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Microplastics (MPs) are commonly found with hydrophobic contaminants in the water column and pose a serious threat to aquatic organisms. The effects of polystyrene microplastics of different particle sizes on the accumulation of triclosan in the gut of Xenopus tropicalis , its toxic effects, and the transmission of resistance genes were evaluated. The results showed that co-exposure to polystyrene (PS-MPs) adsorbed with triclosan (TCS) caused the accumulation of triclosan in the intestine with the following accumulation capacity: TCS + 5 µm PS group > TCS group > TCS + 20 µm PS group > TCS + 0.1 µm PS group. All experimental groups showed increased intestinal inflammation and antioxidant enzyme activity after 28 days of exposure to PS-MPs and TCS of different particle sizes. The TCS + 20 µm PS group exhibited the highest upregulated expression of pro-inflammatory factors (IL- 10, IL -1β). The TCS + 20 µm group showed the highest increase in enzyme activity compared to the control group. PS-MPs and TCS, either alone or together, altered the composition of the intestinal microbial community. In addition, the presence of more antibiotic resistance genes than triclosan resistance genes significantly increased the expression of tetracycline resistance and sulfonamide resistance genes, which may be associated with the development of intestinal inflammation and oxidative stress. This study refines the aquatic ecotoxicity assessment of TCS adsorbed by MPs and provides informative information for the management and control of microplastics and non-antibiotic bacterial inhibitors. [Display omitted] [ABSTRACT FROM AUTHOR]

Xenopus oocytes are encompassed by a layer of follicular cells that contribute to oocyte growth and meiosis in relation to oocyte maturation. However, the effects of the interaction between follicular cells and the oocyte surface on meiotic processes are unclear. Here, we investigated Xenopus follicular cell function using oocyte signaling and heterologousexpressing capabilities. We found that oocytes deprotected from their surrounding layer of follicular cells and expressing the epidermal growth factor (EGF) receptor (EGFR) and the Grb7 adaptor undergo accelerated prophase I to metaphase II meiosis progression upon stimulation by EGF. This unusual maturation unravels atypical spindle formation but is rescued by inhibiting integrin β1 or Grb7 binding to the EGFR. In addition, we determined that oocytes surrounded by their follicular cells expressing EGFR–Grb7 exhibit normal meiotic resumption. These oocytes are protected from abnormal meiotic spindle formation through the recruitment of O-GlcNAcylated Grb7, and OGT (O-GlcNAc transferase), the enzyme responsible for O-GlcNAcylation processes, in the integrin β1–EGFR complex. Folliculated oocytes can be forced to adopt an abnormal phenotype and exclusive Grb7 Y338 and Y188 phosphorylation instead of O-GlcNAcylation under integrin activation. Furthermore, an O-GlcNAcylation increase (by inhibition of O-GlcNAcase), the glycosidase that removes O-GlcNAc moieties, or decrease (by inhibition of OGT) amplifies oocyte spindle defects when follicular cells are absent highlighting a control of the meiotic spindle by the OGT–O-GlcNAcase duo. In summary, our study provides further insight into the role of the follicular cell layer in oocyte meiosis progression. [ABSTRACT FROM AUTHOR]

TRESK (K2P18.1) possesses unique structural proportions within the K2P background potassium channel family. The previously described TRESK regulatory mechanisms are based on the long intracellular loop between the second and the third transmembrane segments (TMS). However, the functional significance of the exceptionally short intracellular C-terminal region (iCtr) following the fourth TMS has not yet been examined. In the present study, we investigated TRESK constructs modified at the iCtr by two-electrode voltage clamp and the newly developed epithelial sodium current ratio (ENaR) method in Xenopus oocytes. The ENaR method allowed the evaluation of channel activity by exclusively using electrophysiology and provided data that are otherwise not readily available under whole-cell conditions. TRESK homodimer was connected with two ENaC (epithelial Na+ channel) heterotrimers, and the Na+ current was measured as an internal reference, proportional to the number of channels in the plasma membrane. Modifications of TRESK iCtr resulted in diverse functional effects, indicating a complex contribution of this region to K+ channel activity. Mutations of positive residues in proximal iCtr locked TRESK in low activity, calcineurin-insensitive state, although this phosphatase binds to distant motifs in the loop region. Accordingly, mutations in proximal iCtr may prevent the transmission of modulation to the gating machinery. Replacing distal iCtr with a sequence designed to interact with the inner surface of the plasma membrane increased the activity of the channel to unprecedented levels, as indicated by ENaR and single channel measurements. In conclusion, the distal iCtr is a major positive determinant of TRESK function. [ABSTRACT FROM AUTHOR]

Direct discharge of aquaculture wastewater may have toxic effects, due to the presence of heavy metals, antibiotics, and even resistant pathogens, but little attention has been given. Here, tanks simulating a wild ecosystem were built to study the effects of long-term exposure to duck wastewater containing oxytetracycline (OTC) and/or arsenic (As) on the growth, physiological function, and gut microbiota evolution of Xenopus tropicalis. The results showed that duck wastewater had no apparent impact on X. tropicalis , but the impact increased significantly (P < 0.05) with exposure to OTC and/or As, especially the impact on body weight and growth rate. Biochemical indicators revealed varying degrees of oxidative stress damage, hepatotoxicity (inflammation, necrosis, and sinusoids), and collagen fibrosis of X. tropicalis in all treated groups after 72 days of exposure, which indirectly inhibited X. tropicalis growth. Moreover, 16S rDNA amplicon sequencing results showed that the gut microbiota structure and metabolic function were perturbed after chronic exposure, which might be the leading cause of growth inhibition. Interestingly, the abundance of intestinal resistance genes (RGs) increased with exposure time owing to the combined direct and indirect effects of stress factors in duck wastewater. Moreover, once the RGs were expressed, the resistance persisted for at least 24 days, especially that conferred by tet A. These results provide evidence of the toxic effects of DW containing OTC (0.1–4.0 mg/L) and/or As (0.3–3.5 µg/L) on amphibians and indicate that it is vital to limit the usage of heavy metals and antibiotics on farms to control the biotoxicity of wastewater. [Display omitted] [ABSTRACT FROM AUTHOR]

Epithelial Na+ channels (ENaCs) and related channels have large extracellular domains where specific factors interact and induce conformational changes, leading to altered channel activity. However, extracellular structural transitions associated with changes in ENaC activity are not well defined. Using crosslinking and two-electrode voltage clamp in Xenopus oocytes, we identified several pairs of functional intersubunit contacts where mouse ENaC activity was modulated by inducing or breaking a disulfide bond between introduced Cys residues. Specifically, crosslinking E499C in the β-subunit palm domain and N510C in the α-subunit palm domain activated ENaC, whereas crosslinking βE499C with αQ441C in the α-subunit thumb domain inhibited ENaC. We determined that bridging βE499C to αN510C or αQ441C altered the Na+ selfinhibition response via distinct mechanisms. Similar to bridging βE499C and αQ441C, we found that crosslinking palm domain αE557C with thumb domain γQ398C strongly inhibited ENaC activity. In conclusion, we propose that certain residues at specific subunit interfaces form microswitches that convey a conformational wave during ENaC gating and its regulation. [ABSTRACT FROM AUTHOR]

