DIMet: an open-source tool for differential analysis of targeted isotope-labeled metabolomics data.

MOTIVATION: Many diseases, such as cancer, are characterized by an alteration of cellular metabolism allowing cells to adapt to changes in the microenvironment. Stable isotope-resolved metabolomics (SIRM) and downstream data analyses are widely used techniques for unraveling cells' metabolic activity to understand the altered functioning of metabolic pathways in the diseased state. While a number of bioinformatic solutions exist for the differential analysis of SIRM data, there is currently no available resource providing a comprehensive toolbox. RESULTS: In this work, we present DIMet, a one-stop comprehensive tool for differential analysis of targeted tracer data. DIMet accepts metabolite total abundances, isotopologue contributions, and isotopic mean enrichment, and supports differential comparison (pairwise and multi-group), time-series analyses, and labeling profile comparison. Moreover, it integrates transcriptomics and targeted metabolomics data through network-based metabolograms. We illustrate the use of DIMet in real SIRM datasets obtained from Glioblastoma P3 cell-line samples. DIMet is open-source, and is readily available for routine downstream analysis of isotope-labeled targeted metabolomics data, as it can be used both in the command line interface or as a complete toolkit in the public Galaxy Europe and Workfow4Metabolomics web platforms. AVAILABILITY AND IMPLEMENTATION: DIMet is freely available at https://github.com/cbib/DIMet, and through https://usegalaxy.eu and https://workflow4metabolomics.usegalaxy.fr. All the datasets are available at Zenodo https://zenodo.org/records/10925786.

A comparative gene expression matrix in Apoe-deficient mice identifies unique and atherosclerotic disease stage-specific gene regulation patterns in monocytes and macrophages.

BACKGROUND AND AIMS: Atherosclerosis is a systemic and chronic inflammatory disease propagated by monocytes and macrophages. Yet, our knowledge on how transcriptome of these cells evolves in time and space is limited. We aimed at characterizing gene expression changes in site-specific macrophages and in circulating monocytes during the course of atherosclerosis. METHODS: We utilized apolipoprotein E-deficient mice undergoing one- and six-month high cholesterol diet to model early and advanced atherosclerosis. Aortic macrophages, peritoneal macrophages, and circulating monocytes from each mouse were subjected to bulk RNA-sequencing (RNA-seq). We constructed a comparative directory that profiles lesion- and disease stage-specific transcriptomic regulation of the three cell types in atherosclerosis. Lastly, the regulation of one gene, Gpnmb, whose expression positively correlated with atheroma growth, was validated using single-cell RNA-seq (scRNA-seq) of atheroma plaque from murine and human. RESULTS: The convergence of gene regulation between the three investigated cell types was surprisingly low. Overall 3245 differentially expressed genes were involved in the biological modulation of aortic macrophages, among which less than 1% were commonly regulated by the remote monocytes/macrophages. Aortic macrophages regulated gene expression most actively during atheroma initiation. Through complementary interrogation of murine and human scRNA-seq datasets, we showcased the practicality of our directory, using the selected gene, Gpnmb, whose expression in aortic macrophages, and a subset of foamy macrophages in particular, strongly correlated with disease advancement during atherosclerosis initiation and progression. CONCLUSIONS: Our study provides a unique toolset to explore gene regulation of macrophage-related biological processes in and outside the atheromatous plaque at early and advanced disease stages.

Catching the Wave: Detecting Strain-Specific SARS-CoV-2 Peptides in Clinical Samples Collected during Infection Waves from Diverse Geographical Locations.

The Coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resulted in a major health crisis worldwide with its continuously emerging new strains, resulting in new viral variants that drive "waves" of infection. PCR or antigen detection assays have been routinely used to detect clinical infections; however, the emergence of these newer strains has presented challenges in detection. One of the alternatives has been to detect and characterize variant-specific peptide sequences from viral proteins using mass spectrometry (MS)-based methods. MS methods can potentially help in both diagnostics and vaccine development by understanding the dynamic changes in the viral proteome associated with specific strains and infection waves. In this study, we developed an accessible, flexible, and shareable bioinformatics workflow that was implemented in the Galaxy Platform to detect variant-specific peptide sequences from MS data derived from the clinical samples. We demonstrated the utility of the workflow by characterizing published clinical data from across the world during various pandemic waves. Our analysis identified six SARS-CoV-2 variant-specific peptides suitable for confident detection by MS in commonly collected clinical samples.

