes are shared by fungi with the same lifestyle in quite different evolutionary orders, thus supporting the view that some genus-specific genes are necessary for specific lifestyles. These lifestyle-specific genes are enriched for key functional classes and often exhibit specialized expression patterns. Genus-specific selection also contributes to lifestyle transitions, and is especially associated with intensity of pathogenesis. Our study, therefore, suggests that fungal adaptation to new lifestyles often requires just a small number of core genes, with gene turnover and positive selection playing complementary roles.

Retracted and Republished from: "Substrate-Specific Differential Gene Expression and RNA Editing in the Brown Rot Fungus Fomitopsis pinicola"

Wood-decaying fungi tend to have characteristic substrate ranges that partly define their ecological niche. Fomitopsis pinicola is a brown rot species of Polyporales that is reported on 82 species of softwoods and 42 species of hardwoods. We analyzed gene expression levels of F. pinicola from submerged cultures with ground wood powder (sampled at 5 days) or solid wood wafers (sampled at 10 and 30 days), using aspen, pine, and spruce substrates (aspen was used only in submerged cultures). Fomitopsis pinicola expressed similar sets of wood-degrading enzymes typical of brown rot fungi across all culture conditions and time points. Nevertheless, differential gene expression was observed across all pairwise comparisons of substrates and time points. Genes exhibiting differential expression encode diverse enzymes with known or potential function in brown rot decay, including laccase, benzoquinone reductase, aryl alcohol oxidase, cytochrome P450s, and various glycoside hydrolases. Comparing transcriptomes from submerged cultures and wood wafers, we found that culture conditions had a greater impact on global expression profiles than substrate wood species. These findings highlight the need for standardization of culture conditions in studies of gene expression in wood-decaying fungi. IMPORTANCE All species of wood-decaying fungi occur on a characteristic range of substrates (host plants), which may be broad or narrow. Understanding the mechanisms that allow fungi to grow on particular substrates is important for both fungal ecology and applied uses of different feedstocks in industrial processes. We grew the wood-decaying polypore Fomitopsis pinicola on three different wood species­aspen, pine, and spruce­under various culture conditions. We found that F. pinicola is able to modify gene expression (transcription levels) across different substrate species and culture conditions. Many of the genes involved encode enzymes with known or predicted functions in wood decay. This study provides clues to how wood-decaying fungi may adjust their arsenal of decay enzymes to accommodate different host substrates.

Mitochondrial-encoded endonucleases drive recombination of protein-coding genes in yeast.

Mitochondrial recombination in yeast is well recognized, yet the underlying genetic mechanisms are not well understood. Recent progress has suggested that mobile introns in mitochondrial genomes (mitogenomes) can facilitate the recombination of their corresponding intron-containing genes through a mechanism known as intron homing. As many mitochondrial genes lack introns, there is a critical need to determine the extent of recombination and underlying mechanism of intron-lacking genes. This study leverages yeast mitogenomes to address these questions. In Saccharomyces cerevisiae, the 3'-end sequences of at least three intron-lacking mitochondrial genes exhibit elevated nucleotide diversity and recombination hotspots. Each of these 3'-end sequences is immediately adjacent to or even fused as overlapping genes with a stand-alone endonuclease. Our findings suggest that SAEs are responsible for recombination and elevated diversity of adjacent intron-lacking genes. SAEs were also evident to drive recombination of intron-lacking genes in Lachancea kluyveri, a yeast species that diverged from S. cerevisiae more than 100 million years ago. These results suggest SAEs as a common driver in recombination of intron-lacking genes during mitogenome evolution. We postulate that the linkage between intron-lacking gene and its adjacent endonuclease gene is the result of co-evolution.

Greater genetic and regulatory plasticity of retained duplicates in Epichloë endophytic fungi.

Gene duplicates can act as a source of genetic material from which new functions arise. Most duplicated genes revert to single copy genes and only a small proportion are retained. However, it remains unclear why some duplicate genes persist in the genome for an extended time. We investigate this question by analysing retained gene duplicates in the fungal genus Epichloë, ascomycete fungi that form close endophytic symbioses with their host grasses. Retained duplicates within this genus have two independent origins, but both long pre-date the origin and diversification of the genus Epichloë. We find that loss of retained duplicates within the genus is frequent and often associated with speciation. Retained duplicates have faster evolutionary rates (Ka) and show relaxed selection (Ka/Ks) compared to single copy genes. Both features are time-dependent. Through comparison of conspecific strains, we find greater evolutionary rates in coding regions and sequence divergence in regulatory regions of retained duplicates than single copy genes, with this pattern more pronounced for strains adapted to different grass host species. Consistent with this sequence divergence in regulatory regions, transcriptome analyses show greater expression variation of retained duplicates than single copy genes. This suggest that cis-regulatory changes make important contributions to the expression patterns of retained duplicates. Coupled with supporting observations from the model yeast Saccharomyces cerevisiae, these data suggest that genetic robustness and regulatory plasticity are common drivers behind the retention of duplicated genes in fungi.

