Mutation-Simulator: fine-grained simulation of random mutations in any genome.

SUMMARY: Mutation-Simulator allows the introduction of various types of sequence alterations in reference sequences, with reasonable compute-time even for large eukaryotic genomes. Its intuitive system for fine-grained control over mutation rates along the sequence enables the mimicking of natural mutation patterns. Using standard file formats for input and output data, it can easily be integrated into any development and benchmarking workflow for high-throughput sequencing applications. AVAILABILITY AND IMPLEMENTATION: Mutation-Simulator is written in Python 3 and the source code, documentation, help and use cases are available on the Github page at https://github.com/mkpython3/Mutation-Simulator. It is free for use under the GPL 3 license.

Radiative Energy Budgets in a Microbial Mat Under Different Irradiance and Tidal Conditions.

Irradiance and temperature variations during tidal cycles modulate microphytobenthic primary production potentially by changing the radiative energy balance of photosynthetic mats between immersion and emersion and thus sediment daily net metabolism. To test the effect of tidal stages on the radiative energy budget, we used microsensor measurements of oxygen, temperature, and scalar irradiance to estimate the radiative energy budget in a coastal photosynthetic microbial mat during immersion (constant water column of 2 cm) and emersion under increasing irradiance. Total absorbed light energy was higher in immersion than emersion, due to a lower reflectance of the microbial mat, while most (> 97%) of the absorbed light energy was dissipated as heat irrespective of tidal conditions. During immersion, the upward heat flux was higher than the downward one, whereas the opposite occurred during emersion. At highest photon irradiance (800 µmol photon m-2 s-1), the sediment temperature increased ~ 2.5 °C after changing the conditions from immersion to emersion. The radiative energy balance showed that less than 1% of the incident light energy (PAR, 400-700 nm) was conserved by photosynthesis under both tidal conditions. At low to moderate incident irradiances, the light use efficiency was similar during the tidal stages. In contrast, we found an ~ 30% reduction in the light use efficiency during emersion as compared to immersion under the highest irradiance likely due to the rapid warming of the sediment during emersion and increased non-photochemical quenching. These changes in the photosynthetic efficiency and radiative energy budget could affect both primary producers and temperature-dependent bacterial activity and consequently daily net metabolism rates having important ecological consequences.

Nitric oxide production by polymorphonuclear leucocytes in infected cystic fibrosis sputum consumes oxygen.

Chronic Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) patients is characterized by persisting mucoid biofilms in hypoxic endobronchial mucus. These biofilms are surrounded by numerous polymorphonuclear leucocytes (PMNs), which consume a major part of present molecular oxygen (O(2)) due to production of superoxide (O(2)(-)). In this study, we show that the PMNs also consume O(2) for production of nitric oxide (NO) by the nitric oxide synthases (NOS) in the infected endobronchial mucus. Fresh expectorated sputum samples (n = 28) from chronically infected CF patients (n = 22) were analysed by quantifying and visualizing the NO production. NO production was detected by optode measurements combined with fluorescence microscopy, flow cytometry and spectrophotometry. Inhibition of nitric oxide synthases (NOS) with N(G) -monomethyl-L-arginine (L-NMMA) resulted in reduced O(2) consumption (P < 0·0008, n = 8) and a lower fraction of cells with fluorescence from the NO-indicator 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) (P < 0·002, n = 8). PMNs stained with DAF-FM and the superoxide indicator hydroethidine (HE) and host cells with inducible NOS (iNOS) were identified in the sputum. In addition, the production of the stable end-products of NO in CF sputum was correlated with the concentration of PMNs; NO(3)(-) (P < 0·04, r = 0·66, n = 10) and NO(2)(-) (P< 0·006, r = 0·78, n = 11). The present study suggests that besides consumption of O(2) for production of reactive oxygen species, the PMNs in CF sputum also consume O(2) for production of NO.

An optode sensor array for long-term in situ oxygen measurements in soil and sediment.

Long-term measurements of molecular oxygen (O) dynamics in wetlands are highly relevant for understanding the effects of water level changes on net greenhouse gas budgets in these ecosystems. However, such measurements have been limited due to a lack of suitable measuring equipment. We constructed an O optode sensor array for long-term in situ measurements in soil and sediment. The new device consists of a 1.3-m-long, cylindrical, spear-shaped rod equipped with 10 sensor spots along the shaft. Each spot contains a thermocouple fixed with a robust fiberoptic O optode made by immobilizing a layer of Pt(II) meso-tetra(pentafluorophenyl)porphine in polystyrene at the end of a 2-mm polymethyl methacrylate plastic fiber. Temperature and O optode readings are collected continuously by a data logger and a multichannel fiberoptic O meter. The construction and measuring characteristics of the sensor array system are presented along with a novel approach for temperature compensation of O optodes. During in situ application over several months in a peat bog, we used the new device to document pronounced variations in O distribution after marked shifts in water level. The measurements showed anoxic conditions below the water level but also diel variations in O concentrations in the upper layer presumably due to rhizospheric oxidation by the main vegetation The new field instrument thus enables new and more detailed insights to the in situ O dynamics in wetlands.

