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Laboratory evolution experiments have led to important findings relating organism adaptation and genomic evolution. However, continuous monitoring of long-term evolution has been lacking for natural systems, limiting our understanding of these processes in situ. Here we characterize the evolutionary dynamics of a lineage of a clinically important opportunistic bacterial pathogen, Pseudomonas aeruginosa, as it adapts to the airways of several individual cystic fibrosis patients over 200.000 bacterial generations, and provide estimates of mutation rates of bacteria in a nat- ural environment. In contrast to predictions based on in vitro evolution experiments, we document limited diversification of the evolving lineage despite a highly structured and complex host environment. Notably, the lineage went through an initial period of rapid adaptation caused by a small number of mutations with plelotropic effects, followed by a period of genetic drift with limited phenotypic change and a genomic signature of negative selection, suggesting that the evolving lineage has reached a major adaptive peak in the fitness landscape. This contrasts with previous findings of continued positive selection from long-term in vitro evolution experiments. The evolved phenotype of the infecting bacteria further suggests that the opportunistic pathogen has transitioned to become a primary pathogen for cystic fibrosis patients. [ABSTRACT FROM AUTHOR]

Background: Expression of the oestrogen receptor (ER) in breast cancer predicts benefit from endocrine therapy. Minimising the frequency of false negative ER status classification is essential to identify all patients with ER positive breast cancers who should be offered endocrine therapies in order to improve clinical outcome. In routine oncological practice ER status is determined by semi-quantitative methods such as immunohistochemistry (IHC) or other immunoassays in which the ER expression level is compared to an empirical threshold[1,2]. The clinical relevance of gene expression-based ER subtypes as compared to IHC-based determination has not been systematically evaluated. Here we attempt to reduce the frequency of false negative ER status classification using two gene expression approaches and compare these methods to IHC based ER status in terms of predictive and prognostic concordance with clinical outcome. Methodology/Principal Findings: Firstly, ER status was discriminated by fitting the bimodal expression of ESR1 to a mixed Gaussian model. The discriminative power of ESR1 suggested bimodal expression as an efficient way to stratify breast cancer; therefore we identified a set of genes whose expression was both strongly bimodal, mimicking ESR expression status, and highly expressed in breast epithelial cell lines, to derive a 23-gene ER expression signature-based classifier. We assessed our classifiers in seven published breast cancer cohorts by comparing the gene expression-based ER status to IHCbased ER status as a predictor of clinical outcome in both untreated and tamoxifen treated cohorts. In untreated breast cancer cohorts, the 23 gene signature-based ER status provided significantly improved prognostic power compared to IHCbased ER status (P = 0.006). In tamoxifen-treated cohorts, the 23 gene ER expression signature predicted clinical outcome (HR = 2.20, P = 0.00035). These complementary ER signature-based strategies estimated that between 15.1% and 21.8% patients of IHC-based negative ER status would be classified with ER positive breast cancer. Conclusion/Significance: Expression-based ER status classification may complement IHC to minimise false negative ER status classification and optimise patient stratification for endocrine therapies. [ABSTRACT FROM AUTHOR]

Background: The hypomethylating agent 5-Azacytidine (5-Aza-CR) is the first drug to prolong overall survival in patients with myelodysplastic syndrome (MDS). Surprisingly, the deoxyribonucleoside analog 5-Aza-2'deoxycytidine (5-Aza-CdR) did not have a similar effect on survival in a large clinical trial. Both drugs are thought to exert their effects after incorporation into DNA by covalent binding of DNA methyltransferase (DNMT). While 5-Aza-CdR is incorporated into only DNA, 5-Aza-CR is also incorporated into RNA. Here, we have analyzed whether this difference in nucleic acid incorporation may influence the capacities of these drugs to regulate the expression of mRNA and microRNAs (miRNA), which may potentially affect the activities of the drugs in patients. Methodology/Principal Findings: A hematopoietic (HL-60; acute myeloid leukemia) and a solid (T24; transitional cell carcinoma) cancer cell line were treated with equitoxic doses of 5-Aza-CR and 5-Aza-CdR for 24 hrs, and the immediate (day 2) and lasting (day 8) effects on RNA expression examined. There was considerable overlap between the RNAs heritably upregulated by both drugs on day 8 but more RNAs were stably induced by the deoxy analog. Both drugs strongly induced expression of cancer testis antigens. On day 2 more RNAs were downregulated by 5-Aza-CR, particularly at higher doses. A remarkable downregulation of miRNAs and a significant upregulation of tRNA synthetases and other genes involved in amino acid metabolism was observed in T24 cells. Conclusions/Significance: Overall, this suggests that significant differences exist in the immediate action of the two drugs, however the dominant pattern of the lasting, and possible heritable changes, is overlapping. [ABSTRACT FROM AUTHOR]