Substrate-Specific Differential Gene Expression and RNA Editing in the Brown Rot Fungus Fomitopsis pinicola.

Wood-decaying fungi tend to have characteristic substrate ranges that partly define their ecological niche. Fomitopsis pinicola is a brown rot species of Polyporales that is reported on 82 species of softwoods and 42 species of hardwoods. We analyzed the gene expression levels and RNA editing profiles of F. pinicola from submerged cultures with ground wood powder (sampled at 5 days) or solid wood wafers (sampled at 10 and 30 days), using aspen, pine, and spruce substrates (aspen was used only in submerged cultures). Fomitopsis pinicola expressed similar sets of wood-degrading enzymes typical of brown rot fungi across all culture conditions and time points. Nevertheless, differential gene expression and RNA editing were observed across all pairwise comparisons of substrates and time points. Genes exhibiting differential expression and RNA editing encode diverse enzymes with known or potential function in brown rot decay, including laccase, benzoquinone reductase, aryl alcohol oxidase, cytochrome P450s, and various glycoside hydrolases. There was no overlap between differentially expressed and differentially edited genes, suggesting that these may provide F. pinicola with independent mechanisms for responding to different conditions. Comparing transcriptomes from submerged cultures and wood wafers, we found that culture conditions had a greater impact on global expression profiles than substrate wood species. In contrast, the suites of genes subject to RNA editing were much less affected by culture conditions. These findings highlight the need for standardization of culture conditions in studies of gene expression in wood-decaying fungi.IMPORTANCE All species of wood-decaying fungi occur on a characteristic range of substrates (host plants), which may be broad or narrow. Understanding the mechanisms that enable fungi to grow on particular substrates is important for both fungal ecology and applied uses of different feedstocks in industrial processes. We grew the wood-decaying polypore Fomitopsis pinicola on three different wood species, aspen, pine, and spruce, under various culture conditions. We examined both gene expression (transcription levels) and RNA editing (posttranscriptional modification of RNA, which can potentially yield different proteins from the same gene). We found that F. pinicola is able to modify both gene expression and RNA editing profiles across different substrate species and culture conditions. Many of the genes involved encode enzymes with known or predicted functions in wood decay. This work provides clues to how wood-decaying fungi may adjust their arsenal of decay enzymes to accommodate different host substrates.

Molecular gated-AlGaN/GaN high electron mobility transistor for pH detection.

A molecular gated-AlGaN/GaN high electron mobility transistor has been developed for pH detection. The sensing surface of the sensor was modified with 3-aminopropyltriethoxysilane to provide amphoteric amine groups, which would play the role of receptors for pH detection. On modification with 3-aminopropyltriethoxysilane, the transistor exhibits good chemical stability in hydrochloric acid solution and is sensitive for pH detection. Thus, our molecular gated-AlGaN/GaN high electron mobility transistor acheived good electrical performances such as chemical stability (remained stable in hydrochloric acid solution), good sensitivity (37.17 µA/pH) and low hysteresis. The results indicate a promising future for high-quality sensors for pH detection.

Extensive Horizontal Transfer and Homologous Recombination Generate Highly Chimeric Mitochondrial Genomes in Yeast.

The frequency of horizontal gene transfer (HGT) in mitochondrial DNA varies substantially. In plants, HGT is relatively common, whereas in animals it appears to be quite rare. It is of considerable importance to understand mitochondrial HGT across the major groups of eukaryotes at a genome-wide level, but so far this has been well studied only in plants. In this study, we generated ten new mitochondrial genome sequences and analyzed 40 mitochondrial genomes from the Saccharomycetaceae to assess the magnitude and nature of mitochondrial HGT in yeasts. We provide evidence for extensive, homologous-recombination-mediated, mitochondrial-to-mitochondrial HGT occurring throughout yeast mitochondrial genomes, leading to genomes that are highly chimeric evolutionarily. This HGT has led to substantial intraspecific polymorphism in both sequence content and sequence divergence, which to our knowledge has not been previously documented in any mitochondrial genome. The unexpectedly high frequency of mitochondrial HGT in yeast may be driven by frequent mitochondrial fusion, relatively low mitochondrial substitution rates and pseudohyphal fusion to produce heterokaryons. These findings suggest that mitochondrial HGT may play an important role in genome evolution of a much broader spectrum of eukaryotes than previously appreciated and that there is a critical need to systematically study the frequency, extent, and importance of mitochondrial HGT across eukaryotes.

