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Recent microbiome research has incorporated a higher number of samples through more participants in a study, longitudinal studies, and metanalysis between studies. Physical limitations in a sequencing machine can result in samples spread across sequencing runs. Here we present the results of sequencing nearly 1000 16S rRNA gene sequences in fecal (stabilized and swab) and oral (swab) samples from multiple human microbiome studies and positive controls that were conducted with identical standard operating procedures. Sequencing was performed in the same center across 18 different runs. The simplified mock community showed limitations in accuracy, while precision (e.g., technical variation) was robust for the mock community and actual human positive control samples. Technical variation was the lowest for stabilized fecal samples, followed by fecal swab samples, and then oral swab samples. The order of technical variation stability was inverse of DNA concentrations (e.g., highest in stabilized fecal samples), highlighting the importance of DNA concentration in reproducibility and urging caution when analyzing low biomass samples. Coefficients of variation at the genus level also followed the same trend for lower variation with higher DNA concentrations. Technical variation across both sample types and the two human sampling locations was significantly less than the observed biological variation. Overall, this research providing comparisons between technical and biological variation, highlights the importance of using positive controls, and provides semi-quantified data to better understand variation introduced by sequencing runs. KEY POINTS: ⢠Mock community and positive control accuracy were lower than precision. ⢠Samples with lower DNA concentration had increased technical variation across sequencing runs. ⢠Biological variation was significantly higher than technical variation due to sequencing runs.
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DNA Bacteriano , Fezes , Microbiota , RNA Ribossômico 16S , Análise de Sequência de DNA , Humanos , RNA Ribossômico 16S/genética , Fezes/microbiologia , Microbiota/genética , Análise de Sequência de DNA/métodos , DNA Bacteriano/genética , Bactérias/genética , Bactérias/classificação , Bactérias/isolamento & purificação , Reprodutibilidade dos Testes , Boca/microbiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
INTRODUCTION: Emery-Dreifuss muscular dystrophy (EDMD) is a disease characterized by skeletal muscle wasting, major tendon contractures, and cardiac conduction defects. Mutations in the gene encoding emerin cause EDMD1. Our previous studies suggested that emerin activation of histone deacetylase 3 (HDAC3) to reduce histone 4-lysine 5 (H4K5) acetylation (ac) is important for myogenic differentiation. METHODS: Pharmacological inhibitors (Nu9056, L002) of histone acetyltransferases targeting acetylated H4K5 were used to test whether increased acetylated H4K5 was responsible for the impaired differentiation seen in emerin-deficient myogenic progenitors. RESULTS: Nu9056 and L002 rescued impaired differentiation in emerin deficiency. SRT1720, which inhibits the nicotinamide adenine dinucleotide (NAD)+ -dependent deacetylase sirtuin 1 (SIRT1), failed to rescue myotube formation. DISCUSSION: We conclude that emerin regulation of HDAC3 activity to affect H4K5 acetylation dynamics is important for myogenic differentiation. Targeting H4K5ac dynamics represents a potential new strategy for ameliorating the skeletal muscle wasting seen in EDMD1.
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Diferenciação Celular/efeitos dos fármacos , Histona Acetiltransferases/antagonistas & inibidores , Distrofia Muscular de Emery-Dreifuss/tratamento farmacológico , Distrofia Muscular de Emery-Dreifuss/patologia , Células-Tronco/efeitos dos fármacos , Tiazóis/uso terapêutico , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Histona Acetiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco/patologia , Tiazóis/farmacologiaRESUMO
The mammalian dorsal root ganglia (DRG) are located on the dorsal roots of the spinal nerves and contain cell bodies of primary sensory neurons. DRG cells have been classified into subpopulations based on their size, morphology, intracellular markers, response to stimuli, and neuropeptides. To understand the connections between DRG chemical heterogeneity and cellular function, we performed optically guided, high-throughput single cell profiling using sequential matrix-assisted laser desorption/ionization mass spectrometry (MS) to detect lipids, peptides, and several proteins in individual DRG cells. Statistical analysis of the resulting mass spectra allows stratification of the DRG population according to cellular morphology and, presumably, major cell types. A subpopulation of small cells contained myelin proteins, which are abundant in Schwann cells, and mass spectra of several larger cells contained peaks matching neurofilament, vimentin, myelin basic protein S, and thymosin beta proteins. Of the over 1000 cells analyzed, approximately 78 % produced putative peptide-rich spectra, allowing the population to be classified into three distinct cell types. Two signals with m/z 4404 and 5487 were exclusively observed in a cell type, but could not be matched to results of our previous liquid chromatography-MS analyses.