The antileukemic activity of decitabine upon PML/RARA-negative AML blasts is supported by all-trans retinoic acid: in vitro and in vivo evidence for cooperation.

The prognosis of AML patients with adverse genetics, such as a complex, monosomal karyotype and TP53 lesions, is still dismal even with standard chemotherapy. DNA-hypomethylating agent monotherapy induces an encouraging response rate in these patients. When combined with decitabine (DAC), all-trans retinoic acid (ATRA) resulted in an improved response rate and longer overall survival in a randomized phase II trial (DECIDER; NCT00867672). The molecular mechanisms governing this in vivo synergism are unclear. We now demonstrate cooperative antileukemic effects of DAC and ATRA on AML cell lines U937 and MOLM-13. By RNA-sequencing, derepression of >1200 commonly regulated transcripts following the dual treatment was observed. Overall chromatin accessibility (interrogated by ATAC-seq) and, in particular, at motifs of retinoic acid response elements were affected by both single-agent DAC and ATRA, and enhanced by the dual treatment. Cooperativity regarding transcriptional induction and chromatin remodeling was demonstrated by interrogating the HIC1, CYP26A1, GBP4, and LYZ genes, in vivo gene derepression by expression studies on peripheral blood blasts from AML patients receiving DAC + ATRA. The two drugs also cooperated in derepression of transposable elements, more effectively in U937 (mutated TP53) than MOLM-13 (intact TP53), resulting in a "viral mimicry" response. In conclusion, we demonstrate that in vitro and in vivo, the antileukemic and gene-derepressive epigenetic activity of DAC is enhanced by ATRA.

Impact of high platelet turnover on the platelet transcriptome: Results from platelet RNA-sequencing in patients with sepsis.

BACKGROUND: Sepsis is associated with high platelet turnover and elevated levels of immature platelets. Changes in the platelet transcriptome and the specific impact of immature platelets on the platelet transcriptome remain unclear. Thus, this study sought to address whether and how elevated levels of immature platelets affect the platelet transcriptome in patients with sepsis. METHODS: Blood samples were obtained from patients with sepsis requiring vasopressor therapy (n = 8) and from a control group of patients with stable coronary artery disease and otherwise similar demographic characteristics (n = 8). Immature platelet fraction (IPF) was determined on a Sysmex XE 2100 analyser and platelet function was tested by impedance aggregometry. RNA from leukocyte-depleted platelets was used for transcriptome analysis by Next Generation Sequencing integrating the use of unique molecular identifiers. RESULTS: IPF (median [interquartile range]) was significantly elevated in sepsis patients (6.4 [5.3-8.7] % vs. 3.6 [2.6-4.6] %, p = 0.005). Platelet function testing revealed no differences in adenosine diphosphate- or thrombin receptor activating peptide-induced platelet aggregation between control and sepsis patients. Putative circular RNA transcripts were decreased in platelets from septic patients. Leukocyte contamination defined by CD45 abundance levels in RNA-sequencing was absent in both groups. Principal component analysis of transcripts showed only partial overlap of clustering with IPF levels. RNA sequencing showed up-regulation of 524 and down-regulation of 118 genes in platelets from sepsis patients compared to controls. Upregulated genes were mostly related to catabolic processes and protein translation. Comparison to published platelet transcriptomes showed a large overlap of changes observed in sepsis and COVID-19 but not with reticulated platelets from healthy donors. CONCLUSIONS: Patients with sepsis appear to have a less degraded platelet transcriptome as indicated by increased levels of immature platelets and decreased levels of putative circular RNA transcripts. The present data suggests that increased protein translation is a characteristic mechanism of systemic inflammation.

Non-coding RNAs underlying the pathophysiological links between type 2 diabetes and pancreatic cancer: A systematic review.

