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CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.

Nonalcoholic fatty liver disease (NAFLD) is a growing cause of chronic liver disease. Using a proxy NAFLD definition of chronic elevation of alanine aminotransferase (cALT) levels without other liver diseases, we performed a multiancestry genome-wide association study (GWAS) in the Million Veteran Program (MVP) including 90,408 cALT cases and 128,187 controls. Seventy-seven loci exceeded genome-wide significance, including 25 without prior NAFLD or alanine aminotransferase associations, with one additional locus identified in European American-only and two in African American-only analyses (P

Microbes alter their transcriptomic profiles in response to the environment. The physiological conditions experienced by a microbial community can thus be inferred using meta-transcriptomic sequencing by comparing transcription levels of specifically chosen genes. However, this analysis requires accurate reference genomes to identify the specific genes from which RNA reads originate. In addition, such an analysis should avoid biases in transcript counts related to differences in organism abundance. In this study we describe an approach to address these difficulties. Sample-specific meta-genomic assembled genomes (MAGs) were used as reference genomes to accurately identify the origin of RNA reads, and transcript ratios of genes with opposite transcription responses were compared to eliminate biases related to differences in organismal abundance, an approach hereafter named the "diametric ratio" method. We used this approach to probe the environmental conditions experienced by Escherichia spp. in the gut of 4 premature infants, 2 of whom developed necrotizing enterocolitis (NEC), a severe inflammatory intestinal disease. We analyzed twenty fecal samples taken from four premature infants (4-6 time points from each infant), and found significantly higher diametric ratios of genes associated with low oxygen levels in samples of infants later diagnosed with NEC than in samples without NEC. We also show this method can be used for examining other physiological conditions, such as exposure to nitric oxide and osmotic pressure. These study results should be treated with caution, due to the presence of confounding factors that might also distinguish between NEC and control infants. Nevertheless, together with benchmarking analyses, we show here that the diametric ratio approach can be applied for evaluating the physiological conditions experienced by microbes in situ. Results from similar studies can be further applied for designing diagnostic methods to detect NEC in its early developmental stages.

Candidate phyla radiation (CPR) bacteria and DPANN (an acronym of the names of the first included phyla) archaea are massive radiations of organisms that are widely distributed across Earth's environments, yet we know little about them. Initial indications are that they are consistently distinct from essentially all other bacteria and archaea owing to their small cell and genome sizes, limited metabolic capacities and often episymbiotic associations with other bacteria and archaea. In this Analysis, we investigate their biology and variations in metabolic capacities by analysis of approximately 1,000 genomes reconstructed from several metagenomics-based studies. We find that they are not monolithic in terms of metabolism but rather harbour a diversity of capacities consistent with a range of lifestyles and degrees of dependence on other organisms. Notably, however, certain CPR and DPANN groups seem to have exceedingly minimal biosynthetic capacities, whereas others could potentially be free living. Understanding of these microorganisms is important from the perspective of evolutionary studies and because their interactions with other organisms are likely to shape natural microbiome function.

Climate change is driving a pervasive global redistribution of the planet's species. Species redistribution poses new questions for the study of ecosystems, conservation science and human societies that require a coordinated and integrated approach. Here we review recent progress, key gaps and strategic directions in this nascent research area, emphasising emerging themes in species redistribution biology, the importance of understanding underlying drivers and the need to anticipate novel outcomes of changes in species ranges. We highlight that species redistribution has manifest implications across multiple temporal and spatial scales and from genes to ecosystems. Understanding range shifts from ecological, physiological, genetic and biogeographical perspectives is essential for informing changing paradigms in conservation science and for designing conservation strategies that incorporate changing population connectivity and advance adaptation to climate change. Species redistributions present challenges for human well-being, environmental management and sustainable development. By synthesising recent approaches, theories and tools, our review establishes an interdisciplinary foundation for the development of future research on species redistribution. Specifically, we demonstrate how ecological, conservation and social research on species redistribution can best be achieved by working across disciplinary boundaries to develop and implement solutions to climate change challenges. Future studies should therefore integrate existing and complementary scientific frameworks while incorporating social science and human-centred approaches. Finally, we emphasise that the best science will not be useful unless more scientists engage with managers, policy makers and the public to develop responsible and socially acceptable options for the global challenges arising from species redistributions.

The number of microbial genomes sequenced each year is expanding rapidly, in part due to genome-resolved metagenomic studies that routinely recover hundreds of draft-quality genomes. Rapid algorithms have been developed to comprehensively compare large genome sets, but they are not accurate with draft-quality genomes. Here we present dRep, a program that reduces the computational time for pairwise genome comparisons by sequentially applying a fast, inaccurate estimation of genome distance, and a slow, accurate measure of average nucleotide identity. dRep achieves a 28 × increase in speed with perfect recall and precision when benchmarked against previously developed algorithms. We demonstrate the use of dRep for genome recovery from time-series datasets. Each metagenome was assembled separately, and dRep was used to identify groups of essentially identical genomes and select the best genome from each replicate set. This resulted in recovery of significantly more and higher-quality genomes compared to the set recovered using co-assembly.