• A long-type PGRP gene was identified and characterized in Xenopus laevis. • Xl-PGRP-L was ubiquitously expressed in all tested tissues. • Xl-PGRP-L was rapidly up-regulated after PGN stimulation. • Recombinant Xl-PGRP-L has Zn2+-dependent amidase activity. • Xl-PGRP-L has antibacterial activity against Edwardsiella tarda and Streptococcus agalactiae in vitro. Peptidoglycan recognition proteins (PGRPs) are a family of multifunctional proteins playing vital roles in PGN metabolism and antibacterial defense, and their functions have been well-characterized in mammals, bony fishes, and insects. However, the information about the functions of amphibian long-type PGRP is rather limited. Here, we identified and cloned a long-type PGRP gene (named Xl-PGRP-L) from African clawed frog, Xenopus laevis. Xl-PGRP-L gene was detected in all orangs/tissues examined, and was rapidly induced in intestine, liver, and lung following the stimulation of PGN. Sequence analysis showed that Xl-PGRP-L possesses four Zn2+-binding residues (His358, Tyr395, His470, and Cys478) required for amidase activity of catalytic PGRPs, and assays for amidase activity revealed that recombinant Xl-PGRP-L cloud degrade PGN in a Zn2+-dependent manner, indicating that Xl-PGRP-L is belonging to catalytic PGRPs. In addition, Xl-PGRP-L have antibacterial activity against Gram-negative bacteria Edwardsiella tarda and Gram-positive bacteria Streptococcus agalactiae. The present investigation represents the first characterization regarding the biological activities of amphibian long-type PGRPs, thus contributes to a better understanding of the functions of tetrapod PGRPs and the molecular mechanisms of amphibian antibacterial defense. [ABSTRACT FROM AUTHOR]

The toxicity of microplastics (MPs) to aquatic animals is closely related to the presence and release kinetics of contained additives, as most plastic products contain various additives. However, the relationship between the occurrence and release of additives from MPs, and their individual or combined toxicity remains unclear. In this study, the nanoscale distribution and release of tetrabromobisphenol A (TBBPA, a common flame retardant with endocrine-disrupting effect) in polystyrene (PS) MPs, and the long-term (60 days) toxicity of TBBPA and MPs containing TBBPA (at doses of 0 %, 1 %, 10 %, w/w) to Xenopus tropicalis tadpoles were investigated. Exposure to 10 μg/L TBBPA alone was the most toxics, while the encapsulation of TBBPA in MPs significantly delayed its lethal toxicity to tadpoles by inhibiting the rapid and extensive release of TBBPA. PS MPs alone and MPs containing 10 % TBBPA exhibited delayed survival toxicity compared to TBBPA alone, whereas PS MPs containing 1 % TBBPA did not show this effect but inhibited growth. These findings suggest that chronic toxicity assessments should be based on long-term (months or even years) exposure experiments due to the encapsulation-controlled slow release of toxic additives. [Display omitted] ● The encapsulation of PS MPs inhibited the release of TBBPA from them. ● TBBPA-containing PS MPs showed a delayed survival toxicity to tadpoles. ● Chronic exposure to TBBPA-containing PS MPs induces a trade-off between tadpole survival and growth. ● TBBPA-containing PS MPs exhibited different dose-dependent hormesis-like effects on tadpoles. [ABSTRACT FROM AUTHOR]

Winged parthenogenetic aphids are mainly responsible for migration and dispersal. Aphid alarm pheromone (E)-β-Farnesene (EBF) has dual effects on repelling and stimulating wing differentiation in aphids. Previous studies have shown that the odorant coreceptor SmisOrco is involved in the perception of EBF by S. miscanthi ; however, its EBF-specific odorant receptor (OR) and the difference between winged and wingless aphids remain unclear. In this study, the Xenopus oocyte expression system and RNAi technology were used to detect the transmission of EBF signals, and it was found that the olfactory receptor SmisOR5 is an EBF-specific OR in S. miscanthi and is specifically highly expressed in the antennae of winged aphids. Furthermore, when OR5 was silenced with dsRNA, the repellent effect of EBF was weakened, and aphids showed more active aimless movements. Therefore, as a specific OR for EBF, the high expression level of SmisOR5 in winged aphids suggests a molecular basis for its high sensitivity to EBF. This study advances our understanding of the molecular mechanisms of aphid EBF perception and provides novel ideas for effective management and prevention of the migration of winged aphids. [Display omitted] • SmisOR5 was an antenna specific protein with a higher expression level in winged aphid. • OR5/Orco expressed in Xenopus oocytes responded to aphid alarm pheromone, E-β-farnesene. • With the EBF response gone of OR5-silenced aphids, more frequent movement was observed. • It suggests that EBF not only repels aphids, but also makes them hesitant to make a move. [ABSTRACT FROM AUTHOR]

The aim of the present work is to propose a new quantitative assessment method (FETAX-score) for determining the degree of Xenopus laevis embryo development intended for use in embryotoxicity studies. Inspired by a similar scoring system used to evaluate developmental delays (young-for-age phenotypes) in rat embryos cultured in vitro, the FETAX-score was established by considering seven morphological features (head, naris, mouth, lower jaw, tentacles, intestine, anus) that are easily evaluable in tadpoles during the late stages of development at the conclusion of the test. Given that X. laevis development is temperature-dependent and that temperatures below 14°C and above 26°C are teratogenic, the FETAX-score was tested in embryos maintained at 17, 20, 23 and 26°C. No abnormalities were observed in any group, while the total score was temperature-related, suggesting that the FETAX-score is sensitive to moderate distress that does not influence general morphology. Intestine and anus were the least sensitive structures to temperature variations. To assess the applicability of the FETAX-score in developmental toxicological studies, we evaluated FETAX-score in tadpoles exposed during the morphogenetic period to Ethanol (Eth) at concentrations of 0, 0.25, 0.5, 1, 1.5, and 2 % v/v. Gross malformations were observed only in tadpoles from the Eth 2 % group. By contrast, data analysis of the other Eth groups showed dose-related reductions in the FETAX-score. Tentacles were the most sensitive structures to Eth-related delays. These results support the use of the FETAX-score to quantitatively assess developmental deviations in FETAX embryotoxicity studies. • Quantitative measurement for developmental degree of Xenopus laevis. • Fine morphological description of key features of X. laevis tadpole. • Description of Eth related young for age phenotype. [ABSTRACT FROM AUTHOR]

Modification of histones provides a dynamic mechanism to regulate chromatin structure and access to DNA. Histone acetylation, in particular, plays a prominent role in controlling the interaction between DNA, histones, and other chromatinassociated proteins. Defects in histone acetylation patterns interfere with normal gene expression and underlie a wide range of human diseases. Here, we utilize Xenopus egg extracts to investigate how changes in histone acetylation influence transcription of a defined gene construct. We show that inhibition of histone deacetylase 1 and 2 (HDAC1/2) specifically counteracts transcription suppression by preventing chromatin compaction and deacetylation of histone residues H4K5 and H4K8. Acetylation of these sites supports binding of the chromatin reader and transcription regulator BRD4. We also identify HDAC1 as the primary driver of transcription suppression and show that this activity is mediated through the Sin3 histone deacetylase complex. These findings highlight functional differences between HDAC1 and HDAC2, which are often considered to be functionally redundant, and provide additional molecular context for their activity. [ABSTRACT FROM AUTHOR]

TransMEMbrane 16A (TMEM16A) is a Ca2+-activated Cl- channel that plays critical roles in regulating diverse physiologic processes, including vascular tone, sensory signal transduction, and mucosal secretion. In addition to Ca2+, TMEM16A activation requires the membrane lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). However, the structural determinants mediating this interaction are not clear. Here, we interrogated the parts of the PI(4,5)P2 head group that mediate its interaction with TMEM16A by using patchand two-electrode voltage-clamp recordings on oocytes from the African clawed frog Xenopus laevis, which endogenously express TMEM16A channels. During continuous application of Ca2+ to excised inside-out patches, we found that TMEM16Aconducted currents decayed shortly after patch excision. Following this rundown, we show that the application of a synthetic PI(4,5)P2 analog produced current recovery. Furthermore, inducible dephosphorylation of PI(4,5)P2 reduces TMEM16A-conducted currents. Application of PIP2 analogs with different phosphate orientations yielded distinct amounts of current recovery, and only lipids that include a phosphate at the 40 position effectively recovered TMEM16A currents. Taken together, these findings improve our understanding of how PI(4,5)P2 binds to and potentiates TMEM16A channels. [ABSTRACT FROM AUTHOR]