Type 2 diabetes is known as a risk factor for pancreatic cancer (PC). Various genetic and environmental factors cause both these global chronic diseases. The mechanisms that define their relationships are complex and poorly understood. Recent studies have implicated that metabolic abnormalities, including hyperglycemia and hyperinsulinemia, could lead to cell damage responses, cell transformation, and increased cancer risk. Hence, these kinds of abnormalities following molecular events could be essential to develop our understanding of this complicated link. Among different molecular events, focusing on shared signaling pathways including metabolic (PI3K/Akt/mTOR) and mitogenic (MAPK) pathways in addition to regulatory mechanisms of gene expression such as those involved in non-coding RNAs (miRNAs, circRNAs, and lncRNAs) could be considered as powerful tools to describe this association. A better understanding of the molecular mechanisms involved in the development of type 2 diabetes and pancreatic cancer would help us to find a new research area for developing therapeutic and preventive strategies. For this purpose, in this review, we focused on the shared molecular events resulting in type 2 diabetes and pancreatic cancer. First, a comprehensive literature review was performed to determine similar molecular pathways and non-coding RNAs; then, the final results were discussed in more detail.

Decitabine Induces Gene Derepression on Monosomic Chromosomes: In Vitro and In Vivo Effects in Adverse-Risk Cytogenetics AML.

Hypomethylating agents (HMA) have become the backbone of nonintensive acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) treatment, also by virtue of their activity in patients with adverse genetics, for example, monosomal karyotypes, often with losses on chromosome 7, 5, or 17. No comparable activity is observed with cytarabine, a cytidine analogue without DNA-hypomethylating properties. As evidence exists for compounding hypermethylation and gene silencing of hemizygous tumor suppressor genes (TSG), we thus hypothesized that this effect may preferentially be reversed by the HMAs decitabine and azacitidine. An unbiased RNA-sequencing approach was developed to interrogate decitabine-induced transcriptome changes in AML cell lines with or without a deletion of chromosomes 7q, 5q or 17p. HMA treatment preferentially upregulated several hemizygous TSG in this genomic region, significantly derepressing endogenous retrovirus (ERV)3-1, with promoter demethylation, enhanced chromatin accessibility, and increased H3K4me3 levels. Decitabine globally reactivated multiple transposable elements, with activation of the dsRNA sensor RIG-I and interferon regulatory factor (IRF)7. Induction of ERV3-1 and RIG-I mRNA was also observed during decitabine treatment in vivo in serially sorted peripheral blood AML blasts. In patient-derived monosomal karyotype AML murine xenografts, decitabine treatment resulted in superior survival rates compared with cytarabine. Collectively, these data demonstrate preferential gene derepression and ERV reactivation in AML with chromosomal deletions, providing a mechanistic explanation that supports the clinical observation of superiority of HMA over cytarabine in this difficult-to-treat patient group. SIGNIFICANCE: These findings unravel the molecular mechanism underlying the intriguing clinical activity of HMAs in AML/MDS patients with chromosome 7 deletions and other monosomal karyotypes.See related commentary by O'Hagan et al., p. 813.

Update on the moFF Algorithm for Label-Free Quantitative Proteomics.

moFF is a modular and operating-system-independent tool for quantitative analysis of label-free mass-spectrometry-based proteomics data. The moFF workflow, comprising matching-between-runs and apex quantification, can be applied to any upstream search engine's output, along with the corresponding Thermo or mzML raw file. We here present moFF 2.0, with improvements in speed through multithreading, the use of a new raw file access library, and a novel filtering approach in the matching-between-runs module. This filter allows moFF to correctly identify features that are present in one run but not in another, as demonstrated using spiked-in iRT peptides. Moreover, moFF 2.0 also provides a new peptide summary export that can be used in downstream statistical analysis. moFF is open source and freely available and can be downloaded from https://github.com/compomics/moFF.

Combinatorial Omics Analysis Reveals Perturbed Lysosomal Homeostasis in Collagen VII-deficient Keratinocytes.

