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skin regeneration of amphibians: a novel model for skin regeneration as adults.adult mammals do not regenerate the dermis of the skin but form a scar after a deep skin injury. since a scar causes serious medical problems, skin regeneration, instead of formation of a scar, has been strongly desired from a clinical point of view. recent studies have suggested multiple origins of myofibroblasts, which are scar-forming cells in skin wound healing of mammals. while amphibians have skin structures that are basically common to mammals as tetrapods, both urodele and anuran amphibi ...201829947057
bapx1 upregulation is associated with ectopic mandibular cartilage development in amphibians.the emergence of novel structures during evolution is crucial for creating variation among organisms, but the underlying processes which lead to the emergence of evolutionary novelties are poorly understood. the gnathostome jaw joint is such a novelty, and the incorporation of bapx1 expression into the intermediate first pharyngeal arch may have played a major role in the evolution of this joint. knockdown experiments revealed that loss of bapx1 function leads to the loss of the jaw joint, becau ...201829942645
musashi and plasticity of xenopus and axolotl spinal cord ependymal cells.the differentiated state of spinal cord ependymal cells in regeneration-competent amphibians varies between a constitutively active state in what is essentially a developing organism, the tadpole of the frog xenopus laevis, and a quiescent, activatable state in a slowly growing adult salamander ambystoma mexicanum, the axolotl. ependymal cells are epithelial in intact spinal cord of all vertebrates. after transection, body region ependymal epithelium in both xenopus and the axolotl disorganizes ...201829535610
morphological and transcriptomic analyses reveal three discrete primary stages of postembryonic development in the common fire salamander, salamandra salamandra.the postembryonic development of amphibians has been characterized as divided into three predominant periods, hereafter named primary developmental stages: premetamorphosis (prem), prometamorphosis (prom), metamorphic climax (meta), and completion of metamorphosis (postm), largely based on examination of anuran development. here, we categorized the postembryonic development of larvae of a poisonous fire salamander (salamandra salamandra) by integrating morphology and gene expression (transcripto ...201829504232
a role for sox9 in post-transcriptional processes: insights from the amphibian oocyte.sox9 is a member of the gene family of sox transcription factors, which is highly conserved among vertebrates. it is involved in different developmental processes including gonadogenesis. in all amniote species examined thus far, sox9 is expressed in the sertoli cells of the male gonad, suggesting an evolutionarily conserved role in testis development. however, in the anamniotes, fishes and amphibians, it is also expressed in the oocyte but the significance of such an expression remains to be el ...201829740094
effects of copper on the early development of xenopus laevis: the case of cuso4 and bordeaux mixture solutions. 201830701063
xenopus: an alternative model system for identifying muco-active agents.the airway epithelium in human plays a central role as the first line of defense against environmental contaminants. most respiratory diseases such as chronic obstructive pulmonary disease (copd), asthma, and respiratory infections, disturb normal muco-ciliary functions by stimulating the hypersecretion of mucus. several muco-active agents have been used to treat hypersecretion symptoms in patients. current muco-active reagents control mucus secretion by modulating either airway inflammation, ch ...201829470529
dicer inactivation stimulates limb regeneration ability in xenopus laevis.the ontogenetic decline of regeneration capacity in the anuran amphibian xenopus makes it an excellent model for regeneration studies. however, the cause of the regeneration ability decline is not fully understood. micrornas regulate animal development and have been indicated in various regeneration situations. however, little is known about the role of micrornas during limb regeneration in xenopus. this study investigates the effect of dicer, an enzyme responsible for microrna maturation, on li ...201829453851
invasive african clawed frogs in california: a reservoir for or predator against the chytrid fungus?amphibian species are experiencing population declines due to infection by the fungal pathogen, batrachochytrium dendrobatidis (bd). the african clawed frog (xenopus laevis), an asymptomatic carrier of bd, has been implicated in the spread of this pathogen through global trade and established invasive populations on several continents. however, research has not explored the relationships of both life stages of this amphibian with bd. while the post-metamorphic individuals may act as a reservoir, ...201829444096
tissue stiffening coordinates morphogenesis by triggering collective cell migration in vivo.collective cell migration is essential for morphogenesis, tissue remodelling and cancer invasion. in vivo, groups of cells move in an orchestrated way through tissues. this movement involves mechanical as well as molecular interactions between cells and their environment. while the role of molecular signals in collective cell migration is comparatively well understood, how tissue mechanics influence collective cell migration in vivo remains unknown. here we investigated the importance of mechani ...201829443958
primordial germ cell transplantation for crispr/cas9-based leapfrogging in xenopus.the creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. crispr/cas9 methods have been adapted for use in the african clawed frog, xenopus, a longstanding model organism for biomedical research. traditional breeding schemes for creating homozygous mutant lines with crispr/cas9-targeted mutagenesis have several time-consuming and laborious steps. to facilitate the creation of mutant embryos, particularly to overcome the obstacles associated with knocking ...201829443056
amphibian zic genes.studies in xenopus laevis have greatly contributed to understanding the roles that the zic family of zinc finger transcription factors play as essential drivers of early development. explant systems that are not readily available in other organisms give xenopus embryos a unique place in these studies, facilitated by the recent sequencing of the xenopus laevis genome. a number of upstream regulators of zic gene expression have been identified, such as inhibition of bmp signaling, as well as calci ...201829442320
isolation and demembranation of xenopus sperm nuclei.the inherent experimental advantages of intact amphibian eggs have been exploited for several decades to advance our understanding of fundamental developmental processes and the cell cycle. characterization of these processes at the molecular level has been greatly advanced by the use of cell-free extracts, which permit the development of biochemically tractable approaches. demembranated xenopus laevis sperm nuclei have been used with cell-free extracts to recapitulate cell cycle progression and ...201829438000