Repair of DNA double-strand breaks by the non-homologous end-joining pathway is initiated by the binding of Ku to DNA ends. Multiple Ku proteins load onto linear DNAs in vitro. However, in cells, Ku loading is limited to ∼1–2 molecules per DNA end. The mechanisms enforcing this limit are currently unclear. Here, we show that the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), but not its protein kinase activity, is required to prevent excessive Ku entry into chromatin. Ku accumulation is further restricted by two mechanisms: a neddylation/FBXL12-dependent process that actively removes loaded Ku molecules throughout the cell cycle and a CtIP/ATM-dependent mechanism that operates in S phase. Finally, we demonstrate that the misregulation of Ku loading leads to impaired transcription in the vicinity of DNA ends. Together, our data shed light on the multiple mechanisms operating to prevent Ku from invading chromatin and interfering with other DNA transactions. [Display omitted] • DNA-PKcs structurally blocks Ku from sliding into chromatin in human and Xenopus • A neddylation/FBXL12-dependent mechanism limits Ku accumulation on chromatin • In S phase, ATM/CtIP overcomes Ku accumulation • Without DNA-PKcs, transcription at the DNA end vicinity is inhibited in a Ku-dependent manner The DNA end binding protein Ku can slide onto naked DNA, but this is limited in cells. Using human cells and Xenopus egg extracts, Bossaert, Moreno, et al. identify DNA-PKcs as the main structural barrier to Ku entry into chromatin, along with two active mechanisms that limit Ku accumulation in the absence of DNA-PKcs. [ABSTRACT FROM AUTHOR]

Various studies have been conducted on the environmental factors that affect the physiology of organisms. Temperature is one of the most important environmental factors that affect the physiology of organisms. Erythropoietic systems have been reported to respond to low environmental temperatures in various animals. A cold exposure model at 5°C in African clawed frogs (< i>Xenopus laevis) was developed by Maekawa et al. (2012), and blood cell count and erythropoiesis were monitored for several days. This model revealed that anemia was induced by the destruction of liver red blood cells and confinement of newly produced red blood cells. Although the causes of cold-induced anemia might be adhesion and phagocytosis due to fluctuations in the membrane proteome, the detailed mechanism remains unclear. Therefore, we examined the kinetics of peripheral erythrocytes during exposure to cold. First, the number of erythrocytes in the peripheral blood and liver was counted using flow cytometry with Sulfo-NHS-LC-biotin or 5- or 6-(N-succinimidyloxycarbonyl) fluorescein 3′,6′-diacetate. They were compared before and after exposure to the frogs at 5°C. Second, erythrocyte surface proteins were trypsinized and transplanted into frogs exposed to 5°C to observe the effects of changes in erythrocyte localization and hepatic confinement. Furthermore, erythrocyte membrane molecules under cold conditions were analyzed by shotgun proteomics. It was able to fractionate by washing with hypotonic hemolysate buffer and selecting the appropriate surfactant, despite the specificity of the nucleated erythrocytes in frogs. This study describes the variability and specificity of erythrocyte membrane molecules, including possible interactions between erythrocytes and vascular endothelial cells, in a cold-exposure model and the results of membrane proteome analysis. [ABSTRACT FROM AUTHOR]

In the present study, using transgenic frogs that express GFP specifically in myeloid cells under the myeloperoxidase enhancer sequence, we found that myeloperoxidase-positive cells are localized in the liver cortex at the late tadpole stages. Immunohistochemical analysis revealed that myelopoiesis in the liver cortex became evident after st. 50 and reached its peak by st. 56. Transplantation experiments indicated that cells with a high density at the liver cortex were derived from the dorso-lateral plate tissue in the neurula embryo. Analysis of smear samples of the cells isolated from collagenase-treated liver tissues of the transgenic tadpoles indicated that myeloid cells were the major population of blood cells in the larval liver and that, in addition to myeloid colonies, erythroid colonies expanded in entire liver after metamorphosis. Cells that were purified from the livers of transgenic tadpoles according to the GFP expression exhibited the multi-lobed nuclei. The results of present study provide evidence that the liver cortex of the Xenopus tadpole is a major site of granulopoiesis. • A transgenic Xenopus line that expresses GFP specifically in myeloid cells has been established. • The GFP-positive cells in the transgenic tadpole were localized in the liver cortex. • These myeloid cells were derived from the dorso-lateral plate tissue in the neurula embryo. • The GFP-positive cells purified from the transgenic tadpole liver exhibited the multi-lobed nuclei. [ABSTRACT FROM AUTHOR]

• PVC microplastics affected the arm regeneration efficiency of Ophiactis virens. • PVC microplastics damaged Xenopus laevis intestines and its swimming behaviour. • In consideration of both models, adverse effects were recorded starting from 1 μg mL−1. • The results suggest the combined use of classic and unconventional toxicity models. • Classic toxicity protocols should incorporate histological and behavioral analyses. In this study, we investigated the effects of PVC microplastics (PVC-MPs) using two different animal models: the brittle star Ophiactis virens, and the African clawed frog Xenopus laevis. This is the first study using an environmental relevant sample of PVC-MPs obtained through mechanical fragmentation of a common PVC plumbing pipe. Exposure experiments on brittle star were performed on the adult stage for a duration of 14 days, while those on African clawed frog were performed on the embryogenic developmental stage according to the standardized FETAX protocol (Frog Embryo Teratogenesis Assay-Xenopus). For both models, different endpoints were analysed: mortality, developmental parameters, behavioural assays and histological analyses on target organs by optical and electronic microscopy. Results showed that the concentration of 0.1 μg mL−1 PVC do not cause any adverse effects in both models (common NOEC concentration), while exposure to 1 μg mL−1 PVC adversely affected at least one species (common LOEC concentration). In particular arm regeneration efficiency was the most affected parameters in O. virens leading to a significantly lower differentiation pattern at 1 μg mL−1 PVC. On the contrary, in X. laevis larvae histopathological analyses and behavioural tests were the most susceptible endpoints, exhibiting several abnormal figures and different swimming speed at 10 μg mL−1 PVC. Histopathological analyses revealed a higher abundance of degenerating cells, pyknotic nuclei and cellular debris in the gut of exposed larvae in respect to control. The comparative analyses performed in this work allowed to characterize the specificity of action of the PVC-MPs on the two species, underlining the importance of exploring a large spectrum of endpoints to offer adequate protection in the emerging fields of microplastic research. [ABSTRACT FROM AUTHOR]

The genome of an early embryo undergoes significant remodelling at the epigenetic, transcriptional, and structural levels. New technological developments have made it possible to study 3D genome organisation in the zygote and early embryo of many different species. Recent studies in human embryos, zebrafish, medaka, and Xenopus have revealed that, similar to previous results in mouse and Drosophila , the zygotic genome is unstructured prior to zygotic genome activation. While these studies show that topologically associating domains are established coincident with zygotic genome activation across species, other 3D genome structures have more varied timing. Here, we review recent studies examining the timing and mechanisms of establishment of 3D genome organisation in the early embryo, and discuss similarities and differences between species. Investigating the establishment of 3D chromatin conformation in early embryos has the potential to reveal novel mechanisms of 3D genome organisation. [ABSTRACT FROM AUTHOR]

Signaling by bone morphogenetic proteins (BMPs) plays pivotal roles in embryogenesis, adult tissue homeostasis, and disease. Recent studies revealed that the well-established WNT agonist R-spondin 2 (RSPO2) is also a BMP receptor (BMP receptor type 1A) antagonist, with roles in early Xenopus embryogenesis and human acute myeloid leukemia (AML). To uncouple the BMP antagonist function from the WNT agonist function and to promote development of AML therapeutics, here we identified a 10-mer peptide (RW) derived from the thrombospondin 1 domain of RSPO2, which specifically pre-vents binding between RSPO2 and BMP receptor type 1A without altering WNT signaling. We also show that a corresponding RW dendrimer (RWd) exhibiting improved half-life relieves inhibition of BMP receptor signaling by RSPO2 in human AML cells, reduces cell growth, and induces differentiation. Moreover, microinjection of RWd in Xenopus embryos ventralizes the dorsoventral embryonic patterning by upregulating BMP signaling without affecting WNT signaling. Our study corroborates the function of RSPO2 as a BMP receptor antagonist and provides a proof of concept for pharmacologically uncoupling BMP antagonist from WNT agonist functions of RSPO2 using the inhibitor peptide RWd with enhanced target selectivity and limited side effects. [ABSTRACT FROM AUTHOR]

