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Altering cellular responses to double-strand breaks in DNA could rebalance CRISPRediting outcomes. Here, the authors use a pooled CRISPR screen to identify inhibition of CDC7 as a strategy to improve HDR outcomes.

Background Immune checkpoint inhibitors (ICI) can achieve durable responses in a subset of patients with locally advanced or metastatic urothelial carcinoma (aUC). The use of tumor genomic profiling in clinical practice may help suggest biomarkers to identify patients most likely to benefit from ICI.Methods We undertook a retrospective analysis of patients treated with an ICI for aUC at a large academic medical center. Patient clinical and histopathological variables were collected. Responses to treatment were assessed for all patients with at least one post-baseline scan or clear evidence of clinical progression following treatment start. Genomic profiling information was also collected for patients when available. Associations between patient clinical/genomic characteristics and objective response were assessed by logistic regression; associations between the characteristics and progression-free survival (PFS) and overall survival (OS) were examined by Cox regression. Multivariable analyses were performed to identify independent prognostic factors.Results We identified 119 aUC patients treated with an ICI from December 2014 to January 2020. Genomic profiling was available for 78 patients. Overall response rate to ICI was 29%, and median OS (mOS) was 13.4 months. Favorable performance status at the start of therapy was associated with improved OS (HR 0.46, p=0.025) after accounting for other covariates. Similarly, the presence of a TERT promoter mutation was an independent predictor of improved PFS (HR 0.38, p=0.012) and OS (HR 0.32, p=0.037) among patients who had genomic profiling available. Patients with both a favorable performance status and a TERT promoter mutation had a particularly good prognosis with mOS of 21.1 months as compared with 7.5 months in all other patients (p=0.03).Conclusions The presence of a TERT promoter mutation was an independent predictor of improved OS in a cohort of aUC patients treated with an ICI who had genomic data available. Most of the clinical and laboratory variables previously shown to be prognostic in aUC patients treated with chemotherapy did not have prognostic value among patients treated with an ICI. Genomic profiling may provide important prognostic information and affect clinical decision making in this patient population. Validation of these findings in prospective patient cohorts is needed.

Vitamin D insufficiency is a worldwide epidemic affecting billions of individuals, including pregnant women and children. Despite its high incidence, the impact of active vitamin D3 (1,25(OH)D3) on embryonic development beyond osteo-regulation remains largely undefined. Here, we demonstrate that 1,25(OH)D3 availability modulates zebrafish hematopoietic stem and progenitor cell (HSPC) production. Loss of Cyp27b1-mediated biosynthesis or vitamin D receptor (VDR) function by gene knockdown resulted in significantly reduced runx1 expression and Flk1+cMyb+ HSPC numbers. Selective modulation in vivo and in vitro in zebrafish indicated that vitamin D3 acts directly on HSPCs, independent of calcium regulation, to increase proliferation. Notably, ex vivo treatment of human HSPCs with 1,25(OH)D3 also enhanced hematopoietic colony numbers, illustrating conservation across species. Finally, gene expression and epistasis analysis indicated that CXCL8 (IL-8) was a functional target of vitamin D3-mediated HSPC regulation. Together, these findings highlight the relevance of developmental 1,25(OH)D3 availability for definitive hematopoiesis and suggest potential therapeutic utility in HSPC expansion.

Sponges (Phylum Porifera) are aquatic sessile metazoans found worldwide in marine and freshwater environments. They are significant in the animal tree of life as one of the earliest-branching metazoan lineages and as filter feeders play crucial ecological roles, particularly in coral reefs, but are susceptible to the effects of climate change. In the face of the current biodiversity crisis, genomic data is crucial for species conservation efforts and predicting their evolutionary potential in response to environmental changes. However, there is a limited availability of culturable sponge species with annotated high-quality genomes to further comprehensive insights into animal evolution, function, and their response to the ongoing global change. Despite the publication of a few high-quality annotated sponge genomes, there remains a gap in resources for culturable sponge species. To address this gap, we provide high quality draft genomes of the two congeneric aquarium species Tethya wilhelma and Tethya minuta , small ball-shaped demosponges that are easily maintained long-term in ex situ culture. As such, they offer promising opportunities as laboratory models to contribute to advancing our understanding of sponge biology and provide valuable resources for studying animal evolution, function, and responses to environmental challenges., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 Wörheide G et al.)

Well-annotated and contiguous genomes are an indispensable resource for understanding the evolution, development, and metabolic capacities of organisms. Sponges, an ecologically important non-bilaterian group of primarily filter-feeding sessile aquatic organisms, are underrepresented with respect to available genomic resources. Here we provide a high-quality and well-annotated genome of Aphrocallistes vastus , a glass sponge (Porifera: Hexactinellida) that forms large reef structures off the coast of British Columbia (Canada). We show that its genome is approximately 80 Mb, small compared to most other metazoans, and contains nearly 2500 nested genes, more than other genomes. Hexactinellida is characterized by a unique skeletal architecture made of amorphous silicon dioxide (SiO 2 ), and we identified 419 differentially expressed genes between the osculum, i.e. the vertical growth zone of the sponge, and the main body. Among the upregulated ones, mineralization-related genes such as glassin, as well as collagens and actins, dominate the expression profile during growth. Silicateins, suggested being involved in silica mineralization, especially in demosponges, were not found at all in the A. vastus genome and suggests that the underlying mechanisms of SiO 2 deposition in the Silicea sensu stricto (Hexactinellida + Demospongiae) may not be homologous., Competing Interests: We declare we have no competing interests., (© 2023 The Authors.)

