ResearchPad - gene-types https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[A tale of textiles: Genetic characterization of historical paper mulberry barkcloth from Oceania]]> https://www.researchpad.co/article/elastic_article_15748 Humans introduced paper mulberry (Broussonetia papyrifera) from Taiwan into the Pacific over 5000 years ago as a fiber source to make barkcloth textiles that were, and still are, important cultural artifacts throughout the Pacific. We have used B. papyrifera, a species closely associated to humans, as a proxy to understand the human settlement of the Pacific Islands. We report the first genetic analysis of paper mulberry textiles from historical and archaeological contexts (200 to 50 years before present) and compare our results with genetic data obtained from contemporary and herbarium paper mulberry samples. Following stringent ancient DNA protocols, we extracted DNA from 13 barkcloth textiles. We confirmed that the fiber source is paper mulberry in nine of the 13 textiles studied using the nuclear ITS-1 marker and by statistical estimates. We detected high genetic diversity in historical Pacific paper mulberry barkcloth with a set of ten microsatellites, showing new alleles and specific genetic patterns. These genetic signatures allow tracing connections to plants from the Asian homeland, Near and Remote Oceania, establishing links not observed previously (using the same genetic tools) in extant plants or herbaria samples. These results show that historic barkcloth textiles are cultural materials amenable to genetic analysis to reveal human history and that these artifacts may harbor evidence of greater genetic diversity in Pacific B. papyrifera in the past.

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<![CDATA[Fine-scale population genetic structure of dengue mosquito vector, <i>Aedes aegypti</i>, in Metropolitan Manila, Philippines]]> https://www.researchpad.co/article/elastic_article_14656 Aedes aegypti is an efficient vector of dengue due to its highly adaptive nature to the urban environment. Although it is observed to have a short dispersal (active) capability, it has been shown to be capable of traveling long distances (passive) via human-mediated transportation. This duality may expand the distribution of the mosquito vector in urbanized areas. In this study, we examined the population genetic structure of Ae. aegypti in a highly urbanized and dengue-endemic region of the Philippines, Metropolitan Manila. Our findings indicated the dual dispersal nature of Ae. aegypti. The use of microsatellites as genetic markers also allowed us to describe the potential long-distance dispersal patterns, possibly through human-aided land transportation via the existing road networks of Metropolitan Manila.

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<![CDATA[Genetic variation and phylogeographic structure of <i>Spodoptera exigua</i> in western China based on mitochondrial DNA and microsatellite markers]]> https://www.researchpad.co/article/elastic_article_14552 The beet armyworm, Spodoptera exigua, is a significant agricultural pest of numerous crops and has caused serious economic losses in China. To effectively control this pest, we analyzed its genetic variation, population genetic structure and demographic history. We used mitochondrial DNA (mtDNA) fragments of the cytochrome oxidase subunit I (COI) and eight nuclear microsatellite loci to investigate genetic diversity and population genetic structure of S. exigua populations at 14 sampling sites in western China. Both mtDNA and microsatellite data indicated low levels of genetic diversity among all populations. A moderate genetic differentiation among some S. exigua populations was detected. Neighbor-joining dendrograms, STRUCTURE, and principal coordinate analysis (PCoA) revealed two genetically distinct groups: the KEL group and the remaining population group. Isolation by distance (IBD) results showed a weak significant correlation between geographic distance and genetic differentiation. Haplotype networks, neutrality testing, and mismatch distribution analysis indicated that the beet armyworm experienced a recent rapid expansion without a recent genetic bottleneck in western China. Thus, the results of this population genetic study can help with the development of strategies for managing this highly migratory pest.

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<![CDATA[Podocyte RNA sequencing reveals Wnt- and ECM-associated genes as central in FSGS]]> https://www.researchpad.co/article/Nff231b2e-f2d8-47eb-acf2-c510faf35a1a