Although selective serotonin reuptake inhibitors are commonly prescribed for prenatal depression, there exists controversy over adverse effects of SSRI use on fetal development. Few studies have adequately isolated outcomes due to SSRI exposure and those due to maternal psychiatric conditions. Here, we directly investigated outcomes of exposure to widely-used SSRIs Fluoxetine and Citalopram on the developing nervous system of Xenopus laevis tadpoles, using an integrative experimental approach. We exposed tadpoles to low doses of Citalopram and Fluoxetine during a critical developmental period and found that different experimental groups displayed opposing behavioral effects. While both groups showed reduced schooling behavior, the Fluoxetine group showed increased seizure susceptibility and reduced startle habituation. In contrast, Citalopram treated tadpoles had decreased seizure susceptibility and increased habituation. Both groups had abnormal dendritic morphology in the optic tectum, a brain area important for behaviors tested. Whole-cell electrophysiological recordings of tectal neurons showed no differences in synaptic function; however, tectal cells from Fluoxetine-treated tadpoles had decreased voltage gated K+ currents while cells in the Citalopram group had increased K+ currents. Both behavioral and electrophysiological findings indicate that cells and circuits in the Fluoxetine treated optic tecta are hyperexcitable, while the Citalopram group exhibits decreased excitability. Taken together, these results show that early developmental exposure to SSRIs is sufficient to induce neurodevelopmental effects, however these effects can be complex and vary depending on the SSRI. This may explain some discrepancies across human studies, and further underscores the importance of serotonergic signaling for the developing nervous system. [ABSTRACT FROM AUTHOR]

Intestinal development takes places in two phases, the initial formation of neonatal (mammals)/larval (anurans) intestine and its subsequent maturation into the adult form. This maturation occurs during postembryonic development when plasma thyroid hormone (T3) level peaks. In anurans such as the highly related Xenopus laevis and Xenopus tropicalis , the larval/tadpole intestine is drastically remodeled from a simple tubular structure to a complex, multi-folded adult organ during T3-dependent metamorphosis. This involved complete degeneration of larval epithelium via programmed cell death and de novo formation of adult epithelium, with concurrent maturation of the muscles and connective tissue. Here, we will summarize our current understanding of the underlying molecular mechanisms, with a focus on more recent genetic and genome-wide studies. • Anuran intestinal metamorphosis involves T3-induced larval cell death and de novo formation of adult stem cells. • TR is essential for both larval cell death and stem cell development but not adult intestinal morphogenesis. • TR-mediated activation of cell cycle is critical role for T3-induced larval epithelial cell death. • A model is proposed for cell cycle activation to affect larval cell fate: apoptosis vs. de-differentiation into stem cells. [ABSTRACT FROM AUTHOR]

The intestinal microbiome might be both a sink and source of resistance genes (RGs). To investigate the impact of environmental stress on the disturbance of exogenous multidrug-resistant bacteria (mARB) within the indigenous microbiome and proliferation of RGs, an intestinal conjugative system was established to simulate the invasion of mARB into the intestinal microbiota in vitro. Oxytetracycline (OTC) and heavy metals (Zn, Cu, Pb), commonly encountered in aquaculture, were selected as typical stresses for investigation. Adenosine 5'-triphosphate (ATP), hydroxyl radical (OH·-) and extracellular polymeric substance (EPS) were measured to investigate their influence on the acceptance of RGs by intestinal bacteria. The results showed that the transfer and diffusion of RGs under typical combined stressors were greater than those under a single stressor. Combined effect of OTC and heavy metals (Zn, Cu) significantly increased the activity and extracellular EPS content of bacteria in the intestinal conjugative system, increasing intI3 and RG abundance. OTC induced a notable inhibitory response in Citrobacter and exerted the proportion of Citrobacter and Carnobacterium in microbiota. The introduction of stressors stimulates the proliferation and dissemination of RGs within the intestinal environment. These results enhance our comprehension of the typical stresses effect on the RGs dispersal in the intestine. [Display omitted] • Combined effect of OTC and heavy metals (Zn, Cu) intensified the enrichment of RGs. • OTC promoted the spread of RGs by altering intI3 proliferation. • Heavy metals and OTC enhanced the resistance by increasing bacterial activity. • Citrobacter showed high reactivity in adjusting microbiome balance under OTC stress. [ABSTRACT FROM AUTHOR]

Skin secretions of the Mexican burrowing toad Rhinophrynus dorsalis (Rhinophrynidae) contain the proline-arginine-rich peptide, rhinophrynin-27 (RP-27; ELRLPEIARPVPEVLPARLPLPALPRN) with insulinotropic and immunomodulatory properties, together with a higher concentration of the biologically inactive form, rhinophrynin-33 (RP-33) that constitutes RP-27 extended from its C-terminus by the hexapeptide KMAKNQ. Determination of the conformation of RP-33 by NMR demonstrates that in both water and in a solvent that promotes protein folding (50% trifluoroethanol-water), the majority of the proline residues are found in a polyproline type II helical region. The peptide adopts a horseshoe (U-shaped) conformation with pronounced bends in the molecule of around 100°–120° at Glu13 and Arg18. The hexapeptide extension adopts a α-helical conformation. When the hexapeptide is excised to generate RP-27, the molecule adopts an L-shaped conformation with a single bend at Glu13. A search of protein sequence databases indicated the P-X-P-XXX-P-XXX-P-X-P motif found in RP-27 and RP-33 occurs in a number of proteins although its functional implications are unclear. The data suggest that RP-33 represents a biosynthetic precursor of RP-27 that is activated by a protease cleaving at a single lysine residue of the type previously identified in Xenopus laevis skin secretions. • Conformation of rhinophrynin-33 is determined by NMR combined to simulated annealing. • The majority of Pro residues are found in the PP-II region of the Ramachandran plot. • The peptide adopts a horseshoe conformation with 100°–120° bends at Glu13 and Arg18. • The hexapeptide extension with respect to RP-27 adopt a α-helical conformation. • RP-33 represents a biosynthetic precursor of RP-27 activated by protease cleaving. [ABSTRACT FROM AUTHOR]

Interferon regulatory factor 1 (IRF1) is an important regulator in controlling the transcription of type I interferon genes, and its functions have been well-characterized in mammals, birds and fish. However, little information is available regarding the function of amphibian IRF1. In this study, an IRF1 gene homolog named as Xt-IRF1 was identified in the Western clawed frog (Xenopus tropicalis), an amphibian model specie widely used for comparative immunology research. Xt-IRF1 and IRF1 in other vertebrates possess similar genomic structure and flanking genes, and were grouped together to form a separate clade in phylogenetic tree. In addition, Xt-IRF1 gene was constitutively expressed in all tissues examined, with the highest expression level observed in spleen, and was inducible after poly(I:C) stimulation. Importantly, the expression of Xt-IRF1 was markedly induced by recombinant type I interferon, and Xt-IRF1 induced a strong activation of both IFNβ and ISRE promoters. The present study opens the door to investigate the roles of IRF1 in amphibians, and thus contributes to a better understanding of the functional evolution of IRFs in lower tetrapods. • The present study represents the first characterization of an IRF1 homolog in amphibians. • Structurally, IRF1 is highly conserved from fish to mammals. • Xenopus IRF1 was ubiquitously expressed in all tested tissues and up-regulated after poly(I:C) stimulation. • Xenopus IRF1 was shown to be a typical interferon-stimulated gene induced by type I interferon. • Xenopus IRF1 can elicit a strong activation of IFNβ and ISRE promoters. [ABSTRACT FROM AUTHOR]