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Gânglios Espinais/química , Lipídeos/análise , Peptídeos/análise , Proteínas/análise , Análise de Célula Única , Animais , Masculino , Espectrometria de Massas , Ratos , Ratos Sprague-DawleyRESUMO
Learning about a moving visual stimulus was examined in zebrafish larvae using an automated imaging system and a t1-t2 design. In three experiments, zebrafish larvae were exposed to one of two inputs at t1 (either a gray bouncing disk or an identical but stationary disk) followed by a common test at t2 (the gray bouncing disk). Using 7days post-fertilization (dpf) larvae and 12 stimulus exposures, Experiment 1 established that these different treatments produced differential responding to the moving disk during testing. Larvae familiar with the moving test stimulus were significantly less likely to be still in its presence than larvae that had been exposed to the identical but stationary stimulus. Experiment 2 confirmed this result in 7dpf larvae and extended the finding to 5 and 6dpf larvae. Experiment 3 found differential responding to the moving test stimulus with 4 or 8 stimulus exposures but not with just one exposure in 7dpf larvae. These results provide evidence for learning in very young zebrafish larvae. The merits and challenges of the t1-t2 framework to study learning are discussed.
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Aprendizagem da Esquiva , Aprendizagem , Animais , Comportamento Animal , Larva , Atividade Motora , Estimulação Luminosa , Fatores de Tempo , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Background and Aims: No recovery related surveillance system exists but given the evidence of effectiveness and growing supply, a house- and resident- level recovery house (RH) surveillance system could be beneficial for data collection on recovery support service (RSS) engagement, and retention; for improved standardization of RH programs and services; and for identification of outcomes associated with long-term recovery. Methods: This study aimed to explore current data collection practices at the resident- and house- level through qualitative focus interviews of RH representatives. The 13 RH interviews were scheduled with 16 RH representative respondents. Results: The most frequently collected resident data was at entry (92%) and departure (85%) and included demographics (n = 5), substance use history (n = 6), treatment and recovery history (n = 5), legal and correctional history (n = 6) and mental health information (n = 7). Recovery support data was collected by 85% of houses. Post-stay data was only collected by four RHs (31%). Conclusion: These results indicate that there is a lack of standardized systematic collection, analysis, and reporting of recovery related data in the RH field. A recovery related surveillance system has the potential to fill this gap and inform and improve standard of resident care to support long-term recovery from substance use disorder.
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Alternative splicing (AS) of Exon 11 of the Insulin Receptor ( INSR ) is highly regulated and disrupted in several human disorders. To better understand INSR exon 11 AS regulation, splicing activity of an INSR exon 11 minigene reporter was measured across a gradient of the AS regulator muscleblind-like 1 protein (MBNL1). The RNA-binding protein Fox-1 (RBFOX1) was added to determine its impact on MBNL1-regulated splicing. The role of the RBFOX1 UGCAUG binding site within intron 11 was assessed across the MBNL1 gradient. Mutating the UGCAUG motif inhibited RBFOX1 regulation of exon 11 and had the unexpected effect of reducing MBNL1 regulation of this exon. Molecular dynamics simulations showed that exon 11 and the adjacent RNA adopts a dynamically stable conformation. Mutation of the RBFOX1 binding site altered RNA structure and dynamics, while a mutation that created an optimal MBNL1 binding site at the RBFOX1 site shifted the RNA back to wild type. An antisense oligonucleotide (ASO) was used to confirm the structure in this region of the pre-mRNA. This example of intronic mutations shifting pre-mRNA structure and dynamics to modulate splicing suggests RNA structure and dynamics should be taken into consideration for AS regulation and therapeutic interventions targeting pre-mRNA.
