Biology subjects

Schneider, D. A.

Publications and source records attributed to Schneider, D. A..

8 recordsLinked to original sources

bioRxiv2025

Quantification and Diagnostic Relevance of Blood and Heme-Mediated Inhibition of Prion Detection by RT-QuIC

Prion diseases are characterized by misfolding of prion protein (PrP) from correctly folded PrPC to a disease-associated form, PrPD. Real-time quaking-induced conversion (RT-QuIC), detects prions by "seeding" reaction mixtures, which contain PrPC, with samples suspected to contain prions, resulting in PrPD amplification. The assay is sensitive to inhibition by tissue constituents, including blood. Heme, a cofactor of hemoglobin (Hb), has been shown to bind PrP in an isoform-specific manner and to affect the stability of other pathogenic amyloids. In the present study, tissue samples from scrapie-positive sheep were used to seed RT-QuIC reactions in the presence of heme--as free hemin, as a cofactor of Hb, and as present in whole blood. At equivalent heme concentrations, the inhibitory action of free heme was the least and that of blood the greatest, suggesting other components of Hb and whole blood have additional inhibitory actions. We also demonstrate that this inhibition of RT-QuIC acts through disruption of the recombinant PrPC assay substrate, rather than destruction of PrPD seeds. Lastly, heme concentrations were measured in several ruminant tissues. Heme levels exceeded inhibitory thresholds in nearly all types of intact tissue but were reduced below inhibitory levels at a 1:1000 dilution of most tissue types, with whole blood being one of a few notable exceptions. Our results suggest that detection of PrPD seeding activity is not precluded by exposure to heme in tissue samples, but that the final heme concentration introduced into the RT-QuIC assay mixture is the critical factor that impacts detection sensitivity.

microbiology↗

bioRxiv2024

Ability of mycobacterial pathogens to establish a persistent infection is lost by deletion of a single gene, rel, regulator of the stringent response

Studies in a mouse model revealed Mycobacterium tuberculosis (Mtb) with a deletion of rel, regulator of the stringent response, could not establish a persistent infection. Studies in cattle with a Mycobacterium. a. paratuberculosis rel deletion mutant revealed inability to establish a persistent infection was associated with development of CD8 cytotoxic T cells (CTL) that kill intracellular bacteria. Further comparative studies ex vivo with Mbv Calmette-Guerin (BCG) and a BCG rel deletion mutant revealed no clear difference in development of CTL in vitro. As reported, a study of the recall response was conducted with cattle vaccinated with either BCG or with BCGrel, to determine if information could be obtained that would show how gene products under control of rel interfere with the CTL response to mycobacterial pathogens in vivo. The study revealed the CTL response elicited by vaccination with BCG was impaired, in comparison with the response elicited by BCGrel. Comparative analysis of the recall response ex vivo revealed the functional impairment was not associated with the timing of appearance of the recall response, expression of IFN-{gamma}, TNF-, IL-17, or IL-22, or molecules that mediate intracellular killing. Further studies are needed to determine how CD8 CTL functional activity is modulated in vivo by gene products regulated by rel.

immunology↗

bioRxiv2023

Vaccination of cattle with a virus-vectored vaccine against a major membrane protein of Mycobacterium a. subsp. paratuberculosis elicits CD8 cytotoxic T cells that kill intracellular bacteria

Infection of cattle with Mycobacterium a. subsp. Paratuberculosis (Map), the causative agent of paratuberculosis, induces an immune response directed toward a 35 kD major membrane protein (MMP) of Map. CD8 cytotoxic T cells (CTL) are elicited when peripheral blood mononuclear cells from healthy cattle are incubated ex vivo with antigen-presenting cells (APC) primed with bacterial recombinant MMP. Ex vivo development of CTL was MHC-restricted and required the presence of both CD4 and CD8 T cells. The gene MAP2121c, encoding MMP, was modified to express a modified form of MMP (p35NN) in a mammalian cell line, also capable of eliciting an ex vivo CTL response. In the present study, the modified gene for p35NN was placed into a BoHV4 vector to determine the potential use of BoHV-4A{Delta}TK-p35NN as a peptide-based vaccine. Subcutaneous vaccination of healthy cattle with BoHV-4A{Delta}TK-p35NN elicited a CTL recall response, as detected ex vivo. Further studies are warranted to conduct a challenge study to determine if CD8 CTL elicited by vaccination with BoHV-4A{Delta}TK-p35NN prevents the establishment of a persistent infection by Map.

