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At least 343 records · Page 19

Analysis of the Phlebiopsis gigantea Genome, Transcriptome and Secretome Provides Insight into Its Pioneer Colonization Strategies of Wood

Collectively classified as white-rot fungi, certain basidiomycetes efficiently degrade the major structural polymers of wood cell walls. A small subset of these Agaricomycetes, exemplified by Phlebiopsis gigantea, is capable of colonizing freshly exposed conifer sapwood despite its high content of extractives, which retards the establishment of other fungal species. The mechanism(s) by which P. gigantea tolerates and metabolizes resinous compounds have not been explored. Here, we report the annotated P. gigantea genome and compare profiles of its transcriptome and secretome when cultured on freshcut versus solvent-extracted loblolly pine wood. The P. gigantea genome contains a conventional repertoire of hydrolase genes involved in cellulose/hemicellulose degradation, whose patterns of expression were relatively unperturbed by the absence of extractives. The expression of genes typically ascribed to lignin degradation was also largely unaffected. In contrast, genes likely involved in the transformation and detoxification of wood extractives were highly induced in its presence. Their products included an ABC transporter, lipases, cytochrome P450s, glutathione S-transferase and aldehyde dehydrogenase. Other regulated genes of unknown function and several constitutively expressed genes are also likely involved in P. gigantea’s extractives metabolism. These results contribute to our fundamental understanding of pioneer colonization of conifer wood and provide insight into the diverse chemistries employed by fungi in carbon cycling processes.

59 BASIC BIOLOGICAL SCIENCES↗

Systems level insights into alternate methane cycling modes in a freshwater lake via community transcriptomics, metabolomics and nano-SIMS analysis

The research conducted as part of this project contributes significantly to the understanding of the microbes and their activities involved in methane metabolism in freshwater lake sediments and in the environment in a more global sense. Significant new insights have been gained into the identity of the species that are most active in methane oxidation. New concepts have been developed based on the new data on how these organisms metabolize methane, impacting not only environmental microbiology but also biotechnology, including biotechnology of next generation biofuels. Novel approaches have been developed for studying functional microbial communities, via holistic approaches, such as metagenomics, metatrancriptomics and metabolite analysis. As a result, a novel outlook has been obtained at how such communities operate in nature. Understanding methane-oxidizing communities in lakes and other environments is of significant benefit to the public, in terms of methane emission mitigation and in terms of potential biotechnological applications.

03 NATURAL GAS↗

Analysis of Plant Pan-Genomes and Transcriptomes with GET_HOMOLOGUES-EST, a Clustering Solution for Sequences of the Same Species

The pan-genome of a species is defined as the union of all the genes and noncoding sequences found in all its individuals. However, constructing a pan-genome for plants with large genomes is daunting both in sequencing cost and the scale of the required computational analysis. A more affordable alternative is to focus on the genic repertoire by using transcriptomic data. Here, the software GET_HOMOLOGUES-EST was benchmarked with genomic and RNA-seq data of 19 Arabidopsis thaliana ecotypes and then applied to the analysis of transcripts from 16 Hordeum vulgare genotypes. The goal was to sample their pan-genomes and classify sequences as core, if detected in all accessions, or accessory, when absent in some of them. The resulting sequence clusters were used to simulate pan-genome growth, and to compile Average Nucleotide Identity matrices that summarize intra-species variation. Although transcripts were found to under-estimate pan-genome size by at least 10%, we concluded that clusters of expressed sequences can recapitulate phylogeny and reproduce two properties observed in A. thaliana gene models: accessory loci show lower expression and higher non-synonymous substitution rates than core genes. Finally, accessory sequences were observed to preferentially encode transposon components in both species, plus disease resistance genes in cultivated barleys, and a variety of protein domains from other families that appear frequently associated with presence/absence variation in the literature. These results demonstrate that pan-genome analyses are useful to explore germplasm diversity.

54 ENVIRONMENTAL SCIENCES↗

A high-resolution single-molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis

Accurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single-molecule long-read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis. We present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 169,000 transcripts—twice that of the best current Arabidopsis transcriptome and including over 1500 novel genes. Seventy-eight percent of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We develop novel methods to determine splice junctions and transcription start and end sites accurately. Mismatch profiles around splice junctions provide a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identify high-confidence transcription start and end sites and remove fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provides higher resolution of transcript expression profiling and identifies cold-induced differential transcription start and polyadenylation site usage. AtRTD3 is the most comprehensive Arabidopsis transcriptome currently. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage analysis from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single-molecule sequencing analysis from any species.

transcription start and end sites↗

Perimatrix of middle ear cholesteatoma: A granulation tissue with a specific transcriptomic signature

