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The freshwater sponge Ephydatia muelleri and its Chlorella-like green algal partner is an emerging model for studying animal:algal endosymbiosis. The sponge host is a highly tractable laboratory organism, and the symbiotic algae are easily cultured. We took advantage of these traits to experimentally interrogate fundamental questions about cellular mechanisms that govern the establishment of durable intracellular partnerships between hosts and symbionts in facultative symbioses. We modified a classical experimental approach to discern the phagocytotic mechanisms that might be co-opted to permit persistent infections, and identified genes differentially expressed in E. muelleri sponges early in the establishment of endosymbiosis. We exposed algal-free E. muelleri to live native algal symbionts, native heat-killed algae, and bacteria, and then performed RNASeq so we could compare patterns of gene expression in each treatment. We contrasted differential gene expression patterns between potential food items (bacteria and heat-killed algae) and the live native Chlorella-like symbiont. We found a relatively small but interesting suite of genes that are uniquely and differentially expressed in the host exposed to live algal symbionts, and a larger number of genes uniquely triggered by host exposure to heat-killed algae. One of the host genes, an ABC transporter that is downregulated in response to live algal symbionts, was further evaluated for its possible role in establishment of the algal symbiosis. We discuss the gene expression profiles associated with host responses to living algal cells in the context of conditions necessary for long-term residency within host cells by phototrophic symbionts as well as the genetic responses to sponge phagocytosis and immune driven pathways.

In many freshwater habitats, green algae form intracellular symbioses with a variety of heterotrophic host taxa including several species of freshwater sponge. These sponges perform important ecological roles in their habitats, and the poriferan:green algae partnerships offers unique opportunities to study the evolutionary origins and ecological persistence of endosymbioses. We examined the association betweenEphydatia muelleriand its chlorophyte partner to identify features of host cellular and genetic responses to the presence of intracellular algal partners.Chlorella-likegreen algal symbionts were isolated from field-collected adultE. muelleritissue harboring algae. The sponge-derived algae were successfully cultured and subsequently used to reinfect aposymbioticE. muelleritissue. We used confocal microscopy to follow the fate of the sponge-derived algae after inoculating algae-freeE. muellerigrown from gemmules to show temporal patterns of symbiont location within host tissue. We also infected aposymbioticE. muelleriwith sponge-derived algae, and performed RNASeq to study differential expression patterns in the host relative to symbiotic states. We compare and contrast our findings with work in other systems (e.g., endosymbioticHydra) to explore possible conserved evolutionary pathways that may lead to stable mutualistic endosymbioses. Our work demonstrates that freshwater sponges offer many tractable qualities to study features of intracellular occupancy and thus meet criteria desired for a model system.

The freshwater sponge Ephydatia muelleri and its Chlorella-like algal partner is an emerging model for studying animal: algal endosymbiosis. The sponge host is a tractable laboratory organism, and the symbiotic algae are easily cultured. We took advantage of these traits to interrogate questions about mechanisms that govern the establishment of durable intracellular partnerships between hosts and symbionts in facultative symbioses. We modified a classical experimental approach to discern the phagocytotic mechanisms that might be co-opted to permit persistent infections, and identified genes differentially expressed in sponges early in the establishment of endosymbiosis. We exposed algal-free E. muelleri to live native algal symbionts and potential food items (bacteria and native heat-killed algae), and performed RNA-Seq to compare patterns of gene expression among treatments. We found a relatively small but interesting suite of genes that are differentially expressed in the host exposed to live algal symbionts, and a larger number of genes triggered by host exposure to heat-killed algae. The upregulated genes in sponges exposed to live algal symbionts were mostly involved in endocytosis, ion transport, metabolic processes, vesicle-mediated transport, and oxidation-reduction. One of the host genes, an ATP-Binding Cassette transporter that is downregulated in response to live algal symbionts, was further evaluated for its possible role in the establishment of the symbiosis. We discuss the gene expression profiles associated with host responses to living algal cells in the context of conditions necessary for long-term residency within host cells by phototrophic symbionts as well as the genetic responses to sponge phagocytosis and immune-driven pathways., This work was supported by the National Science Foundation (Award #1555440) to A.L.H. and M.S.H., the Gordon and Betty Moore Foundation (Award #9332) to A.L.H. and A.R., and by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health (#P20GM103423).

