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  • 1
    In: Cells, MDPI AG, Vol. 11, No. 24 ( 2022-12-08), p. 3970-
    Abstract: The evolutionary conserved NEAT1-MALAT1 gene cluster generates large noncoding transcripts remaining nuclear, while tRNA-like transcripts (mascRNA, menRNA) enzymatically generated from these precursors translocate to the cytosol. Whereas functions have been assigned to the nuclear transcripts, data on biological functions of the small cytosolic transcripts are sparse. We previously found NEAT1−/− and MALAT1−/− mice to display massive atherosclerosis and vascular inflammation. Here, employing selective targeted disruption of menRNA or mascRNA, we investigate the tRNA-like molecules as critical components of innate immunity. CRISPR-generated human ΔmascRNA and ΔmenRNA monocytes/macrophages display defective innate immune sensing, loss of cytokine control, imbalance of growth/angiogenic factor expression impacting upon angiogenesis, and altered cell–cell interaction systems. Antiviral response, foam cell formation/oxLDL uptake, and M1/M2 polarization are defective in ΔmascRNA/ΔmenRNA macrophages, defining first biological functions of menRNA and describing new functions of mascRNA. menRNA and mascRNA represent novel components of innate immunity arising from the noncoding genome. They appear as prototypes of a new class of noncoding RNAs distinct from others (miRNAs, siRNAs) by biosynthetic pathway and intracellular kinetics. Their NEAT1-MALAT1 region of origin appears as archetype of a functionally highly integrated RNA processing system.
    Type of Medium: Online Resource
    ISSN: 2073-4409
    Language: English
    Publisher: MDPI AG
    Publication Date: 2022
    detail.hit.zdb_id: 2661518-6
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  • 2
    In: Health Physics, Ovid Technologies (Wolters Kluwer Health), Vol. 119, No. 1 ( 2020-7), p. 109-117
    Abstract: Little is known about the mutational impact of ionizing radiation (IR) exposure on a genome-wide level in mammalian tissues. Recent advancements in sequencing technology have provided powerful tools to perform exome-wide analyses of genetic variation. This also opened up new avenues for studying and characterizing global genomic IR-induced effects. However, genotypes generated by next generation sequencing (NGS) studies can contain errors, which may significantly impact the power to detect signals in common and rare variant analyses. These genotyping errors are not explicitly detected by the standard Genotype Analysis ToolKit (GATK) and Variant Quality Score Recalibration (VQSR) tool and thus remain a potential source of false-positive variants in whole exome sequencing (WES) datasets. In this context, the transition-transversion ratio (Ti/Tv) is commonly used as an additional quality check. In case of IR experiments, this is problematic when Ti/Tv itself might be influenced by IR treatment. It was the aim of this study to determine a suitable threshold for variant filters for NGS datasets from irradiated cells in order to achieve high data quality using Ti/Tv, while at the same time being able to investigate radiation-specific effects on the Ti/Tv ratio for different radiation doses. By testing a variety of filter settings and comparing the obtained results with publicly available datasets, we observe that a coverage filter setting of depth (DP) 3 and genotype quality (GQ) 20 is sufficient for high quality single nucleotide variants (SNVs) calling in an analysis combining GATK and VSQR and that Ti/Tv values are a consistent and useful indicator for data quality assessment for all tested NGS platforms. Furthermore, we report a reduction in Ti/Tv in IR-induced mutations in primary human gingiva fibroblasts (HGFs), which points to an elevated proportion of transversions among IR-induced SNVs and thus might imply that mismatch repair (MMR) plays a role in the cellular damage response to IR-induced DNA lesions.
    Type of Medium: Online Resource
    ISSN: 1538-5159 , 0017-9078
    RVK:
    Language: English
    Publisher: Ovid Technologies (Wolters Kluwer Health)
    Publication Date: 2020
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  • 3
    In: Emerging Microbes & Infections, Informa UK Limited, Vol. 12, No. 2 ( 2023-12-08)
    Type of Medium: Online Resource
    ISSN: 2222-1751
    Language: English
    Publisher: Informa UK Limited
    Publication Date: 2023
    detail.hit.zdb_id: 2681359-2
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  • 4
    In: Frontiers in Pharmacology, Frontiers Media SA, Vol. 12 ( 2021-5-7)
    Abstract: Organic cation transporter 1 (OCT1, SLC22A1) is localized in the sinusoidal membrane of human hepatocytes and mediates hepatic uptake of weakly basic or cationic drugs and endogenous compounds. Common amino acid substitutions in OCT1 were associated with altered pharmacokinetics and efficacy of drugs like sumatriptan and fenoterol. Recently, the common splice variant rs35854239 has also been suggested to affect OCT1 function. rs35854239 represents an 8 bp duplication of the donor splice site at the exon 7-intron 7 junction. Here we quantified the extent to which this duplication affects OCT1 splicing and, as a consequence, the expression and the function of OCT1. We used pyrosequencing and deep RNA-sequencing to quantify the effect of rs35854239 on splicing after minigene expression of this variant in HepG2 and Huh7 cells and directly in human liver samples. Further, we analyzed the effects of rs35854239 on OCT1 mRNA expression in total, localization and activity of the resulting OCT1 protein, and on the pharmacokinetics of sumatriptan and fenoterol. The 8 bp duplication caused alternative splicing in 38% (deep RNA-sequencing) to 52% (pyrosequencing) of the minigene transcripts when analyzed in HepG2 and Huh7 cells. The alternatively spliced transcript encodes for a truncated protein that after transient transfection in HEK293 cells was not localized in the plasma membrane and was not able to transport the OCT1 model substrate ASP + . In human liver, however, the alternatively spliced OCT1 transcript was detectable only at very low levels (0.3% in heterozygous and 0.6% in homozygous carriers of the 8 bp duplication, deep RNA-sequencing). The 8 bp duplication was associated with a significant reduction of OCT1 expression in the human liver, but explained only 9% of the general variability in OCT1 expression and was not associated with significant changes in the pharmacokinetics of sumatriptan and fenoterol. Therefore, the rs35854239 variant only partially changes splicing, causing moderate changes in OCT1 expression and may be of only limited therapeutic relevance.