BackgroundEstablishment of the human gut microbiota begins at birth. This early-life microbiota development can impact host physiology during infancy and even across an entire life span. However, the functional stability and population structure of the gut microbiota during initial colonization remain poorly understood. Metaproteomics is an emerging technology for the large-scale characterization of metabolic functions in complex microbial communities (gut microbiota).ResultsWe applied a metagenome-informed metaproteomic approach to study the temporal and inter-individual differences of metabolic functions during microbial colonization of preterm human infants' gut. By analyzing 30 individual fecal samples, we identified up to 12,568 protein groups for each of four infants, including both human and microbial proteins. With genome-resolved matched metagenomics, proteins were confidently identified at the species/strain level. The maximum percentage of the proteome detected for the abundant organisms was ~45%. A time-dependent increase in the relative abundance of microbial versus human proteins suggested increasing microbial colonization during the first few weeks of early life. We observed remarkable variations and temporal shifts in the relative protein abundances of each organism in these preterm gut communities. Given the dissimilarity of the communities, only 81 microbial EggNOG orthologous groups and 57 human proteins were observed across all samples. These conserved microbial proteins were involved in carbohydrate, energy, amino acid and nucleotide metabolism while conserved human proteins were related to immune response and mucosal maturation. We identified seven proteome clusters for the communities and showed infant gut proteome profiles were unstable across time and not individual-specific. Applying a gut-specific metabolic module (GMM) analysis, we found that gut communities varied primarily in the contribution of nutrient (carbohydrates, lipids, and amino acids) utilization and short-chain fatty acid production.ConclusionsOverall, this study reports species-specific proteome profiles and metabolic functions of human gut microbiota during early colonization. In particular, our work contributes to reveal microbiota-associated shifts and variations in the metabolism of three major nutrient sources and short-chain fatty acid during colonization of preterm infant gut.

Locoregional delivery of chimeric antigen receptor (CAR) T cells has resulted in objective responses in adults with glioblastoma, but the feasibility and tolerability of this approach is yet to be evaluated for pediatric central nervous system (CNS) tumors. Here we show that engineering of a medium-length CAR spacer enhances the therapeutic efficacy of human erb-b2 receptor tyrosine kinase 2 (HER2)-specific CAR T cells in an orthotopic xenograft medulloblastoma model. We translated these findings into BrainChild-01 (NCT03500991), an ongoing phase 1 clinical trial at Seattle Children's evaluating repetitive locoregional dosing of these HER2-specific CAR T cells to children and young adults with recurrent/refractory CNS tumors, including diffuse midline glioma. Primary objectives are assessing feasibility, safety and tolerability; secondary objectives include assessing CAR T cell distribution and disease response. In the outpatient setting, patients receive infusions via CNS catheter into either the tumor cavity or the ventricular system. The initial three patients experienced no dose-limiting toxicity and exhibited clinical, as well as correlative laboratory, evidence of local CNS immune activation, including high concentrations of CXCL10 and CCL2 in the cerebrospinal fluid. This interim report supports the feasibility of generating HER2-specific CAR T cells for repeated dosing regimens and suggests that their repeated intra-CNS delivery might be well tolerated and activate a localized immune response in pediatric and young adult patients. In an interim analysis of a phase 1 trial, repeated intracranial infusions of HER2-specific CAR T cells were well tolerated with no observed dose-limiting toxicities in three young adult patients with CNS tumors., Author(s): Nicholas A. Vitanza [sup.1] [sup.2] , Adam J. Johnson [sup.1] [sup.3] , Ashley L. Wilson [sup.1] [sup.3] , Christopher Brown [sup.3] [sup.4] , Jason K. Yokoyama [sup.1] [sup.3] , [...]

The impact of inherited genetic variation on gene expression in humans is well-established. The majority of known expression quantitative trait loci (eQTLs) impact expression of local genes (cis-eQTLs). More research is needed to identify effects of genetic variation on distant genes (trans-eQTLs) and understand their biological mechanisms. One common trans-eQTLs mechanism is "mediation" by a local (cis) transcript. Thus, mediation analysis can be applied to genome-wide SNP and expression data in order to identify transcripts that are "cis-mediators" of trans-eQTLs, including those "cis-hubs" involved in regulation of many trans-genes. Identifying such mediators helps us understand regulatory networks and suggests biological mechanisms underlying trans-eQTLs, both of which are relevant for understanding susceptibility to complex diseases. The multitissue expression data from the Genotype-Tissue Expression (GTEx) program provides a unique opportunity to study cis-mediation across human tissue types. However, the presence of complex hidden confounding effects in biological systems can make mediation analyses challenging and prone to confounding bias, particularly when conducted among diverse samples. To address this problem, we propose a new method: Genomic Mediation analysis with Adaptive Confounding adjustment (GMAC). It enables the search of a very large pool of variables, and adaptively selects potential confounding variables for each mediation test. Analyses of simulated data and GTEx data demonstrate that the adaptive selection of confounders by GMAC improves the power and precision of mediation analysis. Application of GMAC to GTEx data provides new insights into the observed patterns of cis-hubs and trans-eQTL regulation across tissue types.