Functional interaction of an axin homolog, conductin, with beta-catenin, APC, and GSK3beta.

Control of stability of beta-catenin is central in the wnt signaling pathway. Here, the protein conductin was found to form a complex with both beta-catenin and the tumor suppressor gene product adenomatous polyposis coli (APC). Conductin induced beta-catenin degradation, whereas mutants of conductin that were deficient in complex formation stabilized beta-catenin. Fragments of APC that contained a conductin-binding domain also blocked beta-catenin degradation. Thus, conductin is a component of the multiprotein complex that directs beta-catenin to degradation and is located downstream of APC. In Xenopus embryos, conductin interfered with wnt-induced axis formation.

Measuring light scattering and absorption in corals with Inverse Spectroscopic Optical Coherence Tomography (ISOCT): a new tool for non-invasive monitoring.

The success of reef-building corals for >200 million years has been dependent on the mutualistic interaction between the coral host and its photosynthetic endosymbiont dinoflagellates (family Symbiodiniaceae) that supply the coral host with nutrients and energy for growth and calcification. While multiple light scattering in coral tissue and skeleton significantly enhance the light microenvironment for Symbiodiniaceae, the mechanisms of light propagation in tissue and skeleton remain largely unknown due to a lack of technologies to measure the intrinsic optical properties of both compartments in live corals. Here we introduce ISOCT (inverse spectroscopic optical coherence tomography), a non-invasive approach to measure optical properties and three-dimensional morphology of living corals at micron- and nano-length scales, respectively, which are involved in the control of light propagation. ISOCT enables measurements of optical properties in the visible range and thus allows for characterization of the density of light harvesting pigments in coral. We used ISOCT to characterize the optical scattering coefficient (µs) of the coral skeleton and chlorophyll a concentration of live coral tissue. ISOCT further characterized the overall micro- and nano-morphology of live tissue by measuring differences in the sub-micron spatial mass density distribution (D) that vary throughout the tissue and skeleton and give rise to light scattering, and this enabled estimates of the spatial directionality of light scattering, i.e., the anisotropy coefficient, g. Thus, ISOCT enables imaging of coral nanoscale structures and allows for quantifying light scattering and pigment absorption in live corals. ISOCT could thus be developed into an important tool for rapid, non-invasive monitoring of coral health, growth and photophysiology with unprecedented spatial resolution.

Author Correction: Measuring light scattering and absorption in corals with Inverse Spectroscopic Optical Coherence Tomography (ISOCT): a new tool for non-invasive monitoring.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genomics, environmental genomics and the issue of microbial species.

A microbial species concept is crucial for interpreting the variation detected by genomics and environmental genomics among cultivated microorganisms and within natural microbial populations. Comparative genomic analyses of prokaryotic species as they are presently described and named have led to the provocative idea that prokaryotes may not form species as we think about them for plants and animals. There are good reasons to doubt whether presently recognized prokaryotic species are truly species. To achieve a better understanding of microbial species, we believe it is necessary to (i) re-evaluate traditional approaches in light of evolutionary and ecological theory, (ii) consider that different microbial species may have evolved in different ways and (iii) integrate genomic, metagenomic and genome-wide expression approaches with ecological and evolutionary theory. Here, we outline how we are using genomic methods to (i) identify ecologically distinct populations (ecotypes) predicted by theory to be species-like fundamental units of microbial communities, and (ii) test their species-like character through in situ distribution and gene expression studies. By comparing metagenomic sequences obtained from well-studied hot spring cyanobacterial mats with genomic sequences of two cultivated cyanobacterial ecotypes, closely related to predominant native populations, we can conduct in situ population genetics studies that identify putative ecotypes and functional genes that determine the ecotypes' ecological distinctness. If individuals within microbial communities are found to be grouped into ecologically distinct, species-like populations, knowing about such populations should guide us to a better understanding of how genomic variation is linked to community function.

Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6.

