Biology subjects

Ziegler, A.

Publications and source records attributed to Ziegler, A..

11 recordsLinked to original sources

bioRxiv2025

Targeting the homeodomain of ceramide-synthase can ameliorate insulin resistance

Multiple studies have linked ceramide accumulation with insulin resistance and diabetes. Ceramide Synthases (CerS) are at the center of ceramide de novo formation. Impaired CerS activity leads to lower ceramide and resolves insulin resistance. Drosophila has only one CerS, named Schlank, which contains a catalytic lag1p motif and, like many CerS, a homeodomain regulating lipid homeostasis. How CerS homeodomains are associated with diabetes has been little studied. Here we demonstrate that, depending on the respective mutation in the CerS homeodomain high sugar diet (HSD)-induced insulin resistance is exacerbated or ameliorated. HSD shifts the profile of sphingolipids towards polyunsaturated longer sphingoid bases, systemic insulin signaling is reduced, as indicated by nuclear accumulation of FoxO, and secretion of insulin-like peptide 2 (DILP2) is impaired. Expression of a CerS variant with a mutation in the nuclear localization signal 2 within its homeodomain in the fat body improves systemic insulin signaling and DILP2 release. Thus, the CerS homeodomain may be a potential target to attenuate insulin resistance.

genetics↗

bioRxiv2025

Dendritic-cell diversity in equine blood revealed by single-cell transcriptomics

Unbiased classification of equine dendritic cells (DC) is necessary to address various research questions such as the role of DC subsets in immune-mediated diseases of horses. We applied single-cell RNA sequencing (scRNA-seq) on DC enriched from the blood of two horses. All main DC subsets were detected by key gene expression, including conventional DC type 1 (cDC1; XCR1) and type 2 (cDC2; FCER1A, CD1E) as well as plasmacytoid DC (pDC; TCF4). In addition, we detected a small cluster of hematopoietic progenitors, as well as transitional DC (tDC; FCER1A, TCF4) and putative DC type 3 (DC3; FLT3, CD163). Our data confirms the previously reported phenotype of equine pDC (Flt3+MHC-IIlowCADM1lowCD172aint), cDC1 (Flt3+MHC-IIhighCADM1highCD172alow-int) and cDC2 (Flt3+MHC-IIhighCADM1intCD172ahigh), while also highlighting considerable CD14 expression for cDC2. Two subclusters of equine cDC2 were found to be enriched in FCER1A or CX3CR1 transcripts (cDC2.1 and cDC2.2, respectively), with suggested enhanced extravasation and T-cell stimulatory capacities of the latter. Conservation of DC subsets across species (horse, pig, human, mouse) was illustrated by enrichment analyses with subset-specific gene signatures and by cross-species data integration with publicly available scRNA-seq datasets. Our atlas of equine blood DC is a valuable resource for comparative analyses, and it forms the foundation for understanding the involvement of distinct DC subsets in infections and immune-mediated pathologies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/644174v2_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@bdce22org.highwire.dtl.DTLVardef@1285715org.highwire.dtl.DTLVardef@16d37c8org.highwire.dtl.DTLVardef@1695121_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Culicoides allergens expressed in insect cells induce sulfidoleukotriene release in peripheral blood leukocytes from horses affected with insect bite hypersensitivity