Gene co-expression networks capture biologically important patterns in gene expression data, enabling functional analyses of genes, discovery of biomarkers, and interpretation of genetic variants. Most network analyses to date have been limited to assessing correlation between total gene expression levels in a single tissue or small sets of tissues. Here, we built networks that additionally capture the regulation of relative isoform abundance and splicing, along with tissue-specific connections unique to each of a diverse set of tissues. We used the Genotype-Tissue Expression (GTEx) project v6 RNA sequencing data across 50 tissues and 449 individuals. First, we developed a framework called Transcriptome-Wide Networks (TWNs) for combining total expression and relative isoform levels into a single sparse network, capturing the interplay between the regulation of splicing and transcription. We built TWNs for 16 tissues and found that hubs in these networks were strongly enriched for splicing and RNA binding genes, demonstrating their utility in unraveling regulation of splicing in the human transcriptome. Next, we used a Bayesian biclustering model that identifies network edges unique to a single tissue to reconstruct Tissue-Specific Networks (TSNs) for 26 distinct tissues and 10 groups of related tissues. Finally, we found genetic variants associated with pairs of adjacent nodes in our networks, supporting the estimated network structures and identifying 20 genetic variants with distant regulatory impact on transcription and splicing. Our networks provide an improved understanding of the complex relationships of the human transcriptome across tissues.

Adenosine-to-inosine (A-to-I) RNA editing is a conserved post-transcriptional mechanism mediated by ADAR enzymes that diversifies the transcriptome by altering selected nucleotides in RNA molecules. Although many editing sites have recently been discovered, the extent to which most sites are edited and how the editing is regulated in different biological contexts are not fully understood. Here we report dynamic spatiotemporal patterns and new regulators of RNA editing, discovered through an extensive profiling of A-to-I RNA editing in 8,551 human samples (representing 53 body sites from 552 individuals) from the Genotype-Tissue Expression (GTEx) project and in hundreds of other primate and mouse samples. We show that editing levels in non-repetitive coding regions vary more between tissues than editing levels in repetitive regions. Globally, ADAR1 is the primary editor of repetitive sites and ADAR2 is the primary editor of non-repetitive coding sites, whereas the catalytically inactive ADAR3 predominantly acts as an inhibitor of editing. Cross-species analysis of RNA editing in several tissues revealed that species, rather than tissue type, is the primary determinant of editing levels, suggesting stronger cis-directed regulation of RNA editing for most sites, although the small set of conserved coding sites is under stronger trans-regulation. In addition, we curated an extensive set of ADAR1 and ADAR2 targets and showed that many editing sites display distinct tissue-specific regulation by the ADAR enzymes in vivo. Further analysis of the GTEx data revealed several potential regulators of editing, such as AIMP2, which reduces editing in muscles by enhancing the degradation of the ADAR proteins. Collectively, our work provides insights into the complex cis- and trans-regulation of A-to-I editing.

The initial microbiome impacts the health and future development of premature infants. Methodological limitations have led to gaps in our understanding of the habitat range and subpopulation complexity of founding strains, as well as how different body sites support microbial growth. Here, we used metagenomics to reconstruct genomes of strains that colonized the skin, mouth, and gut of two hospitalized premature infants during the first month of life. Seven bacterial populations, considered to be identical given whole-genome average nucleotide identity of >99.9%, colonized multiple body sites, yet none were shared between infants. Gut-associated Citrobacter koseri genomes harbored 47 polymorphic sites that we used to define 10 subpopulations, one of which appeared in the gut after 1 wk but did not spread to other body sites. Differential genome coverage was used to measure bacterial population replication rates in situ. In all cases where the same bacterial population was detected in multiple body sites, replication rates were faster in mouth and skin compared to the gut. The ability of identical strains to colonize multiple body sites underscores the habit flexibility of initial colonists, whereas differences in microbial replication rates between body sites suggest differences in host control and/or resource availability. Population genomic analyses revealed microdiversity within bacterial populations, implying initial inoculation by multiple individual cells with distinct genotypes. Overall, however, the overlap of strains across body sites implies that the premature infant microbiome can exhibit very low microbial diversity.

The Candidate Phyla Radiation (CPR) is a large group of bacteria, the scale of which approaches that of all other bacteria. CPR organisms are inferred to depend on other community members for many basic cellular building blocks and all appear to be obligate anaerobes. To date, there has been no evidence for any significant respiratory capacity in an organism from this radiation. Here we report a curated draft genome for 'Candidatus Parcunitrobacter nitroensis' a member of the Parcubacteria (OD1) superphylum of the CPR. The genome encodes versatile energy pathways, including fermentative and respiratory capacities, nitrogen and fatty acid metabolism, as well as the first complete electron transport chain described for a member of the CPR. The sequences of all of these enzymes are highly divergent from sequences found in other organisms, suggesting that these capacities were not recently acquired from non-CPR organisms. Although the wide respiration-based repertoire points to a different lifestyle compared to other CPR bacteria, we predict similar obligate dependence on other organisms or the microbial community. The results substantially expand the known metabolic potential of CPR bacteria, although sequence comparisons indicate that these capacities are very rare in members of this radiation.

The potentially critical stage of initial gut colonization in premature infants occurs in the hospital environment, where infants are exposed to a variety of hospital-associated bacteria. Because few studies of microbial communities are strain-resolved, we know little about the extent to which specific strains persist in the hospital environment and disperse among infants. To study this, we compared 304 near-complete genomes reconstructed from fecal samples of 21 infants hospitalized in the same intensive care unit in two cohorts, over 3 years apart. The genomes represent 159 distinct bacterial strains, only 14 of which occurred in multiple infants. Enterococcus faecalis and Staphylococcus epidermidis, common infant gut colonists, exhibit diversity comparable to that of reference strains, inline with introduction of strains from infant-specific sources rather than a hospital strain pool. Unlike other infants, a pair of sibling infants shared multiple strains, even after extensive antibiotic administration, suggesting overlapping strain-sources and/or genetic selection drive microbiota similarities. Interestingly, however, five strains were detected in infants hospitalized three years apart. Three of these were also detected in multiple infants in the same year. This finding of a few widely dispersed and persistent bacterial colonizers despite overall low potential for strain dispersal among infants has implications for understanding and directing healthy colonization.