preparation of cellular extracts from xenopus eggs and embryos.cell-free cytoplasmic extracts prepared from xenopus eggs have been used extensively to recapitulate and characterize intracellular events in vitro. egg extracts can be induced to transit the cell cycle and reconstitute assembly of dynamic structures including the interphase nucleus and the mitotic spindle. in this protocol, methods are described for preparing crude cytoplasmic extracts from xenopus eggs and embryos that are arrested in metaphase of the cell cycle. the basic protocol uses unfert ...201829437998
fluorescence in situ hybridization of cryosectioned xenopus oocytes.xenopus laevis oocytes are widely used to study mechanisms of rna function and biogenesis. while the large size of xenopus oocytes is amenable to both biochemical and imaging approaches, the relative opacity of the yolk-rich cytoplasm has limited high-resolution imaging of endogenous rnas. here, we present a protocol that combines multi-probe fluorescence in situ hybridization with cryosectioning to provide a highly sensitive means of imaging endogenous oocyte rnas.201829437997
assembly of spindles and asters in xenopus egg extracts.here, we provide methods for assembly of mitotic spindles and interphase asters in xenopus laevis egg extract, and compare them to spindles and asters in the egg and zygote. classic "cycled" spindles are made by adding sperm nuclei to metaphase-arrested cytostatic factor (csf) extract and inducing entry into interphase extract to promote nucleus formation and dna replication. interphase nuclei are then converted to cycled spindles arrested in metaphase by addition of csf extract. kinetochores as ...201829437996
plant cation-chloride cotransporters (ccc): evolutionary origins and functional insights.genomes of unicellular and multicellular green algae, mosses, grasses and dicots harbor genes encoding cation-chloride cotransporters (ccc). ccc proteins from the plant kingdom have been comparatively less well investigated than their animal counterparts, but proteins from both plants and animals have been shown to mediate ion fluxes, and are involved in regulation of osmotic processes. in this review, we show that ccc proteins from plants form two distinct phylogenetic clades (ccc1 and ccc2). s ...201829415511
threonine 67 is a key component in the coupling of the nss amino acid transporter kaat1. 201829409909
molecular cloning, spatiotemporal and functional expression of gaba receptor subunits rdl1 and rdl2 of the rice stem borer chilo suppressalis.insect γ-aminobutyric acid (gaba) receptor (gabar) is one of the major targets of insecticides. in the present study, cdnas (csrdl1a and csrdl2s) encoding the two isoforms of rdl subunits were cloned from the rice stem borer chilo suppressalis. transcripts of both genes demonstrated similar expression patterns in different tissues and developmental stages, although csrdl2s was ∼2-fold more abundant than csrdl1a throughout all development stages. to investigate the function of channels formed by ...201829408355
tooth germ initiation patterns in a developing dentition: an in vivo study of xenopus laevis tadpoles.the transparency of soft tissue in xenopus laevis tadpoles and the anterior-posterior orientation of their developing tooth germs in the upper jaw offer a unique opportunity for the in vivo charting of the first 15-20 days of the developing dentition. twenty-two x. laevis tadpoles were anesthetized daily and their mouths opened to record the first appearance, position, and development of tooth germs in the upper jaw. the initiation patterns revealed considerable variability between animals, and ...201829399866
the phosphorylation site t613 in the β-subunit of rat epithelial na+ channel (enac) modulates channel inhibition by nedd4-2.the epithelial na+ channel (enac) is a heteromeric channel composed of three subunits (α, β, γ). at the c-terminus of each subunit, a py-motif allows binding of the ubiquitin ligase nedd4-2 which plays a key role in promoting enac retrieval from the plasma membrane. phosphorylation of nedd4-2 by the serum and glucocorticoid-inducible kinase 1 (sgk1) reduces nedd4-2 binding to the py-motifs. in β and γenac, threonine residues (βt613, γt623) belong to an extracellular signal-regulated kinase (erk) ...201829397423
a multichannel computer-driven system to raise aquatic embryos under selectable hypoxic conditions.the formation of a functional cardiovascular system is an essential step in the early vertebrate embryo. nevertheless, the effect of hypoxia on the developmental program of organisms was studied rarely. in particular, this holds true for vertebrate embryos that depend on a functional placenta for proper development and had not been studied in this respect due to the obvious limitation. we established a protocol to culture aquatic embryos, which enabled us to culture a high number of xenopus embr ...201829392150
stac3 enhances expression of human cav1.1 in xenopus oocytes and reveals gating pore currents in hypopp mutant channels.mutations of cav1.1, the pore-forming subunit of the l-type ca2+ channel in skeletal muscle, are an established cause of hypokalemic periodic paralysis (hypopp). however, functional assessment of hypopp mutant channels has been hampered by difficulties in achieving sufficient plasma membrane expression in cells that are not of muscle origin. in this study, we show that coexpression of stac3 dramatically increases the expression of human cav1.1 (plus α2-δ1b and β1a subunits) at the plasma membran ...201829386226
protein immunodepletion and complementation in xenopus laevis egg extracts.the xenopus egg extract system has been widely used to study cell cycle events, including dna replication, nuclear envelope formation, spindle assembly, chromosome condensation and kinetochore formation. the functional roles of the proteins involved in these processes can be determined by immunodepleting a protein of interest from the extract. as immunodepletion may result in co-depletion of other proteins, the protein of interest can be added back to the extract to verify its function. addition ...201829382808
quantitative proteomics after spinal cord injury (sci) in a regenerative and a nonregenerative stage in the frog xenopus laevis.the capacity to regenerate the spinal cord after an injury is a coveted trait that only a limited group of nonmammalian organisms can achieve. in xenopus laevis, this capacity is only present during larval or tadpole stages, but is absent during postmetamorphic frog stages. this provides an excellent model for comparative studies between a regenerative and a nonregenerative stage to identify the cellular and molecular mechanisms that explain this difference in regenerative potential. here, we us ...201829358338
a model for investigating developmental eye repair in xenopus laevis.vertebrate eye development is complex and requires early interactions between neuroectoderm and surface ectoderm during embryogenesis. in the african clawed frog, xenopus laevis, individual eye tissues such as the retina and lens can undergo regeneration. however, it has been reported that removal of either the specified eye field at the neurula stage or the eye during tadpole stage does not induce replacement. here we describe a model for investigating xenopus developmental eye repair. we found ...201829357285