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Purpose: Traditional CT acquisition planning is based on scout projection images from planar anterior-posterior and lateral projections where the radiographer estimates organ locations. Alternatively, a new scout method utilizing ultra-low dose helical CT (3D Landmark Scan) offers cross-sectional imaging to identify anatomic structures in conjunction with artificial intelligence based Anatomic Landmark Detection (ALD) for automatic CT acquisition planning. The purpose of this study is to quantify changes in scan length and radiation dose of CT examinations planned using 3D Landmark Scan and ALD and performed on next generation wide volume CT versus examinations planned using traditional scout methods. We additionally aim to quantify changes in radiation dose reduction of scans planned with 3D Landmark Scan and performed on next generation wide volume CT. Methods: Single-center retrospective analysis of consecutive patients with prior CT scan of the same organ who underwent clinical CT using 3D Landmark Scan and automatic scan planning. Acquisition length and dose-length-product (DLP) were collected. Data was analyzed by paired t-tests. Results: 104 total CT examinations (48.1â¯% chest, 15.4â¯% abdomen, 36.5â¯% chest/abdomen/pelvis) on 61 individual consecutive patients at a single center were retrospectively analyzed. 79.8â¯% of scans using 3D Landmark Scan had reduction in acquisition length compared to the respective prior acquisition. Median acquisition length using 3D Landmark Scan was 26.7â¯mm shorter than that using traditional scout methods (p < 0.001) with a 23.3â¯% median total radiation dose reduction (245.6 (IQR 150.0-400.8) mGy cm vs 320.3 (IQR 184.1-547.9) mGy cm). CT dose index similarly was overall decreased for scans planned with 3D Landmark and ALD and performed on next generation CT versus traditional methods (4.85 (IQR 3.8-7) mGy vs. 6.70 (IQR 4.43-9.18) mGy, respectively, p < 0.001). Conclusion: Scout imaging using reduced dose 3D Landmark Scan images and Anatomic Landmark Detection reduces acquisition range in chest, abdomen, and chest/abdomen/pelvis CT scans. This technology, in combination with next generation wide volume CT reduces total radiation dose.
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Alternative splicing (AS) is a dynamic RNA processing step that produces multiple RNA isoforms from a single pre-mRNA transcript and contributes to the complexity of the cellular transcriptome and proteome. This process is regulated through a network of cis-regulatory sequence elements and trans-acting factors, most-notably RNA binding proteins (RBPs). The muscleblind-like (MBNL) and RNA binding fox-1 homolog (RBFOX) are two well characterized families of RBPs that regulate fetal to adult AS transitions critical for proper muscle, heart, and central nervous system development. To better understand how the concentration of these RBPs influences AS transcriptome wide, we engineered a MBNL1 and RBFOX1 inducible HEK-293 cell line. Modest induction of exogenous RBFOX1 in this cell line modulated MBNL1-dependent AS outcomes in 3 skipped exon events, despite significant levels of endogenous RBFOX1 and RBFOX2. Due to background RBFOX levels, we conducted a focused analysis of dose-dependent MBNL1 skipped exon AS outcomes and generated transcriptome wide dose-response curves. Analysis of this data demonstrates that MBNL1-regulated exclusion events may require higher concentrations of MBNL1 protein to properly regulate AS outcomes compared to inclusion events and that multiple arrangements of YGCY motifs can produce similar splicing outcomes. These results suggest that rather than a simple relationship between the organization of RBP binding sites and a specific splicing outcome, that complex interaction networks govern both AS inclusion and exclusion events across a RBP gradient.