BACKGROUND: Dickkopf-1 (Dkk-1) is a head inducer secreted from the vertebrate head organizer and induces anterior development by antagonizing Wnt signaling. Although several families of secreted antagonists have been shown to inhibit Wnt signal transduction by binding to Wnt, the molecular mechanism of Dkk-1 action is unknown. The Wnt family of secreted growth factors initiates signaling via the Frizzled (Fz) receptor and its candidate coreceptor, LDL receptor-related protein 6 (LRP6), presumably through Fz-LRP6 complex formation induced by Wnt. The significance of the Fz-LRP6 complex in signal transduction remains to be established. RESULTS: We report that Dkk-1 is a high-affinity ligand for LRP6 and inhibits Wnt signaling by preventing Fz-LRP6 complex formation induced by Wnt. Dkk-1 binds neither Wnt nor Fz, nor does it affect Wnt-Fz interaction. Dkk-1 function in head induction and Wnt signaling inhibition strictly correlates with its ability to bind LRP6 and to disrupt the Fz-LRP6 association. LRP6 function and Dkk-1 inhibition appear to be specific for the Wnt/Fz beta-catenin pathway. CONCLUSIONS: Our results demonstrate that Dkk-1 is an LRP6 ligand and inhibits Wnt signaling by blocking Wnt-induced Fz-LRP6 complex formation. Our findings thus reveal a novel mechanism for Wnt signal modulation. LRP6 is a Wnt coreceptor that appears to specify Wnt/Fz signaling to the beta-catenin pathway, and Dkk-1, distinct from Wnt binding antagonists, may be a specific inhibitor for Wnt/beta-catenin signaling. Our findings suggest that Wnt-Fz-LRP6 complex formation, but not Wnt-Fz interaction, triggers Wnt/beta-catenin signaling.

The Wnt/Ca2+ pathway: a new vertebrate Wnt signaling pathway takes shape.

Members of the vertebrate Wnt family have been subdivided into two functional classes according to their biological activities. Some Wnts signal through the canonical Wnt-1/wingless pathway by stabilizing cytoplasmic beta-catenin. By contrast other Wnts stimulate intracellular Ca2+ release and activate two kinases, CamKII and PKC, in a G-protein-dependent manner. Moreover, putative Wnt receptors belonging to the Frizzled gene family have been identified that preferentially couple to the two prospective pathways in the absence of ectopic Wnt ligand and that might account for the signaling specificity of the Wnt pathways. As Ca2+ release was the first described feature of the noncanonical pathway, and as Ca2+ probably plays a key role in the activation of CamKII and PKC, we have named this Wnt pathway the Wnt/Ca2+ pathway.

The Wnt/Wg signal transducer beta-catenin controls fibronectin expression.

beta-Catenin stabilizes the cadherin cell adhesion complex but, as a component of the Wnt/Wg signaling pathway, also controls gene expression by forming a heterodimer with a transcription factor of the LEF-TCF family. We demonstrate that the substrate adhesion molecule fibronectin is a direct target of Wnt/Wg signaling. Nuclear depletion of beta-catenin following cadherin transfection in Xenopus fibroblasts resulted in downregulation of fibronectin expression which was restored by activating the Wnt/Wg signaling cascade via LiCl treatment or transfection of either Xwnt-8 or beta-catenin. We isolated the Xenopus fibronectin gene (FN) promoter and found four putative LEF-TCF binding sites. By comparing the activities of different fibronectin gene reporter constructs in fibroblasts and cadherin transfectants, the LEF-TCF site at position -368 was identified as a Wnt/Wg response element. LEF-1-related proteins were found in nuclei of the fibroblasts but were absent in a kidney epithelial cell line. Consistent with the lack of these transcription factors, the FN promoter was silent in the epithelial cells but was activated upon transfection of LEF-1. Wild-type Xenopus Tcf-3 (XTcf-3) was unable to activate FN promoter reporter constructs, while a mutant lacking the groucho binding region behaved like LEF-1. In contrast to XTcf-3, LEF-1 does not interact with groucho proteins, which turn TCFs into activators or repressors (J. Roose, M. Molenaar, J. Hurenkamp, J. Peterson, H. Brantjes, P. Moerer, M. van de Wetering, O. Destreé, and H. Clevers, Nature 395:608-612, 1998). Together these data provide evidence that expressing LEF-1 enables fibroblasts, in contrast to epithelial cells, to respond to the Wnt/Wg signal via beta-catenin in stimulating fibronectin gene transcription. Our findings further promote the idea that due to its dual function, beta-catenin regulates the balance between cell-cell and cell-substrate adhesion.

Cadherin transfection of Xenopus XTC cells downregulates expression of substrate adhesion molecules.