robustly cycling xenopus laevis cell-free extracts in teflon chambers.a central advantage of studying xenopus laevis is the manipulability of its cell-free extracts, which perform the cell cycle in vitro. however, these extracts are known to be experimentally temperamental and will often complete at most one or two cycles. over the course of developing systems for imaging cell cycle events in extracts in real time, we unexpectedly found that when standard xenopus extracts are placed in teflon tubes, they cycle extremely robustly; in one series of experiments, over ...201829475997
centromere and kinetochore assembly in xenopus laevis egg extract.during cell division, chromosomes must be equally segregated to daughter cells. centromeres, the primary interaction site between chromosomes and microtubules, mediate faithful chromosome segregation during mitosis. functional studies of centromere proteins in cells have proven difficult, as mutation or deletion of most centromeric proteins often results in cell lethality. in this protocol, sperm chromatin or reconstituted chromatin arrays, together with xenopus laevis egg extracts, are used to ...201829475995
thyroid hormone levels and temperature during development alter thermal tolerance and energetics of xenopus laevis larvae.environmental variation induced by natural and anthropogenic processes including climate change may threaten species by causing environmental stress. anuran larvae experiencing environmental stress may display altered thyroid hormone (th) status with potential implications for physiological traits. therefore, any capacity to adapt to environmental changes through plastic responses provides a key to determining species vulnerability to environmental variation. we investigated whether developmenta ...201830464840
cloning and functional characterisation of the duplicated rdl subunits from the pea aphid, acyrthosiphon pisum.the insect gaba receptor, rdl (resistance to dieldrin), is a cys-loop ligand-gated ion channel (cyslgic) that plays a central role in neuronal signaling, and is the target of several classes of insecticides. many insects studied to date possess one rdl gene; however, there is evidence of two rdls in aphids. to characterise further this insecticide target from pests that cause millions of dollars' worth of crop damage each year, we identified the complete cyslgic gene superfamily of the pea aphid ...201830065178
α1-fangs: protein ligands selective for the α-bungarotoxin site of the α1-nicotinic acetylcholine receptor.nicotinic acetylcholine receptors (nachrs) are pentameric ligand-gated ion channels that play a central role in neuronal and neuromuscular signal transduction. here, we have developed fang ligands, fibronectin antibody-mimetic nicotinic acetylcholine receptor-generated ligands, using mrna display. we generated a 1 trillion-member primary e10fniii library to target a stabilized α1 nicotinic subunit (α211). this library yielded 270000 independent potential protein binding ligands. the lead sequenc ...201830059207
altered thyroid hormone levels affect body condition at metamorphosis in larvae of xenopus laevis.chemical, physical and biological environmental stressors may affect the endocrine system, such as the thyroid hormone (th) axis in larval amphibians with consequences for energy partitioning among development, growth and metabolism. we studied the effects of two th level affecting compounds, exogenous l-thyroxine (t4 ) and sodium perchlorate (sp), on various measures of development and body condition in larvae of the african clawed frog (xenopus laevis). we calculated the scaled mass index, hep ...201830058724
tight junction-associated protein gef-h1 in the neighbours of dividing epithelial cells is essential for adaptation of cell-cell membrane during cytokinesis.animal cells divide by a process called cytokinesis which relies on the constriction of a contractile actomyosin ring leading to the production of two daughter cells. cytokinesis is an intrinsic property of cells which occurs even for artificially isolated cells. during division, isolated cells undergo dramatic changes in shape such as rounding and membrane deformation as the division furrow ingresses. however, cells are often embedded in tissues and thus are surrounded by neighbouring cells. ho ...201830056063
nosip functions during vertebrate eye and cranial cartilage development.the nitric oxide synthase interacting protein (nosip) has been associated with diverse human diseases including psychological disorders. in line, early neurogenesis of mouse and xenopus is impaired upon nosip deficiency. nosip knockout mice show craniofacial defects and the down-regulation of nosip in the mouse and xenopus leads to microcephaly. until now, the exact underlying molecular mechanisms of these malformations were still unknown.201830055071
a cyclic nucleotide-gated channel, hvcngc2-3, is activated by the co-presence of na⁺ and k⁺ and permeable to na⁺ and k⁺ non-selectively.cyclic nucleotide-gated channels (cngcs) have been postulated to contribute significantly in plant development and stress resistance. however, their electrophysiological properties remain poorly understood. here, we characterized barley cngc2-3 (hvcngc2-3) by the two-electrode voltage-clamp technique in the xenopus laevis oocyte heterologous expression system. current was not observed in x. laevis oocytes injected with hvcngc2-3 complementary rna (crna) in a bathing solution containing either na ...201830049942
inward- and outward-facing homology modeling of human concentrative nucleoside transporter 3 (hcnt3) predicts an elevator-type transport mechanism.the human slc28 family of concentrative (na+-dependent) nucleoside transporters has three members, hcnt1, hcnt2 and hcnt3. previously, we have used heterologous expression in xenopus laevis oocytes in combination with an engineered cysteine-less hcnt3 protein hcnt3(c-) to undertake systematic substituted cysteine accessibility method (scam) analysis of the transporter using the membrane-impermeant thiol reactive reagent p-chloromercuribenzene sulfonate (pcmbs). a continuous sequence of more than ...201830096006
apoptosis propagates through the cytoplasm as trigger waves.apoptosis is an evolutionarily conserved form of programmed cell death critical for development and tissue homeostasis in animals. the apoptotic control network includes several positive feedback loops that may allow apoptosis to spread through the cytoplasm in self-regenerating trigger waves. we tested this possibility in cell-free xenopus laevis egg extracts and observed apoptotic trigger waves with speeds of ~30 micrometers per minute. fractionation and inhibitor studies implicated multiple f ...201830093599
the age-regulated zinc finger factor znf367 is a new modulator of neuroblast proliferation during embryonic neurogenesis.global population aging is one of the major social and economic challenges of contemporary society. during aging the progressive decline in physiological functions has serious consequences for all organs including brain. the age-related incidence of neurodegenerative diseases coincides with the sharp decline of the amount and functionality of adult neural stem cells. recently, we identified a short list of brain age-regulated genes by means of next-generation sequencing. among them znf367 codes ...201830087422