Metagenomic studies recently uncovered form II/III RubisCO genes, originally thought to only occur in archaea, from uncultivated bacteria of the candidate phyla radiation (CPR). There are no isolated CPR bacteria and these organisms are predicted to have limited metabolic capacities. Here we expand the known diversity of RubisCO from CPR lineages. We report a form of RubisCO, distantly similar to the archaeal form III RubisCO, in some CPR bacteria from the Parcubacteria (OD1), WS6 and Microgenomates (OP11) phyla. In addition, we significantly expand the Peregrinibacteria (PER) II/III RubisCO diversity and report the first II/III RubisCO sequences from the Microgenomates and WS6 phyla. To provide a metabolic context for these RubisCOs, we reconstructed near-complete (>93%) PER genomes and the first closed genome for a WS6 bacterium, for which we propose the phylum name Dojkabacteria. Genomic and bioinformatic analyses suggest that the CPR RubisCOs function in a nucleoside pathway similar to that proposed in Archaea. Detection of form II/III RubisCO and nucleoside metabolism gene transcripts from a PER supports the operation of this pathway in situ. We demonstrate that the PER form II/III RubisCO is catalytically active, fixing CO2 to physiologically complement phototrophic growth in a bacterial photoautotrophic RubisCO deletion strain. We propose that the identification of these RubisCOs across a radiation of obligately fermentative, small-celled organisms hints at a widespread, simple metabolic platform in which ribose may be a prominent currency.

Oxidative damage to mitochondria (MT) is a major mechanism for aging and neurodegeneration. We have developed a novel synthetic antioxidant, XJB-5-131, which directly targets MT, the primary site and primary target of oxidative damage. XJB-5-131 prevents the onset of motor decline in an HdhQ(150/150) mouse model for Huntington's disease (HD) if treatment starts early. Here, we report that XJB-5-131 attenuates or reverses disease progression if treatment occurs after disease onset. In animals with well-developed pathology, XJB-5-131 promotes weight gain, prevents neuronal death, reduces oxidative damage in neurons, suppresses the decline of motor performance or improves it, and reduces a graying phenotype in treated HdhQ(150/150) animals relative to matched littermate controls. XJB-5-131 holds promise as a clinical candidate for the treatment of HD.

Nitrogen, sulfur and carbon fluxes in the terrestrial subsurface are determined by the intersecting activities of microbial community members, yet the organisms responsible are largely unknown. Metagenomic methods can identify organisms and functions, but genome recovery is often precluded by data complexity. To address this limitation, we developed subsampling assembly methods to re-construct high-quality draft genomes from complex samples. We applied these methods to evaluate the interlinked roles of the most abundant organisms in biogeochemical cycling in the aquifer sediment. Community proteomics confirmed these activities. The eight most abundant organisms belong to novel lineages, and two represent phyla with no previously sequenced genome. Four organisms are predicted to fix carbon via the Calvin-Benson-Bassham, Wood-Ljungdahl or 3-hydroxyproprionate/4-hydroxybutarate pathways. The profiled organisms are involved in the network of denitrification, dissimilatory nitrate reduction to ammonia, ammonia oxidation and sulfate reduction/oxidation, and require substrates supplied by other community members. An ammonium-oxidizing Thaumarchaeote is the most abundant community member, despite low ammonium concentrations in the groundwater. This organism likely benefits from two other relatively abundant organisms capable of producing ammonium from nitrate, which is abundant in the groundwater. Overall, dominant members of the microbial community are interconnected through exchange of geochemical resources.

Five closely related populations of bacteria from the Candidate Phylum (CP) Peregrinibacteria, part of the bacterial Candidate Phyla Radiation (CPR), were sampled from filtered groundwater obtained from an aquifer adjacent to the Colorado River near the town of Rifle, CO, USA. Here, we present the first complete genome sequences for organisms from this phylum. These bacteria have small genomes and, unlike most organisms from other lineages in the CPR, have the capacity for nucleotide synthesis. They invest significantly in biosynthesis of cell wall and cell envelope components, including peptidoglycan, isoprenoids via the mevalonate pathway, and a variety of amino sugars including perosamine and rhamnose. The genomes encode an intriguing set of large extracellular proteins, some of which are very cysteine-rich and may function in attachment, possibly to other cells. Strain variation in these proteins is an important source of genotypic variety. Overall, the cell envelope features, combined with the lack of biosynthesis capacities for many required cofactors, fatty acids, and most amino acids point to a symbiotic lifestyle. Phylogenetic analyses indicate that these bacteria likely represent a new class within the Peregrinibacteria phylum, although they ultimately may be recognized as members of a separate phylum. We propose the provisional taxonomic assignment as 'Candidatus Peribacter riflensis', Genus Peribacter, Family Peribacteraceae, Order Peribacterales, Class Peribacteria in the phylum Peregrinibacteria.

