Quantifying the role of genome size and repeat content in adaptive variation and the architecture of flowering time in Amaranthus tuberculatus.

Genome size variation, largely driven by repeat content, is poorly understood within and among populations, limiting our understanding of its significance for adaptation. Here we characterize intraspecific variation in genome size and repeat content across 186 individuals of Amaranthus tuberculatus, a ubiquitous native weed that shows flowering time adaptation to climate across its range and in response to agriculture. Sequence-based genome size estimates vary by up to 20% across individuals, consistent with the considerable variability in the abundance of transposable elements, unknown repeats, and rDNAs across individuals. The additive effect of this variation has important phenotypic consequences-individuals with more repeats, and thus larger genomes, show slower flowering times and growth rates. However, compared to newly-characterized gene copy number and polygenic nucleotide changes underlying variation in flowering time, we show that genome size is a marginal contributor. Differences in flowering time are reflected by genome size variation across sexes and marginally, habitats, while polygenic variation and a gene copy number variant within the ATP synthesis pathway show consistently stronger environmental clines than genome size. Repeat content nonetheless shows non-neutral distributions across the genome, and across latitudinal and environmental gradients, demonstrating the numerous governing processes that in turn influence quantitative genetic variation for phenotypes key to plant adaptation.

Phased Assembly of Neo-Sex Chromosomes Reveals Extensive Y Degeneration and Rapid Genome Evolution in Rumex hastatulus.

Y chromosomes are thought to undergo progressive degeneration due to stepwise loss of recombination and subsequent reduction in selection efficiency. However, the timescales and evolutionary forces driving degeneration remain unclear. To investigate the evolution of sex chromosomes on multiple timescales, we generated a high-quality phased genome assembly of the massive older (<10 MYA) and neo (<200,000 yr) sex chromosomes in the XYY cytotype of the dioecious plant Rumex hastatulus and a hermaphroditic outgroup Rumex salicifolius. Our assemblies, supported by fluorescence in situ hybridization, confirmed that the neo-sex chromosomes were formed by two key events: an X-autosome fusion and a reciprocal translocation between the homologous autosome and the Y chromosome. The enormous sex-linked regions of the X (296 Mb) and two Y chromosomes (503 Mb) both evolved from large repeat-rich genomic regions with low recombination; however, the complete loss of recombination on the Y still led to over 30% gene loss and major rearrangements. In the older sex-linked region, there has been a significant increase in transposable element abundance, even into and near genes. In the neo-sex-linked regions, we observed evidence of extensive rearrangements without gene degeneration and loss. Overall, we inferred significant degeneration during the first 10 million years of Y chromosome evolution but not on very short timescales. Our results indicate that even when sex chromosomes emerge from repetitive regions of already-low recombination, the complete loss of recombination on the Y chromosome still leads to a substantial increase in repetitive element content and gene degeneration.

Characterization of hOAT4 and mOat5 as functional orthologs for renal anion uptake and efflux transport.

Organic anions (OA) are compounds including drugs or toxicants that are negatively charged at physiological pH and are typically transported by Organic Anion Transporters (OATs). Human OAT4 (SLC22A11) is expressed in the apical membrane of renal proximal tubules. Although there is no rodent ortholog of hOAT4, rodents express Oat5 (Slc22a19), an anion exchanger that is also localized to the apical membrane of renal proximal tubule cells. The purpose of this study was to determine the functional similarity between mouse Oat5 and human OAT4. Chinese hamster ovary (CHO) cells expressing SLC22A11 or Slc22a19 were used to assess the transport characteristics of radiolabeled ochratoxin (OTA). We determined the kinetics of OTA transport; the resulting Kt and Jmax values were very similar for both hOAT4 and mOat5: Kt 3.9 and 7.2 µM, respectively, & Jmax 4.4 and 3.9 pmol/cm2, respectively. For the profile of OTA inhibition by OAs, IC50 values were determined for several clinically important drugs and toxicants. The resulting IC50 values ranged from 9 µM for indomethacin to ~600 µM for the diuretic hydrochlorothiazide. We measured the efflux of OTA from preloaded cells; both hOAT4 and mOat5 supported the efflux of OTA. These data support the hypothesis that OAT4 and Oat5 are functional orthologs and share selectivity for OTA both for reabsorption and secretion. Significance Statement This study compares the selectivity profile between human OAT4 and mouse Oat5. Our data revealed a similar selectivity profile for OTA reabsorption and secretion by these two transporters, thereby supporting the hypothesis that hOAT4 and mOat5, while not genetic orthologs, behave as functional orthologs for both uptake and efflux. These data will be instrumental in selecting an appropriate animal model when studying the renal disposition of anionic drugs and toxicants.