Objectives/Hypothesis: To establish comprehensive transcriptomic profiles of cholesteatoma perimatrix tissue and granulation tissue from chronic otitis media (COM) that did not develop cholesteatoma, which can indicate molecular pathways involved in the cholesteatoma perimatrix pathology and invasiveness. Study Design: Retrospective Case Series. Methods: Transcriptome data were obtained from cholesteatoma perimatrix tissue and COM granulation tissue by an Illumina iScan microarray. Differentially expressed genes (DEGs) were subsequently analyzed using both bioinformatical functional annotation and network analysis. Expression of candidate genes (MMP9 and LCN2) was validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) on a larger group of samples. Results: Analysis of the transcriptome led to the identification of 169 differentially expressed genes between investigated tissues. Bioinformatic analysis suggested that most significant biological processes involving DEGs were previously described in cholesteatoma pathology. Network analysis identified ERBB2, TFAP2A, and TP63 as major hubs of the DEGs molecular network. Furthermore, it was observed that the cellular component most significantly enriched in DEGs was extracellular space containing 47 DEGs. Using qRT-PCR, it was confirmed that mRNA levels of the major extracellular hub (MMP9) are increased, whereas its interacting molecule (LCN2) mRNA levels were decreased in cholesteatoma perimatrix tissue compared to COM granulation tissue. Conclusions: The current study approach offers an overall look at molecular mechanisms that describe the cholesteatoma entity by focusing exclusively on the perimatrix processes in comparison to COM granulation tissue. The observed differences in gene expression between cholesteatoma perimatrix and COM granulation tissue could suggest novel markers potentially influenced by the perimatrix–matrix molecular interplay, which is not present in COM without cholesteatoma. Level of Evidence: NA. Laryngoscope, 130:E220–E227, 2020. © 2019 The American Laryngological, Rhinological and Otological Society, Inc.

60 APPLIED LIFE SCIENCES↗

Pairwise comparisons of ten porcine tissues identify differential transcriptional regulation at the gene, isoform, promoter and transcription start site level

Highlights: •Transcriptome sequencing yielded 223 mill porcine RNA-seq reads, and 59,000 transcribed locations. •Establishment of unique transcription profiles for ten porcine tissues including four brain tissues. •Comparison of transcription profiles at gene, isoform, promoter and transcription start site level. •Highlights a high level of regulation of neuro-related genes at both gene, isoform, and TSS level. •Our results emphasize the pig as a valuable animal model with respect to human biological issues. -- Abstract: The transcriptome is the absolute set of transcripts in a tissue or cell at the time of sampling. In this study RNA-Seq is employed to enable the differential analysis of the transcriptome profile for ten porcine tissues in order to evaluate differences between the tissues at the gene and isoform expression level, together with an analysis of variation in transcription start sites, promoter usage, and splicing. Totally, 223 million RNA fragments were sequenced leading to the identification of 59,930 transcribed gene locations and 290,936 transcript variants using Cufflinks with similarity to approximately 13,899 annotated human genes. Pairwise analysis of tissues for differential expression at the gene level showed that the smallest differences were between tissues originating from the porcine brain. Interestingly, the relative level of differential expression at the isoform level did generally not vary between tissue contrasts. Furthermore, analysis of differential promoter usage between tissues, revealed a proportionally higher variation between cerebellum (CBE) versus frontal cortex and cerebellum versus hypothalamus (HYP) than in the remaining comparisons. In addition, the comparison of differential transcription start sites showed that the number of these sites is generally increased in comparisons including hypothalamus in contrast to other pairwise assessments. A comprehensive analysis of one of the tissue contrasts, i.e. cerebellum versus heart for differential variation at the gene, isoform, and transcription start site (TSS), and promoter level showed that several of the genes differed at all four levels. Interestingly, these genes were mainly annotated to the “electron transport chain” and neuronal differentiation, emphasizing that “tissue important” genes are regulated at several levels. Furthermore, our analysis shows that the “across tissue approach” has a promising potential when screening for possible explanations for variations, such as those observed at the gene expression levels.