immunology↗

bioRxiv2022

Distinct states of nucleolar stress induced by anti-cancer drugs

Ribosome biogenesis is a vital and energy-consuming cellular function occurring primarily in the nucleolus. Cancer cells have an especially high demand for ribosomes to sustain continuous proliferation. This study evaluated the impact of existing anticancer drugs on the nucleolus by screening a library of anticancer compounds for drugs that induce nucleolar stress. For a readout, a novel parameter termed "nucleolar normality score" was developed that measures the ratio of the fibrillar center and granular component proteins in the nucleolus and nucleoplasm. Multiple classes of drugs were found to induce nucleolar stress, including DNA intercalators, inhibitors of mTOR/PI3K, heat shock proteins, proteasome, and cyclin-dependent kinases (CDKs). Each class of drugs induced morphologically and molecularly distinct states of nucleolar stress accompanied by changes in nucleolar biophysical properties. In-depth characterization focused on the nucleolar stress induced by inhibition of transcriptional CDKs, particularly CDK9, the main CDK that regulates RNA Pol II. Multiple CDK substrates were identified in the nucleolus, including RNA Pol I - recruiting protein Treacle, which was phosphorylated by CDK9 in vitro. These results revealed a concerted regulation of RNA Pol I and Pol II by transcriptional CDKs. Our findings exposed many classes of chemotherapy compounds that are capable of inducing nucleolar stress, and we recommend considering this in anticancer drug development. Types of nucleolar stresses identified in this study O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/517150v3_ufig1.gif" ALT="Figure 1"> View larger version (80K): org.highwire.dtl.DTLVardef@18724b6org.highwire.dtl.DTLVardef@17b60e7org.highwire.dtl.DTLVardef@117004dorg.highwire.dtl.DTLVardef@114ecec_HPS_FORMAT_FIGEXP M_FIG (1) DNA intercalators and RNA Pol inhibitors induced canonical nucleolar stress manifested by partial dispersion of granular component (GC) and segregation of rDNA and fibrillar center (FC) components UBF, Treacle, and POLR1A within nucleolar stress caps. (2) Inhibition of mTOR and PI3K growth pathways induced a metabolic suppression of function accompanied by the decrease in nucleolar normality score, size, and rRNA production, without dramatic re-organization of nucleolar anatomy. (3) Inhibitors targeting HSP90 and proteasome induced proteotoxicity, resulting in the disruption of protein homeostasis and the accumulation of misfolded and/or undegraded proteins. These effects were accompanied by a decrease in nucleolar normality score, rRNA output, and in some cases formation of protein aggregates (aggresomes) inside the nucleolus. (4) Inhibition of transcriptional CDK activity led to the disruption of interactions between rDNA, RNA Pol I, and GC proteins. This resulted in almost complete nucleolar dissolution, leaving behind an extended bare rDNA scaffold with only a few associated FC proteins remaining. UBF and PolI-recruiting protein Treacle remained associated with the rDNA, while POLR1A and GC dispersed in the nucleoplasm. rRNA production ceased and the nucleolar normality score was greatly reduced. C_FIG

cell biology↗

bioRxiv2021

miR-486 is an epigenetic modulator of Duchenne muscular dystrophy pathologies

Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle disorder resulting in muscle weakness and cardiomyopathy. MicroRNAs have been shown to play essential roles in muscle development, metabolism, and disease pathologies. We demonstrated that miR-486 expression is reduced in DMD muscles and its expression levels correlate with dystrophic disease severity. MicroRNA-486 knockout mice developed disrupted myofiber architecture, decreased myofiber size, decreased locomotor activity, increased cardiac fibrosis, and metabolic defects that were exacerbated on the dystrophic mdx5cv background. We integrated RNA-sequencing and chimeric eCLIP-sequencing data to identify direct in vivo targets of miR-486 and associated dysregulated gene signatures in skeletal muscle. In comparison to our DMD mouse muscle transcriptomes, we identified several of these miR-486 muscle targets including known modulators of dystrophinopathy disease symptoms. Together, our studies identify miR-486 as a driver of muscle remodeling in DMD, a useful biomarker for dystrophic disease progression, and highlight chimeric eCLIP-sequencing as a useful tool to identify direct in vivo microRNA target transcripts. O_FIG O_LINKSMALLFIG WIDTH=122 HEIGHT=200 SRC="FIGDIR/small/448387v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@13eeaf5org.highwire.dtl.DTLVardef@1b3b2dborg.highwire.dtl.DTLVardef@a36782org.highwire.dtl.DTLVardef@1e0b95f_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2019

Differential pulmonary immunopathologic response of domestic sheep (Ovis aries) and bighorn sheep (Ovis canadensis) to Mycoplasma ovipneumoniae infection: a retrospective study