Lack of association between methylenetetrahydrofolate dehydrogenase 1 G1958A polymorphism and prostate cancer risk: a meta-analysis.

The methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) polymorphism G1958A has been extensively investigated as a potential risk factor for prostate cancer (PCa), but the results have thus far been inconclusive. This meta-analysis was performed to derive a more precise estimation of the association. A comprehensive search was conducted to identify all case-control studies of MTHFD1 G1958A polymorphism and PCa risk. We used odds ratios (ORs) to assess the strength of the association, and 95% confidence intervals (CIs) give a sense of the precision of the estimate. Statistical analyses were performed using Review Manage version 5.0 and Stata 10.0. A total of six available studies were considered in the present meta-analysis, with 7,493 patients and 36,941 controls. When all groups were pooled, there was no evidence that G1958A had significant association with PCa under additive, recessive, dominant, and allelic models. This meta-analysis suggests that MTHFD1 G1958A polymorphism might not be a risk factor for PCa. However, further large-scale and well-designed case-control studies are necessary to validate the risk identified in the present meta-analysis.

Genome-Wide Association Study Identifies Pharmacogenomic Variants Associated With Metformin Glycemic Response in African American Patients With Type 2 Diabetes.

OBJECTIVE: Metformin is the most common treatment for type 2 diabetes (T2D). However, there have been no pharmacogenomic studies for T2D in which a population of color was used in the discovery analysis. This study sought to identify genomic variants associated with metformin response in African American patients with diabetes. RESEARCH DESIGN AND METHODS: Patients in the discovery set were adult, African American participants from the Diabetes Multi-omic Investigation of Drug Response (DIAMOND), a cohort study of patients with T2D from a health system serving southeast Michigan. DIAMOND participants had genome-wide genotype data and longitudinal electronic records of laboratory results and medication fills. The genome-wide discovery analysis identified polymorphisms correlated to changes in glycated hemoglobin (HbA1c) levels among individuals on metformin monotherapy. Lead associations were assessed for replication in an independent cohort of African American participants from Kaiser Permanente Northern California (KPNC) and in European American participants from DIAMOND. RESULTS: The discovery set consisted of 447 African American participants, whereas the replication sets included 353 African American KPNC participants and 466 European American DIAMOND participants. The primary analysis identified a variant, rs143276236, in the gene ARFGEF3, which met the threshold for genome-wide significance, replicated in KPNC African Americans, and was still significant in the meta-analysis (P = 1.17 × 10-9). None of the significant discovery variants replicated in European Americans DIAMOND participants. CONCLUSIONS: We identified a novel and biologically plausible genetic variant associated with a change in HbA1c levels among African American patients on metformin monotherapy. These results highlight the importance of diversity in pharmacogenomic studies.

Validation of human telomere length multi-ancestry meta-analysis association signals identifies POP5 and KBTBD6 as human telomere length regulation genes.

Genome-wide association studies (GWAS) have become well-powered to detect loci associated with telomere length. However, no prior work has validated genes nominated by GWAS to examine their role in telomere length regulation. We conducted a multi-ancestry meta-analysis of 211,369 individuals and identified five novel association signals. Enrichment analyses of chromatin state and cell-type heritability suggested that blood/immune cells are the most relevant cell type to examine telomere length association signals. We validated specific GWAS associations by overexpressing KBTBD6 or POP5 and demonstrated that both lengthened telomeres. CRISPR/Cas9 deletion of the predicted causal regions in K562 blood cells reduced expression of these genes, demonstrating that these loci are related to transcriptional regulation of KBTBD6 and POP5. Our results demonstrate the utility of telomere length GWAS in the identification of telomere length regulation mechanisms and validate KBTBD6 and POP5 as genes affecting telomere length regulation.

European and multi-ancestry genome-wide association meta-analysis of atopic dermatitis highlights importance of systemic immune regulation.