Current understanding of microorganism-virus interactions, which shape the evolution and functioning of Earth's ecosystems, is based primarily on cultivated organisms. Here we investigate thousands of viral and microbial genomes recovered using a cultivation-independent approach to study the frequency, variety and taxonomic distribution of viral defence mechanisms. CRISPR-Cas systems that confer microorganisms with immunity to viruses are present in only 10% of 1,724 sampled microorganisms, compared with previous reports of 40% occurrence in bacteria and 81% in archaea. We attribute this large difference to the lack of CRISPR-Cas systems across major bacterial lineages that have no cultivated representatives. We correlate absence of CRISPR-Cas with lack of nucleotide biosynthesis capacity and a symbiotic lifestyle. Restriction systems are well represented in these lineages and might provide both non-specific viral defence and access to nucleotides.

The tree of life is one of the most important organizing principles in biology(1). Gene surveys suggest the existence of an enormous number of branches(2), but even an approximation of the full scale of the tree has remained elusive. Recent depictions of the tree of life have focused either on the nature of deep evolutionary relationships(3-5) or on the known, well-classified diversity of life with an emphasis on eukaryotes(6). These approaches overlook the dramatic change in our understanding of life's diversity resulting from genomic sampling of previously unexamined environments. New methods to generate genome sequences illuminate the identity of organisms and their metabolic capacities, placing them in community and ecosystem contexts(7,8). Here, we use new genomic data from over 1,000 uncultivated and little known organisms, together with published sequences, to infer a dramatically expanded version of the tree of life, with Bacteria, Archaea and Eukarya included. The depiction is both a global overview and a snapshot of the diversity within each major lineage. The results reveal the dominance of bacterial diversification and underline the importance of organisms lacking isolated representatives, with substantial evolution concentrated in a major radiation of such organisms. This tree highlights major lineages currently underrepresented in biogeochemical models and identifies radiations that are probably important for future evolutionary analyses.

The subterranean world hosts up to one-fifth of all biomass, including microbial communities that drive transformations central to Earth's biogeochemical cycles. However, little is known about how complex microbial communities in such environments are structured, and how inter-organism interactions shape ecosystem function. Here we apply terabase-scale cultivation-independent metagenomics to aquifer sediments and groundwater, and reconstruct 2,540 draft-quality, near-complete and complete strain-resolved genomes that represent the majority of known bacterial phyla as well as 47 newly discovered phylum-level lineages. Metabolic analyses spanning this vast phylogenetic diversity and representing up to 36% of organisms detected in the system are used to document the distribution of pathways in coexisting organisms. Consistent with prior findings indicating metabolic handoffs in simple consortia, we find that few organisms within the community can conduct multiple sequential redox transformations. As environmental conditions change, different assemblages of organisms are selected for, altering linkages among the major biogeochemical cycles.

Chromatin comprises nucleosomes as well as nonnucleosomal histone-DNA particles. Prenucleosomes are rapidly formed histone-DNA particles that can be converted into canonical nucleosomes by a motor protein such as ACF. Here we show that the prenucleosome is a stable conformational isomer of the nucleosome. It consists of a histone octamer associated with ∼ 80 base pair (bp) of DNA, which is located at a position that corresponds to the central 80 bp of a nucleosome core particle. Monomeric prenucleosomes with free flanking DNA do not spontaneously fold into nucleosomes but can be converted into canonical nucleosomes by an ATP-driven motor protein such as ACF or Chd1. In addition, histone H3K56, which is located at the DNA entry and exit points of a canonical nucleosome, is specifically acetylated by p300 in prenucleosomes relative to nucleosomes. Prenucleosomes assembled in vitro exhibit properties that are strikingly similar to those of nonnucleosomal histone-DNA particles in the upstream region of active promoters in vivo. These findings suggest that the prenucleosome, the only known stable conformational isomer of the nucleosome, is related to nonnucleosomal histone-DNA species in the cell.

Little is known about the biogeography or stability of sediment-associated microbial community membership because these environments are biologically complex and generally difficult to sample. High-throughput-sequencing methods provide new opportunities to simultaneously genomically sample and track microbial community members across a large number of sampling sites or times, with higher taxonomic resolution than is associated with 16 S ribosomal RNA gene surveys, and without the disadvantages of primer bias and gene copy number uncertainty. We characterized a sediment community at 5 m depth in an aquifer adjacent to the Colorado River and tracked its most abundant 133 organisms across 36 different sediment and groundwater samples. We sampled sites separated by centimeters, meters and tens of meters, collected on seven occasions over 6 years. Analysis of 1.4 terabase pairs of DNA sequence showed that these 133 organisms were more consistently detected in saturated sediments than in samples from the vadose zone, from distant locations or from groundwater filtrates. Abundance profiles across aquifer locations and from different sampling times identified organism cohorts that comprised subsets of the 133 organisms that were consistently associated. The data suggest that cohorts are partly selected for by shared environmental adaptation.