IntroductionVasa previa is a condition where fetal blood vessels run unprotected in the membranes, outside the umbilical cord, and cross the internal opening of the cervix. During rupture of membranes, these vessels can rupture and put the baby at serious risk of severe blood loss and death. Numerous studies are being conducted to improve diagnostic modalities and establish clear management plans to improve pregnancy outcomes. However, the lack of a standardised set of outcomes for studies on vasa previa makes it difficult to compare study findings and draw meaningful conclusions. Through this project, we will be developing a core outcome set for studies on pregnant women with vasa previa (COVasP).Methods and analysisThe development of COVasP will involve five steps. The first will be a systematic review, in which we will generate a long list of outcomes based on published studies in pregnancies complicated with vasa previa. The second will involve in-depth interviews with current and former patients, their family members and healthcare providers who care for these patients. This will be followed by a two-round Delphi survey, which will aim to narrow down the long list of outcomes into those considered important by four groups of ‘stakeholders’: (1) patients, family members and patient advocates/representatives, (2) healthcare providers, (3) researchers, epidemiologists and methodologists and (4) other stakeholders directly or indirectly involved in the management of these pregnancies such as administrators, guideline developers and policymakers. The fourth step will involve a face-to-face consensus meeting using a nominal group approach to establish a finalised core outcome set. The final step will involve measuring and defining the identified outcomes using a combination of systematic reviews and Delphi surveys.Ethics and disseminationThis study as well as consent forms for stakeholder participation have received approval from the Mount Sinai Hospital Research Ethics Board (REB number 18-0173-E) on 05 September 2018 and the Human Research Ethics Committee at The University of Technology Sydney, Australia on 30 July 2019 (UTS HREC reference number ETH19-3718). All progress will be documented on the international prospective register of systematic reviews and Core Outcome Measures in Effectiveness Trials databases.Registration detailshttp://www.comet-initiative.org/studies/details/1117.

Background/purpose Owing to the frequency of gastrostomy tube placement in children and the numerous regimens used to start feeds after placement we attempted to see if it matters if the initial feeds after a gastrostomy tube placement are provided in a bolus or continuous manner. Methods Using a prospective randomized trial, children were randomized to initial bolus or continuous chimney feeding after gastrostomy tube placement. Feeding tolerance and complications related to the gastrostomy tube were collected for 4 weeks after placement. Results Demographics were similar in the two groups. Times to goal feeds were similar in both groups, but in the first two weeks more feeding modifications were required in the bolus group. Other than the rate of leakage during the second week after placement which occurred more in the bolus group, all other clinical outcomes were similar in the two groups. Conclusions Other than minor, clinically insignificant differences noted above, the method of initial feeding after a gastrostomy tube placement does not affect feeding tolerance or gastrostomy tube complication in the first month after placement. Level of evidence Therapeutic, level II.

Background Clinical studies looking at interventions to optimize pregnancy and long-term outcomes for women with cardiac disease and their babies are inconsistent in their reporting of clinical outcomes, making it difficult to compare results across studies and draw meaningful conclusions. The development of a core outcome set (COS)—a standardized, minimum set of outcomes that must be collected and reported in all studies—is a practical solution to this problem. Methods/design We will follow a five-step process in developing a COS for studies on pregnant women with cardiac disease. First, a systematic literature review will identify all reported outcomes (including patient-reported outcomes) and definitions. Second, semi-structured interviews with stakeholders involved in the care of pregnant women with cardiac disease will determine their perspective and add new outcomes that they consider important. Third, an international electronic Delphi survey will narrow outcomes obtained through the first two steps, in an attempt to arrive at a consensus. Fourth, a face-to-face consensus meeting will deliberate to finalize the COS. Finally, measurement tools and definitions for included outcomes will be determined through a series of literature reviews and Delphi surveys. Discussion This protocol provides an overview of the steps involved in the development of a COS that must be reported in studies involving pregnant women with cardiac disease, in an attempt to harmonize outcome reporting and ensure the validity of study results that will not only inform clinical practice and future research but also encourage the development of COS in other areas of medicine. COMET core outcome set registration http://www.cometinitiative.org/studies/details/834