• Mechanical cues can be manipulated indentation, microaspiration, and stretch. • Mechanical cues can pattern tissues and drive differentiation in many cell types. • Accessibility and ease of manipulation make Xenopus embryos ideal for mechanobiology. Features of amphibian embryos that have served so well to elucidate the genetics of vertebrate development also enable detailed analysis of the physics that shape morphogenesis and regulate development. Biophysical tools are revealing how genes control mechanical properties of the embryo. The same tools that describe and control mechanical properties are being turned to reveal how dynamic mechanical information and feedback regulate biological programs of development. In this review we outline efforts to explore the various roles of mechanical cues in guiding cilia biology, axonal pathfinding, goblet cell regeneration, epithelial-to-mesenchymal transitions in neural crest, and mesenchymal-to-epithelial transitions in heart progenitors. These case studies reveal the power of Xenopus experimental embryology to expose pathways integrating mechanical cues with programs of development, organogenesis, and regeneration. [ABSTRACT FROM AUTHOR]

Extracellular adenosine triphosphate (ATP) participates in maintaining the vascular tone in the CNS, particularly in the retina, via the tonic activity of ligand gated activated P2X1 receptors. P2X1 receptors are characterized by their high affinity for ATP and their strong desensitization to concentrations of ATP that are 200-fold lower than their EC 50. The mechanism behind P2X1 tonic activity remains unclear. In this study, we expressed human P2X1 (hP2X1) homomeric receptors in Xenopus oocytes to explore the relationship between ATP release from oocytes at rest, hP2X1, and Ca2+-activated Cl− channels. Our results indicate that Xenopus oocytes release ATP at rest via vesicular exocytosis, and this process is a constitutive phenomenon independent of extracellular Ca2+. Our results also indicate that hP2X1 receptors are able to sustain a tonic activity of Ca2+-activated Cl− channels. In the presence of extracellular Ca2+ the activity of hP2X1 receptors is greatly amplified by its coupling with Ca2+-activated Cl− channels. Future studies addressing the relationship between hP2X1 receptors and Ca2+-activated Cl− channels in vascular smooth muscle cells should provide information about additional mechanisms that regulate the vascular tone and their potential as pharmaceutical targets. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries. • Ca2+-independent vesicular release of ATP in Xenopus oocytes • Vesicular release of ATP is blocked by brefeldin. • ATP release can persistently activate hP2X1 receptors. • ATP-dependent tonic activity of hP2X1 is amplified by Ca2+-activated Cl− channels [ABSTRACT FROM AUTHOR]

The S -specific pollen rejection response in Nicotiana depends on the interaction between S-RNase and a suite of SLF proteins. However, the biochemical pathway requires other essential proteins. One of them is the stigmatic protein NaStEP, which belongs to the Kunitz-type protease inhibitor family. Within the pollen tubes, NaStEP is a positive regulator of HT-B stability, likely inhibiting its degradation and, additionally, interacts with NaSIPP, a mitochondrial phosphate carrier. To gain a deeper understanding of the biochemical role of NaStEP in pollen rejection, we evaluated whether the activity of NaStEP as protease inhibitor is specific to a particular type of protease and whether it has the function of a voltage-dependent channel (VDC) blocker. Our findings indicate that, in vitro, NaStEP inhibits a subtilisin-like protease in an irreversible manner , but not other proteases, such as thermolysin and papain. Furthermore, we found that subtilisin processes the native NaStEP (24 kDa) into two lower molecular weight peptides of 21 and 14 kDa. Moreover, when we incubated NaStEP along with Xenopus leavis oocytes expressing the voltage-dependent potassium channel Kv 1.3, the current was blocked, indicating that NaStEP acts as a VDC blocker. These data allow us to propose NaStEP acts as a key molecule with two functions, one protecting HT-B from degradation by inhibiting a subtilisin-like protease and the second one by forming a complex with a mitochondrial VDC that could destabilize the mitochondria to trigger cell death, which would reinforce S -specific pollen rejection in Nicotiana. • NaStEP specifically and irreversibly inhibits a subtilisin-like protease. • A subtilisin-like processes the native NaStEP into two lower molecular weight peptides. • NaStEP also blocks the current of the voltage-dependent potassium channel Kv 1.3 when expressed in Xenopus oocytes. [ABSTRACT FROM AUTHOR]

• lhx2/9 are expressed in the forebrain, in complementary domains to slit1 / sema3a. • etv1 mRNA expression coincides with mRNA expression of slit1 and sema3a. • Lhx2 is an indirect repressor of these guidance genes while Etv1 is an activator. • Lhx2/9 and Etv1 may regulate the levels and boundaries of slit1 / sema3a expression. During neural network development, growing axons read a map of guidance cues expressed in the surrounding tissue that lead the axons toward their targets. In particular, Xenopus retinal ganglion axons use the cues Slit1 and Semaphorin 3a (Sema3a) at a key guidance decision point in the mid-diencephalon in order to continue on to their midbrain target, the optic tectum. The mechanisms that control the expression of these cues, however, are poorly understood. Extrinsic Fibroblast Growth Factor (Fgf) signals are known to help coordinate the development of the brain by regulating gene expression. Here, we propose Lhx2/9 and Etv1 as potential downstream effectors of Fgf signalling to regulate slit1 and sema3a expression in the Xenopus forebrain. We find that lhx2/9 and etv1 mRNAs are expressed complementary to and within slit1 / sema3a expression domains, respectively. Our data indicate that Lhx2 functions as an indirect repressor in that lhx2 overexpression within the forebrain downregulates the mRNA expression of both guidance genes, and in vitro lhx2/9 overexpression decreases the activity of slit1 and sema3a promoters. The Lhx2-VP16 constitutive activator fusion reduces sema3a promoter function, and the Lhx2-En constitutive repressor fusion increases slit1 induction. In contrast, etv1 gain of function transactivates both guidance genes in vitro and in the forebrain. Based on these data, together with our previous work, we hypothesize that Fgf signalling promotes both slit1 and sema3a expression in the forebrain through Etv1, while using Lhx2/9 to limit the extent of expression, thereby establishing the proper boundaries of guidance cue expression. [ABSTRACT FROM AUTHOR]

The paraoxonase (PON) family comprises three highly conserved members: PON1, PON2, and PON3. They are orthologs of Caenorhabditis elegans MEC-6, an endoplasmic reticulum- resident chaperone that has a critical role in proper assembly and surface expression of the touch-sensing degenerin channel in nematodes. We have shown recently that MEC-6 and PON2 negatively regulate functional expression of the epithelial Na+ channel (ENaC), suggesting that the chaperone function is conserved within this family. We hypothesized that otherPONfamily members also modulate ion channel expression. Pon3 is specifically expressed in the aldosterone-sensitive distal tubules in the mouse kidney. We found here that knocking down endogenous Pon3 in mouse cortical collecting duct cells enhanced Na+ transport, which was associated with increased γENaC abundance. We further examined Pon3 regulation of ENaC in two heterologous expression systems, Fisher rat thyroid cells and Xenopus oocytes. Pon3 coimmunoprecipitated with each of the three ENa Csubunits in Fisher rat thyroid cells. As a result of this interaction, the wholecell and surface abundance of ENaCα andγ subunits was reduced by Pon3. When expressed in oocytes, Pon3 inhibited ENaC-mediated amiloride-sensitive Na+ currents, in part by reducing the surface expression of ENaC. In contrast, Pon3 did not alter the response of ENaC to chymotrypsin-mediated proteolytic activation or [2-(trimethylammonium)ethyl]methanethiosulfonate- induced activation of αβS518Cγ, suggesting that Pon3 does not affect channel open probability. Together, our results suggest that PON3 regulates ENaC expression by inhibiting its biogenesis and/or trafficking. [ABSTRACT FROM AUTHOR]