Objectives: These data were collected to generate a novel reference metagenome for the sponge Halichondria panicea and its microbiome for subsequent differential expression analyses., Data Description: These data include raw sequences from four separate sequencing runs of the metagenome of a single individual of Halichondria panicea-one Illumina MiSeq (2 × 300 bp, paired-end) run and three Oxford Nanopore Technologies (ONT) long-read sequencing runs, generating 53.8 and 7.42 Gbp respectively. Comparing assemblies of Illumina, ONT and an Illumina-ONT hybrid revealed the hybrid to be the 'best' assembly, comprising 163 Mbp in 63,555 scaffolds (N50: 3084). This assembly, however, was still highly fragmented and only contained 52% of core metazoan genes (with 77.9% partial genes), so it was also not complete. However, this sponge is an emerging model species for field and laboratory work, and there is considerable interest in genomic sequencing of this species. Although the resultant assemblies from the data presented here are suboptimal, this data note can inform future studies by providing an estimated genome size and coverage requirements for future sequencing, sharing additional data to potentially improve other suboptimal assemblies of this species, and outlining potential limitations and pitfalls of the combined Illumina and ONT approach to novel genome sequencing., (© 2022. The Author(s).)

The evolutionary origin of metazoan cell types such as neurons and muscles is not known. Using whole-body single-cell RNA sequencing in a sponge, an animal without nervous system and musculature, we identified 18 distinct cell types. These include nitric oxide–sensitive contractile pinacocytes, amoeboid phagocytes, and secretory neuroid cells that reside in close contact with digestive choanocytes that express scaffolding and receptor proteins. Visualizing neuroid cells by correlative x-ray and electron microscopy revealed secretory vesicles and cellular projections enwrapping choanocyte microvilli and cilia. Our data show a communication system that is organized around sponge digestive chambers, using conserved modules that became incorporated into the pre- and postsynapse in the nervous systems of other animals.

Here, we present a karyotype, a chromosome-scale genome assembly, and a genome annotation from the ctenophore Hormiphora californensis (Ctenophora: Cydippida: Pleurobrachiidae). The assembly spans 110 Mb in 44 scaffolds and 99.47% of the bases are contained in 13 scaffolds. Chromosome micrographs and Hi-C heatmaps support a karyotype of 13 diploid chromosomes. Hi-C data reveal three large heterozygous inversions on chromosome 1, and one heterozygous inversion shares the same gene order found in the genome of the ctenophore Pleurobrachia bachei. We find evidence that H. californensis and P. bachei share thirteen homologous chromosomes, and the same karyotype of 1n = 13. The manually curated PacBio Iso-Seq-based genome annotation reveals complex gene structures, including nested genes and trans-spliced leader sequences. This chromosome-scale assembly is a useful resource for ctenophore biology and will aid future studies of metazoan evolution and phylogenetics., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

Cobalt is one of the main components of metal hip prostheses and cobalt nanoparticles (CoNPs) produced from wear cause inflammation, bone lyses and cytotoxicity at high concentrations. Cobalt ions mimic hypoxia in the presence of normal oxygen levels, and activate hypoxic signalling by stabilising hypoxia inducible transcription factor 1α (HIF1α). This study aimed to assess in vitro the functional role of HIF1α in CoNP induced cellular cytotoxicity. HIF1α, lysosomal pH, tumour necrosis factor α and interleukin 1β expression were analysed in THP-1 macrophages treated with CoNP (0, 10 and 100 μg/mL). HIF1α knock out assays were performed using small interfering RNA to assess the role of HIF1α in CoNP-induced cytotoxicity. Increasing CoNP concentration increased lysosomal activity and acidity in THP-1 macrophages. Higher doses of CoNP significantly reduced cell viability, stimulated caspase 3 activity and apoptosis. Reducing HIF1αactivity increased the pro-inflammatory activity of tumour necrosis factorαand interleukin 1β,but had no significant impact on cellular cytotoxicity. This suggests that whilst CoNP promotes cytotoxicity and cellular inflammation, the apoptotic mechanism is not dependent on HIF1α.

Diverse physiological groups congregate into environmental corrosive biofilms, yet the interspecies interactions between these corrosive physiological groups are seldom examined. We, therefore, explored Fe 0 -dependent cross-group interactions between acetogens and methanogens from lake sediments. On Fe 0 , acetogens were more corrosive and metabolically active when decoupled from methanogens, whereas methanogens were more metabolically active when coupled with acetogens. This suggests an opportunistic (win-loss) interaction on Fe 0 between acetogens (loss) and methanogens (win). Clostridia and Methanobacterium were the major candidates doing acetogenesis and methanogenesis after four transfers (metagenome sequencing) and the only groups detected after 11 transfers (amplicon sequencing) on Fe 0 . Since abiotic H 2 failed to explain the high metabolic rates on Fe 0 , we examined whether cell exudates (spent media filtrate) promoted the H 2 -evolving reaction on Fe 0 above abiotic controls. Undeniably, spent media filtrate generated three- to four-fold more H 2 than abiotic controls, which could be partly explained by thermolabile enzymes and partly by non-thermolabile constituents released by cells. Next, we examined the metagenome for candidate enzymes/shuttles that could catalyze H 2 evolution from Fe 0 and found candidate H 2 -evolving hydrogenases and an almost complete pathway for flavin biosynthesis in Clostridium. Clostridial ferredoxin-dependent [FeFe]-hydrogenases may be catalyzing the H 2 -evolving reaction on Fe 0 , explaining the significant H 2 evolved by spent media exposed to Fe 0 . It is typical of Clostridia to secrete enzymes and other small molecules for lytic purposes. Here, they may secrete such molecules to enhance their own electron uptake from extracellular electron donors but indirectly make their H 2 -consuming neighbors- Methanobacterium -fare five times better in their presence. The particular enzymes and constituents promoting H 2 evolution from Fe 0 remain to be determined. However, we postulate that in a static environment like corrosive crust biofilms in lake sediments, less corrosive methanogens like Methanobacterium could extend corrosion long after acetogenesis ceased, by exploiting the constituents secreted by acetogens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Palacios, Francis and Rotaru.)

