Evaluation of absolute quantitation by nonlinear regression in probe-based real-time PCR

Background  

In real-time PCR data analysis, the cycle threshold (CT) method is currently the gold standard. This method is based on an assumption of equal PCR efficiency in all reactions, and precision may suffer if this condition is not met. Nonlinear regression analysis (NLR) or curve fitting has therefore been suggested as an alternative to the cycle threshold method for absolute quantitation. The advantages of NLR are that the individual sample efficiency is simulated by the model and that absolute quantitation is possible without a standard curve, releasing reaction wells for unknown samples. However, the calculation method has not been evaluated systematically and has not previously been applied to a TaqMan platform. Aim: To develop and evaluate an automated NLR algorithm capable of generating batch production regression analysis.     

PFN2 and NAA80 cooperate to efficiently acetylate the N-terminus of actin

The actin cytoskeleton is of profound importance to cell shape, division, and intracellular force generation. Profilins bind to globular (G-)actin and regulate actin filament formation. Although profilins are well-established actin regulators, the distinct roles of the dominant profilin, profilin 1 (PFN1), versus the less abundant profilin 2 (PFN2) remain enigmatic. In this study, we use interaction proteomics to discover that PFN2 is an interaction partner of the actin N-terminal acetyltransferase NAA80, and further confirm this by analytical ultracentrifugation. Enzyme assays with NAA80 and different profilins demonstrate that PFN2 binding specifically increases the intrinsic catalytic activity of NAA80. NAA80 binds PFN2 through a proline-rich loop, deletion of which abrogates PFN2 binding. Small-angle X-ray scattering shows that NAA80, actin, and PFN2 form a ternary complex and that NAA80 has partly disordered regions in the N-terminus and the proline-rich loop, the latter of which is partly ordered upon PFN2 binding. Furthermore, binding of PFN2 to NAA80 via the proline-rich loop promotes binding between the globular domains of actin and NAA80, and thus acetylation of actin. However, the majority of cellular NAA80 is stably bound to PFN2 and not to actin, and we propose that this complex acetylates G-actin before it is incorporated into filaments. In conclusion, we reveal a functionally specific role of PFN2 as a stable interactor and regulator of the actin N-terminal acetyltransferase NAA80, and establish the modus operandi for NAA80-mediated actin N-terminal acetylation, a modification with a major impact on cytoskeletal dynamics.     

SARS-CoV-2 infects human adult donor eyes and hESC-derived ocular epithelium

1. Download : Download high-res image (205KB)
2. Download : Download full-size image

     

Divergence,gene flow,and the origin of leapfrog geographic distributions: The history of colour pattern variation in Phyllobates poison‐dart frogs

The geographic distribution of phenotypic variation among closely related populations is a valuable source of information about the evolutionary processes that generate and maintain biodiversity. Leapfrog distributions, in which phenotypically similar populations are disjunctly distributed and separated by one or more phenotypically distinct populations, represent geographic replicates for the existence of a phenotype, and are therefore especially informative. These geographic patterns have mostly been studied from phylogenetic perspectives to understand how common ancestry and divergent evolution drive their formation. Other processes, such as gene flow between populations, have not received as much attention. Here, we investigate the roles of divergence and gene flow between populations in the origin and maintenance of a leapfrog distribution in Phyllobates poison frogs. We found evidence for high levels of gene flow between neighbouring populations but not over long distances, indicating that gene flow between populations exhibiting the central phenotype may have a homogenizing effect that maintains their similarity, and that introgression between ‘leapfroging’ taxa has not played a prominent role as a driver of phenotypic diversity in Phyllobates. Although phylogenetic analyses suggest that the leapfrog distribution was formed through independent evolution of the peripheral (i.e. leapfrogging) populations, the elevated levels of gene flow between geographically close populations poise alternative scenarios, such as the history of phenotypic change becoming decoupled from genome‐averaged patterns of divergence, which we cannot rule out. These results highlight the importance of incorporating gene flow between populations into the study of geographic variation in phenotypes, both as a driver of phenotypic diversity and as a confounding factor of phylogeographic inferences.     

Mapping the degradation pathway of a disease-linked aspartoacylase variant

Canavan disease is a severe progressive neurodegenerative disorder that is characterized by swelling and spongy degeneration of brain white matter. The disease is genetically linked to polymorphisms in the aspartoacylase (ASPA) gene, including the substitution C152W. ASPA C152W is associated with greatly reduced protein levels in cells, yet biophysical experiments suggest a wild-type like thermal stability. Here, we use ASPA C152W as a model to investigate the degradation pathway of a disease-causing protein variant. When we expressed ASPA C152W in Saccharomyces cerevisiae, we found a decreased steady state compared to wild-type ASPA as a result of increased proteasomal degradation. However, molecular dynamics simulations of ASPA C152W did not substantially deviate from wild-type ASPA, indicating that the native state is structurally preserved. Instead, we suggest that the C152W substitution interferes with the de novo folding pathway resulting in increased proteasomal degradation before reaching its stable conformation. Systematic mapping of the protein quality control components acting on misfolded and aggregation-prone species of C152W, revealed that the degradation is highly dependent on the molecular chaperone Hsp70, its co-chaperone Hsp110 as well as several quality control E3 ubiquitin-protein ligases, including Ubr1. In addition, the disaggregase Hsp104 facilitated refolding of aggregated ASPA C152W, while Cdc48 mediated degradation of insoluble ASPA protein. In human cells, ASPA C152W displayed increased proteasomal turnover that was similarly dependent on Hsp70 and Hsp110. Our findings underscore the use of yeast to determine the protein quality control components involved in the degradation of human pathogenic variants in order to identify potential therapeutic targets.     

ADAPTIONISM—30 YEARS AFTER GOULD AND LEWONTIN

Gould and Lewontin's 30-year-old critique of adaptionism fundamentally changed the discourse of evolutionary biology. However, with the influx of new ideas and scientific traditions from genomics into evolutionary biology, the old adaptionist controversies are being recycled in a new context. The insight gained by evolutionary biologists, that functional differences cannot be equated to adaptive changes, has at times not been appreciated by the genomics community. In this comment, I argue that even in the presence of both functional data and evidence for selection from DNA sequence data, it is still difficult to construct strong arguments in favor of adaptation. However, despite the difficulties in establishing scientific arguments in favor of specific historic evolutionary events, there is still much to learn about evolution from genomic data.     

Robustness of the Estimator of the Index of Dispersion for DNA Sequences

If substitutions in DNA sequences follow a Poisson process, the ratio of the variance in the number of substitutions to the mean number of substitutions (the index of dispersion) should equal 1. In this paper, the robustness of the commonly applied estimator of the index of dispersion in replacement sites and silent sites to various assumptions regarding DNA evolution is explored using simulation methods. The estimate of the index of dispersion may be strongly biased if the assumptions of the model of substitution are violated. However, the results of this study support the conclusions of studies by Gillespie and Ohta that the process of substitution in replacement sites is overdispersed. This result contradicts those of a recent study and shows that the high index of dispersion for replacement sites is not an artifact caused by the method of estimation.