Biology subjects

Fabian, R.

Publications and source records attributed to Fabian, R..

4 recordsLinked to original sources

bioRxiv2024

The golden Syrian hamster (Mesocricetus auratus) as a model to decipher relevant pathogenic aspects of sheep-associated malignant catarrhal fever

Malignant catarrhal fever (MCF) is an often fatal sporadic gammaherpesvirus-induced disease of ruminants with global relevance. Ovine gammaherpesvirus-2 (OvHV-2), with sheep as reservoir host, is a major cause of MCF in susceptible species. Despite extensive research on the molecular aspects of the disease, its pathogenesis is not yet fully understood. The present study re-established the Syrian golden hamster (Mesocricetus auratus) as amenable animal model of MCF and applied complementary in situ approaches to confirm recent findings in natural disease that could shed new light on pathogenetic aspects of MCF. These showed that systemic OvHV-2 infection is associated with T cell and macrophage dominated mononuclear infiltrates and vasculitis in various organs. Both T cells and monocytes/macrophages harbor the virus, and infected leukocytes are abundant in the infiltrates. The results also indicate that OvHV-2 has a broader target cell spectrum, including vascular endothelial cells and selected squamous epithelia. The former supports the interpretation that the inflammatory processes develop due to circulating activated, infected T cells and monocytes that home to tissues and emigrate from vessels prone to leukocyte emigration, possibly with direct interaction between virus infected leukocytes and endothelial cells. The latter supports the hypothesis of a graft versus host disease scenario, without viral cytopathic effect on epithelial cells but infiltration of the mucosa by infected T cells and macrophages. The disease processes are accompanied by evidence of expansion of the T cell compartments and the monocyte/macrophage pool in lymphatic tissues and bone marrow.

pathology↗

bioRxiv2019

A precisely-titratable, variation-suppressed transcriptional controller to enable genetic discovery

Methods to express genes conditionally into phenotype remain central to biological experimentation and biotechnology. Current methods enable either on/off or imprecisely controlled graded gene expression. We developed a "well-tempered" controller, WTC846, for precisely adjustable, graded and growth condition independent conditional expression of genes in Saccharomyces cerevisiae. In WTC846 strains, the controlled genes are expressed from a strong, native promoter engineered to be repressed by the prokaryotic TetR protein and induced by tetracycline and analogues. A second instance of this promoter drives TetR itself. This autorepression loop exhibits low cell-to-cell variation in gene expression and allows precise adjustment of the steady state abundance of any protein with inducer. A second, constitutively expressed zeroing repressor abolishes basal expression in the absence of inducer. WTC846-controlled, stable (Cdc42, Tpi1) and unstable (Ipl1) proteins recapitulated known knockout and overexpression phenotypes. WTC846::CDC20 strains enabled inducer regulated cell cycle synchronization. WTC846 alleles of CDC28, TOR1, PBR1 and PMA1 exhibited expected gene dosage-dependent growth rates and morphological phenotypes, and WTC846::WHI5 strains exhibited inducer controlled differences in cell volume. WTC846 controlled genes comprise a new kind of "expression clamped" allele, for which variation in expression is minimized and gene dosage can be set by the experimenter across the range of cellular protein abundances. In yeast, we expect WTC846 alleles to find use in assessment of phenotypes now incompletely penetrant due to variable dosage of the causative protein, and in genome-wide epistasis screens. Implementation in higher cells should enable experiments now impossible due to cell-to-cell variation and imprecise control.

molecular biology↗

bioRxiv2019

Development and Optimisation of a Defined High Cell Density Yeast Medium

Saccharomyces cerevisiae cells grown in a small volume of a defined media neither reach the desired cell density nor grow at a fast enough rate to scale down the volume and increase the sample number of classical biochemical assays, as the detection limit of the readout often requires a high number of cells as an input. To ameliorate this problem, we developed and optimised a new high cell density (HCD) medium for S. cerevisiae. Starting from a widely-used synthetic medium composition, we systematically varied the concentrations of all components without the addition of other compounds. We used response surface methodology (RSM) to develop and optimise the five components of the medium: glucose, yeast nitrogen base, amino acids, mono-sodium glutamate and inositol. We monitored growth, cell number and cell size to ensure that the optimisation was towards a greater density of cells rather than just towards an increase in biomass (i.e larger cells). Cells grown in the final medium, HCD, exhibit growth more similar to the complex medium YPD than to the synthetic medium SD, while the final cell density prior to the diauxic shift is increased about three- and tenfold, respectively. We found normal cell-cycle behaviour throughout the growth phases by monitoring DNA content and protein expression using fluorescent reporters. We also ensured that HCD media could be used with a variety of strains and that they allow selection for all common yeast auxotrophic markers.

microbiology↗

bioRxiv2017

Preventing photomorbidity in long-term multi-color fluorescence imaging of S. cerevisiae and S. pombe

Time-lapse imaging using multiple fluorescent reporters is an essential tool to study molecular processes in single cells. However, exposure to even moderate doses of visible excitation light can disturb cellular physiology and alter the quantitative behavior of the cells under study. Here, we set out to develop guidelines to avoid the confounding effects of excitation light in multi-color long-term imaging. We use widefield fluorescence microscopy to measure the effect of the administered excitation light on growth (here called photomorbidity) in yeast. We find that photomorbidity is determined by the cumulative light dose at each wavelength, but independent of the way excitation light is applied. Importantly, photomorbidity possesses a threshold light dose below which no effect is detectable (NOEL, no-observed-effect level). We found, that the suitability of fluorescent proteins for live-cell imaging at the respective excitation light NOEL is equally determined by the cellular autofluorescence and the fluorescent protein brightness. Last, we show that photomorbidity of multiple wavelengths is additive and imaging conditions absent of photomorbidity can be predicted. Our findings enable researchers to find imaging conditions with minimal impact on physiology and can provide a framework for how to approach photomorbidity in other organisms.

cell biology↗