The genomes of non-bilaterian metazoans are key to understanding the molecular basis of early animal evolution. However, a full comprehension of how animal-specific traits, such as nervous systems, arose is hindered by the scarcity and fragmented nature of genomes from key taxa, such as Porifera. Ephydatia muelleri is a freshwater sponge found across the northern hemisphere. Here, we present its 326 Mb genome, assembled to high contiguity (N50: 9.88 Mb) with 23 chromosomes on 24 scaffolds. Our analyses reveal a metazoan-typical genome architecture, with highly shared synteny across Metazoa, and suggest that adaptation to the extreme temperatures and conditions found in freshwater often involves gene duplication. The pancontinental distribution and ready laboratory culture of E. muelleri make this a highly practical model system which, with RNAseq, DNA methylation and bacterial amplicon data spanning its development and range, allows exploration of genomic changes both within sponges and in early animal evolution.

The history of animal evolution, and the relative placement of extant animal phyla in this history is, in principle, testable from phylogenies derived from molecular sequence data. Though datasets have increased in size and quality in the past years, the contribution of individual genes (and ultimately amino acid sites) to the final phylogeny is unequal across genes. Here we demonstrate that removing a small fraction of sites strongly favoring one topology can produce a highly-supported tree of an alternate topology. We explore this approach using a dataset for animal phylogeny, and create a highly-supported tree with a monophyletic group of sponges and ctenophores, a topology not usually recovered. Because of the high sensitivity of such an analysis to gene selection, and because most gene sets are neither standardized nor representative of the entire genome, researchers should be diligent about making intermediate analyses available with their phylogenetic studies. Effort is needed to ensure these datasets are maximally informative, by ensuring all genes are systematically sampled across relevant species. From there, it could be determined whether any gene or gene sets introduce bias, and then deal with those biases appropriately., Competing Interests: The authors declare there are no competing interests., (©2020 Francis and Canfield.)

To date, five ctenophore species' mitochondrial genomes have been sequenced, and each contains open reading frames (ORFs) that if translated have no identifiable orthologs. ORFs with no identifiable orthologs are called unidentified reading frames (URFs). If truly protein-coding, ctenophore mitochondrial URFs represent a little understood path in early-diverging metazoan mitochondrial evolution and metabolism. We sequenced and annotated the mitochondrial genomes of three individuals of the beroid ctenophore Beroe forskalii and found that in addition to sharing the same canonical mitochondrial genes as other ctenophores, the B. forskalii mitochondrial genome contains two URFs. These URFs are conserved among the three individuals but not found in other sequenced species. We developed computational tools called pauvre and cuttlery to determine the likelihood that URFs are protein coding. There is evidence that the two URFs are under negative selection, and a novel Bayesian hypothesis test of trinucleotide frequency shows that the URFs are more similar to known coding genes than noncoding intergenic sequence. Protein structure and function prediction of all ctenophore URFs suggests that they all code for transmembrane transport proteins. These findings, along with the presence of URFs in other sequenced ctenophore mitochondrial genomes, suggest that ctenophores may have uncharacterized transmembrane proteins present in their mitochondria., Competing Interests: The authors declare that they have no competing Interests., (© 2020 Schultz et al.)

Severe injury and hemorrhagic shock (HS) result in multiple changes to hematopoietic differentiation, which contribute to the development of immunosuppression and multiple organ failure (MOF). Understanding the changes that take place during the acute injury phase may help predict which patients will develop MOF and provide potential targets for therapy. Obtaining bone marrow from humans during the acute injury phase is difficult so published data are largely derived from peripheral blood samples, which infer bone marrow changes that reflect the sustained inflammatory response. This preliminary and opportunistic study investigated leucopoietic changes in rat bone marrow 6 h following traumatic injury and HS. Terminally anesthetized male Porton Wistar rats were allocated randomly to receive a sham operation (cannulation with no injury) or femoral fracture and HS. Bone marrow cells were flushed from rat femurs and immunophenotypically stained with specific antibody panels for lymphoid (CD45R, CD127, CD90, and IgM) or myeloid (CD11b, CD45, and RP-1) lineages. Subsequently, cell populations were fluorescence-activated cell sorted for morphological assessment. Stage-specific cell populations were identified using a limited number of antibodies, and leucopoietic changes were determined 6 h following trauma and HS. Myeloid subpopulations could be identified by varying levels CD11b expression, CD45, and RP-1. Trauma and HS resulted in a significant reduction in total CD11b + myeloid cells including both immature (RP-1(-)) and mature (RP-1+) granulocytes. Multiple B-cell lymphoid subsets were identified. The total percentage of CD90+ subsets remained unchanged following trauma and HS, but there was a reduction in the numbers of maturing CD90(-) cells suggesting movement into the periphery. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry., (© 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.)