Loss of podocyte differentiation can cause nephrotic-range proteinuria and Focal and Segmental Glomerulosclerosis (FSGS). As specific therapy is still lacking, FSGS frequently progresses to end-stage renal disease. The exact molecular mechanisms of FSGS and gene expression changes in podocytes are complex and widely unknown as marker changes have mostly been assessed on the glomerular level. To gain a better insight, we isolated podocytes of miR-193a overexpressing mice, which suffer from FSGS due to suppression of the podocyte master regulator Wt1. We characterised the podocytic gene expression changes by RNAseq and identified many novel candidate genes not linked to FSGS so far. This included strong upregulation of the receptor tyrosine kinase EphA6 and a massive dysregulation of circadian genes including the loss of the transcriptional activator Arntl. By comparison with podocyte-specific changes in other FSGS models we found a shared dysregulation of genes associated with the Wnt signaling cascade, while classical podocyte-specific genes appeared widely unaltered. An overlap with gene expression screens from human FSGS patients revealed a strong enrichment in genes associated with extra-cellular matrix (ECM) and metabolism. Our data suggest that FSGS progression might frequently depend on pathways that are often overlooked when considering podocyte homeostasis.

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<![CDATA[The genetic diversity and population structure of Sophora alopecuroides (Faboideae) as determined by microsatellite markers developed from transcriptome]]> https://www.researchpad.co/article/N8ed88142-6689-430c-b82a-b033b4ff58ac

Sophora alopecuroides (Faboideae) is an endemic species, mainly distributed in northwest China. However, the limited molecular markers range for this species hinders breeding and genetic studies. A total of 20,324 simple sequence repeat (SSR) markers were identified from 118,197 assembled transcripts and 18 highly polymorphic SSR markers were used to explore the genetic diversity and population structure of S. alopecuroides from 23 different geographical populations. A relatively low genetic diversity was found in S. alopecuroides based on mean values of the number of effective alleles (Ne = 1.81), expected heterozygosity (He = 0.39) and observed heterozygosity (Ho = 0.55). The results of AMOVA indicated higher levels of variation within populations than between populations. Bayesian-based cluster analysis, principal coordinates analysis and Neighbor-Joining phylogeny analysis roughly divided all genotypes into four major groups with some admixtures. Meanwhile, geographic barriers would have restricted gene flow between the northern and southern regions (separated by Tianshan Mountains), wherein the two relatively ancestral and independent clusters of S. alopecuroides occur. History trade and migration along the Silk Road would together have promoted the spread of S. alopecuroides from the western to the eastern regions of the northwest plateau in China, resulting in the current genetic diversity and population structure. The transcriptomic SSR markers provide a valuable resource for understanding the genetic diversity and population structure of S. alopecuroides, and will assist effective conservation management.

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<![CDATA[Global transcriptional regulation of innate immunity by ATF-7 in C. elegans]]> https://www.researchpad.co/article/5c784faad5eed0c4840072b2

The nematode Caenorhabditis elegans has emerged as a genetically tractable animal host in which to study evolutionarily conserved mechanisms of innate immune signaling. We previously showed that the PMK-1 p38 mitogen-activated protein kinase (MAPK) pathway regulates innate immunity of C. elegans through phosphorylation of the CREB/ATF bZIP transcription factor, ATF-7. Here, we have undertaken a genomic analysis of the transcriptional response of C. elegans to infection by Pseudomonas aeruginosa, combining genome-wide expression analysis by RNA-seq with ATF-7 chromatin immunoprecipitation followed by sequencing (ChIP-Seq). We observe that PMK-1-ATF-7 activity regulates a majority of all genes induced by pathogen infection, and observe ATF-7 occupancy in regulatory regions of pathogen-induced genes in a PMK-1-dependent manner. Moreover, functional analysis of a subset of these ATF-7-regulated pathogen-induced target genes supports a direct role for this transcriptional response in host defense. The genome-wide regulation through PMK-1– ATF-7 signaling reveals a striking level of control over the innate immune response to infection through a single transcriptional regulator.

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<![CDATA[A novel polymorphism in the fatty acid desaturase 2 gene (Fads2): A possible role in the basal metabolic rate]]> https://www.researchpad.co/article/5c818e86d5eed0c484cc247a

