Biology subjects

Aidlen, D.

Publications and source records attributed to Aidlen, D..

6 recordsLinked to original sources

bioRxiv2026

Processing and Analyzing High-Throughput Microfluidic Enzymology Data: A Practical Guide To Rate Fitting and Quality Control

High-throughput enzymology enables quantitative characterization of enzyme function across hundreds to thousands of sequence variants and experimental conditions. Nevertheless, the scale and complexity of these datasets create substantial challenges for analysis and quality control. High-Throughput Microfluidic Enzyme Kinetics (HT-MEK), for example, generates large microscopy datasets that must pass through multiple analytical stages, including image processing, initial-rate fitting, and kinetic modeling. Choices or errors made at any stage can propagate into the final kinetic parameters without being evident from fit statistics alone. Here, we provide a broadly applicable guide for analyzing high-throughput enzymology data from the HT-MEK platform, using Michaelis-Menten kinetics as a representative example. We first outline the conceptual workflow from raw fluorescence measurements to estimates of kcat, KM, and kcat/KM. We then discuss common experimental and analytical failure modes and present a framework for deciding when data should be refitted, filtered, qualified, or repeated. Finally, we provide a step-by-step workflow using Mercury, an open-source Python framework that integrates scalable HT-MEK data processing with traceable quality control and diagnostic visualization. This workflow preserves the connection between reported parameters and their underlying measurements and can be adapted to other kinetic models and biochemical assays.

biochemistry↗

bioRxiv2026

Coupling high-throughput protease enzymology with viral replication reveals biochemical constraints of viral fitness

Proteases govern essential biological processes and are key drug targets, yet how protease sequence variation quantitatively reshapes biochemical parameters and constrains biological fitness remains poorly understood. Here, we integrate high-throughput in vitro enzymology with cellular assays to link protease sequence, biochemistry, and fitness. We extend a microfluidic platform for high-throughput protease enzymology (HT-MEKpro), which is broadly applicable across protease families and catalytic classes, enabling measurement of catalytic turnover (kcat), Michaelis constant (KM), inhibitor potency (IC50), and relative substrate specificity for 102-103 variants. Applied to the SARS-CoV-2 main protease (Mpro), HT-MEKpro generated parallel catalytic and inhibitory landscapes for >400 variants. Integration with viral replication and in-cell cleavage assays reveals that variants with altered substrate specificity fail to support replication, suggesting imbalanced polyprotein processing as a constraint on viral fitness. More broadly, these data can enable mechanistically grounded modeling of protease sequence-property relationships and inform strategies for pharmacological modulation beyond active-site inhibition.

biochemistry↗

bioRxiv2026

Positive germline selection of mtDNA mutations: evidence from the oocyte.

Purifying selection is the removal of detrimental mutations. Nuclear DNA mutations are purged by removing mutant individuals, embryos, or germline cells. Because mtDNA is present in numerous copies per cell, purifying selection of mtDNA mutations introduces an additional layer of complexity. The intracellular mutant fraction can change due to clonal expansion/reduction of mutant mtDNA molecules in germline cells. Different research groups have reported either negative (purifying), or positive (destructive) germline mtDNA selection. In this study, we use recently published high-fidelity data on mtDNA mutations in individual human oocytes from the Makova laboratory (Arbeithuber et al., 2025) to resolve this disagreement. To assess selection, we used the "selective expansion" metric St, which compares the average weighted rate of clonal expansion in a set of mutations tested for selection (e.g. non-synonymous mutations) to that of the set of synonymous mutations. We found that, on average, noncoding oocyte mutations in the control region expand faster than synonymous mutations, i.e., are under positive selection. Intriguingly, mutations in the coding region, the detrimental ones in particular, were also on average under positive selection. The prescence of average positive selection does not preclude purifying selection against individual mutations; it only indicates that positive selection dominates in aggregate mutational dynamics. Positive selection impacts the dynamics of de novo germline mutations that arise in primordial germ cells or oocytes and have not yet been inherited by the next generation. Contrastly, mutations that are passed to subsequent generations are under average purifying selection. We suggest that initial positive selection begets subsequent purifying selection by increasing intracellular mutant fractions to levels at which their deleterious effects become phenotypically apparent and can be efficiently removed.

genetics↗

bioRxiv2022

Reanalysis of mtDNA mutations of human primordial germ cells (PGCs) reveals significant contamination with NUMTs, and challenges predominantly purifying selection in late PGCs