gli2 is required for the induction and migration of xenopus laevis neural crest.the neural crest (nc) is a multipotent migratory embryonic population that is formed during late gastrulation and gives rise to a wide array of derivatives, including cells from the peripheral nervous system (pns), the craniofacial bones and cartilages, peripheral glial cells, and melanocyte cells, among others. in this work we analyzed the role of the hedgehog signaling pathway effector gli2 in xenopus nc. we provide evidence that the gli2 gene is expressed in the prospective, premigratory and ...201830086335
spatial and temporal analysis of pcp protein dynamics during neural tube closure.planar cell polarity (pcp) controls convergent extension and axis elongation in all vertebrates. although asymmetric localization of pcp proteins is central to their function, we understand little about pcp protein localization during convergent extension. here, we use quantitative live imaging to simultaneously monitor cell intercalation behaviors and pcp protein dynamics in the xenopus laevis neural plate epithelium. we observed asymmetric enrichment of pcp proteins, but more interestingly, we ...201830080139
tectal crfr1 receptors modulate food intake and feeding behavior in the south african clawed frog xenopus laevis.the optic tectum and superior colliculus rapidly inhibit food intake when a visual threat is present. previous work indicates that crf, acting on crfr1 receptors, may play a role in tectal inhibition of feeding behavior and food intake. here we test the hypothesis that tectal crfr1 receptors modulate food intake and feeding behavior in juvenile xenopus laevis. we performed five experiments to test the following questions: 1) does tectal crf injection decrease food intake/feeding behavior? 2) doe ...201830077740
differential modulation of human gabac-ρ1 receptor by sulfur-containing compounds structurally related to taurine.the amino acid taurine (2-aminoethanesulfonic acid) modulates inhibitory neurotransmitter receptors. this study aimed to determine if the dual action of taurine on gabac-ρ1r relates to its structure. to address this, we tested the ability of the structurally related compounds homotaurine, hypotaurine, and isethionic acid to modulate gabac-ρ1r.201830075755
a simple decision to move in response to touch reveals basic sensory memory and mechanisms for variable response times.short-term working memory and decision-making are usually studied in the cerebral cortex; in many models of simple decision making, sensory signals build slowly and noisily to threshold to initiate a motor response after long, variable delays. when touched, hatchling frog tadpoles decide whether to swim; we define the long and variable delays to swimming and use whole-cell recordings to uncover the neurons and processes responsible. firing in sensory and sensory pathway neurons is short latency, ...201830074236
disruption of amino acid homeostasis by novel asct2 inhibitors involves multiple targets.the glutamine transporter asct2 (slc1a5) is actively investigated as an oncological target, but the field lacks efficient asct2 inhibitors. a new group of asct2 inhibitors, 2-amino-4-bis(aryloxybenzyl)aminobutanoic acids (aaba), were developed recently and shown to suppress tumor growth in preclinical in vivo models. to test its specificity, we deleted asct2 in two human cancer cell lines. surprisingly, growth of parental and asct2-knockout cells was equally sensitive to aaba compounds. aaba com ...201830072900
ras-dva small gtpases lost during evolution of amniotes regulate regeneration in anamniotes.in contrast to amniotes (reptiles, birds and mammals), anamniotes (fishes and amphibians) can effectively regenerate body appendages such as fins, limbs and tails. why such a useful capability was progressively lost in amniotes remains unknown. as we have hypothesized recently, one of the reasons for this could be loss of some genes regulating the regeneration in evolution of amniotes. here, we demonstrate the validity of this hypothesis by showing that genes of small gtpases ras-dva1 and ras-dv ...201830158598
isolation and primary culture methods of adult and larval myogenic cells from xenopus laevis.during amphibian metamorphosis, larval-to-adult conversion of the myogenic system occurs and there are two distinct types of muscle stem cells; larval myogenic cells have a death-fate by apoptosis in the presence of thyroid hormone t3, and adult myogenic cells have a life-fate under the same conditions. here, we describe isolation and culture methods for adult and larval myogenic cells from the frog, xenopus laevis. both types of cultured myogenic cells undergo cell division and cell differentia ...201830151775
isolation and culture of amphibian (xenopus laevis) sub-capsular liver and bone marrow cells.the x. laevis sub-capsular liver is thought to be the principal hematopoietic site of xenopodinae species from early development and, in case of certain species, into adulthood. the xenopus bone marrow appears to be comprised of precursor cells committed to myeloid lineages, such as macrophage- and granulocyte-progenitor cells. with alarming increases in the contribution of pathogenic infections to the global amphibian declines, now more than ever a better understanding of the mechanisms control ...201830151774
x-fact: xenopus-fast clearing technique.accessibility and imaging of cell compartments in big specimens are crucial for cellular biological research but also a matter of contention. confocal imaging and tissue clearing on whole organs allow for 3d imaging of cellular structures after being subjected to in-toto immunohistochemistry. lately, the passive clarity technique (pact) has been adapted to clear and immunolabel large specimens or individual organs of several aquatic species. we recently demonstrated tissue clearing on one-week o ...201830151770
dye electroporation and imaging of calcium signaling in xenopus nervous system.electroporation is an efficient method of transferring charged macromolecules into living cells in order to study their morphology, function, and connectivity within neuronal networks. labeling cells with fluorophore-coupled macromolecules can be used to trace projections of whole neuronal ensembles, as well as the fine morphology of single cells. here, we present a protocol to visualize pre- and postsynaptic components of a sensory relay synapse in the brain, using the olfactory system of xenop ...201830151769
quantitative proteomics for xenopus embryos ii, data analysis.the oocytes, embryos, and cell-free lysates of the frog xenopus laevis have emerged as powerful models for quantitative proteomic experiments. in the accompanying paper (chapter 13) we describe how to prepare samples and acquire multiplexed proteomics spectra from those. as an illustrative example we use a 10-stage developmental time series from the egg to stage 35 (just before hatching). here, we outline how to convert the ~700,000 acquired mass spectra from this time series into protein expres ...201830151768
quantitative proteomics of xenopus embryos i, sample preparation.xenopus oocytes and embryos are model systems optimally suited for quantitative proteomics. this is due to the availability of large amount of protein material and the ease of physical manipulation. furthermore, facile in vitro fertilization provides superbly synchronized embryos for cell cycle and developmental stages. here, we detail protocols developed over the last few years for sample preparation of multiplexed proteomics with tmt-tags followed by quantitative mass spectrometry analysis usi ...201830151767