    Type of Medium: Online Resource
    ISSN: 1663-9812
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2021
    detail.hit.zdb_id: 2587355-6
    SSG: 15,3
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  • 5
    Online Resource
    Online Resource
    Frontiers Media SA ; 2023
    In:  Frontiers in Microbiology Vol. 14 ( 2023-6-26)
    In: Frontiers in Microbiology, Frontiers Media SA, Vol. 14 ( 2023-6-26)
    Abstract: Eukaryotic algae in the top few centimeters of fellfield soils of ice-free Maritime Antarctica have many important effects on their habitat, such as being significant drivers of organic matter input into the soils and reducing the impact of wind erosion by soil aggregate formation. To better understand the diversity and distribution of Antarctic terrestrial algae, we performed a pilot study on the surface soils of Meseta , an ice-free plateau mountain crest of Fildes Peninsula, King George Island, being hardly influenced by the marine realm and anthropogenic disturbances. It is openly exposed to microbial colonization from outside Antarctica and connected to the much harsher and dryer ice-free zones of the continental Antarctic. A temperate reference site under mild land use, SchF , was included to further test for the Meseta algae distribution in a contrasting environment. Methods We employed a paired-end metabarcoding analysis based on amplicons of the highly variable nuclear-encoded ITS2 rDNA region, complemented by a clone library approach. It targeted the four algal classes, Chlorophyceae, Trebouxiophyceae, Ulvophyceae, and Xanthophyceae, representing key groups of cold-adapted soil algae. Results A surprisingly high diversity of 830 algal OTUs was revealed, assigned to 58 genera in the four targeted algal classes. Members of the green algal class Trebouxiophyceae predominated in the soil algae communities. The major part of the algal biodiversity, 86.1% of all algal OTUs, could not be identified at the species level due to insufficient representation in reference sequence databases. The classes Ulvophyceae and Xanthophyceae exhibited the most unknown species diversity. About 9% of the Meseta algae species diversity was shared with that of the temperate reference site in Germany. Discussion In the small portion of algal OTUs for which their distribution could be assessed, the entire ITS2 sequence identity with references shows that the soil algae likely have a wide distribution beyond the Polar regions. They probably originated from soil algae propagule banks in far southern regions, transported by aeolian transport over long distances. The dynamics and severity of environmental conditions at the soil surface, determined by high wind currents, and the soil algae’s high adaptability to harsh environmental conditions may account for the high similarity of soil algal communities between the northern and southern parts of the Meseta .
    Type of Medium: Online Resource
    ISSN: 1664-302X
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2023
    detail.hit.zdb_id: 2587354-4
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  • 6
    Online Resource
    Online Resource
    Frontiers Media SA ; 2022
    In:  Frontiers in Bioengineering and Biotechnology Vol. 10 ( 2022-8-25)
    In: Frontiers in Bioengineering and Biotechnology, Frontiers Media SA, Vol. 10 ( 2022-8-25)
    Abstract: Exogenous glucocorticoids increase the risk for osteoporosis, but the role of endogenous glucocorticoids remains elusive. Here, we describe the generation and validation of a loss- and a gain-of-function model of the cortisol producing enzyme 11β-HSD1 ( HSD11B1 ) to modulate the endogenous glucocorticoid conversion in SCP-1 cells — a model for human mesenchymal stem cells capable of adipogenic and osteogenic differentiation. CRISPR-Cas9 was successfully used to generate a cell line carrying a single base duplication and a 5 bp deletion in exon 5, leading to missense amino acid sequences after codon 146. These inactivating genomic alterations were validated by deep sequencing and by cloning with subsequent capillary sequencing. 11β-HSD1 protein levels were reduced by 70% in the knockout cells and cortisol production was not detectable. Targeted chromosomal integration was used to stably overexpress HSD11B1 . Compared to wildtype cells, HSD11B1 overexpression resulted in a 7.9-fold increase in HSD11B1 mRNA expression, a 5-fold increase in 11β-HSD1 protein expression and 3.3-fold increase in extracellular cortisol levels under adipogenic differentiation. The generated cells were used to address the effects of 11β-HSD1 expression on adipogenic and osteogenic differentiation. Compared to the wildtype, HSD11B1 overexpression led to a 3.7-fold increase in mRNA expression of lipoprotein lipase (LPL) and 2.5-fold increase in lipid production under adipogenic differentiation. Under osteogenic differentiation, HSD11B1 knockout led to enhanced alkaline phosphatase (ALP) activity and mRNA expression, and HSD11B1 overexpression resulted in a 4.6-fold and 11.7-fold increase in mRNA expression of Dickkopf-related protein 1 (DKK1) and LPL , respectively. Here we describe a HSD11B1 loss- and gain-of-function model in SCP-1 cells at genetic, molecular and functional levels. We used these models to study the effects of endogenous cortisol production on mesenchymal stem cell differentiation and demonstrate an 11β-HSD1 dependent switch from osteogenic to adipogenic differentiation. These results might help to better understand the role of endogenous cortisol production in osteoporosis on a molecular and cellular level.