IntroductionInsect bite hypersensitivity (IBH) is an IgE mediated dermatitis in horses caused by bites of Culicoides spp. The allergens are salivary gland proteins from these insects and nine major allergens from Culicoides obsoletus have been identified and expressed in E. coli. However, proteins expressed in procaryotic systems have limitations in cellular assays, particularly in functional assays assessing the allergen-induced release of mediators in vitro, such as sulfidoleukotrienes (sLT) from basophils. Aims of the study were to produce functional Culicoides allergens in insect cells, to assess their allergenicity using a sLT release assay and to relate the sLT release with IgE sensitization to the respective allergens using ELISA. MethodsEight major Culicoides obsoletus allergens (Cul o 1P, Cul o 2P, Cul o 3, Cul o 5, Cul o 7, Cul o 8, Cul o 9 and Cul o 11) were expressed in insect cells and purified. sLT release from peripheral blood leukocytes (PBL) following stimulation with the eight Culicoides allergens was measured in 28 IBH-affected and 24 healthy control horses. Allergen-specific serum IgE levels was determined by ELISA. ResultsThe eight major allergens were successfully expressed in insect cells and purified. All allergens induced a significantly higher sLT release from PBL of IBH-affected horses compared to healthy controls. There was a high correlation and substantial to excellent agreement between sLT release and serum IgE levels for six Culicoides allergens, while for two the agreement was moderate. Positivity rates in IBH horses were usually higher in IgE serology but more false positive results were obtained. The allergens performing best in both assays were Cul o 3, Cul o 8 and Cul o 9, with very high specificity and good sensitivity. DiscussionInsect cell expressed Culicoides recombinant allergens are functionally relevant and will open new opportunities for the study of Culicoides hypersensitivity not only in horses, but also potentially in human patients or other species. They will also greatly improve IBH diagnostics using cellular assays as well as IgE serology.

immunology↗

bioRxiv2024

Discovery and functional analysis of a novel ALPK1 variant causing ROSAH syndrome

ROSAH syndrome is an autosomal dominant autoinflammatory disorder characterised by visual disturbance caused by pathogenic variation in the protein kinase ALPK1. Only two such variants have been reported to cause ROSAH syndrome to date: 66 out of 67 patients harbour the Thr237Met variant, while a single patient carries a Tyr254Cys variant. Here we identify a family in which ROSAH syndrome is caused by a Ser277Phe variant in ALPK1. The phenotypic variability in this family is high, with four of the seven individuals legally blind. Hypohidrosis, splenomegaly and arthritis were present in several family members. In contrast to wildtype ALPK1, which is activated specifically by the bacterial metabolite ADP-heptose during bacterial infection, ALPK1[Ser277Phe] was also activated by the human metabolites UDP-mannose and ADP-ribose, even more strongly than the ALPK1[Thr237Met] variant. However, unlike ALPK1[Thr237Met], ALPK1[Ser277Phe] could additionally be activated by GDP-mannose. These observations can explain why these ALPK1 variants are active in cells in the absence of ADP-heptose and hence why patients have episodes of autoinflammation. Examination of the three-dimensional structure of ALPK1 revealed that the sidechains of Ser277 and Tyr254 interact but mutational analysis established that this interaction is not critical for the integrity of the ADP-heptose binding site. Instead, it is replacement of Ser277 by a large hydrophobic phenylalanine residue or the replacement of Tyr254 by a much smaller cysteine residue that is responsible for altering the specificity of the ADP-heptose-binding pocket. The characterisation of ALPK1 variants that cause ROSAH syndrome suggests ways in which drugs that inhibit these disease-causing variants selectively can be developed.

genetics↗

bioRxiv2024

Supramolecular Enzyme-Peptide Gels for Localized Therapeutic Biocatalysis

Enzyme therapeutics are often compromised by poor accumulation within target tissues, necessitating repeated systemic dosing that can exacerbate side-effects like immunogenicity. We report an injectable supramolecular enzyme-peptide gel platform for prolonged local enzyme retention in vivo. The gel is based on CATCH(+/-) (Co-Assembling Tags based on CHarge-complementarity), cationic and anionic peptide pairs that form {beta}-sheet fibrillar gels upon mixing. Five disparate enzymes recombinantly fused to CATCH(-) peptide integrate into CATCH(+/-) fibrils during assembly, resulting in five single-enzyme and three dual-enzyme catalytically-active enzyme-peptide gels. CATCH gels enable titratable catalytic activity and prolonged localized retention. Gels of uricase, an FDA-approved but systemically-ineffective enzyme therapy for gout, rapidly dissolve gout crystals, with low risk of anaphylaxis after repeat dosing. Dual-enzyme gels of uricase and anti-inflammatory indoleamine 2,3-dioxygenase outperformed either single-enzyme formulation by concomitantly dissolving the crystals and suppressing pro-inflammatory neutrophil functions. Modular and tunable enzyme content, coupled with prolonged retention, establish CATCH enzyme-peptide gels as a generalizable vehicle for effective local therapeutic biocatalysis.