Ctenophores, also known as comb jellies, live across extremely broad ranges of temperature and hydrostatic pressure in the ocean. Because various ctenophore lineages adapted independently to similar environmental conditions, Phylum Ctenophora is an ideal system for the study of protein adaptation to extreme environments in a comparative framework. We present such a study here, using a phylogenetically-informed method to compare sequences of four essential metabolic enzymes across gradients of habitat depth and temperature. This method predicts convergent adaptation to these environmental parameters at the amino acid level, providing a novel view of protein adaptation to extreme environments and demonstrating the power and relevance of phylogenetic comparison applied to multi-species transcriptomic datasets from early-diverging metazoa. Across all four enzymes analyzed, 46 amino acid sites were associated with depth-adaptation, 59 with temperature-adaptation, and 56 with both. Sites predicted to be depth- and temperature-adaptive occurred consistently near Rossmann fold cofactor binding motifs and disproportionately in solvent-exposed regions of the protein. These results suggest that the hydrophobic effect and ligand binding may mediate efficient enzyme function at different hydrostatic pressures and temperatures. Using predicted adaptive site maps, such mechanistic hypotheses can now be tested via mutagenesis., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

Background: More than 3,000 species of octocorals (Cnidaria, Anthozoa) inhabit an expansive range of environments, from shallow tropical seas to the deep-ocean floor. They are important foundation species that create coral "forests," which provide unique niches and 3-dimensional living space for other organisms. The octocoral genus Renilla inhabits sandy, continental shelves in the subtropical and tropical Atlantic and eastern Pacific Oceans. Renilla is especially interesting because it produces secondary metabolites for defense, exhibits bioluminescence, and produces a luciferase that is widely used in dual-reporter assays in molecular biology. Although several anthozoan genomes are currently available, the majority of these are hexacorals. Here, we present a de novo assembly of an azooxanthellate shallow-water octocoral, Renilla muelleri., Findings: We generated a hybrid de novo assembly using MaSuRCA v.3.2.6. The final assembly included 4,825 scaffolds and a haploid genome size of 172 megabases (Mb). A BUSCO assessment found 88% of metazoan orthologs present in the genome. An Augustus ab initio gene prediction found 23,660 genes, of which 66% (15,635) had detectable similarity to annotated genes from the starlet sea anemone, Nematostella vectensis, or to the Uniprot database. Although the R. muelleri genome may be smaller (172 Mb minimum size) than other publicly available coral genomes (256-448 Mb), the R. muelleri genome is similar to other coral genomes in terms of the number of complete metazoan BUSCOs and predicted gene models., Conclusions: The R. muelleri hybrid genome provides a novel resource for researchers to investigate the evolution of genes and gene families within Octocorallia and more widely across Anthozoa. It will be a key resource for future comparative genomics with other corals and for understanding the genomic basis of coral diversity., (© The Author(s) 2019. Published by Oxford University Press.)

Resolving the relationships of animals (Metazoa) is crucial to our understanding of the origin of key traits such as muscles, guts, and nerves. However, a broadly accepted metazoan consensus phylogeny has yet to emerge. In part, this is because the genomes of deeply diverging and fast-evolving lineages may undergo significant gene turnover, reducing the number of orthologs shared with related phyla. This can limit the usefulness of traditional phylogenetic methods that rely on alignments of orthologous sequences. Phylogenetic analysis of gene content has the potential to circumvent this orthology requirement, with binary presence/absence of homologous gene families representing a source of phylogenetically informative characters. Applying binary substitution models to the gene content of 26 complete animal genomes, we demonstrate that patterns of gene conservation differ markedly depending on whether gene families are defined by orthology or homology, that is, whether paralogs are excluded or included. We conclude that the placement of some deeply diverging lineages may exceed the limit of resolution afforded by the current methods based on comparisons of orthologous protein sequences, and novel approaches are required to fully capture the evolutionary signal from genes within genomes., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The origin of novel traits can promote expansion into new niches and drive speciation. Ctenophores (comb jellies) are unified by their possession of a novel cell type: the colloblast, an adhesive cell found only in the tentacles. Although colloblast-laden tentacles are fundamental for prey capture among ctenophores, some species have tentacles lacking colloblasts and others have lost their tentacles completely. We used transcriptomes from 36 ctenophore species to identify gene losses that occurred specifically in lineages lacking colloblasts and tentacles. We cross-referenced these colloblast- and tentacle-specific candidate genes with temporal RNA-Seq during embryogenesis in Mnemiopsis leidyi and found that both sets of candidates are preferentially expressed during tentacle morphogenesis. We also demonstrate significant upregulation of candidates from both data sets in the tentacle bulb of adults. Both sets of candidates were enriched for an N-terminal signal peptide and protein domains associated with secretion; among tentacle candidates we also identified orthologs of cnidarian toxin proteins, presenting tantalizing evidence that ctenophore tentacles may secrete toxins along with their adhesive. Finally, using cell lineage tracing, we demonstrate that colloblasts and neurons share a common progenitor, suggesting the evolution of colloblasts involved co-option of a neurosecretory gene regulatory network. Together these data offer an initial glimpse into the genetic architecture underlying ctenophore cell-type diversity.

The Neoproterozoic Era (1000-541 million years ago, Ma) was characterized by dramatic environmental and evolutionary change, including at least two episodes of extensive, low-latitude glaciation, potential changes in the redox structure of the global ocean, and the origin and diversification of animal life. How these different events related to one another remains an active area of research, particularly how these environmental changes influenced, and were influenced by, the earliest evolution of animals. Animal multicellularity is estimated to have evolved in the Tonian Period (1000-720 Ma) and represents one of at least six independent acquisitions of complex multicellularity, characterized by cellular differentiation, three-dimensional body plans, and active nutrient transport. Compared with the other instances of complex multicellularity, animals represent the only clade to have evolved from wall-less, phagotrophic flagellates, which likely placed unique cytological and trophic constraints on the evolution of animal multicellularity. Here, we compare recent molecular clock estimates with compilations of the chromium isotope, micropaleontological, and organic biomarker records, suggesting that, as of now, the origin of animals was not obviously correlated to any environmental-ecological change in the Tonian Period. This lack of correlation is consistent with the idea that the evolution of animal multicellularity was primarily dictated by internal, developmental constraints and occurred independently of the known environmental-ecological changes that characterized the Neoproterozoic Era., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology.)

[This corrects the article DOI: 10.1371/journal.pbio.2005359.].

