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Mitochondrial DNA (mtDNA) is present in multiple copies within cells and is required for mitochondrial ATP generation. Even within individual cells, mtDNA copies can differ in their sequence, a state known as heteroplasmy. The principles underlying dynamic changes in the degree of heteroplasmy remain incompletely understood, due to the inability to monitor this phenomenon in real time. Here, we employ mtDNA-based fluorescent markers, microfluidics, and automated cell tracking, to follow mtDNA variants in live heteroplasmic yeast populations at the single-cell level. This approach, in combination with direct mtDNA tracking and data-driven mathematical modeling reveals asymmetric partitioning of mtDNA copies during cell division, as well as limited mitochondrial fusion and fission frequencies, as critical driving forces for mtDNA variant segregation. Given that our approach also facilitates assessment of segregation between intact and mutant mtDNA, we anticipate that it will be instrumental in elucidating the mechanisms underlying the purifying selection of mtDNA., Competing Interests: Disclosure and competing interests statement The authors declare no competing interests., (© 2024. The Author(s).)

The femoral artery (FA) is the largest vessel in the hindlimb circulation and its proper tone regulation ensures adequate blood supply to muscle tissue. We investigated whether an alanine mutation of the targeting subunit of myosin-light-chain-phosphatase (MLCP), MYPT1, at threonine 696 (MYPT1-T696A/+), decisive for enzyme acivity, affects the responsiveness of young and old FAs (y-FAs and o-FAs) to activation of nitric-oxide/soluble-guanylate-cyclase/protein-kinase-G cascade (NO/sGC/PKG). Contractile responses of the vessels were measured by wire myography. Phosphorylation of the regulatory myosin-light-chain at serine 19 (MLC 20 -S19), the myosin-light-chain-phosphatase targeting subunit, MYPT1-T696, the PKG-sensitive site of MYPT1 at S695 (MYPT1-S695) and S668 (MYPT1-S668), and the regulatory phosphorylation of eNOS at S1177 (eNOS-S1177) were determined in arterial homogenates by Western blot. In FAs of all ages, the MYPT1-T696A-mutation did not alter vessel diameter and the contractile reactivity to the thromboxaneA 2 -analogue, U46619 and the RhoA kinase inhibitor, Y27632. In contrast, the mutation T696 into alanine attenuated the relaxing effect of exogenous NO (DEA-NONOate) in y-FAs. The effect of a direct sGC activation by cinaciguat was also attenuated in both age groups of MYPT1-T696A/+, but strongly in o-FA. The MYPT1-T696A-mutation also attenuated acetylcholine-induced relaxation, but only in o-FAs. Similary, the alanine mutation attenuated the acetylcholine effect on MLC 20 -S19- and MYPT1-T696 only in WT o-FAs. Interestingly, neither eNOS-S1177 nor the phosphorylation of the PKG phosphospecific sites, MYPT1-S695 and MYPT1-S668 were altered by MYPT1-T696A-mutation or aging. These findings suggest that the alanine mutation of MYPT1-T696 reduces the ability of the NO/cGMP/PKG-system to relax FAs in aging., Competing Interests: Declaration of competing interest Hereby I declare in relation to the following Manuscript entitled “Alanine mutation of the targeting subunit of the myosin phosphatase, MYPT1 at threonine 696 reduces cGMP responsiveness of mouse femoral arteries” no potential conflicts of interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Chromosomal inversions are structural mutations that can play a prominent role in adaptation and speciation. Inversions segregating across species boundaries (trans-species inversions) are often taken as evidence for ancient balancing selection or adaptive introgression, but can also be due to incomplete lineage sorting. Using whole-genome resequencing data from 18 populations of 11 recognized munia species in the genus Lonchura (N = 176 individuals), we identify four large para- and pericentric inversions ranging in size from 4 to 20 Mb. All four inversions cosegregate across multiple species and predate the numerous speciation events associated with the rapid radiation of this clade across the prehistoric Sahul (Australia, New Guinea) and Bismarck Archipelago. Using coalescent theory, we infer that trans-specificity is improbable for neutrally segregating variation despite substantial incomplete lineage sorting characterizing this young radiation. Instead, the maintenance of all three autosomal inversions (chr1, chr5, and chr6) is best explained by selection acting along ecogeographic clines not observed for the collinear parts of the genome. In addition, the sex chromosome inversion largely aligns with species boundaries and shows signatures of repeated positive selection for both alleles. This study provides evidence for trans-species inversion polymorphisms involved in both adaptation and speciation. It further highlights the importance of informing selection inference using a null model of neutral evolution derived from the collinear part of the genome., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Ticks are important vectors of human and animal pathogens, but many questions remain unanswered regarding their taxonomy. Molecular sequencing methods have allowed research to start understanding the evolutionary history of even closely related tick species. Ixodes inopinatus is considered a sister species and highly similar to Ixodes ricinus, an important vector of many tick-borne pathogens in Europe, but identification between these species remains ambiguous with disagreement on the geographic extent of I. inopinatus. In 2018-2019, 1583 ticks were collected from breeding great tits (Parus major) in southern Germany, of which 45 were later morphologically identified as I. inopinatus. We aimed to confirm morphological identification using molecular tools. Utilizing two genetic markers (16S rRNA, TROSPA) and whole genome sequencing of specific ticks (n = 8), we were able to determine that German samples, morphologically identified as I. inopinatus, genetically represent I. ricinus regardless of previous morphological identification, and most likely are not I. ricinus/I. inopinatus hybrids. Further, our results showed that the entire mitochondrial genome, let alone singular mitochondrial genes (i.e., 16S), is unable to distinguish between I. ricinus and I. inopinatus. Our results suggest that I. inopinatus is geographically isolated as a species (northern Africa and potentially southern Spain and Portugal) and brings into question whether I. inopinatus exists in central Europe. Our results highlight the probable existence of I. inopinatus and the power of utilizing genomic data in answering questions regarding tick taxonomy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

There is limited information about the species of rumen fluke (Family Paramphistomidae) in the Caribbean. However, knowledge of species distribution is needed to better understand disease risk and epidemiology. Morphological identification is challenging with more recent DNA sequencing enabling a better understanding of rumen fluke distribution. In this study, rumen fluke specimens, collected between 2015 and 2016 from cattle on the island of St. Kitts, West Indies, were analysed. The ribosomal internal transcribed spacer 2 (ITS-2) region of rDNA was amplified using generic trematode primers. Results from Sanger sequencing were compared to reference sequences in GenBank and indicated the species was Cotylophoron cotylophorum with 100% sequence identity and 91% query cover. The ITS2 sequences were then compared to previously published ITS2 sequences for the Cotylophoron genus. When all the St. Kitts C. cotylophorum ITS2 sequences were compared with all other Cotylophoron sequences from India, Kenya, and Zimbabwe, three variable nucleotide sites, resulting in five unique haplotypes, were identified. Nine ITS2 sequences shared haplotype 1, which included all those from St. Kitts and single representatives from India and Kenya, potentially indicating global movement of this species., Competing Interests: Declaration of Competing Interest The authors declare that there were no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Objective: Small arteries from different organs vary with regard to the mechanisms that regulate vasoconstriction. This study investigated the impact of advanced age on the regulation of vasoconstriction in isolated human small arteries from kidney cortex and periintestinal mesenteric tissue., Methods: Renal and mesenteric tissues were obtained from patients (mean age 71 ± 9 years) undergoing elective surgery. Furthermore, intrarenal and mesenteric arteries from young and aged mice were studied. Arteries were investigated by small vessel myography and western blot., Results: Human intrarenal arteries (h-RA) showed higher stretch-induced tone and higher reactivity to α 1 adrenergic receptor stimulation than human mesenteric arteries (h-MA). Rho-kinase (ROK) inhibition resulted in a greater decrease in Ca 2+ and depolarization-induced tone in h-RA than in h-MA. Basal and α 1 adrenergic receptor stimulation-induced phosphorylation of the regulatory light chain of myosin (MLC 20 ) was higher in h-RA than in h-MA. This was associated with higher ROK-dependent phosphorylation of the regulatory subunit of myosin light-chain-phosphatase (MLCP), MYPT1-T853. In h-RA phosphorylation of ribosomal S6-kinase II (RSK2-S227) was significantly higher than in h-MA. Stretch-induced tone and RSK2 phosphorylation was also higher in interlobar arteries (m-IAs) from aged mice than in respective vessels from young mice and in murine mesenteric arteries (m-MA) from both age groups., Conclusion: Vasoconstriction in human intrarenal arteries shows a greater ROK-dependence than in mesenteric arteries. Activation of RSK2 may contribute to intrarenal artery tone dysregulation associated with aging. Compared with h-RA, h-MA undergo age-related remodeling leading to a reduction of the contractile response to α 1 adrenergic stimulation., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Human land modification is a known driver of animal-to-human transmission of infectious agents (zoonotic spillover). Infection prevalence in the reservoir is a key predictor of spillover, but landscape-level associations between the intensity of land modification and infection rates in wildlife remain largely untested. Bat-borne coronaviruses have caused three major disease outbreaks in humans: severe acute respiratory syndrome (SARS), Middle East respiratory syndrome, and coronavirus disease 2019 (COVID-19). We statistically link high-resolution land modification data with bat coronavirus surveillance records and show that coronavirus prevalence significantly increases with the intensity of human impact across all climates and levels of background biodiversity. The most significant contributors to the overall human impact are agriculture, deforestation, and mining. Regions of high predicted bat coronavirus prevalence coincide with global disease hotspots, suggesting that infection prevalence in wildlife may be an important factor underlying links between human land modification and zoonotic disease emergence.