The article 'Industrial antifoam agents impair ethanol fermentation and induce stress responses in yeast cells' was originally published Online First without open access. After publication in volume 101, issue 22, page 8237-8248, the author decided to opt for Open Choice and to make the article an open access publication. [ABSTRACT FROM AUTHOR]

There has been limited success with blood-based biomarkers of neurodegeneration. One perceived reason is that blood has no direct contact to the brain. Recently developed blood-based biomarkers of tau-degradation have shown promise as potential tools for peripheral assessment of neurodegeneration; however, factors contributing to the levels of these in blood are poorly understood. Using multiple linear regression analysis in cross-sectional data from an observational cohort (n = 5626), the aim was to examine which factors correlate to the serological levels of two novel biomarkers measuring truncated tau fragments (Tau-A and Tau-C) in serum. Platelets, albumin and several modifiable risk factors, including Body Mass Index, high density lipoprotein and white blood cell count were associated with the serum level of tau fragments. The factors associated with tau in serum may promote neurodegeneration and alter the permeability of the Blood Brain Barrier through chronic inflammation and vascular dysfunction. These data are of key importance for understanding the mechanism of release and subsequent peripheral processing of tau from the brain and will assist in the development of future blood-based biomarkers. [ABSTRACT FROM AUTHOR]

The majority of the nearly 10,000 described species of green algae are photoautotrophs; however, some species have lost their ability to photosynthesize and become obligate heterotrophs that rely on parasitism for survival. Two high-quality genomes of the heterotrophic algae Prototheca zopfii Pz20 and Pz23 were obtained using short- and long-read genomic as well as transcriptomic data. The genome sizes were 31.2 Mb and 31.3 Mb, respectively, and contig N50 values of 1.99 Mb and 1.26 Mb. Although P. zopfii maintained its plastid genome, the transition to heterotrophy led to a reduction in both plastid and nuclear genome size, including the loss of photosynthesis-related genes from both the nuclear and plastid genomes and the elimination of genes encoding for carotenoid oxygenase and pheophorbide an oxygenase. The loss of genes, including basic leucine-zipper (bZIP) transcription factors, flavin adenine dinucleotide-linked oxidase, and helicase, could have played a role in the transmission of autotrophy to heterotrophs and in the processes of abiotic stress resistance and pathogenicity. A total of 66 (1.37%) and 73 (1.49%) genes were identified as potential horizontal gene transfer events in the two P. zopfii genomes, respectively. Genes for malate synthase and isocitrate lyase, which are horizontally transferred from bacteria, may play a pivotal role in carbon and nitrogen metabolism as well as the pathogenicity of Prototheca and non-photosynthetic organisms. The two high-quality P. zopfii genomes provide new insights into their evolution as obligate heterotrophs and pathogenicity., Importance: The genus Prototheca , characterized by its heterotrophic nature and pathogenicity, serves as an exemplary model for investigating pathobiology. The limited understanding of the protothecosis infectious disease is attributed to the lack of genomic resources. Using HiFi long-read sequencing, both nuclear and plastid genomes were generated for two strains of P. zopfii . The findings revealed a concurrent reduction in both plastid and nuclear genome size, accompanied by the loss of genes associated with photosynthesis, carotenoid oxygenase, basic leucine-zipper (bZIP) transcription factors, and others. The analysis of horizontal gene transfer revealed the presence of 1.37% and 1.49% bacterial genes, including malate synthase and isocitrate lyase, which play crucial roles in carbon and nitrogen metabolism, as well as pathogenicity and obligate heterotrophy. The two high-quality P. zopfii genomes represent valuable resources for investigating their adaptation and evolution as obligate heterotrophs, as well as for developing future prevention and treatment strategies against protothecosis., Competing Interests: The authors declare no conflict of interest.