bioengineering↗

bioRxiv2022

Comparison of calling pipelines for whole genome sequencing: an empirical study demonstrating the importance of mapping and alignment

Rapid advances in high-throughput DNA sequencing technologies have enabled the conduct of whole genome sequencing (WGS) studies, and several bioinformatics pipelines have become available. The aim of this study was the comparison of 6 WGS data pre-processing pipelines, involving two mapping and alignment approaches (GATK utilizing BWA-MEM2 2.2.1, and DRAGEN 3.8.4) and three variant calling pipelines (GATK 4.2.4.1, DRAGEN 3.8.4 and DeepVariant 1.1.0). We sequenced one genome in a bottle (GIAB) sample 70 times in different runs, and one GIAB trio in triplicate. The truth set of the GIABs was used for comparison, and performance was assessed by computation time, F1 score, precision, and recall. In the mapping and alignment step, the DRAGEN pipeline was faster than the GATK with BWA-MEM2 pipeline. DRAGEN showed systematically higher F1 score, precision, and recall values than GATK for single nucleotide variations (SNVs) and Indels in simple-to-map, complex-to-map, coding and non-coding regions. In the variant calling step, DRAGEN was fastest. In terms of accuracy, DRAGEN and DeepVariant performed similarly and both superior to GATK, with slight advantages for DRAGEN for Indels and for DeepVariant for SNVs. The DRAGEN pipeline showed the lowest Mendelian inheritance error fraction for the GIAB trios. Mapping and alignment played a key role in variant calling of WGS, with the DRAGEN outperforming GATK.

bioinformatics↗

bioRxiv2019

2D-HELS-AA MS Seq: Direct sequencing of tRNA reveals its different isoforms and multiple dynamic base modifications

We report a direct method for sequencing tRNAPhe without cDNA by combining 2-dimensional hydrophobic RNA end-labeling with an anchor-based algorithm in mass spectrometry-based sequencing (2D-HELS-AA MS Seq). The entire tRNAPhe was sequenced and the identity, location and abundance of all 11 base modifications were determined. Changes in ratios of wybutosine and its depurinated form under different conditions were quantified, pointing to the ability of our technology to determine dynamic changes of nucleotide modifications. Two truncated isoforms at 3CCA tail of the tRNAPhe (75 nt CC, 80% and 74 nt C, 3%) were identified in addition to the 76 nt tRNAPhe with a full-length 3CCA tail (17%). We also discovered a new isoform with A-G transitions at both the 44 and 45 positions in the tRNAPhe variable loop.\n\nOne Sentence SummaryDirect 2D-HELS-AA MS Seq of tRNA reveals different isoforms and base modifications

biochemistry↗

bioRxiv2019

A general LC/MS-based RNA sequencing method for direct analysis of multiple-base modifications in RNA mixtures

A complete understanding of the structural and functional potential of RNA requires understanding of chemical modifications and noncanonical bases; this in turn requires advances in current sequencing methods to be able to sequence not only canonical ribonucleotides, but at the same time directly sequence these nonstandard moieties. Here, we present the first direct and modification type-independent RNA sequencing method via integration of a hydrophobic end-labeling strategy with of 2-D mass-retention time LC/MS analysis to allow de novo sequencing of RNA mixtures and enhance sample usage efficiency. Our method can directly read out the complete sequence, while identifying, locating, and quantifying base modifications accurately in both single and mixed RNA samples containing multiple different modifications at single-base resolution. Our method can also quantify stoichiometry/percentage of modified RNA vs. its canonical counterpart RNA, simulating a real biological sample where modifications exist but may not be 100% at a particular site of the RNA. This method is a critical step towards fully sequencing real complex cellular RNA samples of any type and containing any modification types and can also be used in the quality control of modified therapeutic RNAs.

biochemistry↗

bioRxiv2018

Evaluating predictive biomarkers for a binary outcome with linear versus logistic regression - Practical recommendations for the choice of the model