Stretch-induced vascular tone is an important element of autoregulatory adaptation of cerebral vasculature to maintain cerebral flow constant despite changes in perfusion pressure. Little is known as to the regulation of tone in senescent basilar arteries. We tested the hypothesis, that thin filament mechanisms in addition to smooth muscle myosin-II regulatory-light-chain-(MLC 20 )-phosphorylation and non-muscle-myosin-II, contribute to regulation of stretch-induced tone. In young BAs (y-BAs) mechanical stretch does not lead to spontaneous tone generation. Stretch-induced tone in y-BAs appeared only after inhibition of NO-release by L-NAME and was fully prevented by treatment with 3 μmol/L RhoA-kinase (ROK) inhibitor Y27632. L-NAME-induced tone was reduced in y-BAs from heterozygous mice carrying a point mutation of the targeting-subunit of the myosin phosphatase, MYPT1 at threonine696 (MYPT1-T696A/+). In y-BAs, MYPT1-T696A-mutation also blunted the ability of L-NAME to increase MLC 20 -phosphorylation. In contrast, senescent BAs (s-BAs; >24 months) developed stable spontaneous stretch-induced tone and pharmacological inhibition of NO-release by L-NAME led to an additive effect. In s-BAs the MYPT1-T696A mutation also blunted MLC 20 -phosphorylation, but did not prevent development of stretch-induced tone. In s-BAs from both lines, Y27632 completely abolished stretch- and L-NAME-induced tone. In s-BAs phosphorylation of non-muscle-myosin-S1943 and PAK1-T423, shown to be down-stream effectors of ROK was also reduced by Y27632 treatment. Stretch- and L-NAME tone were inhibited by inhibition of non-muscle myosin (NM-myosin) by blebbistatin. We also tested whether the substrate of PAK1 the thin-filament associated protein, caldesmon is involved in the regulation of stretch-induced tone in advanced age. BAs obtained from heterozygotes Cald1 +/- mice generated stretch-induced tone already at an age of 20-21 months old BAs (o-BA). The magnitude of stretch-induced tone in Cald1 +/- o-BAs was similar to that in s-BA. In addition, truncation of caldesmon myosin binding Exon2 (CaD-▵Ex2 -/- ) did not accelerate stretch-induced tone. Our study indicates that in senescent cerebral vessels, mechanisms distinct from MLC 20 phosphorylation contribute to regulation of tone in the absence of a contractile agonist. While in y-and o-BA the canonical pathways, i.e., inhibition of MLCP by ROK and increase in pMLC 20 , predominate, tone regulation in senescence involves ROK regulated mechanisms, involving non-muscle-myosin and thin filament linked mechanisms involving caldesmon., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Lubomirov, Schroeter, Hasse, Frohn, Metzler, Bust, Pryymachuk, Hescheler, Grisk, Chalovich, Smyth, Pfitzer and Papadopoulos.)

This work explored the mechanism of augmented stress-induced vascular reactivity of senescent murine femoral arteries (FAs). Mechanical and pharmacological reactivity of young (12-25 weeks, y-FA) and senescent (>104 weeks, s-FAs) femoral arteries was measured by wire myography. Expression and protein phosphorylation of selected regulatory proteins were studied by western blotting. Expression ratio of the Exon24 in/out splice isoforms of the regulatory subunit of myosin phosphatase, MYPT1 (MYPT1-Exon24 in/out), was determined by polymerase chain reaction (PCR). While the resting length-tension relationship showed no alteration, the stretch-induced-tone increased to 8.3 ± 0.9 mN in s-FA versus only 4.6 ± 0.3 mN in y-FAs. Under basal conditions, phosphorylation of the regulatory light chain of myosin at S19 was 19.2 ± 5.8% in y-FA versus 49.2 ± 12.6% in s-FA. Inhibition of endogenous NO release raised tone additionally to 10.4 ± 1.2 mN in s-FA, whereas this treatment had a negligible effect in y-FAs (4.8 ± 0.3 mN). In s-FAs, reactivity to NO donor was augmented (pD 2  = -4.5 ± 0.3 in y-FA vs. -5.2 ± 0.1 in senescent). Accordingly, in s-FAs, MYPT1-Exon24-out-mRNA, which is responsible for expression of the more sensitive to protein-kinase G, leucine-zipper-positive MYPT1 isoform, was increased. The present work provides evidence that senescent murine s-FA undergoes vascular remodelling associated with increases in stretch-activated contractility and sensitivity to NO/cGMP/PKG system., (© 2021 The Authors. Basic & Clinical Pharmacology & Toxicology published by John Wiley & Sons Ltd on behalf of Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Identification of fusion genes in clinical routine is mostly based on cytogenetics and targeted molecular genetics, such as metaphase karyotyping, fluorescence in situ hybridization and reverse-transcriptase polymerase chain reaction. However, sequencing technologies are becoming more important in clinical routine as processing time and costs per sample decrease. To evaluate the performance of fusion gene detection by RNAsequencing compared to standard diagnostic techniques, we analyzed 806 RNA-sequencing samples from patients with acute myeloid leukemia using two state-of-the-art software tools, namely Arriba and FusionCatcher. RNA-sequencing detected 90% of fusion events that were reported by routine with high evidence, while samples in which RNA-sequencing failed to detect fusion genes had overall lower and inhomogeneous sequence coverage. Based on properties of known and unknown fusion events, we developed a workflow with integrated filtering strategies for the identification of robust fusion gene candidates by RNA-sequencing. Thereby, we detected known recurrent fusion events in 26 cases that were not reported by routine and found discrepancies in evidence for known fusion events between routine and RNA-sequencing in three cases. Moreover, we identified 157 fusion genes as novel robust candidates and comparison to entries from ChimerDB or Mitelman Database showed novel recurrence of fusion genes in 14 cases. Finally, we detected the novel recurrent fusion gene NRIP1- MIR99AHG resulting from inv(21)(q11.2;q21.1) in nine patients (1.1%) and LTN1-MX1 resulting from inv(21)(q21.3;q22.3) in two patients (0.25%). We demonstrated that NRIP1-MIR99AHG results in overexpression of the 3' region of MIR99AHG and the disruption of the tricistronic miRNA cluster miR-99a/let-7c/miR-125b-2. Interestingly, upregulation of MIR99AHG and deregulation of the miRNA cluster, residing in the MIR99AHG locus, are known mechanisms of leukemogenesis in acute megakaryoblastic leukemia. Our findings demonstrate that RNA-sequencing has a strong potential to improve the systematic detection of fusion genes in clinical applications and provides a valuable tool for fusion discovery.