Fatty acyl composition of cell membrane lipids, particularly the abundance of highly unsaturated docosahexaenoic fatty acid (22:6n-3, DHA), is likely to be an important predictor of basal metabolic rate (BMR). Our study was performed using two lines of laboratory mice divergently selected for either high or low BMR. We describe a novel single nucleotide polymorphism in the Fads2 gene encoding Δ6-desaturase, a key enzyme in the metabolic pathways of polyunsaturated fatty acids (PUFAs). The allele frequencies of Fads2 were significantly different in both lines of mice. The analysis of genetic distances revealed that the genetic differentiation between the two studied lines developed significantly faster at the Fads2 locus than it did at neutral loci. Such a pattern suggests that the Fads2 polymorphism is related to the variation in BMR, i.e. the direct target of selection. The Fads2 polymorphism significantly affected abundance of several PUFAs; however, the differences in PUFA composition between lines were compatible with the difference in frequency of Fads2 alleles only for DHA. We hypothesize that the polymorphism in the Fads2 gene affects the BMR through modification of DHA abundance in cell membranes. This may be the first example of a significant link between a polymorphism in a gene responsible for fatty acyl composition and variation in BMR.

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<![CDATA[ARR22 overexpression can suppress plant Two-Component Regulatory Systems]]> https://www.researchpad.co/article/5c6b2616d5eed0c4842892c3

In plants, several developmental processes are co-coordinated by cytokinins via phosphorylation dependent processes of the Two-Component System (TCS). An outstanding challenge is to track phosphorelay flow from cytokinin perception to its molecular outputs, of which gene activation plays a major role. To address this issue, a kinetic-based reporter system was expounded to track TCS phosphorelay activity in vivo that can distinguish between basal and cytokinin dependent effects of overexpressed TCS members. The TCS phosphorelay can be positively activated by cytokinin and inhibited by pharmaceuticals or naturally interfering components. In this case we took advantage of the phosphohistidine-phosphatase Arabidopsis Response Regulator (ARR) 22 and investigated its phosphocompetition with other TCS members in regulating promoters of ARR5 and WUS in Arabidopsis thaliana cell culture protoplasts. In congruency with the proposed function of ARR22, overexpression of ARR22 blocked the activation of all B-type ARRs in this study in a TCS dependent manner. Furthermore, this effect could not be mimicked by A-type response regulator overexpression or compensated by AHP overexpression. Compared to other reporter assays, ours mimicked effects previously observed only in transgenic plants for all of the TCS proteins studied, suggesting that it is possible to expose phosphocompetition. Thus, our approach can be used to investigate gene signaling networks involving the TCS by leveraging ARR22 as a TCS inhibitor along with B-type ARR overexpression.

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<![CDATA[Discovery of gene regulatory elements through a new bioinformatics analysis of haploid genetic screens]]> https://www.researchpad.co/article/5c59fef9d5eed0c48413586a

The systematic identification of regulatory elements that control gene expression remains a challenge. Genetic screens that use untargeted mutagenesis have the potential to identify protein-coding genes, non-coding RNAs and regulatory elements, but their analysis has mainly focused on identifying the former two. To identify regulatory elements, we conducted a new bioinformatics analysis of insertional mutagenesis screens interrogating WNT signaling in haploid human cells. We searched for specific patterns of retroviral gene trap integrations (used as mutagens in haploid screens) in short genomic intervals overlapping with introns and regions upstream of genes. We uncovered atypical patterns of gene trap insertions that were not predicted to disrupt coding sequences, but caused changes in the expression of two key regulators of WNT signaling, suggesting the presence of cis-regulatory elements. Our methodology extends the scope of haploid genetic screens by enabling the identification of regulatory elements that control gene expression.

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<![CDATA[Vgsc-interacting proteins are genetically associated with pyrethroid resistance in Aedes aegypti]]> https://www.researchpad.co/article/5c59feaed5eed0c4841352e6

