Biology subjects

Huisman, M.

Publications and source records attributed to Huisman, M..

5 recordsLinked to original sources

bioRxiv2018

Centenarian Controls Increase Variant Effect-sizes by an average two-fold in an Extreme Case-Extreme Control Analysis of Alzheimer’s Disease

The detection of genetic loci associated with Alzheimers disease (AD) requires large numbers of cases and controls because variant effect-sizes are mostly small. We hypothesized that variant effect-sizes should increase when individuals who represent the extreme ends of a disease spectrum are considered, as their genomes are assumed to be maximally enriched or depleted with disease-associated genetic variants.\n\nWe used 1,073 extensively phenotyped AD cases with relatively young age at onset as extreme cases (66.3{+/-}7.9 years), 1,664 age-matched controls (66.0{+/-}6.5 years) and 255 cognitively healthy centenarians as extreme controls (101.4{+/-}1.3 years). We estimated the effect-size of 29 variants that were previously associated with AD in genome-wide association studies.\n\nComparing extreme AD-cases with centenarian-controls increased the variant effect-size relative to published effect-sizes by on average 1.90-fold (SE=0.29, p=9.0x10-4). The effect-size increase was largest for the rare high-impact TREM2 (R74H) variant (6.5-fold), and significant for variants in/near ECHDC3 (4.6-fold), SLC24A4-RIN3 (4.5-fold), NME8 (3.8-fold), PLCG2 (3.3-fold), APOE-{varepsilon}2 (2.2-fold) and APOE-{varepsilon}4 (2.0-fold). Comparing extreme phenotypes enabled us to replicate the AD association for 10 variants (p<0.05) in relatively small samples. The increase in effect-sizes depended mainly on using centenarians as extreme controls: the average variant effect-size was not increased in a comparison of extreme AD cases and age-matched controls (0.94-fold, p=6.8x10-1), suggesting that on average the tested genetic variants did not explain the extremity of the AD-cases. Concluding, using centenarians as extreme controls in AD case-controls studies boosts the variant effect-size by on average two-fold, allowing the replication of disease-association in relatively small samples.

genetics↗

bioRxiv2018

An Automated Bayesian Pipeline for Rapid Analysis of Single-Molecule Binding Data

Single-molecule binding assays enable the study of how molecular machines assemble and function. Current algorithms can identify and locate individual molecules, but require tedious manual validation of each spot. Moreover, no solution for high-throughput analysis of single-molecule binding data exists. Here, we describe an automated pipeline to analyze single-molecule data over a wide range of experimental conditions. We benchmarked the pipeline by measuring the binding properties of the well-studied, DNA-guided DNA endonuclease, TtAgo, an Argonaute protein from the Eubacterium Thermus thermophilus. We also used the pipeline to extend our understanding of TtAgo by measuring the proteins binding kinetics at physiological temperatures and for target DNAs containing multiple, adjacent binding sites.

molecular biology↗

bioRxiv2017

Fluorescence polarization control for on-off switching of single molecules at cryogenic temperatures

Light microscopy allowing sub-diffraction limited resolution has been among the fastest developing techniques at the interface of biology, chemistry and physics. Intriguingly no theoretical limit exists on how far the underlying measurement uncertainty can be lowered. In particular data fusion of large amounts of images can reduce the measurement error to match the resolution of structural methods like cryo-electron microscopy. Fluorescence, although reliant on a reporter molecule and therefore not the first choice to obtain ultra resolution structures, brings highly specific labeling of molecules in a large assemble to the table and inherently allows the detection of multiple colors, which enable the interrogation of multiple molecular species at the same time in the same sample. Here we discuss the problems to be solved in the coming years to aim for higher resolution and describe what polarization depletion of fluorescence at cryogenic temperatures can contribute for fluorescence imaging of biological samples like whole cells.

biophysics↗

bioRxiv2017

Single-molecule FISH in Drosophila muscle reveals location dependent mRNA composition of megaRNPs

Single-molecule fluorescence in-situ hybridization (smFISH) provides direct access to the spatial relationship between nucleic acids and specific subcellular locations. The ability to precisely localize a messenger RNA can reveal key information about its regulation. Although smFISH is well established in cell culture or thin sections, methods for its accurate application to tissues are lacking. The utility of smFISH in thick tissue sections must overcome several challenges, including probe penetration of fixed tissue, accessibility of target mRNAs for probe hybridization, high fluorescent background, spherical aberration along the optical axis, and image segmentation of organelles. Here we describe how we overcame these obstacles to study mRNA localization in Drosophila larval muscle samples that approach 50 m thickness. We use sample-specific optimization of smFISH, particle identification based on maximum likelihood testing, and 3-dimensional multiple-organelle segmentation. The latter allows using independent thresholds for different regions of interest within an image stack. Our approach therefore facilitates accurate measurement of mRNA location in thick tissues.

cell biology↗

bioRxiv2017

Reconstructing Spatial Transport Distributions In The Nuclear Pore Complex From 2D Images -- How Reliable Is It?

Imaging single molecules in living cells and reconstituted cell systems has resulted in a new understanding of the dynamics of nuclear pore complex functions over the last decade. It does, however, fall short on providing insights into the functional relationships between the pore and nucleocytoplasmic cargo in three-dimensional space. This limited ability is the result of insufficient resolution of optical microscopes along the optical axis and limited fluorescent signal due to the short timescales involved in nuclear transport (fractions of a second). To bypass current technological limitations, it was suggested that highly time-resolved 2D single molecule data could be interpreted as projected cargo locations and could subsequently be transformed into a spatial cargo distribution by assuming cylindrical symmetry 1. Such cargo distributions would provide valuable insights into the NPC-mediated transport in cells. This method, termed 3D-SPEED, has attracted large interest inside and beyond the nuclear pore field, but has also been sharply critiqued for a lack of critical evaluation. Here we present such an evaluation, testing the robustness, reconstruction quality and model-dependency.

cell biology↗