Embryonic cell fate specification and axis patterning requires integration of several signaling pathways that orchestrate region-specific gene expression. The transcription factor signal transducer and activator of transcription 3 (Stat3) plays important roles during early development, but it is unclear how Stat3 is activated. Here, using Xenopus as a model, we analyzed the post-translational regulation and functional consequences of Stat3 activation in dorsoventral axis patterning. We show that Stat3 phosphorylation, lysine methylation, and transcriptional activity increase before gastrulation and induce ventral mesoderm formation. Down syndrome critical region gene 6 (DSCR6), a RIPPLY family member that induces dorsal mesoderm by releasing repressive polycomb group proteins from chromatin, bound to the Stat3 C-terminal region and antagonized its transcriptional and ventralizing activities by interfering with its lysine methylation. Enhancer of zeste 2 polycombrepressive complex 2 subunit (Ezh2) also bound to this region; however, its methyltransferase activity was required for Stat3 methylation and activation. Loss of Ezh2 resulted in dorsalization of ventral mesoderm and formation of a secondary axis. Furthermore, interference with Ezh2 phosphorylation also prevented Stat3 lysine methylation and transcriptional activity. Thus, inhibition of either Ezh2 phosphorylation or Stat3 lysine methylation compensated for the absence of DSCR6 function. These results reveal that DSCR6 and Ezh2 critically and posttranslationally regulate Stat3 transcriptional activity. Ezh2 promotes Stat3 activation in ventral mesoderm formation independently of epigenetic regulation, whereas DSCR6 specifies dorsal fate by counteracting this ventralizing activity. This antagonism helps pattern the mesoderm along the dorsoventral axis, representing a critical facet of cell identity regulation during development. [ABSTRACT FROM AUTHOR]

Oocytes are arrested in prophase I. In vertebrates, meiotic resumption is triggered by hormonal stimulation that results in cAMP-dependent protein kinase (PKA) downregulation leading to Cdk1 activation. Yet the pathways connecting PKA to Cdk1 remain unclear. Here, we identify molecular events triggered by PKA downregulation occurring upstream of Cdk1 activation. We describe a two-step regulation controlling cyclin B1 and Mos accumulation, which depends on both translation and stabilization. Cyclin B1 accumulation is triggered by PKA inhibition upstream of Cdk1 activation, while its translation requires Cdk1 activity. Conversely, Mos translation initiates in response to the hormone, but the protein accumulates only downstream of Cdk1. Furthermore, two successive translation waves take place, the first controlled by PKA inhibition and the second by Cdk1 activation. Notably, Arpp19, an essential PKA effector, does not regulate the early PKA-dependent events. This study elucidates how PKA downregulation orchestrates multiple pathways that converge toward Cdk1 activation and induce the oocyte G2/M transition. [Display omitted] • Multiple molecular pathways connect PKA downregulation to Cdk1 activation • Cyclin B1 and Mos accumulation is opposingly controlled by a two-step mechanism • Two waves of translation occur during the G2/M transition Santoni et al. identify multiple pathways connecting PKA downregulation to Cdk1 activation and controlling G2/M transition in oocytes. Among the events taking place upstream of Cdk1 activation, they highlight the interplay between protein translation and stabilization in controlling the accumulation of critical cell-cycle regulators, including Mos and cyclin B1. [ABSTRACT FROM AUTHOR]

Morphogenetic movements during animal development involve repeated making and breaking of cell-cell contacts. Recent biophysical models of cell-cell adhesion integrate adhesion molecule interactions and cortical cytoskeletal tension modulation, describing equilibrium states for established contacts. We extend this emerging unified concept of adhesion to contact formation kinetics, showing that aggregating Xenopus embryonic cells rapidly achieve Ca2+-independent low-contact states. Subsequent transitions to cadherin-dependent high-contact states show rapid decreases in contact cortical F-actin levels but slow contact area growth. We developed a biophysical model that predicted contact growth quantitatively from known cellular and cytoskeletal parameters, revealing that elastic resistance to deformation and cytoskeletal network turnover are essential determinants of adhesion kinetics. Characteristic time scales of contact growth to low and high states differ by an order of magnitude, being at a few minutes and tens of minutes, respectively, thus providing insight into the timescales of cell-rearrangement-dependent tissue movements. [Display omitted] • Xenopus gastrula cell-cell attachment kinetics show two states of adhesive contact • A low Ca2+-independent contact state precedes a high, stable cadherin-dependent one • Cadherin-dependent adhesion is associated with a rapid loss of F-actin at contacts • Relatively slow contact growth is explained by cytoskeletal elasticity and turnover Parent et al. show that mutual attachment of embryonic cells in Xenopus proceeds from a rapidly attained, unstable, Ca2+-independent low-contact state to stable cadherin-dependent adhesion. The transition is driven by the rapid loss of F-actin cytoskeleton at the contact, and its slow rate is explained by cytoskeletal elasticity and turnover. [ABSTRACT FROM AUTHOR]

The oocyte-to-embryo transition (OET) is regulated by maternal products stored in the oocyte cytoplasm, independent of transcription. How maternal products are precisely remodeled to dictate the OET remains largely unclear. In this work, we discover the dynamic solubility phase transition of maternal RNAs during Xenopus OET. We have identified 863 maternal transcripts that transition from a soluble state to a detergent-insoluble one after oocyte maturation. These RNAs are enriched in the animal hemisphere, and many of them encode key cell cycle regulators. In contrast, 165 transcripts, including nearly all Xenopus germline RNAs and some vegetally localized somatic RNAs, undergo an insoluble-to-soluble phase transition. This phenomenon is conserved in zebrafish. Our results demonstrate that the phase transition of germline RNAs influences their susceptibility to RNA degradation machinery and is mediated by the remodeling of germ plasm. This work thus identifies important remodeling mechanisms that act on RNAs to control vertebrate OET. [Display omitted] • Many maternal RNAs transition between soluble and insoluble states during the OET • Germline RNAs undergo soluble-to-insoluble phase transition during the OET • Insoluble germline RNAs are resistant to RNA degradation machinery • Degradation of Velo/Buc during the OET allows solubilization of germline RNAs Hwang et al. report that many maternal RNAs can transition between the soluble and insoluble phases during vertebrate oocyte-to-embryo transition (OET). In the case of germline RNAs, they undergo an insoluble-to-soluble phase transition during the OET as a result of the remodeling of germ plasm. [ABSTRACT FROM AUTHOR]

Peroxidase genes (Prdx) encode a family of antioxidant proteins, which can protect cells from oxidative damage by reducing various cellular peroxides. This study investigated the spatiotemporal expression patterns of gene members in this family during the early development of Xenopus tropicalis. Real-time quantitative PCR showed that all members of this gene family have a distinct temporal expression pattern during the early development of X. tropicalis embryos. Additionally, whole mount in situ hybridization revealed that individual prdx genes display differential expression patterns, with overlapping expression in lymphatic vessels, pronephros, proximal tubule, and branchial arches. This study provides a basis for further study of the function of the prdx gene family. [ABSTRACT FROM AUTHOR]

Many retinal diseases are associated with pathological cell swelling, but the underlying etiology remains to be established. A key component of the volume-sensitive machinery, the transient receptor potential vanilloid 4 (TRPV4) ion channel, may represent a sensor and transducer of cell swelling, but the molecular link between the swelling and TRPV4 activation is unresolved. Here, our results from experiments using electrophysiology, cell volumetric measurements, and fluorescence imaging conducted in murine retinal cells and Xenopus oocytes indicated that cell swelling in the physiological range activated TRPV4 in Müller glia and Xenopus oocytes, but required phospholipaseA 2 (PLA2) activity exclusively in Müller cells. Volumedependent TRPV4 gating was independent of cytoskeletal rearrangements and phosphorylation. Our findings also revealed that TRPV4-mediated transduction of volume changes is dependent by its N terminus, more specifically by its distal-most part. Weconclude that the volume sensitivity and function of TRPV4 in situ depend critically on its functional and cell type-specific interactions. [ABSTRACT FROM AUTHOR]

