Pervasive contingency and entrenchment in a billion years of Hsp90 evolution [preprint]

Interactions among mutations within a protein have the potential to make molecular evolution contingent and irreversible, but the extent to which epistasis actually shaped historical evolutionary trajectories is unclear. We addressed this question by identifying all amino acid substitutions that occurred during the billion-year evolutionary history of the heat shock protein 90 (Hsp90) ATPase domain beginning from a deep eukaryotic ancestor to modern Saccharomyces cerevisiae and then precisely measuring their fitness effects when introduced into both extant and reconstructed ancestral Hsp90 proteins. We find a pervasive influence of epistasis: of 98 derived states that evolved during history, most were deleterious at times before they happened, and the vast majority also became subsequently entrenched, with the ancestral state becoming deleterious after its substitution. This epistasis was primarily caused by specific interactions among sites rather than a general permissive or restrictive effect on the protein\u27s tolerance to mutation. Our results show that epistasis continually opens and closes windows of mutational opportunity over evolutionary timescales, producing histories and biological states that reflect the transient internal constraints imposed by a protein\u27s fleeting sequence states

Improved methods for detection of β-galactosidase (lacZ) activity in hard tissue

The ß-galactosidase gene (lacZ) of Escherichia coli is widely used as a reporter gene. The expression of lacZ can be detected by enzyme-based histochemical staining using chromogenic substrates such as 5-bromo-4-chloro-3-indolyl-ß-D: -galactoside (X-gal). Because the enzymatic activity of lacZ is vulnerable to high temperatures and acid treatment for demineralization, detection of lacZ on paraffinized sections is difficult, especially for hard tissues, which require demineralization before sectioning in paraffin. To circumvent this problem, whole-mount X-gal staining before sectioning is performed. However, detection of lacZ activity in the center of larger portions of hard whole adult tissues is challenging. In this study, focusing on fixation procedures, we determined the conditions conducive to improved detection of lacZ activity in deeper areas of whole tissues. We used an annexin a5 (Anxa5)-lacZ reporter mouse model in which the Anxa5 expression in hard tissue is indicated by lacZ activity. We found that lacZ activity could be detected throughout the periodontal ligament of adult mice when fixed in 100% acetone, whereas it was not detected in the periodontal ligament around the root apex fixed in glutaraldehyde and paraformaldehyde. This staining could not be detected in wild-type mice. Acetone maintains the lacZ activity within 48 h of fixation at both 4°C and at room temperature. In conclusion, acetone is the optimal fixative to improve permeability for staining of lacZ activity in large volumes of adult hard tissues

The Combined Effect of Oseltamivir and Favipiravir on Influenza A Virus Evolution

Influenza virus inflicts a heavy death toll annually and resistance to existing antiviral drugs has generated interest in the development of agents with novel mechanisms of action. Favipiravir is an antiviral drug that acts by increasing the genome-wide mutation rate of influenza A virus (IAV). Potential synergistic benefits of combining oseltamivir and favipiravir have been demonstrated in animal models of influenza, but the population-level effects of combining the drugs are unknown. In order to elucidate the underlying evolutionary processes at play, we performed genome-wide sequencing of IAV experimental populations subjected to serial passaging in vitro under a combined protocol of oseltamivir and favipiravir. We describe the interplay between mutation, selection, and genetic drift that ultimately culminates in population extinction. In particular, selective sweeps around oseltamivir resistance mutations reduce genome-wide variation while deleterious mutations hitchhike to fixation given the increased mutational load generated by favipiravir. This latter effect reduces viral fitness and accelerates extinction compared with IAV populations treated with favipiravir alone, but risks spreading both established and newly emerging mutations, including possible drug resistance mutations, if transmission occurs before the viral populations are eradicated