A predictive biomarker can forecast whether a patient benefits from a specific treatment under study. To establish predictiveness of a biomarker, a statistical interaction between the biomarker status and the treatment group concerning the clinical outcome needs to be shown. In clinical trials looking at a binary outcome, linear or logistic regression models may be used to evaluate the interaction, but the effects in the two models are different and differently interpreted. Specifically, the effects are estimated as absolute risk reductions (ARRs) and odds ratios (ORs) in the linear and logistic model, thus measuring the effect on an additive and multiplicative scale, respectively.\n\nWe derived the relationship between the effects of the linear and the logistic regression model allowing for translations between the effect estimates between both models. In addition, we performed a comprehensive simulation study to compare the power of the two models under a variety of scenarios in different study designs. In general, the differences in power to detect interaction were minor, and visible differences were detected in rather unrealistic scenarios of effect size combinations and were usually in favor of the logistic model.\n\nBased on our results and theoretical considerations, we recommend to 1) estimate logistic regression models because of their statistical properties, 2) test for interaction effects and 3) calculate and report both ARRs and ORs from these using the formulae provided.

epidemiology↗

bioRxiv2018

Pathogenic DDX3X mutations impair RNA metabolism and neurogenesis during fetal cortical development

De novo germline mutations in the RNA helicase DDX3X account for 1-3% of unexplained intellectual disability (ID) cases in females, and are associated with autism, brain malformations, and epilepsy. Yet, the developmental and molecular mechanisms by which DDX3X mutations impair brain function are unknown. Here we use human and mouse genetics, and cell biological and biochemical approaches to elucidate mechanisms by which pathogenic DDX3X variants disrupt brain development. We report the largest clinical cohort to date with DDX3X mutations (n=78), demonstrating a striking correlation between recurrent dominant missense mutations, polymicrogyria, and the most severe clinical outcomes. We show that Ddx3x controls cortical development by regulating neuronal generation and migration. Severe DDX3X missense mutations profoundly disrupt RNA helicase activity and induce ectopic RNA-protein granules and aberrant translation in neural progenitors and neurons. Together, our study demonstrates novel mechanisms underlying DDX3X syndrome, and highlights roles for RNA-protein aggregates in the pathogenesis of neurodevelopmental disease.

genetics↗

bioRxiv2017

Low-level mitochondrial heteroplasmy modulates DNA replication, glucose metabolism and lifespan in mice

Mutations in mitochondrial DNA (mtDNA) lead to heteroplasmy, i.e. the intracellular coexistence of wild-type and mutant mtDNA strands, which impact a wide spectrum of diseases but also physiological processes, including endurance exercise performance in athletes. However, the phenotypic consequences of limited levels of naturally-arising heteroplasmy have not been experimentally studied to date. We hence generated a conplastic mouse strain carrying the mitochondrial genome of a AKR/J mouse strain (B6-mtAKR) together with a C57BL/6J nuclear genomic background, leading to >20% heteroplasmy in the origin of light-strand DNA replication (OriL). These conplastic mice demonstrate a shorter lifespan as well as dysregulation of multiple metabolic pathways, culminating in impaired glucose metabolism, compared to wild-type C57BL/6J mice carrying lower levels of heteroplasmy. Our results indicate that physiologically relevant differences in mtDNA heteroplasmy levels at a single, functionally important site impair metabolic health and lifespan in mice.\n\nHighlightsO_LIWe identify heteroplasmy of the adenine-repeat variation (9 to 13A) in nt5172 in the origin of light-strand DNA replication (OriL) in inbred mice.\nC_LIO_LIB6-mtAKR mice carry >20% 12A heteroplasmy in the OriL, while B6 mice carry only [~] 10% heteroplasmy.\nC_LIO_LIThe level of 12A heteroplasmy correlates to mtDNA copy number, glucose metabolism, and lifespan in mice.\nC_LIO_LIGiven the established role of mtDNA heteroplasmy in regards to endurance exercise performance in athletes, these findings may impact our understanding of metabolism and aging in humans.\nC_LI

genetics↗