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Processamento Alternativo , Proteínas de Ligação a RNA , Humanos , Processamento Alternativo/genética , Células HEK293 , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Splicing de RNA , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Proteínas Repressoras/genéticaRESUMO
Economically viable production of cellulosic biofuels requires operation at high solids loadings-on the order of 15 wt%. To this end we characterize Nature's ability to deconstruct and utilize mid-season switchgrass at increasing solid loadings using an anaerobic methanogenic microbiome. This community exhibits undiminished fractional carbohydrate solubilization at loadings ranging from 30 g/L to 150 g/L. Metaproteomic interrogation reveals marked increases in the abundance of specific carbohydrate-active enzyme classes. Significant enrichment of auxiliary activity family 6 enzymes at higher solids suggests a role for Fenton chemistry. Stress-response proteins accompanying these reactions are similarly upregulated at higher solids, as are ß-glucosidases, xylosidases, carbohydrate-debranching, and pectin-acting enzymes-all of which indicate that removal of deconstruction inhibitors is important for observed undiminished solubilization. Our work provides insights into the mechanisms by which natural microbiomes effectively deconstruct and utilize lignocellulose at high solids loadings, informing the future development of defined cultures for efficient bioconversion.
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Lignina , Microbiota , Anaerobiose , Carboidratos , Lignina/metabolismoRESUMO
The quantification of lipopolysaccharide (LPS) shed by bacteria within aqueous samples is typically performed by binding LPS to a protein called Factor C within a lysate prepared from the blood of horseshoe crabs (Limulus amebocyte lysate (LAL)). How the state of aggregation of LPS impacts Factor C activation, however, is not understood, particularly in the presence of select salts and non-ionic surfactants that are commonly incorporated into pharmaceutical formulations. To address this open question, herein we report on the aggregation status of LPS in aqueous solution, characterized using angle-dependent static and dynamic light scattering with and without chelating salts and polysorbate surfactants, and its correlation with activation of Factor C. Because the aggregation status of LPS is kinetically controlled, care was taken to compare LPS aggregation and activity using identically prepared samples. By plotting LPS activity versus the LPS aggregate size distribution over varied solution conditions, we found a positive correlation between LPS aggregate sizes between 30 and 50â¯nm and LAL activity. Overall, our results support the hypothesis that activation of Factor C is dependent of LPS aggregate size, and that the modulating effects of salts and surfactants on activation of Factor C is associated with changes in the LPS aggregation.
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Proteínas de Artrópodes/antagonistas & inibidores , Precursores Enzimáticos/antagonistas & inibidores , Lipopolissacarídeos/química , Lipopolissacarídeos/farmacologia , Proteínas de Artrópodes/metabolismo , Precursores Enzimáticos/metabolismo , Escherichia coli/química , Lipopolissacarídeos/antagonistas & inibidores , Estrutura Molecular , Tamanho da Partícula , Serina Endopeptidases/metabolismo , Propriedades de Superfície , Tensoativos/química , Tensoativos/farmacologia , Água/químicaRESUMO
Microbial community behavior is coupled to a set of genetically-regulated chemical signals that correlate with cell density - the quorum sensing (QS) system - and there is growing appreciation that the QS-regulated behavior of bacteria is chemically, spatially, and temporally complex. In addition, while it has been known for some time that different species use different QS networks, we are beginning to appreciate that different strains of the same bacterial species also differ in their QS networks. Here we combine mass spectrometric imaging (MSI) and confocal Raman microscopy (CRM) approaches to investigate co-cultures involving different strains (FRD1 and PAO1C) of the same species (Pseudomonas aeruginosa) as well as those involving different species (P. aeruginosa and E. coli). Combining MSI and CRM makes it possible to supersede the limits imposed by individual imaging approaches and enables the spatial mapping of individual bacterial species and their microbial products within a mixed bacterial community growing in situ on surfaces. MSI is used to delineate the secretion of a specific rhamnolipid surfactant as well as alkyl quinolone (AQ) messengers between FRD1 and PAO1C strains of P. aeruginosa, showing that the spatial distribution and production rate of AQ messengers in PAO1C far outstrips that of FRD1. In the case of multiple species, CRM is used to show that the prolific secretion of AQs by the PAO1C strain of P. aeruginosa is used to mediate its interaction with co-cultured E. coli.