60 APPLIED LIFE SCIENCES↗

Analysis of Global Gene Expression in Brachypodium distachyon Reveals Extensive Network Plasticity in Response to Abiotic Stress

Brachypodium distachyon is a close relative of many important cereal crops. Abiotic stress tolerance has a significant impact on productivity of agriculturally important food and feedstock crops. Analysis of the transcriptome of Brachypodium after chilling, high-salinity, drought, and heat stresses revealed diverse differential expression of many transcripts. Weighted Gene Co-Expression Network Analysis revealed 22 distinct gene modules with specific profiles of expression under each stress. Promoter analysis implicated short DNA sequences directly upstream of module members in the regulation of 21 of 22 modules. Functional analysis of module members revealed enrichment in functional terms for 10 of 22 network modules. Analysis of condition-specific correlations between differentially expressed gene pairs revealed extensive plasticity in the expression relationships of gene pairs. Photosynthesis, cell cycle, and cell wall expression modules were down-regulated by all abiotic stresses. Modules which were up-regulated by each abiotic stress fell into diverse and unique gene ontology GO categories. This study provides genomics resources and improves our understanding of abiotic stress responses of Brachypodium.

59 BASIC BIOLOGICAL SCIENCES↗

Transcriptome-driven integrative exploration of functional state of ureter tissue affected by CAKUT

Aims: (1) to identify the most dysregulated genes in ureter tissue affected by congenital anomalies of the kidney and urinary tract (CAKUT) and to extract the biological meaning of these markers; (2) to describe the key molecular networks in CAKUT and to provide expression validation of the genes selected from these networks. Main methods: Transcriptome data was obtained from ureter samples of CAKUT patients and controls by Illumina iScan microarray. Identification of differentially expressed genes was coupled with subsequent bioinformatics analyses. Expression of candidate genes was validated by qRT-PCR. Key findings: Analysis of the transcriptome led to the identification of 78 commonly dysregulated genes in CAKUT tissue compared to controls. Integrative bioinformatic analyses of differentially expressed genes identified 7 major networks. The targets for qRT-PCR validation were selected as members of the major molecular networks in CAKUT, which had both, the significant high fold change and biological relevance for CAKUT. By qRT-PCR the substantial increase of LCN2, PROM1, SOSTDC1, and decrease of INA, RASD1 and TAC3 mRNA levels was confirmed. Significance: Since CAKUT is a leading cause of end-stage renal disease in children, the search for molecular targets for postnatal therapy is of particular interest. Data described in this study represents the gene expression profile and significant molecular networks specific to human ureter affected by CAKUT. The discovery of impaired molecular factors and processes is the step towards the uncovering of the key mechanisms that reflect CAKUT postnatally and could lead to the affected tissue deterioration and end organ damage. © 2018 Elsevier Inc.

60 APPLIED LIFE SCIENCES↗

Creb5 controls its own expression and directly induces the joint interzone regulatory program

Prior studies have indicated that the transcription factor Creb5 is expressed in the joint interzone, which contains the progenitors for all synovial joint tissues in both mouse and human embryos. In the absence of Creb5 function, most synovial joint interzones fail to form and the cartilage templates in the long bones remain fused. This earlier work did not clarify whether Creb5 initiates a cascade of signaling molecules, such as growth and differentiation factor 5 (Gdf5) and Wnt-family members, that in turn induce the formation of the joint interzone, or instead directly activates the expression of joint interzone markers. In the present study, an integrative analysis of the transcriptome, chromatin accessibility, and Creb5-occupancy in joint progenitors revealed that Creb5 directly binds to both its own two promoters and to the regulatory regions of Gdf5 and Sfrp2, each of whose expression in the joint interzone is Creb5-dependent. Functional enhancer analysis indicated that Creb5 binding sites in either the two Creb5 promoters, or in Gdf5 and Sfrp2 regulatory elements are necessary for these sequences to drive transgene expression in the developing synovial joints. While Creb5 directly drives Gdf5 and Sfrp2 expression in the inner joint interzone, Creb5 activates Barx1 expression specifically in the outer joint interzone. Our findings indicate that Creb5 initiates a regulatory network that both promotes the formation of synovial joints, and subsequently activates distinct transcriptional targets in the inner versus the outer regions of the joint interzone, thus regionalizing gene expression in the developing joint.

Zhang, Cheng-Hai↗

High-throughput Single-Cell Proteomics and Transcriptomics from the Same Cells with a Nanoliter-Scale Spin-Transfer Approach

Single-cell multiomic platforms provide a comprehensive snapshot of cellular states and cell types by offering critical insights into the spatiotemporal regulation of biomolecular networks at a systems level, thereby defining the basis of multicellularity. Here, we introduce nanoSPINS, an advanced platform that enables high-throughput profiling and integrative analysis of the transcriptome and proteome from the same single cells using RNA sequencing and isobaric labeling LC-MS-based proteomics, respectively. NanoSPINS can efficiently transfer mRNA-containing droplets across two microarrays via a centrifugation-based approach, while proteins are retained on the initial platform. Benchmarking of nanoSPINS on two cell lines demonstrates its ability to generate global proteomic and transcriptomic profiles that align well with previously established methodologies/platforms. The incorporation of isobaric TMTpro labeling into this single-cell multiomics platform significantly enhances the throughput of single-cell proteomic analyses. Through the high-throughput quantification of the proteome and transcriptome, nanoSPINS not only facilitates the identification of molecular features at both mRNA and protein level but also provides larger sample sizes for improved statistical power in clustering and differential abundance. Given the broad applicability of single-cell multiomics in biological research and clinical settings, we believe nanoSPINS represents a powerful platform for the characterization of heterogeneous cell populations.