The Metabolism and Disposition of Brepocitinib in Humans and Characterization of the Formation Mechanism of an Aminopyridine Metabolite.

Brepocitinib is an oral once-daily Janus kinase 1 and Tyrosine kinase 2 selective inhibitor currently in development for the treatment of several autoimmune disorders. Mass balance and metabolic profiles were determined using accelerator mass spectrometry in six healthy male participants following a single oral 60 mg dose of 14C-brepocitinib (∼300 nCi). The average mass balance recovery was 96.7% ± 6.3%, with the majority of dose (88.0% ± 8.0%) recovered in urine and 8.7% ± 2.1% of the dose recovered in feces. Absorption of brepocitinib was rapid, with maximal plasma concentrations of total radioactivity and brepocitinib achieved within 0.5 hours after dosing. Circulating radioactivity consisted primarily of brepocitinib (47.8%) and metabolite M1 (37.1%) derived from hydroxylation at the C5' position of the pyrazole ring. Fractional contributions to metabolism via cytochrome P450 enzymes were determined to be 0.77 for CYP3A4/5 and 0.14 for CYP1A2 based on phenotyping studies in human liver microsomes. However, additional clinical studies are required to understand the potential contribution of CYP1A1. Approximately 83% of the dose was eliminated as N-methylpyrazolyl oxidative metabolites, with 52.1% of the dose excreted as M1 alone. Notably, M1 was not observed as a circulating metabolite in earlier metabolic profiling of human plasma from a multiple ascending dose study with unlabeled brepocitinib. Mechanistic studies revealed that M1 was highly unstable in human plasma and phosphate buffer, undergoing chemical oxidation leading to loss of the 5-hydroxy-1-methylpyrazole moiety and formation of aminopyrimidine cleavage product M2. Time-dependent inhibition and trapping studies with M1 yielded insights into the mechanism of this unusual and unexpected instability. SIGNIFICANCE STATEMENT: This study provides a detailed understanding of the disposition and metabolism of brepocitinib, a JAK1/TYK2 inhibitor for atopic dermatitis, in humans as well as characterization of clearance pathways and pharmacokinetics of brepocitinib and its metabolites.

Girls are good at STEM: Opening minds and providing evidence reduces boys' stereotyping of girls' STEM ability.

Girls and women face persistent negative stereotyping within STEM (science, technology, engineering, mathematics). This field intervention was designed to improve boys' perceptions of girls' STEM ability. Boys (N = 667; mostly White and East Asian) aged 9-15 years in Canadian STEM summer camps (2017-2019) had an intervention or control conversation with trained camp staff. The intervention was a multi-stage persuasive appeal: a values affirmation, an illustration of girls' ability in STEM, a personalized anecdote, and reflection. Control participants discussed general camp experiences. Boys who received the intervention (vs. control) had more positive perceptions of girls' STEM ability, d = 0.23, an effect stronger among younger boys. These findings highlight the importance of engaging elementary-school-aged boys to make STEM climates more inclusive.

Learning physics-based reduced-order models from data using nonlinear manifolds.