    Type of Medium: Online Resource
    ISSN: 2296-4185
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2022
    detail.hit.zdb_id: 2719493-0
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  • 7
    Online Resource
    Online Resource
    MDPI AG ; 2020
    In:  International Journal of Molecular Sciences Vol. 21, No. 16 ( 2020-08-12), p. 5778-
    In: International Journal of Molecular Sciences, MDPI AG, Vol. 21, No. 16 ( 2020-08-12), p. 5778-
    Abstract: While ionizing radiation (IR) is a powerful tool in medical diagnostics, nuclear medicine, and radiology, it also is a serious threat to the integrity of genetic material. Mutagenic effects of IR to the human genome have long been the subject of research, yet still comparatively little is known about the genome-wide effects of IR exposure on the DNA-sequence level. In this study, we employed high throughput sequencing technologies to investigate IR-induced DNA alterations in human gingiva fibroblasts (HGF) that were acutely exposed to 0.5, 2, and 10 Gy of 240 kV X-radiation followed by repair times of 16 h or 7 days before whole-genome sequencing (WGS). Our analysis of the obtained WGS datasets revealed patterns of IR-induced variant (SNV and InDel) accumulation across the genome, within chromosomes as well as around the borders of topologically associating domains (TADs). Chromosome 19 consistently accumulated the highest SNVs and InDels events. Translocations showed variable patterns but with recurrent chromosomes of origin (e.g., Chr7 and Chr16). IR-induced InDels showed a relative increase in number relative to SNVs and a characteristic signature with respect to the frequency of triplet deletions in areas without repetitive or microhomology features. Overall experimental conditions and datasets the majority of SNVs per genome had no or little predicted functional impact with a maximum of 62, showing damaging potential. A dose-dependent effect of IR was surprisingly not apparent. We also observed a significant reduction in transition/transversion (Ti/Tv) ratios for IR-dependent SNVs, which could point to a contribution of the mismatch repair (MMR) system that strongly favors the repair of transitions over transversions, to the IR-induced DNA-damage response in human cells. Taken together, our results show the presence of distinguishable characteristic patterns of IR-induced DNA-alterations on a genome-wide level and implicate DNA-repair mechanisms in the formation of these signatures.
    Type of Medium: Online Resource
    ISSN: 1422-0067
    Language: English
    Publisher: MDPI AG
    Publication Date: 2020
    detail.hit.zdb_id: 2019364-6
    SSG: 12
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  • 8
    In: Journal of Clinical Medicine, MDPI AG, Vol. 12, No. 1 ( 2023-01-01), p. 335-
    Abstract: During the past few years, unexpected developments have driven studies in the field of clinical immunology. One driver of immense impact was the outbreak of a pandemic caused by the novel virus SARS-CoV-2. Excellent recent reviews address diverse aspects of immunological re-search into cardiovascular diseases. Here, we specifically focus on selected studies taking advantage of advanced state-of-the-art molecular genetic methods ranging from genome-wide epi/transcriptome mapping and variant scanning to optogenetics and chemogenetics. First, we discuss the emerging clinical relevance of advanced diagnostics for cardiovascular diseases, including those associated with COVID-19—with a focus on the role of inflammation in cardiomyopathies and arrhythmias. Second, we consider newly identified immunological interactions at organ and system levels which affect cardiovascular pathogenesis. Thus, studies into immune influences arising from the intestinal system are moving towards therapeutic exploitation. Further, powerful new research tools have enabled novel insight into brain–immune system interactions at unprecedented resolution. This latter line of investigation emphasizes the strength of influence of emotional stress—acting through defined brain regions—upon viral and cardiovascular disorders. Several challenges need to be overcome before the full impact of these far-reaching new findings will hit the clinical arena.
    Type of Medium: Online Resource
    ISSN: 2077-0383
    Language: English
    Publisher: MDPI AG
    Publication Date: 2023
    detail.hit.zdb_id: 2662592-1
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