Coralline hydroxyapatite/calcium carbonate (CHACC) is a biodegradable and osteoconductive bone graft material with promising clinical performance. CHACC has been shown to support proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells (MSCs) in vitro and demonstrated to work as a functional scaffold for bone formation in vivo. Umbilical cord matrix is a more accessible and abundant tissue source of MSCs, but its osteogenic capacity in comparison to human bone marrow when cultured on CHACC has not yet been demonstrated. In this study, we assessed the osteogenic differentiation capacity of human MSCs, isolated from bone marrow and umbilical cord matrix and characterised by flow cytometry, when cultured on 200-300  μ m CHACC granules. The 3D cultures were characterised by brightfield and scanning electron microscopy (SEM). Osteogenic potential was assessed by immunocytochemistry and qPCR for key markers of bone differentiation (alkaline phosphatase, runx2, type I collagen, and osteocalcin). By day 1, the MSCs had enveloped the surface of the CHACC granules to form organoids, and by day 7, cells had proliferated to bridge nearby organoids. Extracellular matrix deposition and osteogenic differentiation were demonstrated by MSCs from both tissue sources at day 21. However, MSCs from bone marrow demonstrated superior osteogenic differentiation capability compared to those from umbilical cord matrix. In conclusion, it is possible to culture and induce osteogenic differentiation of umbilical cord matrix MSCs on CHACC. Further research is required to optimise the osteogenicity of umbilical cord matrix MSCs to release their full potential as a readily available, accessible, and abundant tissue source for bone tissue engineering.

Placozoans are a phylum of nonbilaterian marine animals currently represented by a single described species, Trichoplax adhaerens, Schulze 1883. Placozoans arguably show the simplest animal morphology, which is identical among isolates collected worldwide, despite an apparently sizeable genetic diversity within the phylum. Here, we use a comparative genomics approach for a deeper appreciation of the structure and causes of the deeply diverging lineages in the Placozoa. We generated a high-quality draft genome of the genetic lineage H13 isolated from Hong Kong and compared it to the distantly related T. adhaerens. We uncovered substantial structural differences between the two genomes that point to a deep genomic separation and provide support that adaptation by gene duplication is likely a crucial mechanism in placozoan speciation. We further provide genetic evidence for reproductively isolated species and suggest a genus-level difference of H13 to T. adhaerens, justifying the designation of H13 as a new species, Hoilungia hongkongensis nov. gen., nov. spec., now the second described placozoan species and the first in a new genus. Our multilevel comparative genomics approach is, therefore, likely to prove valuable for species distinctions in other cryptic microscopic animal groups that lack diagnostic morphological characters, such as some nematodes, copepods, rotifers, or mites., Competing Interests: The authors have declared that no competing interests exist.

Animals have a carefully orchestrated relationship with oxygen. When exposed to low environmental oxygen concentrations, and during periods of increased energy expenditure, animals maintain cellular oxygen homeostasis by enhancing internal oxygen delivery, and by enabling the anaerobic production of ATP. These low-oxygen responses are thought to be controlled universally across animals by the hypoxia-inducible factor (HIF). We find, however, that sponge and ctenophore genomes lack key components of the HIF pathway. Since sponges and ctenophores are likely sister to all remaining animal phyla, the last common ancestor of extant animals likely lacked the HIF pathway as well. Laboratory experiments show that the marine sponge Tethya wilhelma maintains normal transcription under oxygen levels down to 0.25% of modern atmospheric saturation, the lowest levels we investigated, consistent with the predicted absence of HIF or any other HIF-like pathway. Thus, the last common ancestor of all living animals could have metabolized aerobically under very low environmental oxygen concentrations., Competing Interests: DM, WF, SV, ML, CE, DC, GW No competing interests declared, (© 2017, Mills et al.)

Scientific drilling has identified a biosphere in marine sediments 1 , which contain many uncultivated microbial groups known only by their DNA sequences 2-4 . Recycling of organic matter in sediments is an important component of biogeochemical cycles because marine sediments are critical for long-term carbon storage 5 . Turnover of carbon is hypothesized to be driven by the secretion of enzymes by microbial organisms 5-7 , which act to break down macromolecules into constitutive monomers that can be transported into cells. As such, the nature of the microbial secretome often influences the function of a community 6 . However, the microbial groups involved in this process and the biochemistry they encode is poorly understood. Here, we show that expressed genes from 5 to 159 meters below the seafloor 8 (mbsf) encode numerous candidate peptidases and carbohydrate-active enzymes ('CAZymes') 9 targeted for secretion. The majority (90-99%) were assigned to Bacteria, of which 12% shared the highest sequence similarity with candidate phyla 10,11 . The remaining putatively secreted proteins shared highest sequence similarity with archaeal and fungal enzymes, which peak in two redox transition zones 12 . In the shallower redox zone at 30 mbsf, 20% of the transcripts encoding putative secreted peptidases were assigned to lineages 7,13,14 of uncultivated Archaea. The target compounds of the predicted secreted proteome show a preference for necromass in the form of microbial cell envelopes as well as plankton and algal detritus. The predicted fungal secreted proteome encodes CAZymes not present in the predicted bacterial or archaeal secreted proteomes, indicating that fungi putatively play a minimal but specialized role in subseafloor carbohydrate recycling.

The squid Sthenoteuthis oualaniensis , formerly Symplectoteuthis oualaniensis , generates light using the luciferin coelenterazine and a unique enzyme, symplectin. Genetic information is limited for bioluminescent cephalopod species, so many proteins, including symplectin, occur in public databases only as sequence isolates with few identifiable homologs. As the distribution of the symplectin/pantetheinase protein family in Metazoa remains mostly unexplored, we have sequenced the transcriptomes of four additional luminous squid, and make use of publicly available but unanalyzed data of other cephalopods, to examine the occurrence and evolution of this protein family. While the majority of spiralians have one or two copies of this protein family, four well-supported groups of proteins are found in cephalopods, one of which corresponds to symplectin. A cysteine that is critical for symplectin functioning is conserved across essentially all members of the protein family, even those unlikely to be used for bioluminescence. Conversely, active site residues involved in pantetheinase catalysis are also conserved across essentially all of these proteins, suggesting that symplectin may have multiple functions including hydrolase activity, and that the evolution of the luminous phenotype required other changes in the protein outside of the main binding pocket., Competing Interests: The authors declare there are no competing interests.