Hybrid zones provide a window into the evolutionary processes governing species divergence. Yet, the contribution of mate choice to the temporal and spatial stability of hybrid zones remains poorly explored. Here, we investigate the effects of assortative mating on hybrid-zone dynamics by means of a mathematical model parameterized with phenotype and genotype data from the hybrid zone between all-black carrion and gray-coated hooded crows. In the best-fit model, narrow clines of the two mating-trait loci were maintained by a moderate degree of assortative mating inducing pre- and postzygotic isolation via positive frequency-dependent selection. Epistasis between the two loci induced hybrid-zone movement in favor of alleles conveying dark plumage followed by a shift in the opposite direction favoring gray-coated phenotypes ∼ 1 200 generations after secondary contact. Unlinked neutral loci diffused near-unimpeded across the zone. These results were generally robust to the choice of matching rule (self-referencing or parental imprinting) and effects of genetic drift. Overall, this study illustrates under which conditions assortative mating can maintain steep clines in mating-trait loci without generalizing to genome-wide reproductive isolation. It further emphasizes the importance of the genetic mating-trait architecture for spatio-temporal hybrid-zone dynamics., (© 2021 The Authors. Evolution © 2021 The Society for the Study of Evolution.)

The actin-, myosin-, and calmodulin-binding protein caldesmon (CaD) is expressed in two splice isoforms: h-CaD, which is an integral part of the actomyosin domain of smooth muscle cells, and l-CaD, which is widely expressed and is involved in many cellular functions. Despite extensive research for many years, CaD's in vivo function has remained elusive. To explore the role of CaD in smooth muscle contraction in vivo, we generated a mutant allele that ablates both isoforms. Heterozygous animals were viable and had a normal life span, but homozygous mutants died perinatally, likely because of a persistent umbilical hernia. The herniation was associated with hypoplastic and dysmorphic abdominal wall muscles. We assessed mechanical parameters in isometrically mounted longitudinal strips of E18.5 urinary bladders and in ring preparations from abdominal aorta using wire myography. Ca2+ sensitivity was higher and relaxation rate was slower in Cald1-/- compared with Cald1+/+ skinned bladder strips. However, we observed no change in the content and phosphorylation of regulatory proteins of the contractile apparatus and myosin isoforms known to affect these contractile parameters. Intact fibers showed no difference in actin and myosin content, regardless of genotype, although KCl-induced force tended to be lower in homozygous and higher in heterozygous mutants than in WTs. Conversely, in skinned fibers, myosin content and maximal force were significantly lower in Cald1-/- than in WTs. In KO abdominal aortas, resting and U46619 elicited force were lower than in WTs. Our results are consistent with the notion that CaD impacts smooth muscle function dually by (1) acting as a molecular brake on contraction and (2) maintaining the structural integrity of the contractile machinery. Most importantly, CaD is essential for resolution of the physiological umbilical hernia and ventral body wall closure., (© 2021 Pütz et al.)

Camouflage is a widespread strategy to increase survival. The cryptic plumage colouration of precocial chicks improves camouflage often through disruptive colouration. Here, we examine whether and how fringed neoptile feathers conceal the outline of chicks. We first conducted a digital experiment to test two potential mechanisms for outline concealment through appendages: (1) reduction of edge intensity and (2) luminance transition. Local Edge Intensity Analysis showed that appendages decreased edge intensity whereas a mean luminance comparison revealed that the appendages created an intermediate transition zone to conceal the object's outline. For edge intensity, the outline diffusion was strongest for a vision system with low spatial acuity, which is characteristic of many mammalian chick predators. We then analysed photographs of young snowy plover (Charadrius nivosus) chicks to examine whether feathers increase outline concealment in a natural setting. Consistent with better camouflage, the outline of digitally cropped chicks with protruding feathers showed lower edge intensities than the outline of chicks without those feathers. However, the observed mean luminance changes did not indicate better concealment. Taken together, our results suggest that thin skin appendages such as neoptile feathers improve camouflage. As skin appendages are widespread, this mechanism may apply to many organisms.

Background: In vertebrate genomes, CpG sites can be clustered into CpG islands, and the amount of methylation in a CpG island can change due to gene regulation processes. Thus, single regulatory events can simultaneously change the methylation states of many CpG sites within a CpG island. This should be taken into account when quantifying the amount of change in methylation, for example in form of a branch length in a phylogeny of cell types., Results: We propose a probabilistic model (the IWE-SSE model) of methylation dynamics that accounts for simultaneous methylation changes in multiple CpG sites belonging to the same CpG island. We further propose a Markov-chain Monte-Carlo (MCMC) method to fit this model to methylation data from cell type phylogenies and apply this method to available data from murine haematopoietic cells and from human cell lines. Combined with simulation studies, these analyses show that accounting for CpG island wide methylation changes has a strong effect on the inferred branch lengths and leads to a significantly better model fit for the methylation data from murine haematopoietic cells and human cell lines., Conclusion: The MCMC based parameter estimation method for the IWE-SSE model in combination with our MCMC based inference method allows to quantify the amount of methylation changes at single CpG sites as well as on entire CpG islands. Accounting for changes affecting entire islands can lead to more accurate branch length estimation in the presence of simultaneous methylation change.

Aim: Constitutive release of NO blunts intrinsic and stimulated contractile activity in cerebral arteries (CA). Here, we explored whether phosphorylation and expression levels of the PKG-sensitive, leucine zipper positive (LZ + ) splice variants of the regulatory subunit of myosin phosphatase (MYPT1) are involved and whether its expression is associated with higher cGMP sensitivity., Methods: Vascular contractility was investigated by wire myography. Phosphorylation of MYPT1 was determined by Western blotting., Results: Constitutive phosphorylation of MYPT1-T696 and T853 was lower and that of S695 and S668 was higher in cerebral arteries from the circulus arteriosus (CA-w) than in femoral arteries (FA), while total MYPT1 expression was not different. In CA-w but not in FA, L-NAME lowered phosphorylation of S695/S668 and increased phosphorylation of T696/T853 and of MLC 20 -S19, plus basal tone. The increase in basal tone was attenuated in CA-w and basilar arteries (BA) from heterozygous MYPT1-T696A/+ mice. Compared to FA, expression of the LZ + -isoform was ~2-fold higher in CA-w coincident with a higher sensitivity to DEA-NONOate, cinaciguat and Y27632 in BA and 8-Br-cGMP (1 μmol/L) in pre-constricted (pCa 6.1) α-toxin permeabilized CAs. In contrast, 6-Bnz-cAMP (10 μmol/L) relaxed BA and FA similarly by ~80%., Conclusion: Our results indicate that (i) regulation of the intrinsic contractile activity in CA involves phosphorylation of MYPT1 at T696 and S695/S668, (ii) the higher NO/cGMP/PKG sensitivity of CAs can be ascribed to the higher expression level of the LZ + -MYPT1 isoform and (iii) relaxation by cAMP/PKA pathway is less dependent on the expression level of the LZ + splice variants of MYPT1., (© 2018 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Cell-specific patterns of gene expression are determined by combinatorial actions of sequence-specific transcription factors at cis-regulatory elements. Studies indicate that relatively simple combinations of lineage-determining transcription factors (LDTFs) play dominant roles in the selection of enhancers that establish cell identities and functions. LDTFs require collaborative interactions with additional transcription factors to mediate enhancer function, but the identities of these factors are often unknown. We have shown that natural genetic variation between individuals has great utility for discovering collaborative transcription factors. Here, we introduce MMARGE (Motif Mutation Analysis of Regulatory Genomic Elements), the first publicly available suite of software tools that integrates genome-wide genetic variation with epigenetic data to identify collaborative transcription factor pairs. MMARGE is optimized to work with chromatin accessibility assays (such as ATAC-seq or DNase I hypersensitivity), as well as transcription factor binding data collected by ChIP-seq. Herein, we provide investigators with rationale for each step in the MMARGE pipeline and key differences for analysis of datasets with different experimental designs. We demonstrate the utility of MMARGE using mouse peritoneal macrophages, liver cells, and human lymphoblastoid cells. MMARGE provides a powerful tool to identify combinations of cell type-specific transcription factors while simultaneously interpreting functional effects of non-coding genetic variation.