Association mapping of factors that condition pyrethroid resistance in Aedes aegypti has consistently identified genes in multiple functional groups. Toward better understanding of the mechanisms involved, we examined high throughput sequencing data (HTS) from two Aedes aegypti aegypti collections from Merida, Yucatan, Mexico treated with either permethrin or deltamethrin. Exome capture enrichment for coding regions and the AaegL5 annotation were used to identify genes statistically associated with resistance. The frequencies of single nucleotide polymorphisms (SNPs) were compared between resistant and susceptible mosquito pools using a contingency χ2 analysis. The -log102 p value) was calculated at each SNP site, with a weighted average determined from all sites in each gene. Genes with -log102 p value) ≥ 4.0 and present among all 3 treatment groups were subjected to gene set enrichment analysis (GSEA). We found that several functional groups were enriched compared to all coding genes. These categories were transport, signal transduction and metabolism, in order from highest to lowest statistical significance. Strikingly, 21 genes with demonstrated association to synaptic function were identified. In the high association group (n = 1,053 genes), several genes were identified that also genetically or physically interact with the voltage-gated sodium channel (VGSC). These genes were eg., CHARLATAN (CHL), a transcriptional regulator, several ankyrin-domain proteins, PUMILIO (PUM), a translational repressor, and NEDD4 (E3 ubiquitin-protein ligase). There were 13 genes that ranked among the top 10%: these included VGSC; CINGULIN, a predicted neuronal gap junction protein, and the aedine ortholog of NERVY (NVY), a transcriptional regulator. Silencing of CHL and NVY followed by standard permethrin bottle bioassays validated their association with permethrin resistance. Importantly, VGSC levels were also reduced about 50% in chl- or nvy-dsRNA treated mosquitoes. These results are consistent with the contribution of a variety of neuronal pathways to pyrethroid resistance in Ae. aegypti.

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<![CDATA[BAP1 expression is prognostic in breast and uveal melanoma but not colon cancer and is highly positively correlated with RBM15B and USP19]]> https://www.researchpad.co/article/5c61e8c4d5eed0c48496f124

BAP1 is a tumor suppressor gene important to the development and prognosis of many cancers, especially uveal melanoma (UM). Its role in more common cancers such as breast and colon cancer is largely unknown. We collected the transcriptome profiling data sets from the TCGA uveal melanoma (TCGA-UVM), breast cancer (TCGA-BRCA), and colon cancer (TCGA-COAD) projects to analyze the expression of BAP1. We found that patients with UM and breast cancer, but not colon cancer, who died had a lower level of BAP1 gene expression compared to surviving patients. Importantly, in breast cancer patients, the lowest BAP1 expression levels corresponded to the dead young patients (age at diagnosis < 46). Since the number of cases in TCGA-BRCA was much higher than TCGA-UVM, we obtained highly correlated genes with BAP1 in invasive breast carcinomas. Then, we tested if these genes are also highly correlated with BAP1 in UM and colon cancer. We found that BAP1 is highly positively correlated with RBM15B and USP19 expression in invasive breast carcinoma, UM, and colon adenocarcinoma. All three genes are located in close proximity on the 3p21 tumor suppressor region that is commonly altered in many cancers.

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<![CDATA[Contribution of the Cpx envelope stress system to metabolism and virulence regulation in Salmonella enterica serovar Typhimurium]]> https://www.researchpad.co/article/5c61e8cfd5eed0c48496f1be

The Cpx-envelope stress system regulates the expression of virulence factors in many Gram-negative pathogens. In Salmonella enterica serovar Typhimurium deletion of the sensor kinase CpxA but not of the response regulator CpxR results in the down regulation of the key regulator for invasion, HilA encoded by the Salmonella pathogenicity island 1 (SPI-1). Here, we provide evidence that cpxA deletion interferes with dephosphorylation of CpxR resulting in increased levels of active CpxR and consequently in misregulation of target genes. 14 potential operons were identified to be under direct control of CpxR. These include the virulence determinants ecotin, the omptin PgtE, and the SPI-2 regulator SsrB. The Tat-system and the PocR regulator that together promote anaerobic respiration of tetrathionate on 1,2-propanediol are also under direct CpxR control. Notably, 1,2-propanediol represses hilA expression. Thus, our work demonstrates for the first time the involvement of the Cpx system in a complex network mediating metabolism and virulence function.

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<![CDATA[Multi-study inference of regulatory networks for more accurate models of gene regulation]]> https://www.researchpad.co/article/5c536a85d5eed0c484a47592

Gene regulatory networks are composed of sub-networks that are often shared across biological processes, cell-types, and organisms. Leveraging multiple sources of information, such as publicly available gene expression datasets, could therefore be helpful when learning a network of interest. Integrating data across different studies, however, raises numerous technical concerns. Hence, a common approach in network inference, and broadly in genomics research, is to separately learn models from each dataset and combine the results. Individual models, however, often suffer from under-sampling, poor generalization and limited network recovery. In this study, we explore previous integration strategies, such as batch-correction and model ensembles, and introduce a new multitask learning approach for joint network inference across several datasets. Our method initially estimates the activities of transcription factors, and subsequently, infers the relevant network topology. As regulatory interactions are context-dependent, we estimate model coefficients as a combination of both dataset-specific and conserved components. In addition, adaptive penalties may be used to favor models that include interactions derived from multiple sources of prior knowledge including orthogonal genomics experiments. We evaluate generalization and network recovery using examples from Bacillus subtilis and Saccharomyces cerevisiae, and show that sharing information across models improves network reconstruction. Finally, we demonstrate robustness to both false positives in the prior information and heterogeneity among datasets.