Berry cell death assessment can become one of the most objective parameters to assess important berry quality traits, such as aroma profiles that can be passed to the wine in the winemaking process. At the moment, the only practical tool to assess berry cell death in the field is using portable near-infrared spectroscopy (NIR) and machine learning (ML) models. This research tested the NIR and ML approach and developed supervised regression ML models using Shiraz and Chardonnay berries and wines from a vineyard located in Yarra Valley, Victoria, Australia. An ML model was developed using NIR measurements from intact berries as inputs to estimate berry cell death (BCD), living tissue (LT) (Model 1). Furthermore, canopy architecture parameters obtained from cover photography of grapevine canopies and computer vision analysis were also tested as inputs to develop ML models to assess BCD and LT (Model 2) and the intensity of sensory descriptors based on visual and aroma profiles of wines for Chardonnay (Model 3) and Shiraz (Model 4). The results showed high accuracy and performance of models developed based on correlation coefficient (R) and slope (b) (M1: R = 0.87, b = 0.82, M2: R = 0.98, b = 0.93, M3: R = 0.99, b = 0.99, M4: R = 0.99, b = 1.00). Models developed based on canopy architecture, and computer vision can be used to automatically estimate the vigor and berry and wine quality traits using proximal remote sensing and with visible cameras as the payload of unmanned aerial vehicles (UAV).

Shigella flexneri is an intracellular bacterial pathogen that invades epithelial cells in the colonic mucosa, leading to bloody diarrhea. A previous study showed that S. flexneri forms biofilms in the presence of bile salts, through an unknown mechanism. Here, we investigated the potential role of adhesin-like autotransporter proteins in S. flexneri biofilm formation. BLAST search analysis revealed that the S. flexneri 2457T genome harbors 4 genes, S1242, S1289, S2406, and icsA, encoding adhesin-like autotransporter proteins. Deletion mutants of the S1242, S1289, S2406 and icsA genes were generated and tested for biofilm formation. Phenotypic analysis of the mutant strains revealed that disruption of icsA abolished bile salt-induced biofilm formation. IcsA is an outer membrane protein secreted at the bacterial pole that is required for S. flexneri actin-based motility during intracellular infection. In extracellular biofilms, IcsA was also secreted at the bacterial pole and mediated bacterial cell-cell contacts and aggregative growth in the presence of bile salts. Dissecting individual roles of bile salts showed that deoxycholate is a robust biofilm inducer compared to cholate. The release of the extracellular domain of IcsA through IcsP-mediated cleavage was greater in the presence of cholate, suggesting that the robustness of biofilm formation was inversely correlated with IcsA processing. Accordingly, deletion of icsP abrogated IcsA processing in biofilms and enhanced biofilm formation.

The purpose of this study was to determine the clinical and non-clinical outcomes that pregnant women with obesity value as pertaining to their health and that of their babies. In this systematic review of the qualitative literature, a search of four databases was conducted for studies using qualitative methods in pregnant women with obesity. From the included studies, the review synthesized relevant themes representing the voiced concerns and perceived benefits of and barriers to the uptake of interventions, to yield patient-reported outcomes (PROs). PROs were categorized into domains according to the published taxonomy of outcomes in medical research. Of the 89 identified studies, 27 were included, none of which were primarily intended to elicit PROs. A total of 256 PROs and 7 distinct themes were identified. Only 13% of PROs represented physiological or clinical outcomes, whereas 21% represented the core area of emotional functioning or well-being, and 15% represented the area of delivery of care. The most frequently voiced concern was inadequate health care provider support (60%), and women considered intervention-specific challenges to be the greatest barriers to the uptake of interventions (34%). This study synthesized the qualitative evidence of concerns that pregnant women with obesity have regarding their pregnancy and postpartum care, as well as specific barriers they perceive to the uptake of interventions. In addition, this study revealed that clinical outcomes, which are most often reported in clinical trials, comprise only a minority of outcomes considered important by these women. A core outcome set that adequately incorporates PROs is required to inform the conduct of future trials in pregnant women with obesity.

DNA cytosine methylation and methyl-cytosine binding domain (MBD) containing proteins are found throughout all vertebrate species studied to date. However, both the presence of DNA methylation and pattern of methylation varies among invertebrate species. Invertebrates generally have only a single MBD protein, MBD2/3, that does not always contain appropriate residues for selectively binding methylated DNA. Therefore, we sought to determine whether sponges, one of the most ancient extant metazoan lineages, possess an MBD2/3 capable of recognizing methylated DNA and recruiting the associated nucleosome remodeling and deacetylase (NuRD) complex. We find that Ephydatia muelleri has genes for each of the NuRD core components including an EmMBD2/3 that selectively binds methylated DNA. NMR analyses reveal a remarkably conserved binding mode, showing almost identical chemical shift changes between binding to methylated and unmethylated CpG dinucleotides. In addition, we find that EmMBD2/3 and EmGATAD2A/B proteins form a coiled-coil interaction known to be critical for the formation of NuRD. Finally, we show that knockdown of EmMBD2/3 expression disrupts normal cellular architecture and development of E. muelleri. These data support a model in which the MBD2/3 methylation-dependent functional role emerged with the earliest multicellular organisms and has been maintained to varying degrees across animal evolution.