We present a novel method for learning reduced-order models of dynamical systems using nonlinear manifolds. First, we learn the manifold by identifying nonlinear structure in the data through a general representation learning problem. The proposed approach is driven by embeddings of low-order polynomial form. A projection onto the nonlinear manifold reveals the algebraic structure of the reduced-space system that governs the problem of interest. The matrix operators of the reduced-order model are then inferred from the data using operator inference. Numerical experiments on a number of nonlinear problems demonstrate the generalizability of the methodology and the increase in accuracy that can be obtained over reduced-order modeling methods that employ a linear subspace approximation.

A group concept mapping and ethnographic study of intensive care rehabilitation culture.

BACKGROUND: Early physical activity and physical rehabilitation are advocated in the critical care unit for patients recovering from critical illness. Despite this, there are still many factors associated with implementation of early physical rehabilitation into routine critical care and practice. One such factor that has been consistently identified is unit culture, yet there is little understanding of how or why the culture of a critical care unit impacts on implementation of early rehabilitation. AIM: To develop a detailed understanding of the cultural barriers and enablers to the promotion and implementation of physical activity and early mobilization in National Health Service (NHS) critical care units in the United Kingdom (UK). STUDY DESIGN: A mixed-methods, two-phase study incorporating online group concept mapping (GCM) and ethnography. GCM will be conducted to provide a multistakeholder co-authored conceptual framework of rehabilitation culture. Ethnographic observations and interviews will be conducted of culture and behaviours in relation to the implementation and promotion of early physical activity and rehabilitation in two NHS critical care units in the North East of England. RESULTS: The results of the Group Concept Mapping and ethnographic observations and interviews will be triangulated to develop a contextual framework of rehabilitation culture in critical care. RELEVANCE TO CLINICAL PRACTICE: This study will provide a detailed understanding of barriers and facilitators in relation to providing a positive rehabilitation culture in the critical care unit.

Mueller maneuver attenuates left atrial phasic volumes and myocardial strain in healthy younger adults.

The left atrium (LA) is a key, but incompletely understood, modulator of left ventricular (LV) filling. Inspiratory negative intrathoracic pressure swings alter cardiac loading conditions, which may impact LA function. We studied acute effects of static inspiratory efforts on LA chamber function, LA myocardial strain, and LV diastolic filling. We included healthy adults (10 males/9 females, 24 ± 4 yr) and used Mueller maneuvers to reduce intrathoracic pressure to -30 cmH2O for 15 s. Over six repeated trials, we used echocardiography to acquire LA- and LV-focused two-dimensional (2-D) images, and mitral Doppler inflow and annular tissue velocity spectra. Images were analyzed for LA and LV chamber volumes, tissue relaxation velocities, transmitral filling velocities, and speckle tracking-derived LA longitudinal strain. Repeated measures were made at baseline, early Mueller, late Mueller, then early release, and late release. In the late Mueller compared with baseline, LV stroke volume decreased by -10 ± 4 mL (P < 0.05) and then returned to baseline upon release; this occurred with a -11 ± 9 mL (P < 0.05) end-diastolic volume reduction. Early diastolic LV filling was attenuated, reflected by decreased tissue relaxation velocity (-2 ± 2 cm/s, P < 0.05), E-wave filling velocity (-13 ± 14 cm/s, P < 0.05), and LA passive emptying volume (-5 ± 5 mL, P < 0.05), each returning to baseline with release. LA maximal volume decreased (-5 ± 5 mL, P < 0.05) during the Mueller maneuver, but increased relative to baseline following release (+4 ± 5 mL, P < 0.05), whereas LA peak positive longitudinal strain decreased (-6 ± 6%, P < 0.05) and then returned to baseline. Attenuated LA and in turn, LV filling may contribute to acute stroke volume reductions experienced during forceful inspiratory efforts.NEW & NOTEWORTHY In healthy younger adults, the Mueller maneuver transiently reduces left atrial filling and passive emptying during the reservoir and conduit phases, respectively. Corresponding reductions are seen in left atrial reservoir and conduit phase longitudinal myocardial strain and strain rate. However, left atrial pump phase active function and mechanics are largely preserved compared with baseline. Rapid changes in LA chamber volumes and myocardial strain with recurrent forceful inspiratory efforts and relaxation may reflect acute LA stress.