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<![CDATA[Sugar and iron: Toward understanding the antibacterial effect of ciclopirox in Escherichia coli]]> https://www.researchpad.co/article/5c42438cd5eed0c4845e0573

New antibiotics are needed against antibiotic-resistant gram-negative bacteria. The repurposed antifungal drug, ciclopirox, equally blocks antibiotic-susceptible or multidrug-resistant Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae clinical isolates, indicating that it is not affected by existing resistance mechanisms. Toward understanding how ciclopirox blocks growth, we screened E. coli mutant strains and found that disruption of genes encoding products involved in galactose salvage, enterobacterial common antigen synthesis, and transport of the iron binding siderophore, enterobactin, lowered the minimum inhibitory concentration of ciclopirox needed to block growth of the mutant compared to the isogenic parent strain. We found that ciclopirox induced enterobactin production and that this effect is strongly affected by the deletion of the galactose salvage genes encoding UDP-galactose 4-epimerase, galE, or galactose-1-phosphate uridylyltransferase, galT. As disruption of ECA synthesis activates the regulation of capsular synthesis (Rcs) phosphorelay, which inhibits bacterial swarming and promotes biofilm development, we test whether ciclopirox prevents activation of the Rcs pathway. Sub-inhibitory concentrations of ciclopirox increased swarming of the E. coli laboratory K12 strain BW25113 but had widely varying effects on swarming or surface motility of clinical isolate E. coli, A. baumannii, and K. pneumoniae. There was no effect of ciclopirox on biofilm production, suggesting it does not target Rcs. Altogether, our data suggest ciclopirox-mediated alteration of lipopolysaccharides stimulates enterobactin production and affects bacterial swarming.

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<![CDATA[Computational characterization of the peptidome in transporter associated with antigen processing (TAP)-deficient cells]]> https://www.researchpad.co/article/5c478ca9d5eed0c484bd3c18

The transporter associated with antigen processing (TAP) is a key element of the major histocompatibility complex (MHC) class I antigen processing and presentation pathway. Nonfunctional TAP complexes impair the translocation of cytosol-derived proteolytic peptides to the endoplasmic reticulum lumen. This drastic reduction in the available peptide repertoire leads to a significant decrease in MHC class I cell surface expression. Using mass spectrometry, different studies have analyzed the cellular MHC class I ligandome from TAP-deficient cells, but the analysis of the parental proteins, the source of these ligands, still deserves an in-depth analysis. In the present report, several bioinformatics protocols were applied to investigate the nature of parental proteins for the previously identified TAP-independent MHC class I ligands. Antigen processing in TAP-deficient cells mainly focused on small, abundant or highly integral transmembrane proteins of the cellular proteome. This process involved abundant proteins of the central RNA metabolism. In addition, TAP-independent ligands were preferentially cleaved from the N- and C-terminal ends with respect to the central regions of the parental proteins. The abundance of glycine, proline and aromatic residues in the C-terminal sequences from TAP-independently processed proteins allows the accessibility and specificity required for the proteolytic activities that generates the TAP-independent ligandome. This limited proteolytic activity towards a set of preferred proteins in a TAP-negative environment would therefore suffice to promote the survival of TAP-deficient individuals.