A prominent feature of the bacterial domain is a radiation of major lineages that are defined as candidate phyla because they lack isolated representatives. Bacteria from these phyla occur in diverse environments and are thought to mediate carbon and hydrogen cycles. Genomic analyses of a few representatives suggested that metabolic limitations have prevented their cultivation. Here we reconstructed 8 complete and 789 draft genomes from bacteria representing >35 phyla and documented features that consistently distinguish these organisms from other bacteria. We infer that this group, which may comprise >15% of the bacterial domain, has shared evolutionary history, and describe it as the candidate phyla radiation (CPR). All CPR genomes are small and most lack numerous biosynthetic pathways. Owing to divergent 16S ribosomal RNA (rRNA) gene sequences, 50-100% of organisms sampled from specific phyla would evade detection in typical cultivation-independent surveys. CPR organisms often have self-splicing introns and proteins encoded within their rRNA genes, a feature rarely reported in bacteria. Furthermore, they have unusual ribosome compositions. All are missing a ribosomal protein often absent in symbionts, and specific lineages are missing ribosomal proteins and biogenesis factors considered universal in bacteria. This implies different ribosome structures and biogenesis mechanisms, and underlines unusual biology across a large part of the bacterial domain.

BackgroundArchaea represent a significant fraction of Earth's biodiversity, yet they remain much less well understood than Bacteria. Gene surveys, a few metagenomic studies, and some single-cell sequencing projects have revealed numerous little-studied archaeal phyla. Certain lineages appear to branch deeply and may be part of a major phylum radiation. The structure of this radiation and the physiology of the organisms remain almost unknown.ResultsWe used genome-resolved metagenomic analyses to investigate the diversity, genomes sizes, metabolic capacities, and potential roles of Archaea in terrestrial subsurface biogeochemical cycles. We sequenced DNA from complex sediment and planktonic consortia from an aquifer adjacent to the Colorado River (USA) and reconstructed the first complete genomes for Archaea using cultivation-independent methods. To provide taxonomic context, we analyzed an additional 151 newly sampled archaeal sequences. We resolved two new phyla within a major, apparently deep-branching group of phyla (a superphylum). The organisms have small genomes, and metabolic predictions indicate that their primary contributions to Earth's biogeochemical cycles involve carbon and hydrogen metabolism, probably associated with symbiotic and/or fermentation-based lifestyles.ConclusionsThe results dramatically expand genomic sampling of the domain Archaea and clarify taxonomic designations within a major superphylum. This study, in combination with recently published work on bacterial phyla lacking cultivated representatives, reveals a fascinating phenomenon of major radiations of organisms with small genomes, novel proteome composition, and strong interdependence in both domains.

Transcriptional activation of eukaryotic genes is accompanied, in general, by a change in the sensitivity of promoter chromatin to endonucleases. The structural basis of this alteration has remained elusive for decades; but the change has been viewed as a transformation of one structure into another, from "closed" to "open" chromatin. In contradistinction to this static and deterministic view of the problem, a dynamical and probabilistic theory of promoter chromatin has emerged as its solution. This theory, which we review here, explains observed variation in promoter chromatin structure at the level of single gene molecules and provides a molecular basis for random bursting in transcription-the conjecture that promoters stochastically transition between transcriptionally conducive and inconducive states. The mechanism of transcriptional regulation may be understood only in probabilistic terms.

BackgroundBacteria of the genus Sulfobacillus are found worldwide as members of microbial communities that accelerate sulfide mineral dissolution in acid mine drainage environments (AMD), acid-rock drainage environments (ARD), as well as in industrial bioleaching operations. Despite their frequent identification in these environments, their role in biogeochemical cycling is poorly understood.ResultsHere we report draft genomes of five species of the Sulfobacillus genus (AMDSBA1-5) reconstructed by cultivation-independent sequencing of biofilms sampled from the Richmond Mine (Iron Mountain, CA). Three of these species (AMDSBA2, AMDSBA3, and AMDSBA4) have no cultured representatives while AMDSBA1 is a strain of S. benefaciens, and AMDSBA5 a strain of S. thermosulfidooxidans. We analyzed the diversity of energy conservation and central carbon metabolisms for these genomes and previously published Sulfobacillus genomes. Pathways of sulfur oxidation vary considerably across the genus, including the number and type of subunits of putative heterodisulfide reductase complexes likely involved in sulfur oxidation. The number and type of nickel-iron hydrogenase proteins varied across the genus, as does the presence of different central carbon pathways. Only the AMDSBA3 genome encodes a dissimilatory nitrate reducatase and only the AMDSBA5 and S. thermosulfidooxidans genomes encode assimilatory nitrate reductases. Within the genus, AMDSBA4 is unusual in that its electron transport chain includes a cytochrome bc type complex, a unique cytochrome c oxidase, and two distinct succinate dehydrogenase complexes.ConclusionsOverall, the results significantly expand our understanding of carbon, sulfur, nitrogen, and hydrogen metabolism within the Sulfobacillus genus.

The number of mRNA and protein molecules expressed from a single gene molecule fluctuates over time. These fluctuations have been attributed, in part, to the random transitioning of promoters between transcriptionally active and inactive states, causing transcription to occur in bursts. However, the molecular basis of transcriptional bursting remains poorly understood. By electron microscopy of single PHO5 gene molecules from yeast, we show that the "activated" promoter assumes alternative nucleosome configurations at steady state, including the maximally repressive, fully nucleosomal, and the maximally non-repressive, nucleosome-free, configuration. We demonstrate that the observed probabilities of promoter nucleosome configurations are obtained from a simple, intrinsically stochastic process of nucleosome assembly, disassembly, and position-specific sliding; and we show that gene expression and promoter nucleosome configuration can be mechanistically coupled, relating promoter nucleosome dynamics and gene expression fluctuations. Together, our findings suggest a structural basis for transcriptional bursting, and offer new insights into the mechanism of transcriptional regulation and the kinetics of promoter nucleosome transitions.