a simple knock-in system for xenopus via microhomology mediated end joining repair.following completion of the genome sequences of xenopus tropicalis and x. laevis, gene targeting techniques have become increasingly important for the further development of xenopus research in the life sciences. gene knockout using programmable nucleases, such as talen and crispr/cas9, has reached a level whereby we can readily and routinely perform loss-of-function analysis of genes of interest in these species. however, there is still room for improvement in gene knock-in techniques owing to ...201830151761
generation and care of xenopus laevis and xenopus tropicalis embryos.robust and efficient protocols for fertilization and early embryo care of xenopus laevis and xenopus tropicalis are essential for experimental success, as well as maintenance and propagation of precious animal stocks. the rapid growth of the national xenopus resource has required effective implementation and optimization of these protocols. here, we discuss the procedures used at the national xenopus resource, which we found helpful for generation and early upkeep of xenopus embryos and tadpoles ...201830151756
husbandry, general care, and transportation of xenopus laevis and xenopus tropicalis.maintenance of optimal conditions such as water parameters, diet, and feeding is essential to a healthy xenopus laevis and xenopus tropicalis colony and thus to the productivity of the lab. our prior husbandry experience as well as the rapid growth of the national xenopus resource has given us a unique insight into identifying and implementing these optimal parameters into our husbandry operations. here, we discuss our standard operating procedures that will be of use to both new and established ...201830151755
evaluating iodide recycling inhibition as a novel molecular initiating event for thyroid axis disruption in amphibians.the enzyme iodotyrosine deiodinase (dehalogenase, iyd) catalyzes iodide recycling and promotes iodide retention in thyroid follicular cells. loss of function or chemical inhibition of iyd reduces available iodide for thyroid hormone synthesis, which leads to hormone insufficiency in tissues and subsequent negative developmental consequences. iyd activity is especially critical under conditions of lower dietary iodine and in low iodine environments. our objective was to evaluate the toxicological ...201830137636
kv12.1 channels are not sensitive to gqpcr-triggered activation of phospholipase cβ.kv12.1 k+ channels are expressed in several brain areas, but no physiological function could be attributed to these subunits so far. as genetically-modified animal models are not available, identification of native kv12.1 currents must rely on characterization of distinct channel properties. recently, it was shown in xenopus laevis oocytes that kv12.1 channels were modulated by membrane pi(4,5)p2. however, it is not known whether these channels are also sensitive to physiologically-relevant pi(4 ...201830136882
developmental dependence for functional eye regrowth in xenopus laevis. 201830136686
photocatalytic degradation of azo dye using core@shell nano-tio2 particles to reduce toxicity.clean and safe water is fundamental for human and environmental health. traditional remediation of textile dye-polluted water with chemical, physical, and biological processes has many disadvantages. due to this, nano-engineered materials are drawing more attention to this area. however, the widespread use of nano-particles for this purpose may lead to photocatalytic degradation of xenobiotics, while increasing the risk of nano-particle-induced ecotoxicity. therefore, we comparatively evaluated ...201830136182
cross-limb communication during xenopus hindlimb regenerative response: non-local bioelectric injury signals.regeneration of damaged body parts requires coordination of size, shape, location and orientation of tissue with the rest of the body. it is not currently known to what extent injury sites communicate with the remaining soma during repair, or what information may emanate from the injury site and reach other regions. we examined the bioelectric properties (resting potential gradients in the epidermis) of xenopus laevis froglets undergoing hindlimb amputation and observed that the contralateral (u ...201830126906
molecular characterization of wdr68 gene in embryonic development of xenopus laevis.wdr68, also known as dcaf7, is a wd40 repeated domain protein highly conserved in eukaryotic organisms in both plants and animals. this protein participates in numerous cellular processes and exerts its function through interaction with other proteins. in the present work, we isolated, sequenced and characterized cdna corresponding to the wdr68 gene in embryos of the amphibian xenopus laevis. syntenic analysis revealed high conservation of the genomic region containing the wdr68 locus in amniote ...201830125741
in vitro expression and functional characterization of npa motifs in aquaporins of nosema bombycis.nosema bombycis contains functional aquaporins (nbaqps), which are key targets for exploring the mechanism of n. bombycis infection; however, the regulation of these nbaqps remains unknown. the two highly conserved asparagine-proline-alanine sequences (npa motifs) play important roles in aqp biogenesis. as part of this study, we constructed a series of nbaqp mutants (nbaqp_npa1, nbaqp_npa2, and nbaqp_npa1,2) and expressed them in bmn cells. the results showed that mutations in either npa motif o ...201830105406
high-throughput screening to identify anesthetic ligands using xenopus laevis tadpoles.general anesthetics are considered among the most significant advances in modern medicine; however, they are also some of the most dangerous commonly administered drugs. despite this, the discovery of novel anesthetics has been slow, with few clinically used agents regardless of their nearly 200-year history. xenopus laevis frogs have a long history as a model organism and provide a vital bridge between in vitro and preclinical mammalian assays. the provided protocols are efficient and cost-effe ...201829588028
mitochondrial ros cause motor deficits induced by synaptic inactivity: implications for synapse pruning.developmental synapse pruning refines burgeoning connectomes. the basic mechanisms of mitochondrial reactive oxygen species (ros) production suggest they select inactive synapses for pruning: whether they do so is unknown. to begin to unravel whether mitochondrial ros regulate pruning, we made the local consequences of neuromuscular junction (nmj) pruning detectable as motor deficits by using disparate exogenous and endogenous models to induce synaptic inactivity en masse in developing xenopus l ...201829587245
unexplained cardiac arrest: a tale of conflicting interpretations of kcnq1 genetic test results.unexplained cardiac arrest (uca) is often the first manifestation of an inherited arrhythmogenic disease. genetic testing in uca is challenging due to the complexities of variant interpretation in the absence of supporting cardiac phenotype. we aimed to investigate if a kcnq1 variant [p.(pro64_pro70del)], previously reported as pathogenic, contributes to the long-qt syndrome phenotype, co-segregates with disease or affects kcnq1 function in vitro.201829582136
