Deep mutational scanning and massively parallel kinetics of plasminogen activator inhibitor-1 functional stability to probe its latency transition

Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor superfamily of proteins, is unique among serine protease inhibitors for exhibiting a spontaneous conformational change to a latent or inactive state. The functional half-life for this transition at physiologic temperature and pH is ∼1 to 2 h. To better understand the molecular mechanisms underlying this transition, we now report on the analysis of a comprehensive PAI-1 variant library expressed on filamentous phage and selected for functional stability after 48 h at 37 °C. Of the 7201 possible single amino acid substitutions in PAI-1, we identified 439 that increased the functional stability of PAI-1 beyond that of the WT protein. We also found 1549 single amino acid substitutions that retained inhibitory activity toward the canonical target protease of PAI-1 (urokinase-like plasminogen activator), whereas exhibiting functional stability less than or equal to that of WT PAI-1. Missense mutations that increase PAI-1 functional stability are concentrated in highly flexible regions within the PAI-1 structure. Finally, we developed a method for simultaneously measuring the functional half-lives of hundreds of PAI-1 variants in a multiplexed, massively parallel manner, quantifying the functional half-lives for 697 single missense variants of PAI-1 by this approach. Overall, these findings provide novel insight into the mechanisms underlying the latency transition of PAI-1 and provide a database for interpreting human PAI-1 genetic variants.     

Cell cycle and developmental control of cortical excitability in Xenopus laevis

Interest in cortical excitability—the ability of the cell cortex to generate traveling waves of protein activity—has grown considerably over the past 20 years. Attributing biological functions to cortical excitability requires an understanding of the natural behavior of excitable waves and the ability to accurately quantify wave properties. Here we have investigated and quantified the onset of cortical excitability in Xenopus laevis eggs and embryos and the changes in cortical excitability throughout early development. We found that cortical excitability begins to manifest shortly after egg activation. Further, we identified a close relationship between wave properties—such as wave frequency and amplitude—and cell cycle progression as well as cell size. Finally, we identified quantitative differences between cortical excitability in the cleavage furrow relative to nonfurrow cortical excitability and showed that these wave regimes are mutually exclusive.     

Lipolysis drives expression of the constitutively active receptor GPR3 to induce adipose thermogenesis

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Negative relationships between species richness and evenness render common diversity indices inadequate for assessing long-term trends in butterfly diversity

Species richness and evenness, the two principle components of species diversity, are frequently used to describe variation in species assemblages in space and time. Compound indices, including variations of both the Shannon–Wiener index and Simpson’s index, are assumed to intelligibly integrate species richness and evenness into all-encompassing measures. However, the efficacy of compound indices is disputed by the possibility of inverse relationships between species richness and evenness. Past studies have assessed relationships between various diversity measures across survey locations for a variety of taxa, often finding species richness and evenness to be inversely related. Butterflies are one of the most intensively monitored taxa worldwide, but have been largely neglected in such studies. Long-term butterfly monitoring programs provide a unique opportunity for analyzing how trends in species diversity relate to habitat and environmental conditions. However, analyzing trends in butterfly diversity first requires an assessment of the applicability of common diversity measures to butterfly assemblages. To accomplish this, we quantified relationships between butterfly diversity measures estimated from 10 years of butterfly population data collected in the North Saskatchewan River Valley in Edmonton, Alberta, Canada. Species richness and evenness were inversely related within the butterfly assemblage. We conclude that species evenness may be used in conjunction with richness to deepen our understandings of assemblage organization, but combining these two components within compound indices does not produce measures that consistently align with our intuitive sense of species diversity.     

Certificates of confidentiality in research: rationale and usage

Certificates of confidentiality (COCs) are a tool to protect researchers from being compelled to release identifying information about their subjects. Whereas institutional review board (IRB) review and informed consent procedures are mandatory tools to protect human subjects, COCs are voluntary. There are limited data about who procures COCs and why, and whether they are useful. Three Institutes of the National Institutes of Health (NIH) provided data on 114 research projects that had received COCs. Eighty-three researchers had procured a single COC and 11 researchers had procured 31 COCs. One hundred and four (91%) of the COCs were obtained by researchers at academic sites, and 17 institutions collectively accounted for 82 COCs. The most commonly cited sources of information about COCs came from colleagues (n = 18, 35%) and previous experience (n = 17, 33%). The most common reasons for procuring a COC were that the research involved genetics (n = 28, 54%), the research could lead to social stigmatization or discrimination (n = 22, 42%), or the research could damage an individual's financial standing, employability, or reputation (n = 21, 40%). These findings show that COCs are often congregated within institutions and by particular individuals. This may be because others are unaware of COCs or because others do not believe they are necessary or useful.     

Absence of DICER in Monocytes and Its Regulation by HIV-1

MicroRNAs (miRNAs) are a class of small RNA molecules that function to control gene expression and restrict viral replication in host cells. The production of miRNAs is believed to be dependent upon the DICER enzyme. Available evidence suggests that in T lymphocytes, HIV-1 can both suppress and co-opt the host''s miRNA pathway for its own benefit. In this study, we examined the state of miRNA production in monocytes and macrophages as well as the consequences of viral infection upon the production of miRNA. Monocytes in general express low amounts of miRNA-related proteins, and DICER in particular could not be detected until after monocytes were differentiated into macrophages. In the case where HIV-1 was present prior to differentiation, the expression of DICER was suppressed. MicroRNA chip results for RNA isolated from transfected and treated cells indicated that a drop in miRNA production coincided with DICER protein suppression in macrophages. We found that the expression of DICER in monocytes is restricted by miR-106a, but HIV-1 suppressed DICER expression via the viral gene Vpr. Additionally, analysis of miRNA expression in monocytes and macrophages revealed evidence that some miRNAs can be processed by both DICER and PIWIL4. Results presented here have implications for both the pathology of viral infections in macrophages and the biogenesis of miRNAs. First, HIV-1 suppresses the expression and function of DICER in macrophages via a previously unknown mechanism. Second, the presence of miRNAs in monocytes lacking DICER indicates that some miRNAs can be generated by proteins other than DICER.     

A spatially aware likelihood test to detect sweeps from haplotype distributions

The inference of positive selection in genomes is a problem of great interest in evolutionary genomics. By identifying putative regions of the genome that contain adaptive mutations, we are able to learn about the biology of organisms and their evolutionary history. Here we introduce a composite likelihood method that identifies recently completed or ongoing positive selection by searching for extreme distortions in the spatial distribution of the haplotype frequency spectrum along the genome relative to the genome-wide expectation taken as neutrality. Furthermore, the method simultaneously infers two parameters of the sweep: the number of sweeping haplotypes and the “width” of the sweep, which is related to the strength and timing of selection. We demonstrate that this method outperforms the leading haplotype-based selection statistics, though strong signals in low-recombination regions merit extra scrutiny. As a positive control, we apply it to two well-studied human populations from the 1000 Genomes Project and examine haplotype frequency spectrum patterns at the LCT and MHC loci. We also apply it to a data set of brown rats sampled in NYC and identify genes related to olfactory perception. To facilitate use of this method, we have implemented it in user-friendly open source software.