Non-coding genetic variation is a major driver of phenotypic diversity and allows the investigation of mechanisms that control gene expression. Here, we systematically investigated the effects of >50 million variations from five strains of mice on mRNA, nascent transcription, transcription start sites, and transcription factor binding in resting and activated macrophages. We observed substantial differences associated with distinct molecular pathways. Evaluating genetic variation provided evidence for roles of ∼100 TFs in shaping lineage-determining factor binding. Unexpectedly, a substantial fraction of strain-specific factor binding could not be explained by local mutations. Integration of genomic features with chromatin interaction data provided evidence for hundreds of connected cis-regulatory domains associated with differences in transcription factor binding and gene expression. This system and the >250 datasets establish a substantial new resource for investigation of how genetic variation affects cellular phenotypes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

It is now widely accepted that genetic processes such as inbreeding depression and loss of genetic variation can increase the extinction risk of small populations. However, it is generally unclear whether extinction risk from genetic causes gradually increases with decreasing population size or whether there is a sharp transition around a specific threshold population size. In the ecological literature, such threshold phenomena are called 'strong Allee effects' and they can arise for example from mate limitation in small populations. In this study, we aim to (i) develop a meaningful notion of a 'strong genetic Allee effect', (ii) explore whether and under what conditions such an effect can arise from inbreeding depression due to recessive deleterious mutations, and (iii) quantify the interaction of potential genetic Allee effects with the well-known mate-finding Allee effect. We define a strong genetic Allee effect as a genetic process that causes a population's survival probability to be a sigmoid function of its initial size. The inflection point of this function defines the critical population size. To characterize survival-probability curves, we develop and analyse simple stochastic models for the ecology and genetics of small populations. Our results indicate that inbreeding depression can indeed cause a strong genetic Allee effect, but only if individuals carry sufficiently many deleterious mutations (lethal equivalents). Populations suffering from a genetic Allee effect often first grow, then decline as inbreeding depression sets in and then potentially recover as deleterious mutations are purged. Critical population sizes of ecological and genetic Allee effects appear to be often additive, but even superadditive interactions are possible. Many published estimates for the number of lethal equivalents in birds and mammals fall in the parameter range where strong genetic Allee effects are expected. Unfortunately, extinction risk due to genetic Allee effects can easily be underestimated as populations with genetic problems often grow initially, but then crash later. Also interactions between ecological and genetic Allee effects can be strong and should not be neglected when assessing the viability of endangered or introduced populations., (© 2016 The Authors. Journal of Animal Ecology © 2016 British Ecological Society.)

Anuran amphibians undergo major morphological transitions during development, but the contribution of their markedly different life-history phases to macroevolution has rarely been analysed. Here we generate testable predictions for coupling versus uncoupling of phenotypic evolution of tadpole and adult life-history phases, and for the underlying expression of genes related to morphological feature formation. We test these predictions by combining evidence from gene expression in two distantly related frogs, Xenopus laevis and Mantidactylus betsileanus, with patterns of morphological evolution in the entire radiation of Madagascan mantellid frogs. Genes linked to morphological structure formation are expressed in a highly phase-specific pattern, suggesting uncoupling of phenotypic evolution across life-history phases. This gene expression pattern agrees with uncoupled rates of trait evolution among life-history phases in the mantellids, which we show to have undergone an adaptive radiation. Our results validate a prevalence of uncoupling in the evolution of tadpole and adult phenotypes of frogs.

Aging causes major alterations of all components of the neurovascular unit and compromises brain blood supply. Here, we tested how aging affects vascular reactivity in basilar arteries from young (<10 weeks; y-BA), old (>22 months; o-BA) and old (>22 months) heterozygous MYPT1-T-696A/+ knock-in mice. In isometrically mounted o-BA, media thickness was increased by ∼10% while the passive length tension relations were not altered. Endothelial denudation or pan-NOS inhibition (100 µmol/L L-NAME) increased the basal tone by 11% in y-BA and 23% in o-BA, while inhibition of nNOS (1 µmol/L L-NPA) induced ∼10% increase in both ages. eNOS expression was ∼2-fold higher in o-BA. In o-BA, U46619-induced force was augmented (pEC 50 ∼6.9 vs. pEC 50 ∼6.5) while responsiveness to DEA-NONOate, electrical field stimulation or nicotine was decreased. Basal phosphorylation of MLC 20 -S19 and MYPT1-T-853 was higher in o-BA and was reversed by apocynin. Furthermore, permeabilized o-BA showed enhanced Ca 2+ -sensitivity. Old T-696A/+ BA displayed a reduced phosphorylation of MYPT1-T696 and MLC 20 , a lower basal tone in response to L-NAME and a reduced eNOS expression. The results indicate that the vascular hypercontractility found in o-BA is mediated by inhibition of MLCP and is partially compensated by an upregulation of endothelial NO release.

Background: Toxoplasma gondii is a worldwide protozoan parasite of felids which can infect almost all warm-blooded animals, including humans. Free-roaming chickens are good indicators of environmental contamination with T. gondii oocysts because they feed from the ground. Previous research has demonstrated a high seroprevalence of T. gondii in domestic animals on St. Kitts but little is known about the genotypes circulating in the environment., Methods: Hearts and brains from 81 free-roaming chickens in St. Kitts were digested and inoculated into 243 Swiss Webster mice in a bioassay. DNA was extracted from digested chicken tissues and the brains of all mice, and screened for T. gondii. Positive samples were genotyped using restriction fragment length polymorphism. Chicken sera were also screened for T. gondii antibodies using a modified agglutination test (MAT)., Results: Overall, 41% (33 out of 81) of chickens were positive for T. gondii either by serology and/or by PCR. Antibodies to T. gondii were detected by MAT in 32% (26 out of 81) of chickens, and T. gondii DNA was detected in mouse brains representing 26% (21 out of 81) of chickens. Genotyping of 21 DNA isolates, using polymorphisms at 10 loci, including SAG1, SAG2 (5'-3' SAG2 and alt.SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico, revealed that 7 were ToxoDB genotype #141, 6 were #1 (Type II), 3 were #13, 3 were #265, one was #264 and one was #2 (Type III). Genotypes #13 and #141 appear to be more virulent., Conclusions: The results of this study highlight the greater genetic diversity of T. gondii circulating in the Caribbean region, with potentially different degrees of virulence to humans.

With the increasing interest in establishing directional etching methods capable of atomic scale resolution for fabricating highly scaled electronic devices, the need for development and characterization of atomic layer etching processes, or generally etch processes with atomic layer precision, is growing. In this work, a flux-controlled cyclic plasma process is used for etching of SiO 2 and Si at the Angstrom-level. This is based on steady-state Ar plasma, with periodic, precise injection of a fluorocarbon (FC) precursor (C 4 F 8 and CHF 3 ) and synchronized, plasma-based Ar + ion bombardment [D. Metzler et al., J. Vac. Sci. Technol., A 32, 020603 (2014) and D. Metzler et al., J. Vac. Sci. Technol., A 34, 01B101 (2016)]. For low energy Ar + ion bombardment conditions, physical sputter rates are minimized, whereas material can be etched when FC reactants are present at the surface. This cyclic approach offers a large parameter space for process optimization. Etch depth per cycle, removal rates, and self-limitation of removal, along with material dependence of these aspects, were examined as a function of FC surface coverage, ion energy, and etch step length using in situ real time ellipsometry. The deposited FC thickness per cycle is found to have a strong impact on etch depth per cycle of SiO 2 and Si but is limited with regard to control over material etching selectivity. Ion energy over the 20-30 eV range strongly impacts material selectivity. The choice of precursor can have a significant impact on the surface chemistry and chemically enhanced etching. CHF 3 has a lower FC deposition yield for both SiO 2 and Si and also exhibits a strong substrate dependence of FC deposition yield, in contrast to C 4 F 8 . The thickness of deposited FC layers using CHF 3 is found to be greater for Si than for SiO 2 . X-ray photoelectron spectroscopy was used to study surface chemistry. When thicker FC films of 11 Å are employed, strong changes of FC film chemistry during a cycle are seen whereas the chemical state of the substrate varies much less. On the other hand, for FC film deposition of 5 Å for each cycle, strong substrate surface chemical changes are seen during an etching cycle. The nature of this cyclic etching with periodic deposition of thin FC films differs significantly from conventional etching with steady-state FC layers since surface conditions change strongly throughout each cycle.