The ability to follow enzyme activity in a cellular context represents a challenging technological frontier that impacts fields ranging from disease pathogenesis to epigenetics. Activity-based probes (ABPs) label the active form of an enzyme via covalent modification of catalytic residues. Here we present an analysis of parameters influencing potency of peptide phosphonate ABPs for trypsin-fold S1A proteases, an abundant and important class of enzymes with similar substrate specificities. We find that peptide length and stability influence potency more than sequence composition and present structural evidence that steric interactions at the prime-side of the substrate-binding cleft affect potency in a protease-dependent manner. We introduce guidelines for the design of peptide phosphonate ABPs and demonstrate their utility in a live-cell labeling application that specifically targets active S1A proteases at the cell surface of cancer cells.

Leg autotomy in spiders is a relatively common occurrence, usually resulting from agonistic interactions with predators or conspecifics. While autotomy has immediate benefits, due to enhanced survival probability, it also potentially decreases future fitness. One possible cost of losing a leg is a reduction in burst running speed, which may affect prey capture and predator avoidance behaviors in wandering spiders. We examined sprint speed in intact and autotomized males and females of the wolf spider Pirata sedentarius from two stream banks in New York in an effort to determine if the sexes differed in either sprint speed or the potential cost of leg loss. Autotomy was fairly common in the field, with 18-22% of spiders missing at least one leg at our two sites. Males and females did not differ in frequency of leg loss. Females were larger than males (both in terms of mass and structural size) and ran significantly faster both before and after autotomy. Sprint speed was uncorrelated with body size (mass, carapace width or leg length) for males, while for females the only significant relationship was that between mass and pre-autotomy speed. Both sexes ran significantly faster when they had all legs compared to their speed after autotomy. We found no difference in speed between spiders which had lost a leg in the field and spiders from which we removed a leg in the laboratory or between intact spiders and spiders collected in the field with a leg missing. Overall, our results for P. sedentarius indicate that: (1) females are faster than males, (2) autotomy significantly reduces sprint speed in the lab for both sexes and (3) reductions in male sprint speed in the lab may result from a combination of autotomy and aging effects, while decreases in female speed are likely due primarily to autotomy.

Tbx1 has been implicated as a candidate gene responsible for defective pharyngeal arch remodeling in DiGeorge/Velocardiofacial syndrome. Tbx[1.sup.+/-] mice mimic aspects of the DiGeorge phenotype with variable penetrance, and null mice display severe pharyngeal hypoplasia. Here, we identify enhancer elements in the Tbx1 gene that are conserved through evolution and mediate tissue-specific expression. We describe the generation of transgenic mice that utilize these enhancer elements to direct Cre recombinase expression in endogenous Tbx1 expression domains. We use these Tbx1-Cre mice to fate map Tbx1-expressing precursors and identify broad regions of mesoderm, including early cardiac mesoderm, which are derived from Tbx1-expressing cells. We test the hypothesis that fibroblast growth factor 8 (Fgf8) functions downstream of Tbx1 by performing tissue-specific inactivation of Fgf8 using Tbx1-Cre mice. Resulting newborn mice display DiGeorge-like congenital cardiovascular defects that involve the outflow tract of the heart. Vascular smooth muscle differentiation in the great vessels is disrupted. This data is consistent with a model in which Tbx1 induces Fgf8 expression in the pharyngeal endoderm, which is subsequently required for normal cardiovascular morphogenesis and smooth muscle differentiation in the aorta and pulmonary artery. Keywords: Fgf8; Tbx1-Cre mice; DiGeorge syndrome

We examined plant climbing by the common striped scorpion Centruroides vittatus to determine which of two hypotheses (the predation avoidance hypothesis or the increased prey availability hypothesis) best explained this behavior. In the field we observed nocturnal scorpion activity for 16 mo during 1992-1993 to quantify climbing behavior and collected data on potential prey abundance on the ground and in vegetation for 5 mo. We also performed a laboratory experiment examining the effects of hunger level on scorpion climbing and activity. Individuals found up in vegetation accounted for 19.3% and 25.2% of scorpions active in 1992 and 1993, respectively. Juveniles were significantly more likely to be found in vegetation than were adults in both years, although juveniles and adults did not differ in the proportion found carrying prey in vegetation. We found significant seasonal variation in prey abundance, with prey density being greatest in September. Prey density was also significantly greater on the ground than in vegetation when all trapped invertebrates were included; however, when we excluded collembolans because of their small size, this difference was no longer significant. Laboratory results indicated that there were no within-treatment differences among age classes/sexes in any behavior (climbing frequency, activity level or maximum height climbed). When hungry, males (but not females or juveniles) climbed higher and juveniles (but not adults) were more active. Hunger level had no effect on climbing frequency for any age class/sex. In combination, the field and laboratory data are most consistent with the predation avoidance hypothesis as the main reason for plant climbing by scorpions.

The synthesis of a novel rigid noncovalent electron transfer model system that juxtaposes chlorin as donor and naphthalene diimide as acceptor was described. Steady-state fluorescence, transient absorption and time-resolved luminescence methods were used to study the electron-transfer ensembles. Experimental results showed that the system is effective in allowing photoinduced electron transfer, although it failed to establish a long-lived charge separated state.