Atopic dermatitis (AD) is a common inflammatory skin condition and prior genome-wide association studies (GWAS) have identified 71 associated loci. In the current study we conducted the largest AD GWAS to date (discovery N = 1,086,394, replication N = 3,604,027), combining previously reported cohorts with additional available data. We identified 81 loci (29 novel) in the European-only analysis (which all replicated in a separate European analysis) and 10 additional loci in the multi-ancestry analysis (3 novel). Eight variants from the multi-ancestry analysis replicated in at least one of the populations tested (European, Latino or African), while two may be specific to individuals of Japanese ancestry. AD loci showed enrichment for DNAse I hypersensitivity and eQTL associations in blood. At each locus we prioritised candidate genes by integrating multi-omic data. The implicated genes are predominantly in immune pathways of relevance to atopic inflammation and some offer drug repurposing opportunities.

Proteome of the Wood Decay Fungus Fomitopsis pinicola Is Altered by Substrate.

The brown rot fungus Fomitopsis pinicola efficiently depolymerizes wood cellulose via the combined activities of oxidative and hydrolytic enzymes. Mass spectrometric analyses of culture filtrates identified specific proteins, many of which were differentially regulated in response to substrate composition.

Evolution of the IL17 receptor family in chordates: a new subfamily IL17REL.

The human interleukin 17 receptor (IL17R) family plays a critical role in inflammatory responses and contributes to the pathology of many autoimmune diseases. So far, five members, IL17RA to IL17RE, have been identified. Recently, some IL17R genes have been identified in non-mammalian species, such as zebrafish IL17RD; however, there are no reports on the evolutionary history of this complex gene family through comparative phylogenetic approaches. Here, we concentrated on the IL17R evolution in chordates. There are two IL17Rs in the genome of the basal chordate amphioxus: IL17RA and IL17RD. After two rounds of whole genome duplications, these two IL17R genes expanded into five early vertebrate IL17R genes, IL17RA to IL17RE. IL17RA and IL17RD are found in most vertebrates, whereas the other three, IL17RB, ILR17RC, and IL17RE, underwent some loss in vertebrates during evolution. Our sequence and structure analyses reveal functional similarities and distinctions between the different IL17Rs. Based on similarity searches for IL17R-like proteins within chordate sequences, a group of IL17RE-like (IL17REL) proteins were identified from mammalians to lower vertebrates. In silico and expression analyses on the novel IL17RELs showed that this group of receptors is highly conserved across species, indicating that IL17REL may represent a unique subfamily of IL17Rs.

Characterization of Bicistronic Transcription in Budding Yeast.

Bicistronic transcripts (operon-like transcripts) have occasionally been reported in eukaryotes, including unicellular yeasts, plants, and humans, despite the fact that they lack trans-splice mechanisms. However, the characteristics of eukaryotic bicistronic transcripts are poorly understood, except for those in nematodes. Here, we describe the genomic, transcriptomic, and ribosome profiling features of bicistronic transcripts in unicellular yeasts. By comparing the expression level of bicistronic transcripts with their monocistronic equivalents, we identify two main categories of bicistronic transcripts: highly and lowly expressed. These two categories exhibit quite different features. First, highly expressed bicistronic transcripts have higher conservation within and between strains and shorter intergenic spacers with higher GC content and less stable secondary structure. Second, genes in highly expressed bicistronic transcripts have lower translation efficiency, with the second gene showing statistically significant lower translation efficiency than the first. Finally, the genes found in these highly expressed bicistronic transcripts tend to be younger, with more recent origins. Together, these results suggest that bicistronic transcripts in yeast are heterogeneous. We further propose that at least some highly expressed bicistronic transcripts appear to play a role in modulating monocistronic translation.IMPORTANCE Operons, where a single mRNA transcript encodes multiple adjacent proteins, are a widespread feature of bacteria and archaea. In contrast, the genes of eukaryotes are generally considered monocistronic. However, a number of studies have revealed the presence of bicistronic transcripts in eukaryotes, including humans. The basic features of these transcripts are largely unknown in eukaryotes, especially in organisms lacking trans-splice mechanisms. Our analyses characterize bicistronic transcripts in one such eukaryotic group, yeasts. We show that highly expressed bicistronic transcripts have unusual features compared to lowly expressed bicistronic transcripts, with several features influencing translational modulation.

Transcanal Endoscopic Ear Surgery for Advanced External Auditory Canal Cholesteatoma in Naim Stage III and IV.