Background: Lytic polysaccharide monooxygenases (LPMOs) are often studied in simple models involving activity measurements of a single LPMO or a blend thereof with hydrolytic enzymes towards an insoluble substrate. However, the contribution of LPMOs to polysaccharide breakdown in complex cocktails of hydrolytic and oxidative enzymes, similar to fungal secretomes, remains elusive. Typically, two starch-specific AA13 LPMOs are encoded by mainly Ascomycota genomes. Here, we investigate the impact of LPMO loss on the growth and degradation of starches of varying resistance to amylolytic hydrolases by Aspergillus nidulans ., Results: Deletion of the genes encoding An AA13A that possesses a CBM20 starch-binding module, An AA13B (lacking a CBM20) or both AA13 genes resulted in reduced growth on solid media with resistant, but not soluble processed potato starch. Larger size and amount of residual starch granules were observed for the AA13-deficient strains as compared to the reference and the impairment of starch degradation was more severe for the strain lacking An AA13A based on a microscopic analysis. After 5 days of growth on raw potato starch in liquid media, the mount of residual starch was about fivefold higher for the AA13 gene deletion strains compared to the reference, which underscores the importance of LPMOs for degradation of especially resistant starches. Proteomic analyses revealed substantial changes in the secretomes of the double AA13 gene deletion, followed by the An AA13A-deficient strain, whereas only a single protein was significantly different in the proteome of the An AA13B-deficient strain as compared to the reference., Conclusions: This study shows that the loss of AA13, especially the starch-binding An AA13A, impairs degradation of resistant potato starch, but has limited impact on less-resistant wheat starch and no impact on processed solubilized starch. The effects of LPMO loss are more pronounced at the later stages of fungal growth, likely due to the accumulation of the less-accessible regions of the substrate. The striking impairment in granular starch degradation due to the loss of a single LPMO from the secretome offers insight into the crucial role played by AA13 in the breakdown of resistant starch and presents a methodological framework to analyse the contribution of distinct LPMOs towards semi-crystalline polysaccharides under in vivo conditions., Competing Interests: Competing interestsThe authors declare no competing interest., (© The Author(s) 2020.)

Obesity and type 2 diabetes (T2D) are metabolic disorders influenced by lifestyle and genetic factors that are characterized by insulin resistance in skeletal muscle, a prominent site of glucose disposal. Numerous genetic variants have been associated with obesity and T2D, of which the majority are located in non-coding DNA regions. This suggests that most variants mediate their effect by altering the activity of gene-regulatory elements, including enhancers. Here, we map skeletal muscle genomic enhancer elements that are dynamically regulated after exposure to the free fatty acid palmitate or the inflammatory cytokine TNFα. By overlapping enhancer positions with the location of disease-associated genetic variants, and resolving long-range chromatin interactions between enhancers and gene promoters, we identify target genes involved in metabolic dysfunction in skeletal muscle. The majority of these genes also associate with altered whole-body metabolic phenotypes in the murine BXD genetic reference population. Thus, our combined genomic investigations identified genes that are involved in skeletal muscle metabolism.

Glycerol is considered as a promising substrate for biotechnological applications and the non-conventional yeast Yarrowia lipolytica has been used extensively for the valorization of this compound. Contrary to S. cerevisiae , Y. lipolytica seems to prefer glycerol over glucose and it has been reported previously that the presence of glycerol can suppress the consumption of glucose in co-substrate fermentations. Based on these observations, we hypothesized glycerol repression-like effects in Y. lipolytica , which are converse to well described carbon repression mechanisms ensuring the prioritized use of glucose ( e.g. , in S. cerevisiae ). We therefore aimed to investigate this effect on the level of transcription. Strains varying in the degree of glucose suppression were chosen and characterized in high-resolution growth screenings, resulting in the detection of different growth phenotypes under glycerol-glucose mixed conditions. Two strains, IBT and W29, were selected and cultivated in chemostats using glucose, glycerol and glucose/glycerol as carbon sources, followed by an RNA-Seq-based transcriptome analysis. We could show that several transporters were significantly higher expressed in W29, which is potentially related to the observed physiological differences. However, most of the expression variation between the strains were regardless of the carbon source applied, and cross-comparisons revealed that the strain-specific carbon source responses underwent in the opposite direction. A deeper analysis of the substrate specific carbon source response led to the identification of several differentially expressed genes with orthologous functions related to signal transduction and transcriptional regulation. This study provides an initial investigation on potentially novel carbon source regulation mechanisms in yeasts., (Copyright © 2019 Lubuta et al.)