One central goal of genome biology is to understand how the usage of the genome differs between organisms. Our knowledge of genome composition, needed for downstream inferences, is critically dependent on gene annotations, yet problems associated with gene annotation and assembly errors are usually ignored in comparative genomics. Here, we analyze the genomes of 68 species across 12 animal phyla and some single-cell eukaryotes for general trends in genome composition and transcription, taking into account problems of gene annotation. We show that, regardless of genome size, the ratio of introns to intergenic sequence is comparable across essentially all animals, with nearly all deviations dominated by increased intergenic sequence. Genomes of model organisms have ratios much closer to 1:1, suggesting that the majority of published genomes of nonmodel organisms are underannotated and consequently omit substantial numbers of genes, with likely negative impact on evolutionary interpretations. Finally, our results also indicate that most animals transcribe half or more of their genomes arguing against differences in genome usage between animal groups, and also suggesting that the transcribed portion is more dependent on genome size than previously thought., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: Fluorescent proteins are optically active proteins found across many clades in metazoans. A fluorescent protein was recently identified in a ctenophore, but this has been suggested to derive from a cnidarian, raising again the question of origins of this group of proteins., Results: Through analysis of transcriptome data from 30 ctenophores, we identified a member of an orthologous group of proteins similar to fluorescent proteins in each of them, as well as in the genome of Mnemiopsis leidyi. These orthologs lack canonical residues involved in chromophore formation, suggesting another function., Conclusions: The phylogenetic position of the ctenophore protein family among fluorescent proteins suggests that this gene was present in the common ancestor of all ctenophores and that the fluorescent protein previously found in a ctenophore actually derives from a siphonophore.

We report the mitochondrial genomes of two freshwater sponges, Spongilla lacustris and Ephydatia cf. muelleri . The genomes contain 14 protein-coding genes and are similar in structure to other published mitochondrial genomes from freshwater sponges. The E. cf. muelleri described here is remarkably similar in coding regions to the published genome, but differs in number and length of hairpin-forming repeats between genes., Competing Interests: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article., (© 2016 The Author(s). Published by Taylor & Francis.)

The biosynthesis of the luciferin coelenterazine has remained a mystery for decades. While not all organisms that use coelenterazine appear to make it themselves, it is thought that ctenophores are a likely producer. Here we analyze the transcriptome data of 24 species of ctenophores, two of which have published genomes. The natural precursors of coelenterazine have been shown to be the amino acids L-tyrosine and L-phenylalanine, with the most likely biosynthetic pathway involving cyclization and further modification of the tripeptide Phe-Tyr-Tyr ("FYY"). Therefore, we searched the ctenophore transcriptome data for genes with the short peptide "FYY" as part of their coding sequence. We recovered a group of candidate genes for coelenterazine biosynthesis in the luminous species which encode a set of highly conserved non-heme iron oxidases similar to isopenicillin-N-synthase. These genes were absent in the transcriptomes and genome of the two non-luminous species. Pairwise identities and substitution rates reveal an unusually high degree of identity even between the most unrelated species. Additionally, two related groups of non-heme iron oxidases were found across all ctenophores, including those which are non-luminous, arguing against the involvement of these two gene groups in luminescence. Important residues for iron-binding are conserved across all proteins in the three groups, suggesting this function is still present. Given the known functions of other members of this protein superfamily are involved in heterocycle formation, we consider these genes to be top candidates for laboratory characterization or gene knockouts in the investigation of coelenterazine biosynthesis.

Tomopteris is a cosmopolitan genus of polychaetes. Many species produce yellow luminescence in the parapodia when stimulated. Yellow bioluminescence is rare in the ocean, and the components of this luminescent reaction have not been identified. Only a brief description, half a century ago, noted fluorescence in the parapodia with a remarkably similar spectrum to the bioluminescence, which suggested that it may be the luciferin or terminal light-emitter. Here, we report the isolation of the fluorescent yellow-orange pigment found in the luminous exudate and in the body of the animals. Liquid chromatography-mass spectrometry revealed the mass to be 270 m/z with a molecular formula of C(15)H(10)O(5), which ultimately was shown to be aloe-emodin, an anthraquinone previously found in plants. We speculate that aloe-emodin could be a factor for resonant-energy transfer or the oxyluciferin for Tomopteris bioluminescence., (© 2014 The Authors. Luminescence published by John Wiley & Sons Ltd.)

During bone maintenance in vivo, estrogen signals through estrogen receptor (ER)-α. The objectives of this study were to investigate the temporal expression of ERα36 and ascertain its functional relevance during osteogenesis in human bone marrow derived stromal cells (BMSC). This was assessed in relation to runt-related transcription factor-2 (runx2), a main modulatory protein involved in bone formation. ERα36 and runx2 subcellular localisation was assessed using immunocytochemistry, and their mRNA expression levels by real time PCR throughout the process of osteogenesis. The osteogenically induced BMSCs demonstrated a rise in ERα36 mRNA during proliferation followed by a decline in expression at day 10, which represents a change in dynamics within the culture between the proliferative stage and the differentiative stage. The mRNA expression profile of runx2 mirrored that of ERα36 and showed a degree subcellular co-localisation with ERα36. This study suggests that ERα36 is involved in the process of osteogenesis in BMSCs, which has implications in estrogen deficient environments., (Copyright © 2014 Elsevier Inc. All rights reserved.)