multi 'omics↗

De novo transcriptome sequencing of Capsicum frutescens . L and comprehensive analysis of salt stress alleviating mechanism by Bacillus atrophaeus WU ‐9

Abstract Salt stress, as one of the most severe environmental stresses, can cause a series of changes in plants. However, the explanation of plant salt stress alleviating mechanism of plant growth–promoting rhizobacteria (PGPR) was hindered by the limited availability of transcriptomic information for salt stress‐treated plants grown in a microorganism‐controlled environment. Our previous reports have selected Bacillus atrophaeus WU‐9 as PGPR significantly alleviating pepper ( Capsicum frutescens . L) salt stress. In this work, the RNA‐seq analysis of salt stress‐treated and untreated plants, grown with and without WU‐9 in a microorganism‐controlled environment, was used to reveal the plant salt stress alleviating mechanisms of WU‐9. Twelve sequencing libraries, prepared by treating with WU‐9 and salt (150 mM NaCl for 36 h), were constructed by RNA‐Seq technique. Non‐inoculated seedlings mainly respond to salt stress through regulation of signal transduction, such as ethylene‐activated signaling pathway, signaling and cell communication, etc. And ethylene signal participated in salt stress response in pepper through regulating defense responses, fruit ripening and senescence. WU‐9 inoculation under salt stress mainly improves salt tolerance and plant growth by regulating salt stress‐responding ethylene and auxin signal transduction, utilization of proline, photosynthesis, antioxidant enzyme activities and cell enlargement. Furthermore, 86 differentially expressed genes and 20 transcription factors were identified as associated with salt stress response and tolerance. Thus, this innovative transcriptomic study identified the salt stress response and alleviation in C. frutescens . L with PGPR inoculation. This result provided novel insights into the salinity alleviation in pepper regulated by PGPR.

Wang, Wenfei↗

Spatial co-transcriptomics reveals discrete stages of the arbuscular mycorrhizal symbiosis

Abstract The symbiotic interaction of plants with arbuscular mycorrhizal (AM) fungi is ancient and widespread. Plants provide AM fungi with carbon in exchange for nutrients and water, making this interaction a prime target for crop improvement. However, plant–fungal interactions are restricted to a small subset of root cells, precluding the application of most conventional functional genomic techniques to study the molecular bases of these interactions. Here we used single-nucleus and spatial RNA sequencing to explore both Medicago truncatula and Rhizophagus irregularis transcriptomes in AM symbiosis at cellular and spatial resolution. Integrated, spatially registered single-cell maps revealed infected and uninfected plant root cell types. We observed that cortex cells exhibit distinct transcriptome profiles during different stages of colonization by AM fungi, indicating dynamic interplay between both organisms during establishment of the cellular interface enabling successful symbiosis. Our study provides insight into a symbiotic relationship of major agricultural and environmental importance and demonstrates a paradigm combining single-cell and spatial transcriptomics for the analysis of complex organismal interactions.

59 BASIC BIOLOGICAL SCIENCES↗

Spatial profiling of the interplay between cell type- and vision-dependent transcriptomic programs in the visual cortex

How early sensory experience during “critical periods” of postnatal life affects the organization of the mammalian neocortex at the resolution of neuronal cell types is poorly understood. We previously reported that the functional and molecular profiles of layer 2/3 (L2/3) cell types in the primary visual cortex (V1) are vision-dependent [S. Chenget al.,Cell185, 311–327.e24 (2022)]. Here, we characterize the spatial organization of L2/3 cell types with and without visual experience. Spatial transcriptomic profiling based on 500 genes recapitulates the zonation of L2/3 cell types along the pial–ventricular axis in V1. By applying multitasking theory, we suggest that the spatial zonation of L2/3 cell types is linked to the continuous nature of their gene expression profiles, which can be represented as a 2D manifold bounded by three archetypal cell types. By comparing normally reared and dark reared L2/3 cells, we show that visual deprivation-induced transcriptomic changes comprise two independent gene programs. The first, induced specifically in the visual cortex, includes immediate-early genes and genes associated with metabolic processes. It manifests as a change in cell state that is orthogonal to cell-type-specific gene expression programs. By contrast, the second program impacts L2/3 cell-type identity, regulating a subset of cell-type-specific genes and shifting the distribution of cells within the L2/3 cell-type manifold. Through an integrated analysis of spatial transcriptomics with single-nucleus RNA-seq data, we describe how vision patterns cortical L2/3 cell types during the critical period.