Background: A large share of agriculturally and horticulturally important plant species are polyploid. Linkage maps are used to locate associations between genes and traits by breeders and geneticists. Linkage map creation for polyploid species is not supported by standard tools. We want to overcome this limitation and validate our results with simulation studies., Results: We developed PERGOLA, a deterministic and heuristic method that addresses this problem. We show that it creates correct linkage groups, marker orders and distances for simulated and real datasets. We compare it to existing tools and demonstrate that it overcomes limitations in ploidy and outperforms them in computational time and mapping accuracy. We represent linkage maps as dendrograms and show that this has advantages in the comparison of different maps., Conclusions: PERGOLA can be used successfully to calculate linkage maps for diploid and polyploid species and outperforms existing tools.

Drosophila suzukii is threatening soft fruit production worldwide due to the females' ability to pierce through the intact skin of ripe fruits and lay eggs inside. Larval consumption and the associated microbial infection cause rapid fruit degradation, thus drastic yield and economic loss. Cultivars that limit the proliferation of flies may be ideal to counter this pest; however, they have not yet been developed or identified. To search for potential breeding material, we investigated the rate of adult D. suzukii emergence from individual fruits (fly emergence) of 107 accessions of Fragaria species that had been exposed to egg-laying D. suzukii females. We found significant variation in fly emergence across strawberries, which correlated with accession and fruit diameter, and to a lesser extent with the strawberry species background. We identified accessions with significantly reduced fly emergence, not explained by their fruit diameter. These accessions constitute valuable breeding material for strawberry cultivars that limit D. suzukii spread.

Background: Association studies are an essential part of modern plant breeding, but are limited for polyploid crops. The increased number of possible genotype classes complicates the differentiation between them. Available methods are limited with respect to the ploidy level or data producing technologies. While genotype classification is an established noise reduction step in diploids, it gains complexity with increasing ploidy levels. Eventually, the errors produced by misclassifications exceed the benefits of genotype classes. Alternatively, continuous genotype values can be used for association analysis in higher polyploids. We associated continuous genotypes to three different traits and compared the results to the output of the genotype caller SuperMASSA. Linear, Bayesian and partial least squares regression were applied, to determine if the use of continuous genotypes is limited to a specific method. A disease, a flowering and a growth trait with h (2) of 0.51, 0.78 and 0.91 were associated with a hexaploid chrysanthemum genotypes. The data set consisted of 55,825 probes and 228 samples., Results: We were able to detect associating probes using continuous genotypes for multiple traits, using different regression methods. The identified probe sets were overlapping, but not identical between the methods. Baysian regression was the most restrictive method, resulting in ten probes for one trait and none for the others. Linear and partial least squares regression led to numerous associating probes. Association based on genotype classes resulted in similar values, but missed several significant probes. A simulation study was used to successfully validate the number of associating markers., Conclusions: Association of various phenotypic traits with continuous genotypes is successful with both uni- and multivariate regression methods. Genotype calling does not improve the association and shows no advantages in this study. Instead, use of continuous genotypes simplifies the analysis, saves computational time and results more potential markers.

The authors studied the effect of the temperature and chemical state of the chamber wall on process performance for atomic layer etching of SiO 2 using a steady-state Ar plasma, periodic injection of a defined number of C 4 F 8 molecules, and synchronized plasma-based Ar + ion bombardment. To evaluate these effects, the authors measured the quartz coupling window temperature. The plasma gas phase chemistry was characterized using optical emission spectroscopy. It was found that although the thickness of the polymer film deposited in each cycle is constant, the etching behavior changed, which is likely related to a change in the plasma gas phase chemistry. The authors found that the main gas phase changes occur after C 4 F 8 injection. The C 4 F 8 and the quartz window react and generate SiF and CO. The emission intensity changes with wall surface state and temperature. Therefore, changes in the plasma gas species generation can lead to a shift in etching performance during processing. During initial cycles, minimal etching is observed, while etching gradually increases with cycle number.

Unlabelled: Simulation programs based on the coalescent efficiently generate genetic data according to a given model of evolution. We present coala, an R package for calling coalescent simulators with a unified syntax. It can execute simulations with several programs, calculate additional summary statistics and combine multiple simulations to create biologically more realistic data., Availability and Implementation: The package is publicly available on CRAN and on https://github.com/statgenlmu/coala under the conditions of the MIT license., Contact: metzler@bio.lmu.de., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

How can antiparasite defence traits evolve even if they do not directly benefit their carriers? An example of such an indirect defence is rebellion of enslaved Temnothorax longispinosus ant workers against their social parasite Temnothorax americanus, a slavemaking ant. Ant slaves have been observed to kill their oppressors' offspring, a behaviour from which the sterile slaves cannot profit directly. Parasite brood killing could, however, reduce raiding pressure on related host colonies nearby. We analyse with extensive computer simulations for the Temnothorax slavemaker system under what conditions a hypothetical rebel allele could invade a host population, and in particular, how host-parasite dynamics and population structure influence the rebel allele's success. Exploring a wide range of model parameters, we only found a small number of parameter combinations for which kin selection or multilevel selection could allow a slave rebellion allele to spread in the host population. Furthermore, we did not detect any cases in which the reduction of raiding pressure in the close vicinity of the slavemaker nest would substantially contribute to the inclusive fitness of rebels. This suggests that slave rebellion is not costly and perhaps a side-effect of some other beneficial trait. In some of our simulations, however, even a costly rebellion allele could spread in the population. This was possible when host-parasite interactions led to a metapopulation dynamic with frequent local extinctions and recolonizations of demes by the offspring of few immigrants., (© 2016 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2016 European Society For Evolutionary Biology.)

To better understand the potential impacts of metal oxide nanoparticles (NPs) on Gram(+) Bacillus subtilis and Gram(-) Escherichia coli (K12) bacteria, eight different nanosized titanium dioxide (TiO2) suspensions with five different concentrations were used. Water quality parameters (pH, temperature, and ionic strength), light sources, and light intensities were also changed to achieve different environmental conditions. The photosensitive TiO2 NPs were found to be harmful to varying degrees under ambient conditions, with antibacterial activity increasing with primary particle sizes from 16 to 20 nm. The presence of light was a significant factor under most conditions tested, presumably due to its role in promoting generation of reactive oxygen species (ROS). However, bacterial growth inhibition was also observed under dark conditions and different water quality parameters, indicating that undetermined mechanisms additional to photocatalytic ROS production were responsible for toxicity. The results also indicated that nano-TiO2 particles in the absence and the presence of photoactivation induced lipid peroxidation and cellular respiration disruption.

Motivation: Coalescent-based simulation software for genomic sequences allows the efficient in silico generation of short- and medium-sized genetic sequences. However, the simulation of genome-size datasets as produced by next-generation sequencing is currently only possible using fairly crude approximations., Results: We present the sequential coalescent with recombination model (SCRM), a new method that efficiently and accurately approximates the coalescent with recombination, closing the gap between current approximations and the exact model. We present an efficient implementation and show that it can simulate genomic-scale datasets with an essentially correct linkage structure., (© The Author 2015. Published by Oxford University Press.)