orthogonality of pyrrolysine trna in the xenopus oocyte.chemical aminoacylation of orthogonal trna allows for the genetic encoding of a wide range of synthetic amino acids without the need to evolve specific aminoacyl-trna synthetases. this method, when paired with protein expression in the xenopus laevis oocyte expression system, can extract atomic scale functional data from a protein structure to advance the study of membrane proteins. the utility of the method depends on the orthogonality of the trna species used to deliver the amino acid. here, w ...201829581437
delay models for the early embryonic cell cycle oscillator.time delays are known to play a crucial role in generating biological oscillations. the early embryonic cell cycle in the frog xenopus laevis is one such example. although various mathematical models of this oscillating system exist, it is not clear how to best model the required time delay. here, we study a simple cell cycle model that produces oscillations due to the presence of an ultrasensitive, time-delayed negative feedback loop. we implement the time delay in three qualitatively different ...201829579091
proteomic characterization of the neural ectoderm fated cell clones in the xenopus laevis embryo by high-resolution mass spectrometry.the molecular program by which embryonic ectoderm is induced to form neural tissue is essential to understanding normal and impaired development of the central nervous system. xenopus has been a powerful vertebrate model in which to elucidate this process. however, abundant vitellogenin (yolk) proteins in cells of the early xenopus embryo interfere with protein detection by high-resolution mass spectrometry (hrms), the technology of choice for identifying these gene products. here, we systematic ...201829578674
the right thalamus may play an important role in anesthesia-awakening regulation in frogs.previous studies have shown that the mammalian thalamus is a key structure for anesthesia-induced unconsciousness and anesthesia-awakening regulation. however, both the dynamic characteristics and probable lateralization of thalamic functioning during anesthesia-awakening regulation are not fully understood, and little is known of the evolutionary basis of the role of the thalamus in anesthesia-awakening regulation.201829576980
incorporation of the δ-subunit into the epithelial sodium channel (enac) generates protease-resistant enacs in xenopus laevis.the epithelial sodium channel (enac) is a critical regulator of vertebrate electrolyte homeostasis. enac is the only constitutively open ion channel in the degenerin/enac protein family, and its expression, membrane abundance, and open probability therefore are tightly controlled. the canonical enac is composed of three subunits (α, β, and γ), but a fourth δ-subunit may replace α and form atypical δβγ-enacs. using xenopus laevis as a model, here we found that mrnas of the α- and δ-subunits are d ...201829576549
effects of trimethoprim-sulfadiazine and detomidine on the function of equine kv 11.1 channels in a two-electrode voltage-clamp (tevc) oocyte model.the long qt syndrome (lqts) is a channelopathy that can lead to severe arrhythmia and sudden cardiac death. pharmacologically induced lqts is caused by interaction between drugs and potassium channels, especially the kv 11.1 channel. due to such interactions, numerous drugs have been withdrawn from the market or are administered with precautions in human medicine. however, some compounds, such as trimethoprim-sulfonamide combinations are still widely used in veterinarian medicine. therefore, we ...201829566261
ribosomal rna biogenesis and its response to chilling stress in oryza sativa.ribosome biogenesis is crucial for plant growth and environmental acclimation. processing of ribosomal rnas (rrnas) is an essential step in ribosome biogenesis and begins with transcription of the rdna. the resulting precursor-rrna (pre-rrna) transcript undergoes systematic processing, where multiple endonucleolytic and exonucleolytic cleavages remove the external and internal transcribed spacers (ets and its). the processing sites and pathways for pre-rrna processing have been deciphered in sac ...201829555785
comparison of etomidate, benzocaine, and ms222 anesthesia with and without subsequent flunixin meglumine analgesia in african clawed frogs (xenopus laevis).often few alternative anesthetics for exotic species are available, due to the small numbers of these animals used in research. in this study, we evaluated the depth and duration of anesthesia in xenopus laevis after their immersion in 3 doses of etomidate (15, 22.5, and 30 mg/l) and in 3 doses of benzocaine (0.1%, 0.5%, and 1%) compared with the 'gold standard,' tricaine methanesulfonate (ms222; 2 g/l). we then chose an optimal dose for each alternative anesthetic according to induction time, d ...201829555009
differential patterns of inhibition of the sugar transporters glut2, glut5 and glut7 by flavonoids.only limited data are available on the inhibition of the sugar transporter glut5 by flavonoids or other classes of bioactives. intestinal glut7 is poorly characterised and no information exists concerning its inhibition. we aimed to study the expression of glut7 in caco-2/tc7 intestinal cells, and evaluate inhibition of glucose transport by glut2 and glut7, and of fructose transport by glut2, glut5 and glut7, by flavonoids. differentiated caco-2/tc7 cell monolayers were used to investigate glut7 ...201829548810
satb1 downregulation induced by oxidative stress participates in trophoblast invasion by regulating β-catenin.preeclampsia (pe) is characterized by abnormal placentation in the early stages of pregnancy. adequate migration and invasion of trophoblasts into the uterine wall and spiral arteries to form a functional maternal-fetal interface are pivotal for normal placentation, but the exact mechanism remains unclear. growing evidence has revealed that special at-rich sequence binding protein 1 (binds to nuclear matrix/scaffold-associating dna) (satb1) is a tumor promoter that participates in cancer cell mi ...201829546272
effects of transportation, transport medium and re-housing on xenopus laevis (daudin).understanding the immediate and longer-term effects of transportation and re-housing in a laboratory species is crucial in order to refine the transfer process, enable the optimal introduction of new animals to a novel environment and to provide a sufficient acclimatisation period before usage. whilst consideration of animal welfare in most model vertebrate species has received attention, little quantitative evidence exists for the optimal care of the common laboratory amphibian xenopus laevis. ...201829545087
large, long range tensile forces drive convergence during xenopus blastopore closure and body axis elongation.indirect evidence suggests that blastopore closure during gastrulation of anamniotes, including amphibians such as xenopus laevis, depends on circumblastoporal convergence forces generated by the marginal zone (mz), but direct evidence is lacking. we show that explanted mzs generate tensile convergence forces up to 1.5 μn during gastrulation and over 4 μn thereafter. these forces are generated by convergent thickening (ct) until the midgastrula and increasingly by convergent extension (ce) there ...201829533180