Science & Technology - Other Topics↗

Metabolic flux, metabolite, and transcript analysis uncover reprogramming of metabolism toward higher seed oil

Overexpression of WRINKLED1 (WRI1), a master regulator of glycolysis and fatty acid biosynthesis, together with DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1), which catalyzes the final step of triacylglycerol assembly, is a promising strategy for enhancing seed oil content. However, how these regulators coordinate system-wide metabolic reprogramming at the levels of gene expression, metabolite pools, and fluxes remains poorly understood. To address this, we performed 13 C-metabolic flux analysis, metabolomics, and transcriptomics on in vitro cultured pennycress (Thlaspi arvense L.) embryos overexpressing the native WRI1 and DGAT1 homologs. Here, in cultured embryos, WRI1/DGAT1 overexpression increased triacylglycerol accumulation by 28% while reducing protein content by 34%, relative to the wild type. Embryos showed ∼20-fold and 50-fold upregulation of WRI1 and DGAT1 along with induction of WRI1 target genes in glycolysis and fatty acid biosynthesis. Genes associated with photosynthesis and Calvin cycle functions were also upregulated, whereas genes encoding ribosomal proteins and seed storage proteins were strongly repressed, consistent with the observed lipid–protein tradeoff. Flux analysis revealed that enhanced triacylglycerol biosynthesis is supported by increased flux through the Rubisco shunt and cytosolic pyruvate kinase, while the oxidative pentose phosphate pathway and malic enzyme contributed little to NADPH or pyruvate supply. Metabolomic profiling revealed extensive perturbations in glycolytic intermediates, tricarboxylic acid cycle metabolites, and amino acids. In plant grown seeds, WRI1/DGAT1 lines also showed a modest but significant increase in total lipid content. Collectively, these findings reveal how WRI1 and DGAT1 reprogram central metabolism to enhance oil accumulation, with relevance to mature seeds.

59 BASIC BIOLOGICAL SCIENCES↗

Investigating the role of the transcriptional regulator Ure2 on the metabolism of Saccharomyces cerevisiae: a multi-omics approach

Ure2 regulates nitrogen catabolite repression in Saccharomyces cerevisiae. Deletion of URE2 induces a physiological state mimicking the nitrogen starvation and autophagic responses. Previous work has shown that deletion of URE2 increases the fermentation rate of some wine-producing strains of S. cerevisiae. In this work, we investigated the effect of URE2 deletion (ΔURE2) on the metabolism of S. cerevisiae. During growth on glucose, the ΔURE2 mutant grew less well than the wild type; however, it produced ethanol at higher rates. To better under the behavior of this mutant, we performed transcriptomics and metabolomics. Analysis of the RNA sequencing results and metabolite levels indicate that the mutant strain exhibited characteristics of both nitrogen starvation and autophagy. In addition, many pyruvate decarboxylase and alcohol dehydrogenase isoforms were expressed at higher rates than the wild type. The mutant also accumulated less trehalose and glycogen, and produced more lipids. These results suggest that URE2 may be a promising target for metabolic engineering in S. cerevisiae and potentially other strains of yeast as well for the production of lipid-based fuels and chemicals.

59 BASIC BIOLOGICAL SCIENCES↗

Multi-omic data integration enables discovery of hidden biological regularities

Rapid growth in size and complexity of biological data sets has led to the ‘Big Data to Knowledge’ challenge. We develop advanced data integration methods for multi-level analysis of genomic, transcriptomic, ribosomal profiling, proteomic and fluxomic data. First, we show that pairwise integration of primary omics data reveals regularities that tie cellular processes together in Escherichia coli: the number of protein molecules made per mRNA transcript and the number of ribosomes required per translated protein molecule. Second, we show that genome-scale models, based on genomic and bibliomic data, enable quantitative synchronization of disparate data types. Integrating omics data with models enabled the discovery of two novel regularities: condition invariant in vivo turnover rates of enzymes and the correlation of protein structural motifs and translational pausing. These regularities can be formally represented in a computable format allowing for coherent interpretation and prediction of fitness and selection that underlies cellular physiology.

59 BASIC BIOLOGICAL SCIENCES↗