There has been limited success with blood-based biomarkers of neurodegeneration. One perceived reason is that blood has no direct contact to the brain. Recently developed blood-based biomarkers of tau-degradation have shown promise as potential tools for peripheral assessment of neurodegeneration; however, factors contributing to the levels of these in blood are poorly understood. Using multiple linear regression analysis in cross-sectional data from an observational cohort (n = 5626), the aim was to examine which factors correlate to the serological levels of two novel biomarkers measuring truncated tau fragments (Tau-A and Tau-C) in serum. Platelets, albumin and several modifiable risk factors, including Body Mass Index, high density lipoprotein and white blood cell count were associated with the serum level of tau fragments. The factors associated with tau in serum may promote neurodegeneration and alter the permeability of the Blood Brain Barrier through chronic inflammation and vascular dysfunction. These data are of key importance for understanding the mechanism of release and subsequent peripheral processing of tau from the brain and will assist in the development of future blood-based biomarkers., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Atopic dermatitis (AD) is caused by a complex interplay between immune and barrier abnormalities. Murine models of AD are essential for preclinical assessments of new treatments. Although many models have been used to simulate AD, their transcriptomic profiles are not fully understood, and a comparison of these models with the human AD transcriptomic fingerprint is lacking., Objective: We sought to evaluate the transcriptomic profiles of 6 common murine models and determine how they relate to human AD skin., Methods: Transcriptomic profiling was performed by using microarrays and quantitative RT-PCR on biopsy specimens from NC/Nga, flaky tail, Flg-mutated, ovalbumin-challenged, oxazolone-challenged, and IL-23-injected mice. Gene expression data of patients with AD, psoriasis, and contact dermatitis were obtained from previous patient cohorts. Criteria of a fold change of 2 or greater and a false discovery rate of 0.05 or less were used for gene arrays., Results: IL-23-injected, NC/Nga, and oxazolone-challenged mice show the largest homology with our human meta-analysis-derived AD transcriptome (37%, 18%, 17%, respectively). Similar to human AD, robust T H 1, T H 2, and also T H 17 activation are seen in IL-23-injected and NC/Nga mice, with similar but weaker inflammation in ovalbumin-challenged mice. Oxazolone-challenged mice show a T H 1-centered reaction, and flaky tail mice demonstrate a strong T H 17 polarization. Flg-mutated mice display filaggrin downregulation without significant inflammation., Conclusion: No single murine model fully captures all aspects of the AD profile; instead, each model reflects different immune or barrier disease aspects. Overall, among the 6 murine models, IL-23-injected mice best simulate human AD; still, the translational focus of the investigation should determine which model is most applicable., (Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

RNA binding proteins (RBPs) are key players in the regulation of gene expression. In this chapter we discuss the main protein-RNA recognition modes used by RBPs in order to regulate multiple steps of RNA processing. We discuss traditional and state-of-the-art technologies that can be used to study RNAs bound by individual RBPs, or vice versa, for both in vitro and in vivo methodologies. To help highlight the biological significance of RBP mediated regulation, online resources on experimentally verified protein-RNA interactions are briefly presented. Finally, we present the major tools to computationally infer RNA binding sites according to the modeling features and to the unsupervised or supervised frameworks that are adopted. Since some RNA binding site search algorithms are derived from DNA binding site search algorithms, we discuss the commonalities and novelties introduced to handle both sequence and structural features uniquely characterizing protein-RNA interactions.

Lipids play a central role in cellular function as constituents of membranes, as signaling molecules, and as storage materials. Although much is known about the role of lipids in regulating specific steps of metabolism, comprehensive studies integrating genome-wide expression data, metabolite levels, and lipid levels are currently lacking. Here, we map condition-dependent regulation controlling lipid metabolism in Saccharomyces cerevisiae by measuring 5636 mRNAs, 50 metabolites, 97 lipids, and 57 (13)C-reaction fluxes in yeast using a three-factor full-factorial design. Correlation analysis across eight environmental conditions revealed 2279 gene expression level-metabolite/lipid relationships that characterize the extent of transcriptional regulation in lipid metabolism relative to major metabolic hubs within the cell. To query this network, we developed integrative methods for correlation of multi-omics datasets that elucidate global regulatory signatures. Our data highlight many characterized regulators of lipid metabolism and reveal that sterols are regulated more at the transcriptional level than are amino acids. Beyond providing insights into the systems-level organization of lipid metabolism, we anticipate that our dataset and approach can join an emerging number of studies to be widely used for interrogating cellular systems through the combination of mathematical modeling and experimental biology.