lithobates catesbeianus (american bullfrog) oocytes: a novel heterologous expression system for aquaporins.xenopus laevis oocytes are a valuable tool for investigating the function of membrane proteins. however, regulations around the world, specifically in brazil, render the import of xenopus laevis frogs impractical, and, in some cases, impossible. here, as an alternative, we evaluate the usefulness of the north american aquatic bullfrog lithobates catesebeianus, which is commercially available in brazil, for the heterologous expression of aquaporin (aqp) proteins. we have developed a method that c ...201829530931
pitx1 regulates cement gland development in xenopus laevis through activation of transcriptional targets and inhibition of bmp signaling.the cement gland in xenopus laevis has long been used as a model to study the interplay of cell signaling and transcription factors during embryogenesis. it has been shown that an intermediate level of bone morphogenetic protein (bmp) signaling is essential for cement gland formation. in addition, several transcription factors have been linked to cement gland development. one of these, the homeodomain-containing protein pitx1, can generate ectopic cement gland formation; however, the mechanisms ...201829530451
frog's dcs have it all in one.although the amphibian xenopus laevis produces antibodies as diversified as those from mammals in the primary repertoire, antibody affinity maturation after immunization is relatively poor and has been associated with a poor b cell selection of aid-mediated hypermutations and lack of germinal centers in the spleen, the only secondary lymphoid organ of this amphibian. in this issue of the european journal of immunology, neely et al., [eur. j. immunol. 2018. 48: 430-440] have unveiled the role of ...201829522243
transcriptome analysis identifies genes involved in sex determination and development of xenopus laevis gonads.development of the gonads is a complex process, which starts with a period of undifferentiated, bipotential gonads. during this period the expression of sex-determining genes is initiated. sex determination is a process triggering differentiation of the gonads into the testis or ovary. sex determination period is followed by sexual differentiation, i.e. appearance of the first testis- and ovary-specific features. in xenopus laevis w-linked dm-domain gene (dm-w) had been described as a master det ...201829518581
c8orf46 homolog encodes a novel protein vexin that is required for neurogenesis in xenopus laevis.neural basic helix-loop helix (bhlh) transcription factors promote progenitor cell differentiation by activation of downstream target genes that coordinate neuronal differentiation. here we characterize a neural bhlh target gene in xenopus laevis, vexin (vxn; previously sbt1), that is homologous to human c8orf46 and is conserved across vertebrate species. c8orf46 has been implicated in cancer progression, but its function is unknown. vxn is transiently expressed in differentiating progenitors in ...201829518376
investigation of higher-order rna g-quadruplex structures in vitro and in living cells by 19f nmr spectroscopy.growing evidence indicates that rna g-quadruplexes have important roles in various processes such as transcription, translation, regulation of telomere length, and formation of telomeric heterochromatin. investigation of rna g-quadruplex structures associated with biological events is therefore essential to understanding the functions of these rna molecules. we recently demonstrated that the sensitivity and simplicity of 19f nmr can be used to directly observe higher-order telomeric g-quadruplex ...201829517770
occurrence of batrachochytrium dendrobatidis in anurans of the mediterranean region of baja california, méxico.chytridiomycosis is caused by the fungal pathogen batrachochytrium dendrobatidis (bd) and is regarded as one of the most significant threats to global amphibian populations. in méxico, bd was first reported in 2003 and has now been documented in 13 states. we visited 33 localities and swabbed 199 wild-caught anurans from 7 species (5 native, 2 exotic) across the mediterranean region of the state of baja california. using quantitative pcr, bd was detected in 94 individuals (47.2% of samples) at 2 ...201829516858
evaluation of the toxic effects of celecoxib on xenopus embryo development.celecoxib is a non-steroidal anti-inflammatory drug that selectively inhibits cyclooxygenase-2 and is prescribed for severe pain and inflammation. the excellent therapeutic effects of celecoxib mean that it is frequently used clinically, including for women of child-bearing age. however, the prenatal effects of this compound have not been studied extensively in vertebrates. the present study examined the developmental toxicity of celecoxib using a frog embryo teratogenic assay-xenopus (fetax). i ...201829505793
booting up the organism during development: pre-behavioral functions of the vertebrate brain in guiding body morphogenesis.a recent study in xenopus laevis embryos showed that the very early brain has important functions long before behavior. while the nascent brain is being constructed, it is required for normal patterning of the muscle and peripheral nerve networks, including those far away from the head. in addition to providing important developmental signals to remote tissues in normal embryogenesis, its presence is also able to render harmless exposure to specific chemicals that normally act as teratogens. the ...201829497473
whole-mount in situ hybridization of xenopus oocytes.whole-mount in situ hybridization (wmish) is a common approach that is used to visualize spatial and temporal gene expression in embryos. in this process, digoxygenin-labeled antisense rna is hybridized to the complementary transcript of interest and rna hybrids are immunohistochemically detected using an alkaline phosphatase-conjugated antibody against digoxigenin. during xenopus laevis oogenesis, certain rnas localize to the animal or vegetal pole laying the foundation for germ cell developmen ...201829496817
identification of circular rnas and their alterations involved in developing male xenopus laevis chronically exposed to atrazine.atrazine (az) is an environmental endocrine disrupting chemical which can affect the development of amphibians. in our past studies, we demonstrated that chronical exposure to 100 μg/l az can cause abnormalities in development and related genes expression of gonads in developing male xenopus laevis (x. laevis) tadpoles. recent studies by others have demonstrated that circular rnas (circrnas) are implicated in multiple developmental anomalies. however, whether circrnas involve in the effects in a ...201829494910
a nurd complex from xenopus laevis eggs is essential for dna replication during early embryogenesis.dna replication in the embryo of xenopus laevis changes dramatically at the mid-blastula transition (mbt), with y rna-independent random initiation switching to y rna-dependent initiation at specific origins. here, we identify xnurd, an mta2-containing assemblage of the nucleosome remodeling and histone deacetylation complex nurd, as an essential factor in pre-mbt xenopus embryos that overcomes a functional requirement for y rnas during dna replication. human nurd complexes have a different subu ...201829490265