The extracellular matrix protein collagen VII is part of the microenvironment of stratified epithelia and critical in organismal homeostasis. Mutations in the encoding gene COL7A1 lead to the skin disorder dystrophic epidermolysis bullosa (DEB), are linked to skin fragility and progressive inflammation-driven fibrosis that facilitates aggressive skin cancer. So far, these changes have been linked to mesenchymal alterations, the epithelial consequences of collagen VII loss remaining under-addressed. As epithelial dysfunction is a principal initiator of fibrosis, we performed a comprehensive transcriptome and proteome profiling of primary human keratinocytes from DEB and control subjects to generate global and detailed images of dysregulated epidermal molecular pathways linked to loss of collagen VII. These revealed downregulation of interaction partners of collagen VII on mRNA and protein level, but also increased abundance of S100 pro-inflammatory proteins in primary DEB keratinocytes. Increased TGF-ß signaling because of loss of collagen VII was associated with enhanced activity of lysosomal proteases in both keratinocytes and skin of collagen VII-deficient individuals. Thus, loss of a single structural protein, collagen VII, has extra- and intracellular consequences, resulting in inflammatory processes that enable tissue destabilization and promote keratinocyte-driven, progressive fibrosis.

An Accessible Proteogenomics Informatics Resource for Cancer Researchers.

Proteogenomics has emerged as a valuable approach in cancer research, which integrates genomic and transcriptomic data with mass spectrometry-based proteomics data to directly identify expressed, variant protein sequences that may have functional roles in cancer. This approach is computationally intensive, requiring integration of disparate software tools into sophisticated workflows, challenging its adoption by nonexpert, bench scientists. To address this need, we have developed an extensible, Galaxy-based resource aimed at providing more researchers access to, and training in, proteogenomic informatics. Our resource brings together software from several leading research groups to address two foundational aspects of proteogenomics: (i) generation of customized, annotated protein sequence databases from RNA-Seq data; and (ii) accurate matching of tandem mass spectrometry data to putative variants, followed by filtering to confirm their novelty. Directions for accessing software tools and workflows, along with instructional documentation, can be found at z.umn.edu/canresgithub. Cancer Res; 77(21); e43-46. ©2017 AACR.

EBF1 binds to EBNA2 and promotes the assembly of EBNA2 chromatin complexes in B cells.

Epstein-Barr virus (EBV) infection converts resting human B cells into permanently proliferating lymphoblastoid cell lines (LCLs). The Epstein-Barr virus nuclear antigen 2 (EBNA2) plays a key role in this process. It preferentially binds to B cell enhancers and establishes a specific viral and cellular gene expression program in LCLs. The cellular DNA binding factor CBF1/CSL serves as a sequence specific chromatin anchor for EBNA2. The ubiquitous expression of this highly conserved protein raises the question whether additional cellular factors might determine EBNA2 chromatin binding selectively in B cells. Here we used CBF1 deficient B cells to identify cellular genes up or downregulated by EBNA2 as well as CBF1 independent EBNA2 chromatin binding sites. Apparently, CBF1 independent EBNA2 target genes and chromatin binding sites can be identified but are less frequent than CBF1 dependent EBNA2 functions. CBF1 independent EBNA2 binding sites are highly enriched for EBF1 binding motifs. We show that EBNA2 binds to EBF1 via its N-terminal domain. CBF1 proficient and deficient B cells require EBF1 to bind to CBF1 independent binding sites. Our results identify EBF1 as a co-factor of EBNA2 which conveys B cell specificity to EBNA2.

MicroRNA Profiling in Aqueous Humor of Individual Human Eyes by Next-Generation Sequencing.