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<![CDATA[Diversification of DNA binding specificities enabled SREBP transcription regulators to expand the repertoire of cellular functions that they govern in fungi]]> https://www.researchpad.co/article/5c33c3afd5eed0c48459e8a0

The Sterol Regulatory Element Binding Proteins (SREBPs) are basic-helix-loop-helix transcription regulators that control the expression of sterol biosynthesis genes in higher eukaryotes and some fungi. Surprisingly, SREBPs do not regulate sterol biosynthesis in the ascomycete yeasts (Saccharomycotina) as this role was handed off to an unrelated transcription regulator in this clade. The SREBPs, nonetheless, expanded in fungi such as the ascomycete yeasts Candida spp., raising questions about their role and evolution in these organisms. Here we report that the fungal SREBPs diversified their DNA binding preferences concomitantly with an expansion in function. We establish that several branches of fungal SREBPs preferentially bind non-palindromic DNA sequences, in contrast to the palindromic DNA motifs recognized by most basic-helix-loop-helix proteins (including SREBPs) in higher eukaryotes. Reconstruction and biochemical characterization of the likely ancestor protein suggest that an intrinsic DNA binding promiscuity in the family was resolved by alternative mechanisms in different branches of fungal SREBPs. Furthermore, we show that two SREBPs in the human commensal yeast Candida albicans drive a transcriptional cascade that inhibits a morphological switch under anaerobic conditions. Preventing this morphological transition enhances C. albicans colonization of the mammalian intestine, the fungus’ natural niche. Thus, our results illustrate how diversification in DNA binding preferences enabled the functional expansion of a family of eukaryotic transcription regulators.

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<![CDATA[Functional divergence of a global regulatory complex governing fungal filamentation]]> https://www.researchpad.co/article/5c3d00ebd5eed0c484036b17

Morphogenetic transitions are prevalent in the fungal kingdom. For a leading human fungal pathogen, Candida albicans, the capacity to transition between yeast and filaments is key for virulence. For the model yeast Saccharomyces cerevisiae, filamentation enables nutrient acquisition. A recent functional genomic screen in S. cerevisiae identified Mfg1 as a regulator of morphogenesis that acts in complex with Flo8 and Mss11 to mediate transcriptional responses crucial for filamentation. In C. albicans, Mfg1 also interacts physically with Flo8 and Mss11 and is critical for filamentation in response to diverse cues, but the mechanisms through which it regulates morphogenesis remained elusive. Here, we explored the consequences of perturbation of Mfg1, Flo8, and Mss11 on C. albicans morphogenesis, and identified functional divergence of complex members. We observed that C. albicans Mss11 was dispensable for filamentation, and that overexpression of FLO8 caused constitutive filamentation even in the absence of Mfg1. Harnessing transcriptional profiling and chromatin immunoprecipitation coupled to microarray analysis, we identified divergence between transcriptional targets of Flo8 and Mfg1 in C. albicans. We also established that Flo8 and Mfg1 cooperatively bind to promoters of key regulators of filamentation, including TEC1, for which overexpression was sufficient to restore filamentation in the absence of Flo8 or Mfg1. To further explore the circuitry through which Mfg1 regulates morphogenesis, we employed a novel strategy to select for mutations that restore filamentation in the absence of Mfg1. Whole genome sequencing of filamentation-competent mutants revealed chromosome 6 amplification as a conserved adaptive mechanism. A key determinant of the chromosome 6 amplification is FLO8, as deletion of one allele blocked morphogenesis, and chromosome 6 was not amplified in evolved lineages for which FLO8 was re-located to a different chromosome. Thus, this work highlights rewiring of key morphogenetic regulators over evolutionary time and aneuploidy as an adaptive mechanism driving fungal morphogenesis.

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<![CDATA[Transcriptional profiling identifies novel regulators of macrophage polarization]]> https://www.researchpad.co/article/5c141ec1d5eed0c484d28154

Macrophages are key inflammatory immune cells that display dynamic phenotypes and functions in response to their local microenvironment. Major advances have occurred in understanding the transcriptional, epigenetic, and functional differences in various macrophage subsets by in vitro modeling and gene expression and epigenetic profiling for biomarker discovery. However, there is still no standardized protocol for macrophage polarization largely due to the lack of thorough validation of macrophage phenotypes following polarization. In addition, transcriptional regulation is recognized as a major mechanism governing differential macrophage polarization programs and as such, many genes have been identified to be associated with each macrophage subset. However, the functional role of many of these genes in macrophage polarization is still unknown. Moreover, the role of other regulatory mechanisms, such as DNA methylation, in macrophage polarization remains poorly understood. Here, we employed an optimized model of human M1 and M2 macrophage polarization which we used for large-scale transcriptional and DNA methylation profiling. We were unable to demonstrate a role for DNA methylation in macrophage polarization, as no significant changes were identified. However, we observed significant changes in the transcriptomes of M1 and M2 macrophages. Additionally, we identified numerous novel differentially regulated genes involved in macrophage polarization, including CYBB and DHCR7 which we show as important regulators of M1 and M2 macrophage polarization, respectively. Taken together, our improved in vitro human M1 and M2 macrophage model provides new understandings of the regulation of macrophage polarization and candidate macrophage biomarkers.