An understanding of ctenophore biology is critical for reconstructing events that occurred early in animal evolution. Toward this goal, we have sequenced, assembled, and annotated the genome of the ctenophore Mnemiopsis leidyi. Our phylogenomic analyses of both amino acid positions and gene content suggest that ctenophores rather than sponges are the sister lineage to all other animals. Mnemiopsis lacks many of the genes found in bilaterian mesodermal cell types, suggesting that these cell types evolved independently. The set of neural genes in Mnemiopsis is similar to that of sponges, indicating that sponges may have lost a nervous system. These results present a newly supported view of early animal evolution that accounts for major losses and/or gains of sophisticated cell types, including nerve and muscle cells.

Background: The lack of genomic resources can present challenges for studies of non-model organisms. Transcriptome sequencing offers an attractive method to gather information about genes and gene expression without the need for a reference genome. However, it is unclear what sequencing depth is adequate to assemble the transcriptome de novo for these purposes., Results: We assembled transcriptomes of animals from six different phyla (Annelids, Arthropods, Chordates, Cnidarians, Ctenophores, and Molluscs) at regular increments of reads using Velvet/Oases and Trinity to determine how read count affects the assembly. This included an assembly of mouse heart reads because we could compare those against the reference genome that is available. We found qualitative differences in the assemblies of whole-animals versus tissues. With increasing reads, whole-animal assemblies show rapid increase of transcripts and discovery of conserved genes, while single-tissue assemblies show a slower discovery of conserved genes though the assembled transcripts were often longer. A deeper examination of the mouse assemblies shows that with more reads, assembly errors become more frequent but such errors can be mitigated with more stringent assembly parameters., Conclusions: These assembly trends suggest that representative assemblies are generated with as few as 20 million reads for tissue samples and 30 million reads for whole-animals for RNA-level coverage. These depths provide a good balance between coverage and noise. Beyond 60 million reads, the discovery of new genes is low and sequencing errors of highly-expressed genes are likely to accumulate. Finally, siphonophores (polymorphic Cnidarians) are an exception and possibly require alternate assembly strategies.

Background: Changes in human bone marrow associated with the systemic inflammatory response to injury are little understood. It was hypothesized that major trauma results in an altered bone marrow leucocyte progenitor profile, with either uniform depletion or the balance between multipotent and committed progenitors varying, depending on whether self-renewal is favoured over differentiation., Methods: Bone marrow aspirate and peripheral blood samples were obtained at definitive surgery in adults with pelvic fractures from blunt trauma (major trauma with Injury Severity Score (ISS) at least 18, or isolated fractures) and control patients undergoing iliac crest bone grafting. ISS, interval to surgery and transfusion in the first 24 h were recorded. Bone marrow aspirate flow cytometry was used to identify haemopoietic progenitor cells (CD34(+) ), multipotent cells (CD34(+) CD45(+) CD38(-) ) and oligopotent cells (CD34(+) CD45(+) CD38(lo/+) and CD34(+) CD45(+) CD38(BRIGHT(++ +)) subsets). Peripheral blood levels of inflammatory markers were measured, and the ratio of immature to mature (CD35(-) /CD35(+) ) granulocytes was determined., Results: The median (range) interval between injury and sampling was 7 (1-21) and 5 (1-21) days in the major trauma and isolated fracture groups respectively. The CD34(+) pool was significantly depleted in the major trauma group (P = 0·017), particularly the CD34(+) CD45(+) CD38(BRIGHT(++ +)) oligopotent pool (P = 0·003). Immature CD35(-) granulocytes increased in bone marrow with increasing injury severity (P = 0·024) and massive transfusion (P = 0·019), and in peripheral blood with increasing interval to surgery (P = 0·005)., Conclusion: Major blunt trauma resulted in changes in the bone marrow CD34(+) progenitor pool. At the point in recovery when these samples were obtained, oligopotent progenitors were lost from the bone marrow, with continued release of immature cells., (Copyright © 2012 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.)

Background Context: An accepted classification of spondylolysis exists. However, etiology remains controversial. There are several reports supporting acquired stress fractures in the etiology of this condition, although radiographic evidence of an acquired fracture in a given individual is very rare., Purpose: To present evidence of development of pars defects during the teenage years., Study Design/setting: Retrospective review of charts., Patient Sample: Two case studies., Methods: Charts and X-rays were reviewed to look for radiographic evidence of development of pars defects in patients who were ultimately found to have spondylolisthesis or spondylolysis., Results: Two cases were found that demonstrated development of a pars fracture. Both patients had radiographic evidence that these fractures developed during their teenage years. Both were active individuals with a consistent level of participation in athletics., Conclusions: Although development of pars defects may be multifactorial, these two cases support the concept that these fractures can be acquired lesions resulting from repetitive stress injury. They also demonstrate that isthmic defects can arise in teenagers, in these cases in an athletic population.

The percutaneous absorption of three highly lipophilic analogs of capsaicin--vanillylnonanamide (VN), olvanil, and NE-21610--was measured in vivo in the CD:VAF rat, and in vitro through excised CD: VAF and SkH:Fz rat skin and human cadaver skin. Absorption and skin metabolism were monitored by radiolabel techniques. The rank order of penetration in all species was VN > olvanil > NE-21610, in accordance with that expected from their physical properties. Rat skin was more permeable than human skin by factors ranging from 4 to 8 for VN, 10 to 20 for olvanil, and approximately 10 to 100 for NE-21610. All three compounds were extensively metabolized during passage through fresh SkH:Fz rat skin, with the primary route of degradation for at least two of the compounds involving hydrolysis of the amide bond (the metabolites of NE-21610 were not identified). For the in vitro studies a range of receptor solutions was employed to determine a set of conditions that best mimicked in vivo absorption. The results with phosphate-buffered saline containing a preservative and 1-6% polyoxyethylene-20 oleyl ether (Oleth-20) were in good agreement with in vivo results for all three compounds for periods up to 24 h post-dose; after this time, in vivo absorption rates declined but in vitro rates remained relatively constant. Buffered saline or saline containing 0.5% bovine serum albumin led to marked underestimates of in vivo penetration for olvanil and NE-21610, whereas a 1:1 ethanol: water solution led to gross overestimates of the in vivo absorption rates for all three compounds.