plasma kallikrein activates the epithelial sodium channel in vitro but is not essential for volume retention in nephrotic mice.recent work has demonstrated that activation of the epithelial sodium channel (enac) by aberrantly filtered serine proteases causes sodium retention in nephrotic syndrome. the aim of this study was to elucidate a potential role of plasma kallikrein (pklk) as a candidate serine protease in this context.201829489063
hydrolytic activity of human nudt16 enzyme on dinucleotide cap analogs and short capped oligonucleotides.human nudt16 (hnudt16) is a member of the nudix family of hydrolases, comprising enzymes catabolizing various substrates including canonical (d)ntps, oxidized (d)ntps, nonnucleoside polyphosphates, and capped mrnas. decapping activity of the xenopus laevis (x29) nudt16 homolog was observed in the nucleolus, with a high specificity toward u8 snorna. subsequent studies have reported cytoplasmic localization of mammalian nudt16 with cap hydrolysis activity initiating rna turnover, similar to dcp2. ...201829483298
a novel rhodopsin phosphodiesterase from salpingoeca rosetta shows light-enhanced substrate affinity.it is since many years textbook knowledge that the concentration of the second messenger cgmp is regulated in animal rod and cone cells by type ii rhodopsins via a g-protein signaling cascade. microbial rhodopsins with enzymatic activity for regulation of cgmp concentration were only recently discovered: in 2014 light-activated guanylyl-cyclase opsins in fungi and in 2017 a novel rhodopsin phosphodiesterase (rhopde) in the protist salpingoeca rosetta (srrhopde). the light regulation of srrhopde, ...201829483295
role of thyroid hormone receptor in amphibian development.the amphibian xenopus laevis has long been used as a model for studying vertebrate cell and developmental biology largely due to the easiness to manipulate this system in vivo and in vitro. while most of the developmental studies have been on xenopus embryogenesis, considerable efforts have been made to understand its metamorphosis, a process mimicking postembryonic development in mammals when many organs mature into their adult forms in the presence of high levels of thyroid hormone (t3). amphi ...201829892830
evaluating thyroid disrupting chemicals in vivo using xenopus laevis.using in vivo animal model systems for chemical screening can permit evaluation of the signaling pathways implicated. xenopus laevis is an ideal model organism to test thyroid axis disruption as thyroid hormones are highly conserved across vertebrates. here, we describe a high-throughput assay using non-feeding embryonic stage transgenic x. laevis (th/bzip) to screen for thyroid disrupting chemicals using a 3 day exposure protocol. we further describe a protocol to detect endocrine disruption of ...201829892825
atrial fibrillation and heart failure-associated remodeling of two-pore-domain potassium (k2p) channels in murine disease models: focus on task-1.understanding molecular mechanisms involved in atrial tissue remodeling and arrhythmogenesis in atrial fibrillation (af) is essential for developing specific therapeutic approaches. two-pore-domain potassium (k2p) channels modulate cellular excitability, and task-1 (k2p3.1) currents were recently shown to alter atrial action potential duration in af and heart failure (hf). finding animal models of af that closely resemble pathophysiological alterations in human is a challenging task. this study ...201829881975
pou3f transcription factor expression during embryonic development highlights distinct pou3f3 and pou3f4 localization in the xenopus laevis kidney.the pou (pit-oct-unc) genes encode a large transcription factor family comprising 6 classes (pou1f to pou6f ) involved in many developmental processes, such as cell commitment and differentiation. the pou3f class contains four members (pou3f1, pou3f2, pou3f3, pou3f4) characterized by expression in ectodermal tissue derivatives, such as nervous system and otic vesicle, during mammalian development. in order to obtain insights into the potential conservation of this class of transcription factors ...201829877572
lrpap1 as a specific marker of proximal pronephric kidney tubuli in xenopus laevis embryos.lrpap1, also known as receptor associated protein (rap) is a small protein of 40 kda associated with six of the seven members of the evolutionary conserved family of ldl receptors. numerous studies showed that lrpap1 has a dual function, initially as a chaperone insuring proper formation of intermolecular disulfide bonds during biogenesis of low density lipoprotein (ldl) receptors and later as an escort protein during trafficking through the endoplasmic reticulum and the early golgi compartment, ...201829877571
genome-wide transcriptomics analysis of genes regulated by gata4, 5 and 6 during cardiomyogenesis in xenopus laevis.the transcription factors gata4, gata5 and gata6 play important roles in heart muscle differentiation. the data presented in this article are related to the research article entitled "genome-wide transcriptomics analysis identifies sox7 and sox18 as specifically regulated by gata4 in cardiomyogenesis" (afouda et al., 2017) [1]. the present study identifies genes regulated by these individual cardiogenic gata factors using genome-wide transcriptomics analysis. we have presented genes that are spe ...201829876429
mechanism of how augmin directly targets the γ-tubulin ring complex to microtubules.microtubules (mts) must be generated from precise locations to form the structural frameworks required for cell shape and function. mts are nucleated by the γ-tubulin ring complex (γ-turc), but it remains unclear how γ-turc gets to the right location. augmin has been suggested to be a γ-turc targeting factor and is required for mt nucleation from preexisting mts. to determine augmin's architecture and function, we purified xenopus laevis augmin from insect cells. we demonstrate that augmin is su ...201829875259
premotor neuron divergence reflects vocal evolution.to identify mechanisms of behavioral evolution, we investigated the hindbrain circuit that generates distinct vocal patterns in two closely related frog species. male xenopus laevis and xenopus petersii produce courtship calls that include a fast trill: trains of ∼60 hz sound pulses. although fast trill rates are similar, x. laevis fast trills have a longer duration and period than those of x. petersii to pinpoint the neural basis of these differences, we used whole-cell patch-clamp recordings i ...201829875228
molecular insights into variable electron transfer in amphibian cryptochrome.cryptochrome proteins are activated by the absorption of blue light, leading to the formation of radical pairs through electron transfer in the active site. recent experimental studies have shown that once some of the amino acid residues in the active site of xenopus laevis cryptochrome dash are mutated, radical-pair formation is still observed. in this study, we computationally investigate electron-transfer pathways in the x. laevis cryptochrome dash by extensively equilibrating a previously es ...201829874607
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