OBJECTIVE: To report the clinical characteristics and treatment outcomes, as well as endoscopic-assisted ear surgery techniques used in patients with advanced external auditory canal cholesteatoma (EACC). STUDY DESIGN: Retrospective case series. SETTING: University hospital. METHODS: From October 2014 to September 2017, adult patients (age > 18) with advanced EACC (Naim's classification: stage III or IV) who underwent transcanal endoscopic ear surgery (TEES) were enrolled. The presenting features, extent of the lesion, and reconstruction techniques used were assessed. The healing time which was defined as the time required to develop a dry, re-epithelialized, and self-cleaning external auditory canal, was compared between stage III and IV. RESULTS: Twenty-three patients were included. EACC was categorized as stage III in 11 ears and stage IV in 12 ears. Cholesteatoma involved the mastoid (30%), middle ear (26%), chorda tympani (22%), temporomandibular joint, antrum, and facial nerve (17% for each). In 96% of patients, a dry and self-cleaning external auditory canal (EAC) was maintained after a mean follow-up of 15 months. The median healing time was 8 weeks in stage III, which was significantly shorter than the 12 weeks required for stage IV (p < 0.05). There was no significant difference in the median healing time between TEES and the canal wall up mastoidectomy for stage IV EACC (14 weeks) performed by the same surgeon over the same period (p > 0.05). CONCLUSIONS: TEES is a feasible and safe technique for the exposure and eradication of advanced EACC. Some critical endoscopic techniques for resecting disease and reconstructing the defect in the EAC and middle ear should be mastered before performing this operation.

Prolonged survival of human hepatocarcinoma cells in the liver of newborn C57BL/6 mice and resulting cellular xenorejection, especially the activation of hepatic natural killer T cells.

OBJECTIVE: To investigate the fate of human hepatocellular carcinoma (HCC) in the livers of newborn mice and the resulting cellular rejection. METHODS: Two HCC cell lines (HepG2 and HCCLM3) labeled with DMAHAS were orthotopically transplanted to newborn and adult mice with or without low-dose cyclosporin A (CsA) treatment (10 mg/kg). The fate of tumor xenografts was examined and the resulting cellular response was investigated. RESULTS: Tumor xenografts survived in newborn mice for > 4 weeks, with a delayed lymphocyte infiltration mediated by CD4+ T, CD8+ T and NK1.1+ cells. In contrast, the xenografts survived in adults < 8-10 days with an acute cellular rejection by CD8+T cells, NK1.1+ cells, macrophages or neutrophils. Orthotopic transplantation of human HCC xenografts elicited a strong cytotoxic response in newborn mice (p < 0.05), and selective T/NK1.1+ cell deletion in vitro suggested that such effector cells were mainly CD8+ T cells. Moreover, tumor xenografts induced a rapid activation of hepatic natural killer T (NKT) cells in both newborn and adult mice with enhanced secretion of IL-4 and IFN-gamma in serum and subsequent NKT-like cytotoxicity. The rapid activation of NKT cells could be efficiently suppressed by low-dose CsA treatment, possibly in a CD1d-independent manner. CONCLUSION: Our data suggest that the livers of newborn mice were more suitable for the survival of xenografts than those of adult mice. Cell-mediated tumor xenorejection in newborn mice was different from that in adults, and hepatic NKT cells may play an important role in early tumor xenorejection.

Evolution of a Record-Setting AT-Rich Genome: Indel Mutation, Recombination, and Substitution Bias.

Genome-wide nucleotide composition varies widely among species. Despite extensive research, the source of genome-wide nucleotide composition diversity remains elusive. Yeast mitochondrial genomes (mitogenomes) are highly A + T rich, and they provide a unique opportunity to study the evolution of AT-biased landscape. In this study, we sequenced ten complete mitogenomes of the Saccharomycodes ludwigii yeast with 8% G + C content, the lowest genome-wide %(G + C) in all published genomes to date. The S. ludwigii mitogenomes have high densities of short tandem repeats but severely underrepresented mononucleotide repeats. Comparative population genomics of these record-setting A + T-rich genomes shows dynamic indel mutations and strong mutation bias toward A/T. Indel mutations play a greater role in genomic variation among very closely related strains than nucleotide substitutions. Indels have resulted in presence-absence polymorphism of tRNAArg (ACG) among S. ludwigii mitogenomes. Interestingly, these mitogenomes have undergone recombination, a genetic process that can increase G + C content by GC-biased gene conversion. Finally, the expected equilibrium G + C content under mutation pressure alone is higher than observed G + C content, suggesting existence of mechanisms other than AT-biased mutation operating to increase A/T. Together, our findings shed new lights on mechanisms driving extremely AT-rich genomes.