Ecological risk of chemicals to aquatic-phase amphibians has historically been evaluated by comparing estimated environmental concentrations in surface water to surrogate toxicity data from fish species. Despite their obvious similarities, there are biological disparities among fish and amphibians that could affect their exposure and response to chemicals. Given the alarming decline in amphibians, in which anthropogenic pollutants play at least some role, investigating the risk of chemicals to amphibians is becoming increasingly important. Here, we evaluate relative sensitivity of fish and larval aquatic-phase amphibians to 45 different pesticides using existing data from three standardized toxicity test designs: (1) amphibian metamorphosis assay (AMA) with the African clawed frog (Xenopus laevis); (2) fish short-term reproduction assay (FSTRA) with the fathead minnow (Pimephales promelas); (3) fish early life stage test (ELS) with fathead minnows or rainbow trout (Oncorhynchus mykiss). The advantage of this dataset over previous work is that the underlying studies are consistent in exposure method, study duration, test species, endpoints measured, and number of concentrations tested. We found very strong positive relationships between fish and frog lowest adverse effect concentrations (LOAEC) for survival [Spearman's rank correlation (r s) = 0.88], body weight (r s = 0.86), and length (r s = 0.89) with only one out of 45 chemicals (propiconazole) exhibiting 100-folder greater sensitivity in frogs relative to fish. While our results suggest comparable toxicity for pesticides between fish and aquatic-phase amphibians under these test conditions, further research with a greater diversity of amphibians and exposure scenarios will help determine the relevance of these results across species and life stages. • Aquatic-phase amphibians and fish exhibit similar sensitivities to pesticides. • Amphibians may be more sensitive than fish to certain fungicides and pyrethroids. • Results address use of fish as surrogates for amphibians in regulatory toxicology. • More amphibian species should be tested to address fish-amphibian surrogacy. [ABSTRACT FROM AUTHOR]

Peptidoglycan recognition proteins (PGRPs) are a family of pattern recognition receptors (PRRs) involved in host antibacterial responses, and their functions have been characterized in most invertebrate and vertebrate animals. However, little information is available regarding the function of frog PGRPs. In this study, a short-type PGRP (termed Xl-PGRP-S) gene was identified in the African clawed frog, Xenopus laevis. The predicted protein of Xl-PGRP-S contains several structural features known in PGRPs, including a typical PGRP domain and two closely spaced conserved cysteines. Xl-PGRP-S gene was constitutively expressed in all tissues examined, with the highest expression level observed in muscle. As a typical PRR, Xl-PGRP-S is inducible after peptidoglycan (PGN) stimulation, and has an ability to bind PGN. In addition, Xl-PGRP-S has been proven to have Zn2+-dependent amidase activity and antibacterial activity against Edwardsiella tarda. The present study represents the first discovery on the function of frog PGRPs, thus contributing to a better understanding of the functional evolution of PGRPs in early tetrapods. • A short-type peptidoglycan recognition protein (PGRP), named as Xl-PGRP-S was identified in Xenopus laevis. • Xl-PGRP-S was predicted to have a PGRP domain and two conserved cysteine. • Xl-PGRP-S is expressed in various organs/tissues of the frog and also in A6 cells. • Xl-PGRP-S can bind with PGN and is inducible following PGN stimulation. • Zn2+-dependent amidase activity and antibacterial activity were observed for Xl-PGRP-S. [ABSTRACT FROM AUTHOR]

Guanine nucleotide exchange factors (GEFs) activate Rho GTPases by accelerating their GDP/GTP exchange. Trio and its paralog Kalirin (Kalrn) are unique members of the Rho-GEFs that harbor three catalytic domains: two functional GEF domains and a serine/threonine kinase domain. The N-terminal GEF domain activates Rac1 and RhoG GTPases, while the C-terminal GEF domain acts specifically on RhoA. Trio and Kalrn have an evolutionary conserved function in morphogenetic processes including neuronal development. De novo mutations in TRIO have lately been identified in patients with intellectual disability, suggesting that this protein family plays an important role in development and disease. Phylogenetic and domain analysis revealed that a Kalrn/Trio ancestor originated in Prebilateria and duplicated in Urbilateria to yield Kalrn and Trio. Only few taxa outside the vertebrates retained both of these highly conserved proteins. To obtain first insights into their redundant or distinct functions in a vertebrate model system, we show for the first time a detailed comparative analysis of trio and kalrn expression in Xenopus laevis development. The mRNAs are maternally transcribed and expression increases starting with neurula stages. Trio and kalrn are detected in mesoderm/somites and different neuronal populations in the neural plate/tube and later also in the brain. However, only trio is expressed in migrating neural crest cells, while kalrn expression is detected in the cranial nerves, suggesting distinct functions. Thus, our expression analysis provides a good basis for further functional studies. • Trio and Kalirin originated by duplication in Urbilateria. • Trio and Kalirin protein sequences are highly conserved in vertebrates. • Xenopus trio and kalrn are expressed in a spatio-temporal manner. • Xenopus trio and kalrn are expressed in mesodermal and neural tissue. • Only trio is expressed in Xenopus neural crest cells. [ABSTRACT FROM AUTHOR]

• Identified GTP binding protein 10 (GTPBP10) as a member of the OBG family of GTPases. • GTPBP10 is differentially expressed throughout development in X. laevis , beginning in the blastula and persisting through the tailbud stages. • GTPBP10 is expressed in the late tailbud stage in the notochord and pronephros. [ABSTRACT FROM AUTHOR]

ALDH16 is a novel family of the aldehyde dehydrogenase (ALDH) superfamily with unique structural characteristics that distinguish it from the other ALDH superfamily members. In addition to structural characteristics, there is an evolutionary-related grouping within the ALDH 16 genes. The ALDH16 isozymes in frog, lower animals, and bacteria possess a critical Cys residue in their active site, which is absent from ALDH16 in mammals and fish. Genomic analysis and plasma metabolomic studies have associated ALDH16A1 with the pathogenesis of gout in humans, although its actual involvement in this disease is poorly understood. Insight into the structure of ALDH16A1 is an important step in deciphering its function in gout. Herein, we report our efforts towards the structural characterization of Xenopus tropicalis ALDH16B1 (the homolog of human ALDH16A1) that was predicted to be catalytically-active. Recombinant ALDH16B1 was expressed in Sf9 cells and purified using affinity and size exclusion chromatography. Crystallization of ALDH16B1 was achieved by vapor diffusion. A data set was collected at 2.5 Å and preliminary crystallographic analysis showed that the frog ALDH16B1 crystals belong to the P 2 1 2 1 2 1 space group with unit cell parameters a = 80.48 Å, b = 89.73 Å, c = 190.92 Å, α = β = γ = 90.00°. Structure determination is currently in progress. • Structural studies of frog ALDH16B1, a homolog of human ALDH16A1. • Overexpression and purification of frog ALDH16B1 in mg quantities. • ALDH16B1 crystallization. • Size exclusion and crystallographic data support a homodimeric structure for ALDH16B1. [ABSTRACT FROM AUTHOR]