The interaction between the soluble form of the granulocyte-macrophage colony stimulating factor receptor and the beta subunit of the receptor when both are expressed in a cell culture is investigated to determine the mechanism of retention of the soluble receptor on the cell membrane. Results reveal that the unique 16-amino acid sequence carboxy terminal of the soluble receptor especially the cysteine at position 323 is responsible for anchoring the receptor to the beta subunit.

A series of pi-electron-rich macrocyclic polyethers incorporating the dioxybenzene (hydroquinone) and/or dioxynaphthalene units were synthesized in good yields by simple two-step processes. These macrocycles can bind bipyridinium-based guests due to a series of cooperative noncovalent bonding interactions. The efficiencies of these self-assembly procedures were observed to depend on the stereoelectronic characteristics of the pi-electron-rich macrocycles.

Epithelial-mesenchymal transformation is a critical event in the development of many organ systems including the heart. Descriptive studies have implicated a number of factors in mediating this transformation, including transforming growth factor [Beta] (TGF[Beta]). We now report that disruption of a TGF[Beta] signal transduction complex by antibodies directed against the Type II TGF[Beta] receptor blocks both the endocardial cell activation and subsequent migration that constitute transformation in the chick atrioventricular (AV) cushion. The Type II receptor was localized to both endothelial and endocardial cells of the chick embryo. Incubation of AV cushion explants from Stage 14, 16, and 18 embryos with antibody resulted in a blockade of AV endocardial cell transformation by greater than 50% as measured by mesenchyme formation. Similarly, the appearance of procollagen Type I, a marker of endocardial cell transformation, was blocked. In addition, within 2 hr after the incubation of activated Stage 18 explants with Type II antibody the rate of migration of transformed cells was decreased by 50%. These data suggest that TGF[Beta] acts directly on AV cushion endocardial cells to stimulate epithelial-mesenchymal transformation and that TGF[Beta] mediates at least two distinct components of AV cushion transformation, activation and migration.

A study was done on the role of carbohydrate modification in the biosynthesis and secretory processing of human granulocyte macrophage-colony stimulating factor (GM-CSF). It was shown that there is no need for either N-linked or O-linked carbohydrate modification in the biosynthesis of GM-CSF. However, the findings confirm that carbohydrate modification is indeed necessary in the secretion of growth factor in human dermal fibroblasts and umbilical vein endothelial cells.

A study was conducted to examine the correlation between the mammalian nuclear pore and the human genome by generating high-resolution, chromosome-wide binding maps of human nucleoporin 93 (Nup93) in the presence and absence of a potent histone deacetylase inhibitor (HDACI). Results showed that inhibition of HDACs lead to significant changes in genomic organization, recruiting regions of transcriptional regulation to mammalian nuclear pore complexes.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2004.04.002 Byline: Christopher B. Brown, Jennifer M. Wenning, Min Min Lu, Douglas J. Epstein, Erik N. Meyers, Jonathan A. Epstein Article History: Received 7 April 2004; Accepted 7 April 2004

Transcriptional regulation implicates the association of genes with the nuclear pore complex (NPC) and nuclear transport factors. The association of components including karyopherins, nuclearporins, and Ran guanine-nucleotide exchange factor (RanGEF) with the Saccharomyces cerevisiae genome of the nuclear transport machinery is examined.

The results of a particular experiment were robust to the particular measure of offspring size and whether offspring data were adjusted for female size variation. The results therefore provide strong evidence in support of the van Noordwijk and de Jong model.

Epithelial-mesenchymal transformation in the atrioventricular (AV) cushion of the tubular heart is a critical step in the formation of the valves and membranous septa. Transforming growth factor [Beta] (TGF [Beta]) ligands are a primary signal of this transformation. To investigate the expression and function of specific Type I TGF [Beta] receptors during AV cushion transformation, we cloned and characterized the chicken homologues of two mammalian activin receptor-like kinases (ALK), ALK2 and ALK5, and generated specific, polyclonal antibodies against the extracellular binding domains of each. Both the chicken ALK2 (ChALK2) and the chicken ALK5 (ChALK5) cDNAs encode proteins that bind TGF [Beta] 1 in the presence of the Type II TGF [Beta] receptor. However, as expected, only ChALK5 stimulated the TGF [Beta]-responsive PAI-1 promoter. These data establish that ChALK2 and ChALK5 are the chicken homologues of the mammalian receptors ALK2 and ALK5. Both ChALK2 and ChALK5 are expressed by AV endocardial cells. AV cushion explants harvested from stage 13-18 embryos were incubated with antisera to ChALK2 or ChALK5. Anti-ChALK2 antisera inhibited mesenchyme formation by 34-50% while neutralizing anti-ChALK5 antisera were without effect. These data demonstrate that ChALK2 can mediate transformation in the AV cushion. Key Words: development; chick; heart; epithelial-mesenchymal transformation; transforming growth factor [Beta] receptors.

An approach for the synthesis of a new axially-chiral tetracationic cyclophanes capable of exhibiting enantiomeric differentiation toward various racemic compounds such as aromatic amino acids phenylalanine, tyrosine, and tryptophan is discussed. To achieve optically active and symmetrical receptors, chiral 3,3'linked-2,2'-dihydroxy-1,1'-binaphthyl spacers were introduced into the tetracationic cyclophane, creating a distance of 8 amstrongs between the two bipyridinium units.