The emergence of self-organizing behavior in ants has been modeled in various theoretical approaches in the past decades. One model explains experimental observations in which Argentine ants (Linepithema humile) selected the shorter of two alternative paths from their nest to a food source (shortest path experiments). This model serves as an important example for the emergence of collective behavior and self-organization in biological systems. In addition, it inspired the development of computer algorithms for optimization problems called ant colony optimization (ACO). In the model, a choice function describing how ants react to different pheromone concentrations is fundamental. However, the parameters of the choice function were not deduced experimentally but freely adapted so that the model fitted the observations of the shortest path experiments. Thus, important knowledge was lacking about crucial model assumptions. A recent study on the Argentine ant provided this information by measuring the response of the ants to varying pheromone concentrations. In said study, the above mentioned choice function was fitted to the experimental data and its parameters were deduced. In addition, a psychometric function was fitted to the data and its parameters deduced. Based on these findings, it is possible to test the shortest path model by applying realistic parameter values. Here we present the results of such tests using Monte Carlo simulations of shortest path experiments with Argentine ants. We compare the choice function and the psychometric function, both with parameter values deduced from the above-mentioned experiments. Our results show that by applying the psychometric function, the shortest path experiments can be explained satisfactorily by the model. The study represents the first example of how psychophysical theory can be used to understand and model collective foraging behavior of ants based on trail pheromones. These findings may be important for other models of pheromone guided ant behavior and might inspire improved ACO algorithms., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Borrelia bavariensis is a recently described agent of Lyme disease within the B. burgdorferi sensu lato species complex and exhibits a strong capacity for human pathogenicity. B. bavariensis strains are widely distributed in Eurasia spanning the distribution range of the tick vectors Ixodes persulcatus and I. ricinus. It has been suggested that B. bavariensis forms two populations, one of which arose through vector adaptation and geographic expansion. We have performed phylogenetic and population genetic analyses with next-generation sequencing data of 26 strains of B. bavariensis targeting the main linear chromosome and two plasmids (lp54, cp26). A very low number of single nucleotide polymorphisms (SNPs) was found in the European population and a deep branching pattern between European and Asian B. bavariensis was observed in all phylogenies. The results confirm the population structure of B. bavariensis and strongly support the hypothesis of clonal expansion of the European population of B. bavariensis. In addition, signals of positive selection identified in the populations further support the hypothesis that the European population of B. bavariensis likely underwent vector adaptation in its recent evolutionary history. Identified genes represent promising candidates for experimental vector adaptation studies. Thus, this species forms a very good model to study vector adaptation, which is known to play an important role in the geographic distribution of B. burgdorferi. Analysis of well known virulence determinants that are attributed to severity of clinical manifestation in B. burgdorferi s.s. revealed no variation within the European population of B. bavariensis, underlining the importance of including various Borrelia species into investigations that aim to understand the pathogenesis of Lyme disease agents., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Background: Toxoplasma gondii is a ubiquitous protozoan parasite capable of infecting all warm-blooded animals including livestock. In these animals, the parasite forms cysts in the tissues which may pose a risk to public health if infected meat is consumed undercooked or raw. The aim of this study was to determine the exposure of livestock to T. gondii in St. Kitts and Nevis., Methods: Sera and/or heart tissue and meat juice were collected from pigs (n = 124), sheep (n = 116) and goats (n = 66) at the St. Kitts Abattoir. Sera and meat juice were screened for reactive antibodies to T. gondii using an in-house ELISA. Heart tissue was screened for T. gondii DNA using quantitative PCR and positive samples were genotyped using RFLP., Results: Antibodies to T. gondii were detected in sera from 48% of pigs, 26% of sheep and 34% of goats tested. Antibodies were also detected in the meat juice from 55% of pig hearts, 22% of sheep hearts and 31% of goat hearts tested. There was a significant positive correlation between serology and meat juice results. T. gondii DNA was detected in heart tissue of 21% of pigs, 16% of sheep and 23% of goats tested. Preliminary PCR-RFLP analysis identified a predominance of the Type III genotype of T. gondii., Conclusions: These results suggest widespread environmental contamination with T. gondii oocysts and that livestock could be a potentially important source of T. gondii infection if their infected meat is consumed (or handled) undercooked.

The evolution of parasite virulence and host defences is affected by population structure. This effect has been confirmed in studies focusing on large spatial scales, whereas the importance of local structure is not well understood. Slavemaking ants are social parasites that exploit workers of another species to rear their offspring. Enslaved workers of the host species Temnothorax longispinosus have been found to exhibit an effective post-enslavement defence behaviour: enslaved workers were observed killing a large proportion of the parasites' offspring. As enslaved workers do not reproduce, they gain no direct fitness benefit from this 'rebellion' behaviour. However, there may be an indirect benefit: neighbouring host nests that are related to 'rebel' nests can benefit from a reduced raiding pressure, as a result of the reduction in parasite nest size due to the enslaved workers' killing behaviour. We use a simple mathematical model to examine whether the small-scale population structure of the host species could explain the evolution of this potentially altruistic defence trait against slavemaking ants. We find that this is the case if enslaved host workers are related to nearby host nests. In a population genetic study, we confirm that enslaved workers are, indeed, more closely related to host nests within the raiding range of their resident slavemaker nest, than to host nests outside the raiding range. This small-scale population structure seems to be a result of polydomy (e.g. the occupation of several nests in close proximity by a single colony) and could have enabled the evolution of 'rebellion' by kin selection., (© 2014 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2014 European Society For Evolutionary Biology.)

A strong demographic Allee effect in which the expected population growth rate is negative below a certain critical population size can cause high extinction probabilities in small introduced populations. But many species are repeatedly introduced to the same location and eventually one population may overcome the Allee effect by chance. With the help of stochastic models, we investigate how much genetic diversity such successful populations harbor on average and how this depends on offspring-number variation, an important source of stochastic variability in population size. We find that with increasing variability, the Allee effect increasingly promotes genetic diversity in successful populations. Successful Allee-effect populations with highly variable population dynamics escape rapidly from the region of small population sizes and do not linger around the critical population size. Therefore, they are exposed to relatively little genetic drift. It is also conceivable, however, that an Allee effect itself leads to an increase in offspring-number variation. In this case, successful populations with an Allee effect can exhibit less genetic diversity despite growing faster at small population sizes. Unlike in many classical population genetics models, the role of offspring-number variation for the population genetic consequences of the Allee effect cannot be accounted for by an effective-population-size correction. Thus, our results highlight the importance of detailed biological knowledge, in this case on the probability distribution of family sizes, when predicting the evolutionary potential of newly founded populations or when using genetic data to reconstruct their demographic history., (Copyright © 2014 by the Genetics Society of America.)

A phenomenon that strongly influences the demography of small introduced populations and thereby potentially their genetic diversity is the demographic Allee effect, a reduction in population growth rates at small population sizes. We take a stochastic modeling approach to investigate levels of genetic diversity in populations that successfully overcame either a strong Allee effect, in which populations smaller than a certain critical size are expected to decline, or a weak Allee effect, in which the population growth rate is reduced at small sizes but not negative. Our results indicate that compared to successful populations without an Allee effect, successful populations with a strong Allee effect tend to (1) derive from larger founder population sizes and thus have a higher initial amount of genetic variation, (2) spend fewer generations at small population sizes where genetic drift is particularly strong, and (3) spend more time around the critical population size and thus experience more genetic drift there. In the case of multiple introduction events, there is an additional increase in diversity because Allee-effect populations tend to derive from a larger number of introduction events than other populations. Altogether, a strong Allee effect can either increase or decrease genetic diversity, depending on the average founder population size. By contrast, a weak Allee effect tends to decrease genetic diversity across the entire range of founder population sizes. Finally, we show that it is possible in principle to infer critical population sizes from genetic data, although this would require information from many independently introduced populations., (Copyright © 2014 by the Genetics Society of America.)

Background: Obtaining an accurate sequence alignment is fundamental for consistently analyzing biological data. Although this problem may be efficiently solved when only two sequences are considered, the exact inference of the optimal alignment easily gets computationally intractable for the multiple sequence alignment case. To cope with the high computational expenses, approximate heuristic methods have been proposed that address the problem indirectly by progressively aligning the sequences in pairs according to their relatedness. These methods however are not flexible to change the alignment of an already aligned group of sequences in the view of new data, resulting thus in compromises on the quality of the deriving alignment. In this paper we present ReformAlign, a novel meta-alignment approach that may significantly improve on the quality of the deriving alignments from popular aligners. We call ReformAlign a meta-aligner as it requires an initial alignment, for which a variety of alignment programs can be used. The main idea behind ReformAlign is quite straightforward: at first, an existing alignment is used to construct a standard profile which summarizes the initial alignment and then all sequences are individually re-aligned against the formed profile. From each sequence-profile comparison, the alignment of each sequence against the profile is recorded and the final alignment is indirectly inferred by merging all the individual sub-alignments into a unified set. The employment of ReformAlign may often result in alignments which are significantly more accurate than the starting alignments., Results: We evaluated the effect of ReformAlign on the generated alignments from ten leading alignment methods using real data of variable size and sequence identity. The experimental results suggest that the proposed meta-aligner approach may often lead to statistically significant more accurate alignments. Furthermore, we show that ReformAlign results in more substantial improvement in cases where the starting alignment is of relatively inferior quality or when the input sequences are harder to align., Conclusions: The proposed profile-based meta-alignment approach seems to be a promising and computationally efficient method that can be combined with practically all popular alignment methods and may lead to significant improvements in the generated alignments.