Tolerogenic dendritic cells (DC) that are maturation-resistant and locked in a semimature state are promising tools in clinical applications for tolerance induction. Different immunomodulatory agents have been shown to induce a tolerogenic DC phenotype, such as the biologically active form of vitamin D (1,25(OH)(2)D(3)), glucocorticoids, and a synergistic combination of both. In this study, we aimed to characterize the protein profile, function and phenotype of DCs obtained in vitro in the presence of 1,25(OH)(2)D(3), dexamethasone (DEX), and a combination of both compounds (combi). Human CD14(+) monocytes were differentiated toward mature DCs, in the presence or absence of 1,25(OH)(2)D(3) and/or DEX. Cells were prefractionated into cytoplasmic and microsomal fractions and protein samples were separated in two different pH ranges (pH 3-7NL and 6-9), analyzed by 2D-DIGE and differentially expressed spots (p < 0.05) were identified after MALDI-TOF/TOF analysis. In parallel, morphological and phenotypical analyses were performed, revealing that 1,25(OH)(2)D(3)- and combi-mDCs are closer related to each other than DEX-mDCs. This was translated in their protein profile, indicating that 1,25(OH)(2)D(3) is more potent than DEX in inducing a tolerogenic profile on human DCs. Moreover, we demonstrate that combining 1,25(OH)(2)D(3) with DEX induces a unique protein expression pattern with major imprinting of the 1,25(OH)(2)D(3) effect. Finally, protein interaction networks and pathway analysis suggest that 1,25(OH)(2)D(3), rather than DEX treatment, has a severe impact on metabolic pathways involving lipids, glucose, and oxidative phosphorylation, which may affect the production of or the response to ROS generation. These findings provide new insights on the molecular basis of DC tolerogenicity induced by 1,25(OH)(2)D(3) and/or DEX, which may lead to the discovery of new pathways involved in DC immunomodulation.

Exposure of insulin-secreting β-cells to inflammatory cytokines or high concentrations of free fatty acids, factors involved in the pathogenesis of type 1 and type 2 diabetes, leads to endoplasmic reticulum (ER) stress, β-cell dysfunction, and eventually apoptotic β-cell death. The aim of this study was to investigate the impact of ER stress on β-cells at the protein level to evaluate the contribution of post-transcriptional and post-translational changes in ER stress-induced β-cell damage. INS-1E cells were exposed in vitro to the ER-stress inducer cyclopiazonic acid (CPA) at two concentrations, and protein changes were evaluated using 2D-DIGE. CPA, 25 μM, led to massive apoptosis, accompanied by a near complete protein translation shut-down. CPA, 6.25 μM, led to adaptation of the β-cells to ER stress. Identification of the differentially expressed proteins in the two conditions led to the discovery of a clear pattern of defense pathways, with post-translational modifications playing a crucial role. Key alterations included inhibition of insulin translation and post-translational modifications in ER chaperones HYOU1 and HSPA5. Also, a central role for 14-3-3 proteins is suggested. In conclusion, INS-1E cells are highly sensitive to ER stress, leading to important post-transcriptional and post-translational modifications that may contribute to β-cell dysfunction and death.

Dendritic cells (DCs) are unique antigen presenting cells, which upon maturation change from a specialized antigen-capturing cell towards a professional antigen presenting cells. In this study, a 2-D DIGE analysis of immature and mature DCs was performed, to identify proteins changing in expression upon maturation. The protein expression profile of immature and mature DCs, derived from CD14(+) peripheral blood monocytes was investigated using two pH ranges (pH 4-7 and 6-9) (n = 4). Ninety one differentially expressed spots (p<0.01) were detected, from which we identified 74 spots (81.32%) corresponding to 41 different proteins. The proteins identified play a role in diverse processes, such as antigen processing/presentation, vesicle transport and cytoskeleton remodeling. In addition, a protein interaction network contained 29 (out of 41) proteins, suggesting that, although they functionally originate from distinct classes, these proteins are acting as a protein-interactome. In conclusion, the proteins shown here to be altered in expression upon maturation are in line with the morphological and functional changes observed during the maturation process, providing a better understanding of the processes involved. This will open new avenues for investigating treatment regimens for immune-associated disorders., (Copyright © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Failure of cells to respond to DNA damage is a primary event associated with mutagenesis and environmental toxicity. To map the transcriptional network controlling the damage response, we measured genomewide binding locations for 30 damage-related transcription factors (TFs) after exposure of yeast to methyl-methanesulfonate (MMS). The resulting 5272 TF-target interactions revealed extensive changes in the pattern of promoter binding and identified damage-specific binding motifs. As systematic functional validation, we identified interactions for which the target changed expression in wild-type cells in response to MMS but was nonresponsive in cells lacking the TF. Validated interactions were assembled into causal pathway models that provide global hypotheses of how signaling, transcription, and phenotype are integrated after damage.