Drug Transporters at the Human Blood-Testis Barrier.

Transporters are involved in the movement of many physiologically important molecules across cell membranes and have a substantial impact on the pharmacological and toxicological effect of xenobiotics. Many transporters have been studied in the context of disposition to, or toxicity in, organs such as the kidney and liver; however, transporters in the testes are increasingly gaining recognition for their role in drug transport across the blood-testis barrier (BTB). The BTB is an epithelial membrane barrier formed by adjacent Sertoli cells (SCs) in the seminiferous tubules that form intercellular junctional complexes to protect developing germ cells from the external environment. Consequently, many charged or large polar molecules cannot cross this barrier without assistance from a transporter. SCs express a variety of drug uptake and efflux transporters to control the flux of endogenous and exogenous molecules across the BTB. Recent studies have identified several transport pathways in SCs that allow certain drugs to circumvent the human BTB. These pathways may exist in other species, such as rodents and nonhuman primates; however, there is (1) a lack of information on their expression and/or localization in these species, and (2) conflicting reports on localization of some transporters that have been evaluated in rodents compared with humans. This review outlines the current knowledge on the expression and localization of pharmacologically relevant drug transporters in human testes and calls attention to the insufficient and contradictory understanding of testicular transporters in other species that are commonly used in drug disposition and toxicity studies. SIGNIFICANCE STATEMENT: While the expression, localization, and function of many xenobiotic transporters have been studied in organs such as the kidney and liver, the characterization of transporters in the testes is scarce. This review summarizes the expression and localization of common pharmacologically-relevant transporters in human testes that have significant implications for the development of drugs that can cross the blood-testis barrier. Potential expression differences between humans and rodents highlighted here suggest rodents may be inappropriate for some testicular disposition and toxicity studies.

In Vitro and In Vivo Models for Drug Transport Across the Blood-Testis Barrier.

The blood-testis barrier (BTB) is a selectively permeable membrane barrier formed by adjacent Sertoli cells (SCs) in the seminiferous tubules of the testes that develops intercellular junctional complexes to protect developing germ cells from external pressures. However, due to this inherent defense mechanism, the seminiferous tubule lumen can act as a pharmacological sanctuary site for latent viruses (e.g., Ebola, Zika) and cancers (e.g., leukemia). Therefore, it is critical to identify and evaluate BTB carrier-mediated drug delivery pathways to successfully treat these viruses and cancers. Many drugs are unable to effectively cross cell membranes without assistance from carrier proteins like transporters because they are large, polar, and often carry a charge at physiologic pH. SCs express transporters that selectively permit endogenous compounds, such as carnitine or nucleosides, across the BTB to support normal physiologic activity, although reproductive toxicants can also use these pathways, thereby circumventing the BTB. Certain xenobiotics, including select cancer therapeutics, antivirals, contraceptives, and environmental toxicants, are known to accumulate within the male genital tract and cause testicular toxicity; however, the transport pathways by which these compounds circumvent the BTB are largely unknown. Consequently, there is a need to identify the clinically relevant BTB transport pathways in in vitro and in vivo BTB models that recapitulate human pharmacokinetics and pharmacodynamics for these xenobiotics. This review summarizes the various in vitro and in vivo models of the BTB reported in the literature and highlights the strengths and weaknesses of certain models for drug disposition studies. SIGNIFICANCE STATEMENT: Drug disposition to the testes is influenced by the physical, physiological, and immunological components of the blood-testis barrier (BTB). But many compounds are known to cross the BTB by transporters, resulting in pharmacological and/or toxicological effects in the testes. Therefore, models that assess drug transport across the human BTB must adequately account for these confounding factors. This review identifies and discusses the benefits and limitations of various in vitro and in vivo BTB models for preclinical drug disposition studies.

Representative Rodent Models for Renal Transporter Alterations in Human Nonalcoholic Steatohepatitis.