PURPOSE: Extracellular microRNAs (miRNAs) in aqueous humor were suggested to have a role in transcellular signaling and may serve as disease biomarkers. The authors adopted next-generation sequencing (NGS) techniques to further characterize the miRNA profile in single samples of 60 to 80 µL human aqueous humor. METHODS: Samples were obtained at the outset of cataract surgery in nine independent, otherwise healthy eyes. Four samples were used to extract RNA and generate sequencing libraries, followed by an adapter-driven amplification step, electrophoretic size selection, sequencing, and data analysis. Five samples were used for quantitative PCR (qPCR) validation of NGS results. Published NGS data on circulating miRNAs in blood were analyzed in comparison. RESULTS: One hundred fifty-eight miRNAs were consistently detected by NGS in all four samples; an additional 59 miRNAs were present in at least three samples. The aqueous humor miRNA profile shows some overlap with published NGS-derived inventories of circulating miRNAs in blood plasma with high prevalence of human miR-451a, -21, and -16. In contrast to blood, miR-184, -4448, -30a, -29a, -29c, -19a, -30d, -205, -24, -22, and -3074 were detected among the 20 most prevalent miRNAs in aqueous humor. Relative expression patterns of miR-451a, -202, and -144 suggested by NGS were confirmed by qPCR. CONCLUSIONS: Our data illustrate the feasibility of miRNA analysis by NGS in small individual aqueous humor samples. Intraocular cells as well as blood plasma contribute to the extracellular aqueous humor miRNome. The data suggest possible roles of miRNA in intraocular cell adhesion and signaling by TGF-ß and Wnt, which are important in intraocular pressure regulation and glaucoma.

An NGS Workflow Blueprint for DNA Sequencing Data and Its Application in Individualized Molecular Oncology.

Next-generation sequencing (NGS) technologies that have advanced rapidly in the past few years possess the potential to classify diseases, decipher the molecular code of related cell processes, identify targets for decision-making on targeted therapy or prevention strategies, and predict clinical treatment response. Thus, NGS is on its way to revolutionize oncology. With the help of NGS, we can draw a finer map for the genetic basis of diseases and can improve our understanding of diagnostic and prognostic applications and therapeutic methods. Despite these advantages and its potential, NGS is facing several critical challenges, including reduction of sequencing cost, enhancement of sequencing quality, improvement of technical simplicity and reliability, and development of semiautomated and integrated analysis workflow. In order to address these challenges, we conducted a literature research and summarized a four-stage NGS workflow for providing a systematic review on NGS-based analysis, explaining the strength and weakness of diverse NGS-based software tools, and elucidating its potential connection to individualized medicine. By presenting this four-stage NGS workflow, we try to provide a minimal structural layout required for NGS data storage and reproducibility.

Autosomal dominant immune dysregulation syndrome in humans with CTLA4 mutations.

The protein cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential negative regulator of immune responses, and its loss causes fatal autoimmunity in mice. We studied a large family in which five individuals presented with a complex, autosomal dominant immune dysregulation syndrome characterized by hypogammaglobulinemia, recurrent infections and multiple autoimmune clinical features. We identified a heterozygous nonsense mutation in exon 1 of CTLA4. Screening of 71 unrelated patients with comparable clinical phenotypes identified five additional families (nine individuals) with previously undescribed splice site and missense mutations in CTLA4. Clinical penetrance was incomplete (eight adults of a total of 19 genetically proven CTLA4 mutation carriers were considered unaffected). However, CTLA-4 protein expression was decreased in regulatory T cells (Treg cells) in both patients and carriers with CTLA4 mutations. Whereas Treg cells were generally present at elevated numbers in these individuals, their suppressive function, CTLA-4 ligand binding and transendocytosis of CD80 were impaired. Mutations in CTLA4 were also associated with decreased circulating B cell numbers. Taken together, mutations in CTLA4 resulting in CTLA-4 haploinsufficiency or impaired ligand binding result in disrupted T and B cell homeostasis and a complex immune dysregulation syndrome.

Characterization and phylogenetic analysis of the mitochondrial genome of Glarea lozoyensis indicates high diversity within the order Helotiales.

BACKGROUND: Glarea lozoyensis is a filamentous fungus used for the industrial production of non-ribosomal peptide pneumocandin B0. In the scope of a whole genome sequencing the complete mitochondrial genome of the fungus has been assembled and annotated. It is the first one of the large polyphyletic Helotiaceae family. A phylogenetic analysis was performed based on conserved proteins of the oxidative phosphorylation system in mitochondrial genomes. RESULTS: The total size of the mitochondrial genome is 45,038 bp. It contains the expected 14 genes coding for proteins related to oxidative phosphorylation,two rRNA genes, six hypothetical proteins, three intronic genes of which two are homing endonucleases and a ribosomal protein rps3. Additionally there is a set of 33 tRNA genes. All genes are located on the same strand. Phylogenetic analyses based on concatenated mitochondrial protein sequences confirmed that G. lozoyensis belongs to the order of Helotiales and that it is most closely related to Phialocephala subalpina. However, a comparison with the three other mitochondrial genomes known from Helotialean species revealed remarkable differences in size, gene content and sequence. Moreover, it was found that the gene order found in P. subalpina and Sclerotinia sclerotiorum is not conserved in G. lozoyensis. CONCLUSION: The arrangement of genes and other differences found between the mitochondrial genome of G. lozoyensis and those of other Helotiales indicates a broad genetic diversity within this large order. Further mitochondrial genomes are required in order to determine whether there is a continuous transition between the different forms of mitochondrial genomes or G. lozoyensis belongs to a distinct subgroup within Helotiales.