Cadherins are discussed not in terms of their adhesive function but rather as morphoregulatory proteins. Changes in gene expression following cadherin transfection of cells in culture or by overexpression in embryos have, until now, not been reported. We established a protocol for stable transfection of Xenopus XTC cells and generated cells bearing high levels of membrane-integrated mouse uvomorulin (E-cadherin) or Xenopus XB-cadherin. These cell lines showed drastically impaired substrate adhesion on fibronectin and laminin. In immunoblot and radioimmunoprecipitation experiments, we found that fibronectin and alpha 3/beta 1 integrin are downregulated. The reduced amounts of proteins result from a decrease of the respective mRNAs as proven by RNase protection assays. Coprecipitations revealed that transfected cadherin molecules are complexed with alpha-catenin and beta-catenin at plasma membranes. However, the alpha-catenin present in the XB-cadherin complex differs immunologically from that found in the uvomorulin complex. When a truncated form of XB-cadherin lacking 38 of the most C-terminal amino acids was expressed in XTC cells, complex formation with endogenous catenins was abolished. In these transfectants, substrate adhesion was not affected. These results prove that complex formation of transfected cadherins in XTC cells with endogenous beta-catenin correlates with altered synthesis of certain substrate adhesion molecules.

In situ methods for assessment of microorganisms and their activities.

Recent technical developments in the field of molecular biology and microsensors are beginning to enable microbiologists to study the abundance, localization and activity of microorganisms in situ. The various new methods on their own bear high potential but it is the combination of studies on structure and function of microbial communities that will yield the most detailed insights in the way microorganisms operate in nature.

XB/U-cadherin mRNA contains cytoplasmic polyadenylation elements and is polyadenylated during oocyte maturation in Xenopus laevis.

Cytoplasmic polyadenylation elements (CPE) are distinct sequence motifs in the 3'-untranslated region of mRNAs. They control translation of these RNAs by cytoplasmic polyadenylation. We show that the mRNA of the cell adhesion molecule XB/U-cadherin contains two CPE motifs. With oocyte maturation this mRNA becomes polyadenylated and increasingly recruited into the polysomal fraction. Our results give evidence that CPEs of the XB/U-cadherin mRNA are responsible for the XB/U-cadherin protein increase during oocyte maturation.

Premature stop codons inactivate the RT6 genes of the human and chimpanzee species.

RT6 is a glycosyl phosphatidylinositol-anchored cell membrane protein, whose expression is restricted to peripheral T cells and intraepithelial lymphocytes. It has attracted interest as a T cell differentiation marker and activation antigen in rats. The only known protein to which RT6 shows significant homology is a recently cloned mono(ADP-ribosyl)transferase of rabbit skeletal muscle which is distantly related also to certain bacterial toxins. Intriguingly, whereas the rat carries a single copy RT6 gene with two known highly divergent alleles, the mouse carries two closely linked, functional RT6 genes that show approximately 85% sequence identity. We have now cloned and sequenced the homologues of the RT6 genes from humans of distinct ethnic backgrounds and of the chimpanzee. Surprisingly, in each case, three premature in-frame stop codons preclude expression of the single copy RT6 gene as a cell surface protein. Otherwise, the RT6 genes of human and chimpanzee exhibit high structural conservation to their rodent counterparts. RNA expression analyses indicate that the RT6 gene is not transcriptionally active in human T cells or any other human tissue analyzed so far. To our knowledge, RT6 represents the first mammalian membrane protein identified that has been lost universally in the human and chimpanzee species due to gene inactivation.

Dominant negative expression of a cytoplasmically deleted mutant of XB/U-cadherin disturbs mesoderm migration during gastrulation in Xenopus laevis.

XB/U-cadherin is a maternal Xenopus cadherin which mediates interblastomere adhesion in early embryogenesis. In order to explore its role in gastrulation, we expressed a cytoplasmic deletion mutant of XB/U-cadherin (XB delta c38) under the control of the CMV promoter in Xenopus embryos. This truncated XB-cadherin fails to form complexes with catenins and does not mediate cell-cell aggregation as shown by transfection of mouse Ltk- cells. Injections of the deletion for XB/U-cadherin into the dorsal-marginal region of four cell stage embryos resulted in a dominant negative expression of the cadherin mutant after MBT. Two different phenotypes were observed in a dose dependent manner: high doses (125-250 pg DNA) led to severe distortions of the gastrulation movement. Involution of the mesoderm was impaired, posterior mesoderm migrated laterally around the blastopore and formed two bands of axial tissue. Low doses (up to 50 pg DNA) resulted in embryos of a posteriorized phenotype with disorganized neural structures. Both phenotypes could be rescued by coinjection of cDNA constructs containing wild-type XB/U-cadherin. Injections of constructs encoding a XB/U-cadherin protein truncated both in its extracellular and cytoplasmic domains yielded normal phenotypes. These results suggest that a proper function of XB/U-cadherin is essential for mesoderm movements during gastrulation.