Alterations in renal elimination processes of glomerular filtration and active tubular secretion by renal transporters can result in adverse drug reactions. Nonalcoholic steatohepatitis (NASH) alters hepatic transporter expression and xenobiotic elimination, but until recently, renal transporter alterations in NASH were unknown. This study investigates renal transporter changes in rodent models of NASH to identify a model that recapitulates human alterations. Quantitative protein expression by surrogate peptide liquid chromatography-coupled mass spectrometry (LC-MS/MS) on renal biopsies from NASH patients was used for concordance analysis with rodent models, including methionine/choline deficient (MCD), atherogenic (Athero), or control rats and Leprdb/db MCD (db/db), C57BL/6J fast-food thioacetamide (FFDTH), American lifestyle-induced obesity syndrome (ALIOS), or control mice. Demonstrating clinical similarity to NASH patients, db/db, FFDTH, and ALIOS showed decreases in glomerular filtration rate (GFR) by 76%, 28%, and 24%. Organic anion transporter 3 (OAT3) showed an upward trend in all models except the FFDTH (from 3.20 to 2.39 pmol/mg protein), making the latter the only model to represent human OAT3 changes. OAT5, a functional ortholog of human OAT4, significantly decreased in db/db, FFDTH, and ALIOS (from 4.59 to 0.45, 1.59, and 2.83 pmol/mg protein, respectively) but significantly increased for MCD (1.67 to 4.17 pmol/mg protein), suggesting that the mouse models are comparable to human for these specific transport processes. These data suggest that variations in rodent renal transporter expression are elicited by NASH, and the concordance analysis enables appropriate model selection for future pharmacokinetic studies based on transporter specificity. These models provide a valuable resource to extrapolate the consequences of human variability in renal drug elimination. SIGNIFICANCE STATEMENT: Rodent models of nonalcoholic steatohepatitis that recapitulate human renal transporter alterations are identified for future transporter-specific pharmacokinetic studies to facilitate the prevention of adverse drug reactions due to human variability.

Renal Transporter Alterations in Patients with Chronic Liver Diseases: Nonalcoholic Steatohepatitis, Alcohol-Associated, Viral Hepatitis, and Alcohol-Viral Combination.

Alterations in hepatic transporters have been identified in precirrhotic chronic liver diseases (CLDs) that result in pharmacokinetic variations causing adverse drug reactions (ADRs). However, the effect of CLD on the expression of renal transporters is unknown despite the overwhelming evidence of kidney injury in CLD patients. This study determines the transcriptomic and proteomic expression profiles of renal drug transporters in patients with defined CLD etiology. Renal biopsies were obtained from patients with a history of CLD etiologies, including nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), alcohol-associated liver disease (ALD), viral hepatitis C (HCV), and combination ALD/HCV. A significant decrease in organic anion transporter (OAT)-3 was identified in NASH, ALD, HCV, and ALD/HCV (1.56 ± 1.10; 1.01 ± 0.46; 1.03 ± 0.43; 0.86 ± 0.57 pmol/mg protein) relative to control (2.77 ± 1.39 pmol/mg protein). Additionally, a decrease was shown for OAT4 in NASH (24.9 ± 5.69 pmol/mg protein) relative to control (43.8 ± 19.9 pmol/mg protein) and in urate transporter 1 (URAT1) for ALD and HCV (1.56 ± 0.15 and 1.65 ± 0.69 pmol/mg protein) relative to control (4.69 ± 4.59 pmol/mg protein). These decreases in organic anion transporter expression could result in increased and prolonged systemic exposure to drugs and possible toxicity. Renal transporter changes, in addition to hepatic transporter alterations, should be considered in dose adjustments for CLD patients for a more accurate disposition profile. It is important to consider a multiorgan approach to altered pharmacokinetics of drugs prescribed to CLD patients to prevent ADRs and improve patient outcomes. SIGNIFICANCE STATEMENT: Chronic liver diseases are known to elicit alterations in hepatic transporters that result in a disrupted pharmacokinetic profile for various drugs. However, it is unknown if there are alterations in renal transporters during chronic liver disease, despite strong indications of renal dysfunction associated with chronic liver disease. Identifying renal transporter expression changes in patients with chronic liver disease facilitates essential investigations on the multifaceted relationship between liver dysfunction and kidney physiology to offer dose adjustments and prevent adverse drug reactions.