The resilience of the mitochondrial genome (mtDNA) to a high mutational pressure depends, in part, on negative purifying selection in the germline. A paradigm in the field has been that such selection, at least in part, takes place in primordial germ cells (PGCs). Specifically, Floros et al. (Nature Cell Biology 20: 144-51) reported an increase in the synonymity of mtDNA mutations (a sign of purifying selection) between pooled early-stage and late-stage PGCs. We re-analyzed Floros et al. pooled PGC data and noticed that their mutational dataset was significantly contaminated with single nucleotide variants (SNVs) derived from a nuclear sequence of mtDNA origin (NUMT) located on chromosome 5. Contamination was caused by co-amplification of the NUMT sequence by cross-specific PCR primers. Importantly, when we removed NUMT-derived SNVs, the evidence of purifying selection was abolished. In addition to pooled PGCs, Floros et al. reported the analysis of single late-stage PGCs, which were amplified with different sets of PCR primers that cannot amplify the NUMT sequence. Accordingly, we found no NUMT-derived SNVs among single PGCs mutations. Interestingly, single PGC mutations show a decrease of synonymity with increased intracellular mutant fraction. This pattern is incompatible with predominantly negative selection. This suggests that germline selection of mtDNA mutations is a complex phenomenon and that the part of this process that takes place in PGCs may be predominantly positive. However counterintuitive, positive germline selection of detrimental mtDNA mutations has been reported previously and potentially may be evolutionarily advantageous.

genomics↗

bioRxiv2021

Bi-phasic dynamics of the mitochondrial DNA mutation m.3243A>G in blood: An unbiased, mutation level-dependent model implies positive selection in the germline.

The A-to-G point mutation at position 3243 in the human mitochondrial genome (m.3243A>G) is the most common pathogenic mtDNA variant responsible for disease in humans. It is widely accepted that m.3243A>G levels decrease in blood with age, and an age correction representing [~]2% annual decline is often applied to account for this change in mutation level. Here we report that recent data indicate the dynamics of m.3243A>G are more complex and depend on the mutation level in blood in a bi-phasic way. Consequently, the traditional 2% correction, which is adequate on average, creates opposite predictive biases at high and low mutation levels. Unbiased age correction is needed to circumvent these drawbacks of the standard model. We propose to eliminate both biases by using an approach where age correction depends on mutation level in a biphasic way to account for the dynamics of m.3243A>G in blood. The utility of this approach was further tested in estimating germline selection of m.3243A>G. The biphasic approach permitted us to uncover patterns consistent with the possibility of positive selection for m.3243A>G. Germline selection of m.3243A>G shows an arching profile by which selection is positive at intermediate mutant fractions and declines at high and low mutant fractions. We conclude that use of this biphasic approach will greatly improve the accuracy of modelling changes in mtDNA mutation frequencies in the germline and in somatic cells during aging.

genomics↗

bioRxiv2020

A mitochondrial mutational signature of temperature and longevity in ectothermic and endothermic vertebrates.

The variation in the mutational spectrum of the mitochondrial genome (mtDNA) among species is not well understood. Recently, we demonstrated an increase in A>G substitutions on a heavy chain (hereafter AH>GH) of mtDNA in aged mammals, interpreting it as a hallmark of age-related oxidative damage. In this study, we hypothesized that the occurrence of AH>GH substitutions may depend on the level of aerobic metabolism, which can be inferred from an organisms body temperature. To test this hypothesis, we used body temperature in endotherms and environmental temperature in ectotherms as proxies for metabolic rate and reconstructed mtDNA mutational spectra for 1350 vertebrate species. Our results showed that temperature was associated with increased rates of AH>GH and asymmetry of AH>GH in different species of ray-finned fishes and within geographically distinct clades of European anchovy. Analysis of nucleotide composition in the most neutral synonymous sites of fishes revealed that warm-water species were expectedly more A-poor and G-rich compared to cold-water species. Finally, we extended our analyses to all vertebrates and observed higher AH>GH and increased asymmetry of AH>GH in warm-blooded (mammals and birds) compared to cold-blooded (Actinopterygii, amphibia, reptilia) vertebrate classes. Overall, our findings suggest that temperature, through its influence on metabolism and oxidative damage, shapes the mutational properties and nucleotide content of the mtDNA in all vertebrates.

evolutionary biology↗