Mycoplasma ovipneumoniae is a respiratory pathogen that can impact domestic sheep (Ovis aries; DS) and bighorn sheep (Ovis canadensis; BHS). Experimental and field data have indicated BHS are more susceptible than DS to developing polymicrobial pneumonia associated with Mycoplasma ovipneumoniae infection. We hypothesized that DS and BHS have a differential immunopathologic pulmonary response to M. ovipneumoniae infection. A retrospective study was performed using formalin-fixed, paraffin-embedded (FFPE) lung tissue from DS and BHS without and with M. ovipneumoniae detected in the lung tissue (n=8 per group). While each M. ovipneumoniae positive lung sample had microscopic changes typical of infection, including hyperplasia of intrapulmonary bronchus-associated lymphoid tissue (BALT) and respiratory epithelium, DS exhibited a more robust and well-organized BALT formation as compared to BHS. Immunohistochemistry was performed with antibodies reactive in FFPE tissues and specific for leukocyte and cytokine markers: T cell marker CD3, B cell markers CD20 and CD79a, macrophage markers CD163 and Iba1, and cytokine IL-17. Digital analysis was used to quantitate chromogen deposition in regions of interest (ROIs), including alveolar and bronchiolar areas, and bronchiolar subregions (epithelium and BALT). Main effects and interaction of species and infection status were analyzed by beta regression and Bonferroni corrections were performed on pairwise comparisons (PBon<0.05 significance). Significant species differences were identified for bronchiolar CD3 (PBon=0.0023) and CD163 (PBon=0.0224), alveolar CD163 (PBon=0.0057), and for IL-17 in each of the ROIs (alveolar: PBon=0.0009; BALT: PBon=0.0083; epithelium: PBon=0.0007). Infected BHS had a higher abundance of bronchiolar CD3 (PBon=0.0005) and CD163 (PBon=0.0162), and alveolar CD163 (PBon=0.0073). While IL-17 significantly increased with infection in BHS BALT (PBon=0.0179) and alveolar (0.0006) ROIs, abundance in DS showed an insignificant decrease in these ROIs and a significant decrease in epithelial abundance (PBon=0.0019). These findings support the hypothesis that DS and BHS have a differential immunopathologic response to M. ovipneumoniae infection.

immunology↗

bioRxiv2019

Cognate epitope recognition by bovine CD4 and CD8 T cells is essential for primary expansion of antigen-specific cytotoxic T-cells following ex vivo stimulation with a candidate Mycobacterium avium subsp. paratuberculosis peptide vaccine.

Studies in cattle show CD8 cytotoxic T cells (CTL), with the ability to kill intracellular bacteria, develop following stimulation of monocyte-depleted peripheral blood mononuclear cells (mdPBMC) with conventional dendritic cells (cDC) and monocyte-derived DC (MoDC) pulsed with MMP, a membrane protein from Mycobacterium avium subsp. paratuberculosis (Map) encoded by MAP2121c. CTL activity was diminished if CD4 T cells were depleted from mdPBMC before antigen (Ag) presentation by cDC and MoDC, suggesting simultaneous cognate recognition of MMP epitopes presented by MHC I and MHC II molecules might be essential for development of CTL activity. To clarify whether cognate recognition is essential for CTL development, studies were conducted with mdPBMC cultures in the presence of monoclonal antibodies (mAbs) specific for MHC class I and MHC class II molecules. The CTL response of mdPBMC to MMP-pulsed DC was completely blocked in the presence of mAbs to both MHC I and II molecules and also blocked in the presence of mAbs to either MHC I or MHC II. The results demonstrate CD4 T-cell help is essential for development of a primary CTL response to MMP, and indicate that cognate recognition is required for delivery of CD4 T-cell help during priming. Of importance, the findings provide support for the importance of CD4 and CD8 T-cell cognate antigen recognition in eliciting CTL responses to vaccines against intracellular pathogens. The methods described herein can be used to elucidate the intracellular interactions between lymphocytes and DC in humans and cattle.

immunology↗

bioRxiv2017

The regulation of tDNA transcription during the directed differentiation of stem cells.

The human genome consists of 625 tDNA copies that encode 51 distinct isoacceptor families. Recent studies demonstrated that changes in chromatin structure during cellular differentiation can alter the expression of these tDNA. However, the mechanism by which tDNA can be differentially regulated remains unclear. Here we used the directed differentiation of pluripotent human embryonic stem cells (hESCs) towards the endoderm lineage as a model system to study the developmental regulation of individual tDNA. We demonstrated a significant change in the Pol III occupancy at 49 tDNA (22 reduced and 27 increased). The regulation of tDNA did not correlate with changes in TFIIIB or TFIIIC occupancy, H3K4me3, or H3K27me3 levels. However, tDNA that had an increase in Pol III binding were preferentially found within strong CTCF-COHESIN chromatin loops. The knockdown of either Ctcf or Rad21 in mouse tail tip fibroblasts had similar effects on changes in tRNA levels. We identified 7 isoacceptors that were differentially expressed during the directed differentiation of hESCs. The open reading frames of the ribosomal protein genes, which are translationally repressed during hESC differentiation, are enriched for codons that are decoded by these downregulated isoacceptors. Thus, translation efficiency during cellular differentiation may be affected by changes in tDNA regulation.

molecular biology↗