Small Change, Big Impact: Reversal of Diastereoselection in Cuprate Conjugate Additions to α,ß-Unsaturated Lactams and Identification of a Competing Mechanism.

A seemingly minor change to a reactant is shown to cause a change in reaction mechanisms. Conjugate addition of organocopper reagents to bicyclic α,ß-unsaturated lactams derived from pyroglutaminol is determined by the nature of the aminal group. Aminals derived from aldehydes give anti addition; those from ketones give syn addition. Divergence in diastereoselection occurs because the substrates react by different mechanisms, ultimately due to a small but significant difference in pyramidalization of the aminal nitrogen.

Phosphoinositide 3-Kinase δ Inhibition Improves Neutrophil Bacterial Killing in Critically Ill Patients at High Risk of Infection.

Acquired neutrophil dysfunction frequently develops during critical illness, independently increasing the risk for intensive care unit-acquired infection. PI3Kδ is implicated in driving neutrophil dysfunction and can potentially be targeted pharmacologically. The aims of this study were to determine whether PI3Kδ inhibition reverses dysfunction in neutrophils from critically ill patients and to describe potential mechanisms. Neutrophils were isolated from blood taken from critically ill patients requiring intubation and mechanical ventilation, renal support, or blood pressure support. In separate validation experiments, neutrophil dysfunction was induced pharmacologically in neutrophils from healthy volunteers. Phagocytosis and bacterial killing assays were performed, and activity of RhoA and protein kinase A (PKA) was assessed. Inhibitors of PI3Kδ, 3-phosphoinositide-dependent protein kinase-1 (PDK1), and PKA were used to determine mechanisms of neutrophil dysfunction. Sixty-six patients were recruited. In the 27 patients (40.9%) with impaired neutrophil function, PI3Kδ inhibition consistently improved function and significantly increased bacterial killing. These findings were validated in neutrophils from healthy volunteers with salbutamol-induced dysfunction and extended to demonstrate that PI3Kδ inhibition restored killing of clinical isolates of nine pathogens commonly associated with intensive care unit-acquired infection. PI3Kδ activation was associated with PDK1 activation, which in turn phosphorylated PKA, which drove phosphorylation and inhibition of the key regulator of neutrophil phagocytosis, RhoA. These data indicate that, in a significant proportion of critically ill patients, PI3Kδ inhibition can improve neutrophil function through PDK1- and PKA-dependent processes, suggesting that therapeutic use of PI3Kδ inhibitors warrants investigation in this setting.

Novel method for kinetic analysis applied to transport by the uniporter OCT2.

The kinetics of solute transport shed light on the roles these processes play in cellular physiology, and the absolute values of the kinetic parameters that quantitatively describe transport are increasingly used to model its impact on drug clearance. However, accurate assessment of transport kinetics is challenging. Although most carrier-mediated transport is adequately described by the Michaelis-Menten equation, its use presupposes that the rates of uptake used in the analysis of maximal rates of transport (Jmax) and half-saturation constants (Kt) reflect true unidirectional rates of influx from known concentrations of substrate. Most experimental protocols estimate the initial rate of transport from net substrate accumulation determined at a single time point (typically between 0.5 and 5 min) and assume it reflects unidirectional influx. However, this approach generally results in systematic underestimates of Jmax and overestimates of Kt; the former primarily due to the unaccounted impact of efflux of accumulated substrate, and the latter due to the influence of unstirred water layers. Here, we describe the bases of these time-dependent effects and introduce a computational model that analyzes the time course of net substrate uptake at several concentrations to calculate Jmax and Kt for unidirectional influx, taking into account the influence of unstirred water layers and mediated efflux. This method was then applied to calculate the kinetics of transport of 1-methyl-4-phenylpryridinium and metformin by renal organic cation transporter 2 as expressed in cultured Chinese hamster ovary cells.NEW & NOTEWORTHY Here, we describe a mathematical model that uses the time course of net substrate uptake into cells from several increasing concentrations to calculate unique kinetic parameters [maximal rates of transport (Jmax) and half-saturation constants (Kt)] of the process. The method is the first to take into consideration the common complicating factors of unstirred layers and carrier-mediated efflux in the experimental determination of transport kinetics.

