Thirty years of brain imaging research has converged to define the brain’s default network—a novel and only recently appreciated brain system that participates in internal modes of cognition Here we synthesize past observations to provide strong evidence that the default network is a specific, anatomically defined brain system preferentially active when individuals are not focused on the external environment Analysis of connectional anatomy in the monkey supports the presence of an interconnected brain system Providing insight into function, the default network is active when individuals are engaged in internally focused tasks including autobiographical memory retrieval, envisioning the future, and conceiving the perspectives of others Probing the functional anatomy of the network in detail reveals that it is best understood as multiple interacting subsystems The medial temporal lobe subsystem provides information from prior experiences in the form of memories and associations that are the building blocks of mental simulation The medial prefrontal subsystem facilitates the flexible use of this information during the construction of self-relevant mental simulations These two subsystems converge on important nodes of integration including the posterior cingulate cortex The implications of these functional and anatomical observations are discussed in relation to possible adaptive roles of the default network for using past experiences to plan for the future, navigate social interactions, and maximize the utility of moments when we are not otherwise engaged by the external world We conclude by discussing the relevance of the default network for understanding mental disorders including autism, schizophrenia, and Alzheimer’s disease

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The Brain's Default Network Anatomy, Function, and Relevance to Disease

We have designed two taxon-selective primers for the internal transcribed spacer (ITS) region in the nuclear ribosomal repeat unit. These primers, ITS1-F and ITS4-B, were intended to be specific to fungi and basidiomycetes, respectively. We have tested the specificity of these primers against 13 species of ascomycetes, 14 of basidiomycetes, and 15 of plants. Our results showed that ITS4-B, when paired with either a 'universal' primer ITS1 or the fungal-specific primer ITS1-F, efficiently amplified DNA from all basidiomycetes and discriminated against ascomycete DNAs. The results with plants were not as clearcut. The ITS1-F/ITS4-B primer pair produced a small amount of PCR product for certain plant species, but the quantity was in most cases less than that produced by the 'universal' ITS primers. However, under conditions where both plant and fungal DNAs were present, the fungal DNA was amplified to the apparent exclusion of plant DNA. ITS1-F/ITS4-B preferential amplification was shown to be particularly useful for detection and analysis of the basidiomycete component in ectomycorrhizae and in rust-infected tissues. These primers can be used to study the structure of ectomycorrhizal communities or the distribution of rusts on alternate hosts.

ITS primers with enhanced specificity for basidiomycetes--application to the identification of mycorrhizae and rusts.

Long-term dietary intake influences the structure and activity of the trillions of microorganisms residing in the human gut, but it remains unclear how rapidly and reproducibly the human gut microbiome responds to short-term macronutrient change. Here we show that the short-term consumption of diets composed entirely of animal or plant products alters microbial community structure and overwhelms inter-individual differences in microbial gene expression. The animal-based diet increased the abundance of bile-tolerant microorganisms (Alistipes, Bilophila and Bacteroides) and decreased the levels of Firmicutes that metabolize dietary plant polysaccharides (Roseburia, Eubacterium rectale and Ruminococcus bromii). Microbial activity mirrored differences between herbivorous and carnivorous mammals, reflecting trade-offs between carbohydrate and protein fermentation. Foodborne microbes from both diets transiently colonized the gut, including bacteria, fungi and even viruses. Finally, increases in the abundance and activity of Bilophila wadsworthia on the animal-based diet support a link between dietary fat, bile acids and the outgrowth of microorganisms capable of triggering inflammatory bowel disease. In concert, these results demonstrate that the gut microbiome can rapidly respond to altered diet, potentially facilitating the diversity of human dietary lifestyles.

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Diet rapidly and reproducibly alters the human gut microbiome

Detailed restriction analyses of many samples often require substantial amounts of time and effort for DNA extraction, restriction digests, Southern blotting, and hybridization. We describe a novel approach that uses the polymerase chain reaction (PCR) for rapid simplified restriction typing and mapping of DNA from many different isolates. DNA fragments up to 2 kilobase pairs in length were efficiently amplified from crude DNA samples of several pathogenic Cryptococcus species, including C. neoformans, C. albidus, C. laurentii, and C. uniguttulatus. Digestion and electrophoresis of the PCR products by using frequent-cutting restriction enzymes produced complex restriction phenotypes (fingerprints) that were often unique for each strain or species. We used the PCR to amplify and analyze restriction pattern variation within three major portions of the ribosomal DNA (rDNA) repeats from these fungi. Detailed mapping of many restriction sites within the rDNA locus was determined by fingerprint analysis of progressively larger PCR fragments sharing a common primer site at one end. As judged by PCR fingerprints, the rDNA of 19 C. neoformans isolates showed no variation for four restriction enzymes that we surveyed. Other Cryptococcus spp. showed varying levels of restriction pattern variation within their rDNAs and were shown to be genetically distinct from C. neoformans. The PCR primers used in this study have also been successfully applied for amplification of rDNAs from other pathogenic and nonpathogenic fungi, including Candida spp., and ought to have wide applicability for clinical detection and other studies.

Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species.

We constructed nine sets of oligonucleotide primers on the basis of the results of DNA hybridization of cloned genes from Neurospora crassa and Aspergillus nidulans to the genomes of select filamentous ascomycetes and deuteromycetes (with filamentous ascomycete affiliations). Nine sets of primers were designed to amplify segments of DNA that span one or more introns in conserved genes. PCR DNA amplification with the nine primer sets with genomic DNA from ascomycetes, deuteromycetes, basidiomycetes, and plants revealed that five of the primer sets amplified a product only from DNA of the filamentous ascomycetes and deuteromycetes. The five primer sets were constructed from the N. crassa genes for histone 3, histone 4, beta-tubulin, and the plasma membrane ATPase. With these five primer sets, polymorphisms were observed in both the size of and restriction enzyme sites in the amplified products from the filamentous ascomycetes. The primer sets described here may provide useful tools for phylogenetic studies and genome analyses in filamentous ascomycetes and deuteromycetes (with ascomycete affiliations), as well as for the rapid differentiation of fungal species by PCR.

Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes.

Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics

The fungi comprise both members of the kingdom Fungi as we now recognize it (Ascomycota, Basidiomycota, Zygomycota, and Chytridiomycota) and fungal-like protists such as the Oomycota and the cellular and acellular slime molds (Myxomycota and Acrasiomycota). Treating this admittedly polyphyletic assemblage as a group is useful because these organisms often fill rather similar roles within ecosystems, and they have traditionally been studied almost exclusively by mycologists and plant pathologists. Throughout this review, Fungi will refer to the Kingdom, fungi to the organisms studied by mycologists. The fungi, as thus defined, are of great importance for the following reasons: (a) They are the primary decomposers in all terrestrial ecosystems; (b) they are important symbiotic associates of vascular plants both in mutualistic and parasitic relationships; (c) they constitute the overwhelming majority of plant pathogens and as such have a tremendous eco-

Fungal Molecular Systematics

We used the polymerase chain reaction (PCR) to amplify specific regions of the nuclear and mitochondrial genomes of fungi using DNA extracted from pure cultures as well as that directly from ectomy...

Identification of indigenous and introduced symbiotic fungi in ectomycorrhizae by amplification of nuclear and mitochondrial ribosomal DNA

Coccidioides immitis, cause of a recent epidemic of "Valley fever" in California, is typical of many eukaryotic microbes in that mating and meiosis have yet to be reported, but it is not clear whether sex is truly absent or just cryptic. To find out, we have undertaken a population genetic study using PCR amplification, screening for single-strand conformation polymorphisms, and direct DNA sequencing to find molecular markers with nucleotide-level resolution. Both population genetic and phylogenetic analyses indicate that C. immitis is almost completely recombining. To our knowledge, this study is the first to find molecular evidence for recombination in a fungus for which no sexual stage has yet been described. These results motivate a directed search for mating and meiosis and illustrate the utility of single-strand conformation polymorphism and sequencing with arbitrary primer pairs in molecular population genetics.

https://www.pnas.org/content/pnas/93/2/770.full.pdf

Molecular markers reveal cryptic sex in the human pathogen Coccidioides immitis

Coccidioides posadasii sp. nov., formerly known as non-California (non-CA) Coccidioides immi- tis, is described. Phylogenetic analyses using single nucleotide polymorphisms, genes, and microsatellites show that C. posadasii represents a divergent, genet- ically recombining monophyletic clade. Coccidioides posadasii can be distinguished from C. immitis by nu- merous DNA polymorphisms, and we show how ei- ther of two microsatellite loci may be used as diag- nostic markers for this species. Growth experiments show that C. posadasii has significantly slower growth rates on high-salt media when compared with C. im- mitis, suggesting that other phenotypic characters may exist.

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Molecular and phenotypic description of Coccidioides posadasii sp. nov., previously recognized as the non-California population of Coccidioides immitis.

Thomas J. White

Papers

Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics

Fungal Molecular Systematics

Identification of indigenous and introduced symbiotic fungi in ectomycorrhizae by amplification of nuclear and mitochondrial ribosomal DNA

Molecular markers reveal cryptic sex in the human pathogen Coccidioides immitis

Molecular and phenotypic description of Coccidioides posadasii sp. nov., previously recognized as the non-California population of Coccidioides immitis.