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Mutations in the gene encoding emerin cause Emery-Dreifuss muscular dystrophy (EDMD). Emerin is an integral inner nuclear membrane protein and a component of the nuclear lamina. EDMD is characterized by skeletal muscle wasting, cardiac conduction defects and tendon contractures. The failure to regenerate skeletal muscle is predicted to contribute to the skeletal muscle pathology of EDMD. We hypothesize that muscle regeneration defects are caused by impaired muscle stem cell differentiation. Myogenic progenitors derived from emerin-null mice were used to confirm their impaired differentiation and analyze selected myogenic molecular pathways. Emerin-null progenitors were delayed in their cell cycle exit, had decreased myosin heavy chain (MyHC) expression and formed fewer myotubes. Emerin binds to and activates histone deacetylase 3 (HDAC3). Here, we show that theophylline, an HDAC3-specific activator, improved myotube formation in emerin-null cells. Addition of the HDAC3-specific inhibitor RGFP966 blocked myotube formation and MyHC expression in wild-type and emerin-null myogenic progenitors, but did not affect cell cycle exit. Downregulation of emerin was previously shown to affect the p38 MAPK and ERK/MAPK pathways in C2C12 myoblast differentiation. Using a pure population of myogenic progenitors completely lacking emerin expression, we show that these pathways are also disrupted. ERK inhibition improved MyHC expression in emerin-null cells, but failed to rescue myotube formation or cell cycle exit. Inhibition of p38 MAPK prevented differentiation in both wild-type and emerin-null progenitors. These results show that each of these molecular pathways specifically regulates a particular stage of myogenic differentiation in an emerin-dependent manner. Thus, pharmacological targeting of multiple pathways acting at specific differentiation stages may be a better therapeutic approach in the future to rescue muscle regeneration in vivo.
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Diferenciação Celular , Histona Desacetilases/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/metabolismo , Desenvolvimento Muscular , Proteínas Nucleares/metabolismo , Células-Tronco/metabolismo , Acetilação , Animais , Butadienos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Flavonoides/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Histonas/metabolismo , Imidazóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Modelos Biológicos , Desenvolvimento Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Nitrilas/farmacologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Células-Tronco/efeitos dos fármacos , Teofilina/farmacologiaRESUMO
Extremely low birth weight (ELBW) infants have high morbidity and mortality, frequently due to invasive infections from bacteria, fungi, and viruses. The microbial communities present in the gastrointestinal tracts of preterm infants may serve as a reservoir for invasive organisms and remain poorly characterized. We used deep pyrosequencing to examine the gut-associated microbiome of 11 ELBW infants in the first postnatal month, with a first time determination of the eukaryote microbiota such as fungi and nematodes, including bacteria and viruses that have not been previously described. Among the fungi observed, Candida sp. and Clavispora sp. dominated the sequences, but a range of environmental molds were also observed. Surprisingly, seventy-one percent of the infant fecal samples tested contained ribosomal sequences corresponding to the parasitic organism Trichinella. Ribosomal DNA sequences for the roundworm symbiont Xenorhabdus accompanied these sequences in the infant with the greatest proportion of Trichinella sequences. When examining ribosomal DNA sequences in aggregate, Enterobacteriales, Pseudomonas, Staphylococcus, and Enterococcus were the most abundant bacterial taxa in a low diversity bacterial community (mean Shannon-Weaver Index of 1.02 ± 0.69), with relatively little change within individual infants through time. To supplement the ribosomal sequence data, shotgun sequencing was performed on DNA from multiple displacement amplification (MDA) of total fecal genomic DNA from two infants. In addition to the organisms mentioned previously, the metagenome also revealed sequences for gram positive and gram negative bacteriophages, as well as human adenovirus C. Together, these data reveal surprising eukaryotic and viral microbial diversity in ELBW enteric microbiota dominated bytypes of bacteria known to cause invasive disease in these infants.