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<![CDATA[The Transcriptional landscape of Streptococcus pneumoniae TIGR4 reveals a complex operon architecture and abundant riboregulation critical for growth and virulence]]> https://www.researchpad.co/article/5c117b9cd5eed0c484699dd5

Efficient and highly organized regulation of transcription is fundamental to an organism’s ability to survive, proliferate, and quickly respond to its environment. Therefore, precise mapping of transcriptional units and understanding their regulation is crucial to determining how pathogenic bacteria cause disease and how they may be inhibited. In this study, we map the transcriptional landscape of the bacterial pathogen Streptococcus pneumoniae TIGR4 by applying a combination of high-throughput RNA-sequencing techniques. We successfully map 1864 high confidence transcription termination sites (TTSs), 790 high confidence transcription start sites (TSSs) (742 primary, and 48 secondary), and 1360 low confidence TSSs (74 secondary and 1286 primary) to yield a total of 2150 TSSs. Furthermore, our study reveals a complex transcriptome wherein environment-respondent alternate transcriptional units are observed within operons stemming from internal TSSs and TTSs. Additionally, we identify many putative cis-regulatory RNA elements and riboswitches within 5’-untranslated regions (5’-UTR). By integrating TSSs and TTSs with independently collected RNA-Seq datasets from a variety of conditions, we establish the response of these regulators to changes in growth conditions and validate several of them. Furthermore, to demonstrate the importance of ribo-regulation by 5’-UTR elements for in vivo virulence, we show that the pyrR regulatory element is essential for survival, successful colonization and infection in mice suggesting that such RNA elements are potential drug targets. Importantly, we show that our approach of combining high-throughput sequencing with in vivo experiments can reconstruct a global understanding of regulation, but also pave the way for discovery of compounds that target (ribo-)regulators to mitigate virulence and antibiotic resistance.

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<![CDATA[Absence of genetic selection in a pathogenic Escherichia coli strain exposed to the manure-amended soil environment]]> https://www.researchpad.co/article/5c141e62d5eed0c484d265cd

Escherichia coli that express curli are more common in subsurface soil drainage when manure is surface applied. However, it is unknown whether this arises from mutations in individual strains leading to curli expression or by selection for individuals already expressing higher levels of curli. To test this, we examined curli production in pathogenic E. coli O157:H7 EDL933 as a function of manure management. Five treatments were investigated: (1) soil only, (2) soil with surface-applied E. coli O157:H7 EDL933 Δstx1-2 (EcO157), (3) soil with incorporated EcO157, (4) soil with surface-applied EcO157-inoculated manure, and (5) soil with incorporated EcO157-inoculated manure. EcO157 was reisolated from soils immediately after application and weekly thereafter for 8 weeks. EcO157 in the surface-applied treatments died faster than their incorporated treatment counterparts. Phenotypic assays revealed differences between treatments as well. Half of surface-applied manure reisolates from week 6 developed a mixed red and white colony morphology on Congo Red plates, indicating changes in curli production that were not seen in other treatments or times. In 37°C growth tests, week 6 reisolates from all treatments except soil surface-applied EcO157 left the lag phase at a significantly greater rate than week 0 isolates. We applied whole genome sequencing technology to interrogate the genetic underpinnings of these phenotypes. Surprisingly, we only found single-nucleotide polymorphisms in two of the 94 resequenced isolates from the different treatments, neither of which correlated with curli phenotype. Likewise, we found no differences in other genomic characteristics that might account for phenotypic differences including the count of gaps and the origin of discarded reads that failed to map to the parental strain. These results suggest there were no systematic genomic differences (i.e. individual-level selection) that correlated with time or treatment. We recommend future research focus on population-level selection of E. coli strains in the manure-amended soil environment.

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