Effect of daily chlorhexidine bathing on hospital-acquired infection

BACKGROUND Results of previous single-center, observational studies suggest that daily bathing of patients with chlorhexidine may prevent hospital-acquired bloodstream infections and the acquisition of multidrug-resistant organisms (MDROs). METHODS We conducted a multicenter, cluster-randomized, nonblinded crossover trial to evaluate the effect of daily bathing with chlorhexidine-impregnated washcloths on the acquisition of MDROs and the incidence of hospital-acquired bloodstream infections. Nine intensive care and bone marrow transplantation units in six hospitals were randomly assigned to bathe patients either with no-rinse 2% chlorhexidine– impregnated washcloths or with nonantimicrobial washcloths for a 6-month period, exchanged for the alternate product during the subsequent 6 months. The incidence rates of acquisition of MDROs and the rates of hospital-acquired bloodstream infections were compared between the two periods by means of Poisson regression analysis. RESULTS A total of 7727 patients were enrolled during the study. The overall rate of MDRO acquisition was 5.10 cases per 1000 patient-days with chlorhexidine bathing versus 6.60 cases per 1000 patient-days with nonantimicrobial washcloths (P=0.03), the equivalent of a 23% lower rate with chlorhexidine bathing. The overall rate of hospital-acquired bloodstream infections was 4.78 cases per 1000 patient-days with chlorhexidine bathing versus 6.60 cases per 1000 patient-days with nonantimicrobial washcloths (P=0.007), a 28% lower rate with chlorhexidine-impregnated washcloths. No serious skin reactions were noted during either study period. CONCLUSIONS Daily bathing with chlorhexidine-impregnated washcloths significantly reduced the risks of acquisition of MDROs and development of hospital-acquired bloodstream infections. (Funded by the Centers for Disease Control and Prevention and Sage Products; ClinicalTrials.gov number, NCT00502476.

An experimental evaluation of drug-induced mutational meltdown as an antiviral treatment strategy [preprint]

The rapid evolution of drug resistance remains a critical public health concern. The treatment of influenza A virus (IAV) has proven particularly challenging, due to the ability of the virus to develop resistance against current antivirals and vaccines. Here we evaluate a novel antiviral drug therapy, favipiravir, for which the mechanism of action in IAV involves an interaction with the viral RNA- dependent RNA polymerase resulting in an effective increase in the viral mutation rate. We utilize an experimental evolution framework, combined with novel population genetic method development for inference from time-sampled data, in order to evaluate the effectiveness of favipiravir against IAV. Evaluating whole genome polymorphism data across fifteen time points under multiple drug concentrations and in controls, we present the first evidence for the ability of viral populations to effectively adapt to low concentrations of favipiravir. In contrast, under high concentrations, we observe population extinction, indicative of mutational meltdown. We discuss the observed dynamics with respect to the evolutionary forces at play and emphasize the utility of evolutionary theory to inform drug development

Complementary genetic and genomic approaches help characterize the linkage group I seed protein QTL in soybean

Background: The nutritional and economic value of many crops is effectively a function of seed protein and oil content. Insight into the genetic and molecular control mechanisms involved in the deposition of these constituents in the developing seed is needed to guide crop improvement. A quantitative trait locus (QTL) on Linkage Group I (LG I) of soybean (Glycine max (L.) Merrill) has a striking effect on seed protein content. Results: A soybean near-isogenic line (NIL) pair contrasting in seed protein and differing in an introgressed genomic segment containing the LG I protein QTL was used as a resource to demarcate the QTL region and to study variation in transcript abundance in developing seed. The LG I QTL region was delineated to less than 8.4 Mbp of genomic sequence on chromosome 20. Using Affymetrix® Soy GeneChip and high-throughput Illumina® whole transcriptome sequencing platforms, 13 genes displaying significant seed transcript accumulation differences between NILs were identified that mapped to the 8.4 Mbp LG I protein QTL region. Conclusions: This study identifies gene candidates at the LG I protein QTL for potential involvement in the regulation of protein content in the soybean seed. The results demonstrate the power of complementary approaches to characterize contrasting NILs and provide genome-wide transcriptome insight towards understanding seed biology and the soybean genome

Clinical Practice Guidelines for Antimicrobial Prophylaxis in Surgery

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140217/1/sur.2013.9999.pd