Patterns and Causes of Signed Linkage Disequilibria in Flies and Plants.

Most empirical studies of linkage disequilibrium (LD) study its magnitude, ignoring its sign. Here, we examine patterns of signed LD in two population genomic data sets, one from Capsella grandiflora and one from Drosophila melanogaster. We consider how processes such as drift, admixture, Hill-Robertson interference, and epistasis may contribute to these patterns. We report that most types of mutations exhibit positive LD, particularly, if they are predicted to be less deleterious. We show with simulations that this pattern arises easily in a model of admixture or distance-biased mating, and that genome-wide differences across site types are generally expected due to differences in the strength of purifying selection even in the absence of epistasis. We further explore how signed LD decays on a finer scale, showing that loss of function mutations exhibit particularly positive LD across short distances, a pattern consistent with intragenic antagonistic epistasis. Controlling for genomic distance, signed LD in C. grandiflora decays faster within genes, compared with between genes, likely a by-product of frequent recombination in gene promoters known to occur in plant genomes. Finally, we use information from published biological networks to explore whether there is evidence for negative synergistic epistasis between interacting radical missense mutations. In D. melanogaster networks, we find a modest but significant enrichment of negative LD, consistent with the possibility of intranetwork negative synergistic epistasis.

Selection on Accessible Chromatin Regions in Capsella grandiflora.

Accurate estimates of genome-wide rates and fitness effects of new mutations are essential for an improved understanding of molecular evolutionary processes. Although eukaryotic genomes generally contain a large noncoding fraction, functional noncoding regions and fitness effects of mutations in such regions are still incompletely characterized. A promising approach to characterize functional noncoding regions relies on identifying accessible chromatin regions (ACRs) tightly associated with regulatory DNA. Here, we applied this approach to identify and estimate selection on ACRs in Capsella grandiflora, a crucifer species ideal for population genomic quantification of selection due to its favorable population demography. We describe a population-wide ACR distribution based on ATAC-seq data for leaf samples of 16 individuals from a natural population. We use population genomic methods to estimate fitness effects and proportions of positively selected fixations (α) in ACRs and find that intergenic ACRs harbor a considerable fraction of weakly deleterious new mutations, as well as a significantly higher proportion of strongly deleterious mutations than comparable inaccessible intergenic regions. ACRs are enriched for expression quantitative trait loci (eQTL) and depleted of transposable element insertions, as expected if intergenic ACRs are under selection because they harbor regulatory regions. By integrating empirical identification of intergenic ACRs with analyses of eQTL and population genomic analyses of selection, we demonstrate that intergenic regulatory regions are an important source of nearly neutral mutations. These results improve our understanding of selection on noncoding regions and the role of nearly neutral mutations for evolutionary processes in outcrossing Brassicaceae species.

Widespread Recombination Suppression Facilitates Plant Sex Chromosome Evolution.