International audience; The Rho associated coiled-coil protein kinase (ROCK) plays crucial roles in development across bilaterian animals. The fact that the Rho/Rock pathway is required to initiate epithelial morphogenesis and thus to establish body plans in bilaterians makes this conserved signaling pathway key for studying the molecular mechanisms that may control early development of basally branching metazoans. The purpose of this study was to evaluate whether or not the main components of this signaling pathway exist in sponges, and if present, to investigate the possible role of the regulatory network in an early branching non-bilaterian species by evaluating ROCK function during Ephydatia muelleri development. Molecular phylogenetic analyses and protein domain predictions revealed the existence of Rho/Rock components in all studied poriferan lineages. Binding assays revealed that both Y-27632 and GSK429286A are capable of inhibiting Em-ROCK activity in vitro. Treatment with both drugs leads to impairment of growth and formation of the basal pinacoderm layer in the developing sponge. Furthermore, inhibition of Em-Rock prevents the establishment of a functional aquiferous system, including the absence of an osculum. In contrast, no effect of ROCK inhibition was observed in juvenile sponges that already possess a fully developed and functional aquiferous system. Thus, the Rho/Rock pathway appears to be essential for the proper development of the freshwater sponge, and may play a role in various cell behaviors (e.g. cell proliferation, cell adhesion and cell motility). Taken together, these data are consistent with an ancestral function of Rho/Rock signaling in playing roles in early developmental processes and may provide a new framework to study the interaction between Wnt signaling and the Rho/Rock pathway. (C) 2016 Elsevier Inc. All rights reserved.

The highly collaborative research sponsored by the NSF-funded Assembling the Porifera Tree of Life (PorToL) project is providing insights into some of the most difficult questions in metazoan systematics. Our understanding of phylogenetic relationships within the phylum Porifera has changed considerably with increased taxon sampling and data from additional molecular markers. PorToL researchers have falsified earlier phylogenetic hypotheses, discovered novel phylogenetic alliances, found phylogenetic homes for enigmatic taxa, and provided a more precise understanding of the evolution of skeletal features, secondary metabolites, body organization, and symbioses. Some of these exciting new discoveries are shared in the papers that form this issue of Integrative and Comparative Biology. Our analyses of over 300 nearly complete 28S ribosomal subunit gene sequences provide specific case studies that illustrate how our dataset confirms new hypotheses of sponge evolution. We recovered monophyletic clades for all 4 classes of sponges, as well as the 4 major clades of Demospongiae (Keratosa, Myxospongiae, Haploscleromorpha, and Heteroscleromorpha), but our phylogeny differs in several aspects from traditional classifications. In most major clades of sponges, families within orders appear to be paraphyletic. Although additional sampling of genes and taxa are needed to establish whether this pattern results from a lack of phylogenetic resolution or from a paraphyletic classification system, many of our results are congruent with those obtained from 18S ribosomal subunit gene sequences and complete mitochondrial genomes. These data provide further support for a revision of the traditional classification of sponges.

Objective The relationship between fatherhood and both psychiatric distress and severity of substance abuse (SA) among men entering SA treatment has not been well explored. This study was designed to (a) examine differences in symptoms of men presenting for SA assessment based on fatherhood status and (b) determine how posttraumatic stress disorder (PTSD) symptoms and severity of SA were associated with parenting for men who were fathers. Methods PTSD symptoms, severity of SA, and parenting data reported on structured questionnaires were collected from 126 men presenting for an SA evaluation at a forensic drug diversion clinic. Results There were no differences in severity of alcohol or drug use between fathers and nonfathers; however, fathers with more PTSD symptoms reported greater severity of alcohol and drug use. Among the fathers, PTSD symptoms correlated significantly and positively with negative parenting behaviors, whereas SA did not. Fathers with more significant PTSD symptoms were more likely to want help with parenting. Conclusions Further exploration of the impact of trauma-related symptoms on the parenting behaviors of substance-abusing men is warranted.