Beneficial mutations can co-occur when population structure slows down adaptation. Here, we consider the process of adaptation in asexual populations distributed over several locations ("islands"). New beneficial mutations arise at constant rate ub, and each mutation has the same selective advantage s>0. We assume that populations evolve within islands according to the successional mutations regime of Desai and Fisher (2007), that is, the time to local fixation of a mutation is short compared to the expected waiting time until the next mutation occurs. To study the rate of adaptation, we introduce an approximate model, the successional mutations (SM) model, which can be simulated efficiently and yields accurate results for a wide range of parameters. In the SM model, mutations fix instantly within islands, and migrants can take over the destination island if they are fitter than the residents. For the special case of a population distributed equally across two islands with population size N, we approximate the model further for small and large migration rates in comparison to the mutation rate. These approximations lead to explicit formulas for the rate of adaptation which fit the original model for a large range of parameter values. For the d island case we provide some heuristics on how to extend the explicit formulas and check these with computer simulations. We conclude that the SM model is a good approximation of the adaptation process in a structured population, at least if mutation or migration is limited., (Copyright © 2013 Elsevier Inc. All rights reserved.)

With the advent of next-generation sequencing technologies, large data sets of several thousand loci from multiple conspecific individuals are available. Such data sets should make it possible to obtain accurate estimates of population genetic parameters, even for complex models of population history. In the analyses of large data sets, it is difficult to consider finite-sites mutation models (FSMs). Here, we use extensive simulations to demonstrate that the inclusion of FSMs is necessary to avoid severe biases in the estimation of the population mutation rate θ, population divergence times, and migration rates. We present a new version of Jaatha, an efficient composite-likelihood method for estimating demographic parameters from population genetic data and evaluate the usefulness of Jaatha in two biological examples. For the first application, we infer the speciation process of two wild tomato species, Solanum chilense and Solanum peruvianum. In our second application example, we demonstrate that Jaatha is readily applicable to NGS data by analyzing genome-wide data from two southern European populations of Arabidopsis thaliana. Jaatha is now freely available as an R package from the Comprehensive R Archive Network (CRAN).

The microbial communities inhabiting the mammalian intestinal tract play an important role in diverse aspects of host biology. However, little is known regarding the forces shaping variation in these communities and their influence on host fitness. To shed light on the contributions of host genetics, transmission and geography to diversity in microbial communities between individuals, we performed a survey of intestinal microbial communities in a panel of 121 house mice derived from eight locations across Western Europe using pyrosequencing of the bacterial 16S rRNA gene. The host factors studied included population structure estimated by microsatellite loci and mitochondrial DNA, genetic distance and geography. To determine whether host tissue (mucosa)-associated communities display properties distinct from those of the lumen, both the caecal mucosa and contents were examined. We identified Bacteroides, Robinsoniella and Helicobacter as the most abundant genera in both the caecal content and mucosa-associated communities of wild house mice. Overall, we found geography to be the most significant factor explaining patterns of diversity in the intestinal microbiota, with a comparatively weaker influence of host population structure and genetic distance. Furthermore, the influence of host genetic distance was limited to the mucosa communities, consistent with this environment being more intimately coupled to the host., (© 2013 Blackwell Publishing Ltd.)

Species introductions to new habitats can cause a decline in the population size of competing native species and consequently also in their genetic diversity. We are interested in why these adverse effects are weak in some cases whereas in others the native species declines to the point of extinction. While the introduction rate and the growth rate of the introduced species in the new environment clearly have a positive relationship with invasion success and impact, the influence of competition is poorly understood. Here, we investigate how the intensity of interspecific competition influences the persistence time of a native species in the face of repeated and ongoing introductions of the nonnative species. We analyze two stochastic models: a model for the population dynamics of both species and a model that additionally includes the population genetics of the native species at a locus involved in its adaptation to a changing environment. Counterintuitively, both models predict that the persistence time of the native species is lowest for an intermediate intensity of competition. This phenomenon results from the opposing effects of competition at different stages of the invasion process: With increasing competition intensity more introduction events are needed until a new species can establish, but increasing competition also speeds up the exclusion of the native species by an established nonnative competitor. By comparing the ecological and the eco-genetic model, we detect and quantify a synergistic feedback between ecological and genetic effects., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Nitrovasodilators and agonists, via an increase in intracellular cyclic nucleotide levels, can induce smooth muscle relaxation without a concomitant decrease in phosphorylation of the regulatory light chains (RLC) of myosin. However, since cyclic nucleotide-induced relaxation is associated with a decrease in intracellular [Ca(2+)], and hence, a decreased activity of MLCK, we tested the hypothesis that the site responsible for the elevated RLC phosphorylation is not Ser19. Smooth muscle strips from gastric fundus were isometrically contracted with ET-1 which induced an increase in monophosphorylation from 9 ± 1 % under resting conditions (PSS) to 36 ± 1 % determined with 2D-PAGE. Electric field stimulation induced a rapid, largely NO-mediated relaxation with a half time of 8 s, which was associated with an initial decline in RLC phosphorylation to 18 % within 2 s and a rebound to 34 % after 30 s whereas relaxation was sustained. In contrast, phosphorylation of RLC at Ser19 probed with phosphospecific antibodies declined in parallel with force. LC/MS and western blot analysis with phosphospecific antibodies against monophosphorylated Thr18 indicate that Thr18 is significantly monophosphorylated during sustained relaxation. We therefore suggest that (i) monophosphorylation of Thr18 rather than Ser19 is responsible for the phosphorylation rebound during sustained EFS-induced relaxation of mouse gastric fundus, and (ii) that relaxation can be ascribed to dephosphorylation of Ser19, the site considered to be responsible for regulation of smooth muscle tone.

We study the trajectory of an allele that affects a polygenic trait selected toward a phenotypic optimum. Furthermore, conditioning on this trajectory we analyze the effect of the selected mutation on linked neutral variation. We examine the well-characterized two-locus two-allele model but we also provide results for diallelic models with up to eight loci. First, when the optimum phenotype is that of the double heterozygote in a two-locus model, and there is no dominance or epistasis of effects on the trait, the trajectories of selected mutations rarely reach fixation; instead, a polymorphic equilibrium at both loci is approached. Whether a polymorphic equilibrium is reached (rather than fixation at both loci) depends on the intensity of selection and the relative distances to the optimum of the homozygotes at each locus. Furthermore, if both loci have similar effects on the trait, fixation of an allele at a given locus is less likely when it starts at low frequency and the other locus is polymorphic (with alleles at intermediate frequencies). Weaker selection increases the probability of fixation of the studied allele, as the polymorphic equilibrium is less stable in this case. When we do not require the double heterozygote to be at the optimum we find that the polymorphic equilibrium is more difficult to reach, and fixation becomes more likely. Second, increasing the number of loci decreases the probability of fixation, because adaptation to the optimum is possible by various combinations of alleles. Summaries of the genealogy (height, total length, and imbalance) and of sequence polymorphism (number of polymorphisms, frequency spectrum, and haplotype structure) next to a selected locus depend on the frequency that the selected mutation approaches at equilibrium. We conclude that multilocus response to selection may in some cases prevent selective sweeps from being completed, as described in previous studies, but that conditions causing this to happen strongly depend on the genetic architecture of the trait, and that fixation of selected mutations is likely in many instances.