The HMG-box transcription factor XTcf-4 demarcates the forebrain-midbrain boundary.

A small subfamily of HMG-box transcription factors, the LEF/TCF group, serves as nuclear transducer of the Wnt-1/Wg signaling cascade. Upon Wnt-1/Wg signaling their members interact with beta-catenin and regulate the expression of Xenopus target genes siamois, twin, nodal related-3 or fibronectin. We have isolated a new HMG-box transcription factor in Xenopus that will be addressed XTcf-4 based on its homology to human and murine Tcf-4. Unlike XTcf-3, which is a maternal gene, and XLef-1 that is expressed after mid blastula transition (Molenaar et al., 1998. Mech. Dev. 75, 151-154), XTcf-4 expression starts at late neurula stage and is restricted to the anterior most midbrain demarcating the forebrain-midbrain boundary. The expression partially overlaps with a broad set of Xenopus Wnt family members in distinct patterns. XTcf-4 transcripts were also found partially co-localized with those of Xaxin, an intracellular antagonist of Wnt-1/Wg signaling.

Expression of Xenopus homologs of the beta-catenin binding protein pontin52.

Identification of pontin52 as an interaction partner of the Wnt/Wg signal transducer beta-catenin implicated a role for this protein in Wnt signaling. Here we describe the isolation of two Xenopus homologs of pontin52, Xpontin and Xreptin, and report the first expression pattern of vertebrate pontin52 homologs. Whole-mount in situ hybridization studies reveal a strong expression of Xpontin in neural crest cells and in later stages in different gastrointestinal organs. Xreptin is also expressed in neural crest cells, in particular in a subpopulation that give raise to the adrenal medulla.

Antagonistic regulation of convergent extension movements in Xenopus by Wnt/beta-catenin and Wnt/Ca2+ signaling.

Convergent extension movements are the main driving force of Xenopus gastrulation. A fine-tuned regulation of cadherin-mediated cell-cell adhesion is thought to be required for this process. Members of the Wnt family of extracellular glycoproteins have been shown to modulate cadherin-mediated cell-cell adhesion, convergent extension movements, and cell differentiation. Here we show that endogenous Wnt/beta-catenin signaling activity is essential for convergent extension movements due to its effect on gene expression rather than on cadherins. Our data also suggest that XLEF-1 rather than XTCF-3 is required for convergent extension movements and that XLEF-1 functions in this context in the Wnt/beta-catenin pathway to regulate Xnr-3. In contrast, activation of the Wnt/Ca2+ pathway blocks convergent extension movements, with potential regulation of the Wnt/beta-catenin pathway at two different levels. PKC, activated by the Wnt/Ca2+ pathway, blocks the Wnt/beta-catenin pathway upstream of beta-catenin and phosphorylates Dishevelled. CamKII, also activated by the Wnt/Ca2+ pathway, inhibits the Wnt/beta-catenin signaling cascade downstream of beta-catenin. Thus, an opposing cross-talk of two distinct Wnt signaling cascades regulates convergent extension movements in Xenopus.

Fritz: a secreted frizzled-related protein that inhibits Wnt activity.

Signaling molecules of the Wnt gene family are involved in the regulation of dorso-ventral, segmental and tissue polarity in Xenopus and Drosophila embryos. Members of the frizzled gene family, such as Drosophila frizzled-2 and rat frizzled-1, have been shown encode Wnt binding activity and to engage intracellular signal transduction molecules known to be part of the Wnt signaling pathway. Here we describe the cloning and characterization of Fritz, a mouse (mfiz) and human (hfiz) gene which codes for a secreted protein that is structurally related to the extracellular portion of the frizzled genes from Drosophila and vertebrates. The Fritz protein antagonizes Wnt function when both proteins are ectopically expressed in Xenopus embryos. In early gastrulation, mouse fiz mRNA is expressed in all three germ layers. Later in embryogenesis fiz mRNA is found in the central and peripheral nervous systems, nephrogenic mesenchyme and several other tissues, all of which are sites where Wnt proteins have been implicated in tissue patterning. We propose a model in which Fritz can interfere with the activity of Wnt proteins via their cognate frizzled receptors and thereby modulate the biological responses to Wnt activity in a multitude of tissue sites.