The institution of a readily-implemented sample screening and data handling procedure for in vitro skin penetration studies yields substantial improvements in sensitivity for distinguishing between formulations, treatments, penetrants, etc. The procedure involves four steps: 1) prescreen the tissue samples to determine their intrinsic permeability; 2) apply treatments using a randomized complete block (RCB) design, with blocking by tissue permeability; 3) apply a variance-stabilizing transformation to the penetration data, followed by outlier testing; and 4) analyze the transformed data according to an RCB analysis of variance, using tissue permeability as the blocking variable. For penetration studies in which high sample variability is a concern, the above procedure commonly yields a sensitivity advantage of several-fold versus alternative methods of comparison.

The relationship of capsaicin desensitization to the antinociception produced by acute capsaicin administration was studied in the rat. Cutaneous application of 1% capsaicin (3 x/day) for 1 day antagonized plasma extravasation evoked by either 5% capsaicin applied to the dorsal surface of the hindpaw or antidromic stimulation of the saphenous nerve. Treatment with either 1% or 5% capsaicin (3 x/day) for up to 4 days failed, however, to produce thermal antinociception as measured by the rat paw-withdrawal procedure. In contrast, intracutaneous injection of 100 micrograms capsaicin produced marked increases in paw-withdrawal response latencies for up to six hours after injection. These data demonstrate a dissociation between block of cutaneous plasma extravasation and thermal antinociception in the rat. The data provide in vivo evidence that desensitization of peripheral nociceptors by capsaicin cannot account for the antinociception observed following acute capsaicin administration. Finally, these data support the notion that some pharmacologic actions of capsaicin in the rat can be dissociated from frank neurotoxicity on the basis of concentration or dose.

The distribution of salicylate to embryonal compartments for in situ and in vitro rat embryos under equivalent exposure conditions, and salicylate disposition in the in vivo mid-gestation embryo and late gestation fetus, were compared. Pregnant Sprague-Dawley CD rats were exposed to steady-state blood levels of salicylate by infusing 14C-salicylic acid iv for a 24 hour period from gestation day 11.5 to 12.5. Cultured Sprague-Dawley rat embryos (in medium consisting of 100% male rat serum) were exposed to the steady-state 14C-salicylate concentration achieved in maternal serum in vivo for the same 24 hour developmental period. At the end of the exposure period radioactivity in visceral yolk sac, extra-embryonic fluid and embryos, and in maternal tissues, was measured. The distribution of salicylate to embryonal tissues was statistically comparable in vivo and in vitro, although the embryos in vitro accumulated slightly (but not significantly) less of the chemical. There was considerable binding of salicylate by maternal serum and culture medium proteins: less than 20% of the chemical was free at the 40 micrograms/ml concentration used in this experiment. Consequently, the salicylate concentration in embryonal compartments appeared to be quite low when compared to the surrounding serum/medium, but was actually equal to or greater than the concentration of unbound salicylate in serum or culture medium. The proportion of free salicylate in serum increased at concentrations higher than 40 micrograms/ml, resulting in somewhat higher concentrations of salicylate in in vitro embryos and extraembryonic fluid (as compared to medium) when cultured in the presence of 200 or 400 micrograms/ml salicylate.(ABSTRACT TRUNCATED AT 250 WORDS)

Immobilization of groups of five to nine male rats for 2-5 days results in a 50% increase in urinary bladder fresh weight compared with normally caged controls. The increase in urinary bladder weight was not due to tissue oedema and was accompanied by epithelial hyperplasia in some urinary bladders. Immobilization did not alter total urine volume, but it did decrease the frequency of urine voiding and doubled the mean urine weight/voiding. Thus, bladder distention caused by the increased volume per voiding caused a rapidly induced increase in bladder tissue growth, and was accompanied by an increase in bladder epithelial cell division.

Consultant pharmacists conducted a drug history and regimen review on one-fourth of the patient population served by a community hospital-based home health department. About one-third of study patients had inappropriately stopped taking their medications; over one-fourth of the study patients had changed their schedule or dose without health professional consultation. Fifteen percent of all medications were duplications, and only 8% of prescription medications were correctly identified by name. Consultant pharmacist activities that could reduce drug-related problems were identified as drug therapy consultation with patients and staff, drug regimen review, drug information, and patient medication education.

The lesions of C2 seen in hyperextension injuries of the cervical spine following motor vehicle accidents, diving accidents, and headlong falls resemble the cervical lesion found in judicial hangings. Although the mechanism of injury in these cervical fractures is different, the distinction is significant. The fracture seen in motor vehicular accidents today usually seems to be one of hyperextension and axial compression rather than hyperextension and distraction. This distinction is responsible for the low incidence of neurologic involvement seen in the fracture caused by motor vehicle accidents. The incidence of face and scalp injuries associated with axis pedicle fractures appears significant. Union of this fracture can generally be expected, and it rarely produces late sequelae. Management of this fracture is generally preferable on an ambulatory basis utilizing a cervicothoracic brace or a halo and caudal support in more unstable fractures. A period of 12 weeks of immobilization is recommended for satisfactory union of this fracture. Occasionally the treatment of this fracture may require traction or operative intervention. If operative intervention is required, an anterior route employing interbody fusion is suggested.