The family Araceae (3790 species, 117 genera) has one of the oldest fossil records among angiosperms. Ecologically, members of this family range from free-floating aquatics (Pistia and Lemna) to tropical epiphytes. Here, we infer some of the macroevolutionary processes that have led to the worldwide range of this family and test how the inclusion of fossil (formerly occupied) geographical ranges affects biogeographical reconstructions. Using a complete genus-level phylogeny from plastid sequences and outgroups representing the 13 other Alismatales families, we estimate divergence times by applying different clock models and reconstruct range shifts under different models of past continental connectivity, with or without the incorporation of fossil locations. Araceae began to diversify in the Early Cretaceous (when the breakup of Pangea was in its final stages), and all eight subfamilies existed before the K/T boundary. Early lineages persist in Laurasia, with several relatively recent entries into Africa, South America, South-East Asia and Australia. Water-associated habitats appear to be ancestral in the family, and DNA substitution rates are especially high in free-floating Araceae. Past distributions inferred when fossils are included differ in nontrivial ways from those without fossils. Our complete genus-level time-scale for the Araceae may prove to be useful for ecological and physiological studies., (© 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.)

Motivation: Today many non-coding RNAs are known to play an active role in various important biological processes. Since RNA's functionality is correlated with specific structural motifs that are often conserved in phylogenetically related molecules, computational prediction of RNA structure should ideally be based on a set of homologous primary structures. But many available RNA secondary structure prediction programs that use sequence alignments do not consider pseudoknots or their estimations consist on a single structure without information on uncertainty., Results: In this article we present a method that takes advantage of the evolutionary history of a group of aligned RNA sequences for sampling consensus secondary structures, including pseudoknots, according to their approximate posterior probability. We investigate the benefit of using evolutionary history and demonstrate the competitiveness of our method compared with similar methods based on RNase P RNA sequences and simulated data., Availability: PhyloQFold, a C + + implementation of our method, is freely available from http://evol.bio.lmu.de/&#95;statgen/software/phyloqfold/.

Norovirus has become an important cause for infectious gastroenteritis. Particularly genotype II.4 (GII.4) has been shown to spread rapidly and causes worldwide pandemics. Emerging new strains evade population immunity and lead to high norovirus prevalence. Chronic infections have been described recently and will become more prevalent with increasing numbers of immunocompromized patients. Here, we studied norovirus evolution in three chronically infected patients, two genotypes II.4 and one II.7. A 719 and 757 nt region was analyzed for GII.4 and GII.7, respectively. This covers the entire hypervariable P2 domain of the VP1 capsid gene. Genetic variability at given and between different time points was assessed. Evolutionary adaptation was analyzed by Bayesian sampling of genealogies. This analysis clearly demonstrated positive selection rather than incidental drift for all three strains. The GII.7 and one GII.4 strain accumulated on average 5-9 mutations per 100 days, most of them non-synonymous. This is a much higher evolutionary rate than observed for noroviruses on a global level. Our data demonstrate that norovirus quasispecies are positively selected in chronically infected patients. The numbers of intraindividual amino acid mutations acquired in the capsid gene are similar to those separating consecutive GII.4 epidemic strains. Evolution in a given, chronically infected individual may thus generate novel genotypes at risk to expedite global evolution particularly for slowly evolving genotypes, as GII.7., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Argonaute proteins are the core components of the microRNP/RISC. The biogenesis and function of microRNAs and endo- and exo- siRNAs are regulated by Ago2, an Argonaute protein with RNA binding and nuclease activities. Currently, there are no in vitro assays suitable for large-scale screening of microRNP/RISC loading modulators. We describe a novel in vitro assay that is based on fluorescence polarization of TAMRA-labeled RNAs loaded to human Ago2. Using this assay, we identified potent small-molecule inhibitors of RISC loading, including aurintricarboxylic acid (IC(50) = 0.47 μM), suramin (IC(50) = 0.69 μM), and oxidopamine HCL (IC(50) = 1.61 μM). Small molecules identified by this biochemical screening assay also inhibited siRNA loading to endogenous Ago2 in cultured cells.

Background: Molecular markers displaying bimodal expression distribution can reveal distinct disease subsets and may serve as prognostic or predictive markers or represent therapeutic targets. Oestrogen (ER) and human epidermal growth factor receptor 2 (HER2) receptors are strongly bimodally expressed genes in breast cancer., Material and Methods: We applied a novel method to identify bimodally expressed genes in 394 triple negative breast cancers (TNBC). We identified 133 bimodally expressed probe sets (128 unique genes), 69 of these correlated to previously reported metagenes that define molecular subtypes within TNBC including basal-like, molecular-apocrine, claudin-low and immune cell rich subgroups but 64 probe sets showed no correlation with these features., Results: The single most prominent functional group among these uncorrelated genes was the X chromosome derived Cancer/Testis Antigens (CT-X) including melanoma antigen family A (MAGE-A) and Cancer/Testis Antigens (CTAG). High expression of CT-X genes correlated with worse survival in multivariate analysis (HR 2.02, 95% CI 1.27-3.20; P=0.003). The only other significant variable was lymph node status. The poor prognosis of patients with high MAGE-A expression was ameliorated by the concomitant high expression of immune cell metagenes (HR 1.87, 95% CI 0.96-3.64; P=0.060), whereas the same immune metagene had lesser prognostic value in TNBC with low MAGE-A expression., Conclusions: MAGE-A antigen defines a very aggressive subgroup of TNBC; particularly in the absence of immune infiltration in the tumour microenvironment. These observations suggest a therapeutic hypothesis; TNBC with MAGE-A expression may benefit the most from further augmentation of the immune response. Novel immune stimulatory drugs such as (anti-cytotoxic T-lymphocyte antigen-4 CTLA-4) directed therapies provide a realistic opportunity to directly test this hypothesis in the clinic., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The genetic structure of social insect populations is influenced by their social organization and dispersal modes. The ant Hypoponera opacior shows diverse reproductive behaviours with regular cycles of outbreeding via winged sexuals and inbreeding via within-nest mating wingless sexuals that reproduce by budding. This unusual life cycle should be reflected in the genetic population structure, and we studied this on different scales using microsatellites. On a macrogeographic scale, populations were considerably structured and migration rates within the Chiricahuas were higher than those in between mountain ranges. On a local scale, our analyses revealed population viscosity through dependent colony foundation and a high genetic diversity with a multicolonial structure. The latter was also evident from recognition trials revealing consistent aggression between non-nestmates. Within-nest matings led to high inbreeding coefficients. Finally, the observed seasonal changes in relatedness can be explained by variation in queen number and differential dispersal of the two reproductive morphs., (© 2011 The Authors. Journal of Evolutionary Biology © 2011 European Society For Evolutionary Biology.)

Introduction: Current prognostic gene expression profiles for breast cancer mainly reflect proliferation status and are most useful in ER-positive cancers. Triple negative breast cancers (TNBC) are clinically heterogeneous and prognostic markers and biology-based therapies are needed to better treat this disease., Methods: We assembled Affymetrix gene expression data for 579 TNBC and performed unsupervised analysis to define metagenes that distinguish molecular subsets within TNBC. We used n = 394 cases for discovery and n = 185 cases for validation. Sixteen metagenes emerged that identified basal-like, apocrine and claudin-low molecular subtypes, or reflected various non-neoplastic cell populations, including immune cells, blood, adipocytes, stroma, angiogenesis and inflammation within the cancer. The expressions of these metagenes were correlated with survival and multivariate analysis was performed, including routine clinical and pathological variables., Results: Seventy-three percent of TNBC displayed basal-like molecular subtype that correlated with high histological grade and younger age. Survival of basal-like TNBC was not different from non basal-like TNBC. High expression of immune cell metagenes was associated with good and high expression of inflammation and angiogenesis-related metagenes were associated with poor prognosis. A ratio of high B-cell and low IL-8 metagenes identified 32% of TNBC with good prognosis (hazard ratio (HR) 0.37, 95% CI 0.22 to 0.61; P < 0.001) and was the only significant predictor in multivariate analysis including routine clinicopathological variables., Conclusions: We describe a ratio of high B-cell presence and low IL-8 activity as a powerful new prognostic marker for TNBC. Inhibition of the IL-8 pathway also represents an attractive novel therapeutic target for this disease.