StreptomeDB: a resource for natural compounds isolated from Streptomyces species.

Bacteria from the genus Streptomyces are very important for the production of natural bioactive compounds such as antibiotic, antitumour or immunosuppressant drugs. Around two-thirds of all known natural antibiotics are produced by these bacteria. An enormous quantity of crucial data related to this genus has been generated and published, but so far no freely available and comprehensive database exists. Here, we present StreptomeDB (http://www.pharmaceutical-bioinformatics.de/streptomedb/). To the best of our knowledge, this is the largest database of natural products isolated from Streptomyces. It contains >2400 unique and diverse compounds from >1900 different Streptomyces strains and substrains. In addition to names and molecular structures of the compounds, information about source organisms, references, biological role, activities and synthesis routes (e.g. polyketide synthase derived and non-ribosomal peptides derived) is included. Data can be accessed through queries on compound names, chemical structures or organisms. Extraction from the literature was performed through automatic text mining of thousands of articles from PubMed, followed by manual curation. All annotated compound structures can be downloaded from the website and applied for in silico screenings for identifying new active molecules with undiscovered properties.

Genome sequence of the fungus Glarea lozoyensis: the first genome sequence of a species from the Helotiaceae family.

The anamorphic fungus Glarea lozoyensis mutant strain 74030 is an overproducer of pneumocandin B(0), which is chemically converted into Cancidas, a potent antibiotic against clinically important fungal pathogens. Pneumocandins are acylated, cyclic hexapeptides with unusual hydroxylated amino acids. With the Glarea lozoyensis genome, the first species from the large polyphyletic family Helotiaceae has been sequenced.

Small-molecule conversion of toxic oligomers to nontoxic ß-sheet-rich amyloid fibrils.

Several lines of evidence indicate that prefibrillar assemblies of amyloid-ß (Aß) polypeptides, such as soluble oligomers or protofibrils, rather than mature, end-stage amyloid fibrils cause neuronal dysfunction and memory impairment in Alzheimer's disease. These findings suggest that reducing the prevalence of transient intermediates by small molecule-mediated stimulation of amyloid polymerization might decrease toxicity. Here we demonstrate the acceleration of Aß fibrillogenesis through the action of the orcein-related small molecule O4, which directly binds to hydrophobic amino acid residues in Aß peptides and stabilizes the self-assembly of seeding-competent, ß-sheet-rich protofibrils and fibrils. Notably, the O4-mediated acceleration of amyloid fibril formation efficiently decreases the concentration of small, toxic Aß oligomers in complex, heterogeneous aggregation reactions. In addition, O4 treatment suppresses inhibition of long-term potentiation by Aß oligomers in hippocampal brain slices. These results support the hypothesis that small, diffusible prefibrillar amyloid species rather than mature fibrillar aggregates are toxic for mammalian cells.

Genome sequence of Streptomyces sp. strain Tü6071.

Streptomyces sp. Tü6071 is a soil-dwelling bacterium which has a highly active isoprenoid biosynthesis. Isoprenoids are important precursors for biopharmaceutical molecules such as antibiotics or anticancer agents, e.g., landomycin. Streptomyces sp. Tü6071 produces the industrially important terpene glycosides phenalinolactones, which have antibacterial activity against several Gram-positive bacteria. The availability of the genome sequence of Streptomyces sp. Tü6071 allows for understanding the biosynthesis of these pharmaceutical molecules and will facilitate rational genome modification to improve industrial use.