Classical models suggest that recombination rates on sex chromosomes evolve in a stepwise manner to localize sexually antagonistic variants in the sex in which they are beneficial, thereby lowering rates of recombination between X and Y chromosomes. However, it is also possible that sex chromosome formation occurs in regions with preexisting recombination suppression. To evaluate these possibilities, we constructed linkage maps and a chromosome-scale genome assembly for the dioecious plant Rumex hastatulus. This species has a polymorphic karyotype with a young neo-sex chromosome, resulting from a Robertsonian fusion between the X chromosome and an autosome, in part of its geographic range. We identified the shared and neo-sex chromosomes using comparative genetic maps of the two cytotypes. We found that sex-linked regions of both the ancestral and the neo-sex chromosomes are embedded in large regions of low recombination. Furthermore, our comparison of the recombination landscape of the neo-sex chromosome to its autosomal homolog indicates that low recombination rates mainly preceded sex linkage. These patterns are not unique to the sex chromosomes; all chromosomes were characterized by massive regions of suppressed recombination spanning most of each chromosome. This represents an extreme case of the periphery-biased recombination seen in other systems with large chromosomes. Across all chromosomes, gene and repetitive sequence density correlated with recombination rate, with patterns of variation differing by repetitive element type. Our findings suggest that ancestrally low rates of recombination may facilitate the formation and subsequent evolution of heteromorphic sex chromosomes.

Biosynthesis, regulation, and engineering of natural products from Lysobacter.

Covering: up to August 2021Lysobacter is a genus of Gram-negative bacteria that was classified in 1987. Several Lysobacter species are emerging as new biocontrol agents for crop protection in agriculture. Lysobacter are prolific producers of new bioactive natural products that are largely underexplored. So far, several classes of structurally interesting and biologically active natural products have been isolated from Lysobacter. This article reviews the progress in Lysobacter natural product research over the past ten years, including molecular mechanisms for biosynthesis, regulation and mode of action, genome mining of cryptic biosynthetic gene clusters, and metabolic engineering using synthetic biology tools.

Physiological Characterization of the Transporter-Mediated Uptake of the Reversible Male Contraceptive H2-Gamendazole Across the Blood-Testis Barrier.

The blood-testis barrier (BTB) is formed by a tight network of Sertoli cells (SCs) to limit the movement of reproductive toxicants from the blood into the male genital tract. Transporters expressed at the basal membranes of SCs also influence the disposition of drugs across the BTB. The reversible, nonhormonal contraceptive, H2-gamendazole (H2-GMZ), is an indazole carboxylic acid analog that accumulates over 10 times more in the testes compared with other organs. However, the mechanism(s) by which H2-GMZ circumvents the BTB are unknown. This study describes the physiologic characteristics of the carrier-mediated process(es) that permit H2-GMZ and other analogs to penetrate SCs. Uptake studies were performed using an immortalized human SC line (hT-SerC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Uptake of H2-GMZ and four analogs followed Michaelis-Menten transport kinetics (one analog exhibited poor penetration). H2-GMZ uptake was strongly inhibited by indomethacin, diclofenac, MK-571, and several analogs. Moreover, H2-GMZ uptake was stimulated by an acidic extracellular pH, reduced at basic pHs, and independent of extracellular Na+, K+, or Cl- levels, which are intrinsic characteristics of OATP-mediated transport. Therefore, the characteristics of H2-GMZ transport suggest that one or more OATPs may be involved. However, endogenous transporter expression in wild-type Chinese hamster ovary (CHO), Madin-Darby canine kidney (MDCK), and human embryonic kidney-293 (HEK-293) cells limited the utility of heterologous transporter expression to identify a specific OATP transporter. Altogether, characterization of the transporters involved in the flux of H2-GMZ provides insight into the selectivity of drug disposition across the human BTB to understand and overcome the pharmacokinetic and pharmacodynamic difficulties presented by this barrier. SIGNIFICANCE STATEMENT: Despite major advancements in female contraceptives, male alternatives, including vasectomy, condom usage, and physical withdrawal, are antiquated and the widespread availability of nonhormonal, reversible chemical contraceptives is nonexistent. Indazole carboxylic acid analogs such as H2-GMZ are promising new reversible, antispermatogenic drugs that are highly effective in rodents. This study characterizes the carrier-mediated processes that permit H2-GMZ and other drugs to enter Sertoli cells and the observations made here will guide the development of drugs that effectively circumvent the BTB.