In a screen for human kinases that regulate Xenopus laevis embryogenesis, we identified Nagk and other components of the UDP-GlcNAc glycosylation salvage pathway as regulators of anteroposterior patterning and Wnt signaling. We find that the salvage pathway does not affect other major embryonic signaling pathways (Fgf, TGFβ, Notch, or Shh), thereby demonstrating specificity for Wnt signaling. We show that the role of the salvage pathway in Wnt signaling is evolutionarily conserved in zebrafish and Drosophila. Finally, we show that GlcNAc is essential for the growth of intestinal enteroids, which are highly dependent on Wnt signaling for growth and maintenance. We propose that the Wnt pathway is sensitive to alterations in the glycosylation state of a cell and acts as a nutritional sensor in order to couple growth/proliferation with its metabolic status. We also propose that the clinical manifestations observed in congenital disorders of glycosylation (CDG) in humans may be due, in part, to their effects on Wnt signaling during development. • The UDP-GlcNAc glycosylation salvage pathway regulates axiation in Xenopus embryos. • Disruption of Xenopus axiation by the salvage pathway is mediated by Wnt signaling. • The salvage pathway regulates Wnt but not Fgf, TGFβ, Notch, or Shh signaling. • Regulation of Wnt signaling by the salvage pathway is evolutionarily conserved. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • GABA receptor (GABAR) β2S and γ2 subunits were first isolated from zebrafish. • The α1, β2S, and γ2 subunits were functionally co-expressed in X. laevis oocytes. • GABA and β-alanine except benzamidine induced inward current in heteromeric channel. • Insecticides significantly inhibited GABA responses in heteromeric channel in oocytes. Abstract The ionotropic GABA A receptor (GABA A R) is the main fast inhibitory post-synaptic receptor and is also an important insecticidal target. Effect of insecticides on fish has attracted intensive attention. However, no systematic study on heteromeric zebrafish GABA A R expressed in oocytes has been reported to date. In this study, the α1 subunit, the β2S subunit lacking 47 amino acid residues compared with the β2L subunit, and the γ2 subunit having five transmembrane domains were isolated from zebrafish Danio rerio. The responses of homomeric and heteromeric (α1, β2S and γ2) channels to agonists and GABA A R-targeted compounds were detected with two-electrode voltage clamp. Dose-dependent responses were observed in heteromeric α1β2S, β2Sγ2, and α1β2Sγ2 GABAR channels with EC 50 values at 21.75, 6291, and 33.69 μM for GABA-induced current and 3.28, 155.5, and 3.79 mM for β-alanine-induced current, respectively. However, no response was induced by benzamidine in all GABAR channels. Abamectin, dieldrin, fluralaner and fipronil could strongly inhibited GABA-induced inward current ≥50% at 10−6 M, while α-endosulfan, flufiprole and ethiprole only inhibited GABA-induced current <50%. This study has clarified the interaction of insecticides with the heteromeric GABA A R channel, which could help us further explore the potential function and toxicological importance of GABA A Rs from D. rerio. [ABSTRACT FROM AUTHOR]

Abstract Owing to the high incidence of multi-drug resistance and challenges posed by the complex and long duration of treatments, Mycobacterium tuberculosis (Mtb) infections remain a significant clinical burden, which would benefit from development of novel immuno-therapeutic-based treatment strategies. Among early immune effectors, invariant or innate-like (i)T cells are attracting attention because of their potential regulatory activity, which can shape anti-mycobacterial immune responses. Unlike conventional T cells, iT cells express a semi-invariant T cell receptor, and respond rapidly and robustly to molecular patterns presented by MHC class I-like molecules. To date, functional studies of iT cells in vivo has been problematic and the role of iT cells in anti- Mtb responses remains unclear. Here, after reviewing the recent literature on anti-mycobacterial iT cell immunity, we describe a novel alternative model system in the amphibian Xenopus laevis tadpoles during infection with Mycobacterium marinum (Mm). X. laevis tadpoles rely mostly on a few distinct prominent innate-like (i)T cell subsets, whose development and function are governed by distinct MHC class I-like molecules. Thus, X. laevis tadpoles provide a convenient and cost-effective in vivo model uniquely suited to investigate the roles of iT cells during mycobacterial infections. We have developed reverse genetics and MHC tetramer technology to characterize this MHC-like/iT system in tadpoles. Our study in X. laevis provides evidence of a conserved convergent function of iT cells in host defenses against mycobacteria between mammals and amphibians. Highlights • X. laevis tadpoles rely on prominent MHC class I-like iT cell-mediated immunity. • Tadpoles are uniquely suited to investigate anti-mycobacterial iT cell immunity. • Tadpoles serve as an alternative model for study of immunity against Mtb. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract In the field of nicotinic acetylcholine receptors (nAChRs), recognized as important therapeutic targets, much effort has been dedicated to the development of nicotinic analogues to agonize or antagonize distinct homo- and heteropentamers nAChR subtypes, selectively. In this work we developed di- and heptavalent nicotinic derivatives based on ethylene glycol (EG) and cyclodextrin cores, respectively. The compounds showed a concentration dependent inhibition of acetylcholine-induced currents on α7 nAChR expressed by Xenopus oocytes. Interesting features were observed with the divalent nicotinic derivatives, acting as antagonists with varied inhibitory concentrations (IC 50) in function of the spacer arm length. The best divalent compounds showed a 16-fold lowered IC 50 compared to the monovalent reference (12 vs 195 µM). Docking investigations provide guidelines to rationalize these experimental findings. [ABSTRACT FROM AUTHOR]

Highlights • SOCS3 and SOCS2 mRNA increased in retinal ganglion cells during axon regeneration. • SOCS3 protein remained unchanged in ganglion cell bodies but increased in axons. • SOCS2 protein did the opposite. • No other orthologs to mammalian SOCS genes changed in expression during regeneration. Abstract In vertebrates from fishes to mammals, optic nerve injury induces increased expression of Suppressor of Cytokine Signaling 3 (SOCS3) mRNA, a modulator of cytokine signaling that is known to inhibit CNS axon regeneration. Unlike amniotes, however, anamniotes successfully regenerate optic axons, despite this increase. To address this seeming paradox, we examined the SOCS3 response to optic nerve injury in the frog, Xenopus laevis , at both the mRNA and protein levels. Far from being only transiently induced, SOCS3 mRNA expression increased throughout regeneration in retinal ganglion cells, but immunostaining and Western blots indicated that this increase was reflected at the protein level in regenerating optic axons but not in ganglion cell bodies. Polysome profiling provided additional evidence that SOCS3 protein levels were regulated post-translationally by demonstrating that the mRNA was efficiently translated in the injured eye. In tumor cells, another member of the SOCS gene family, SOCS2 , is known to mediate SOCS3 degradation by targeting it for proteasomal degradation. Unlike the SOCS2 response in mammalian optic nerve injury, SOCS2 expression increased in Xenopus retinal ganglion cells after injury, at both the mRNA and protein levels; it was, however, largely absent from both uninjured and regenerating optic axons. We propose a similar degradation mechanism may be spatially restricted in Xenopus to keep SOCS3 protein levels sufficiently in check within ganglion cell bodies, where SOCS3 would otherwise inhibit transcription of genes needed for regeneration, but allow them to rise within the axons, where SOCS3 has pro-regenerative effects. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract Competitive antagonists (CAs) of ionotropic GABA receptors (GABARs) reportedly exhibit insecticidal activity and have potential for development as novel insecticides for overcoming emerging resistance to traditional GABAR-targeting insecticides. Our previous studies demonstrated that 4,5-disubstituted 3-isoxazolols or 3-isothiazolols are an important class of insect GABAR CAs. In the present study, we synthesized a series of 4-aryl-5-carbamoyl-3-isoxazolols and examined their antagonism of insect GABARs expressed in Xenopus oocytes. Several of these 3-isoxazolols exhibited potent antagonistic activities against housefly and common cutworm GABARs, with IC 50 values in the low-micromolar range in both receptors. 4-(3-Amino-4-methylphenyl)-5-carbamoyl-3-isoxazolol (3u) displayed the highest antagonism, with IC 50 values of 2.0 and 0.9 μM in housefly and common cutworm GABARs, respectively. Most of the synthesized 3-isoxazolols showed moderate larvicidal activities against common cutworms, with more than 50% mortality at 100 μg/g. These results indicate that 4-monocyclic aryl-5-carbamoyl-3-isoxazolol is a promising scaffold for insect GABAR CA discovery and provide important information for the design and development of GABAR-targeting insecticides with a novel mode of action. [ABSTRACT FROM AUTHOR]

Abstract The neural crest (NC) is a transient embryonic cell population that migrates extensively during development. Ric-8A, a guanine nucleotide exchange factor (GEF) for different Gα subunits regulates cranial NC (CNC) cell migration in Xenopus through a mechanism that still remains to be elucidated. To properly migrate, CNC cells establish an axis of polarization and undergo morphological changes to generate protrusions at the leading edge and retraction of the cell rear. Here, we aim to study the role of Ric-8A in cell polarity during CNC cell migration by examining whether its signaling affects the localization of GTPase activity in Xenopus CNC using GTPase-based probes in live cells and aPKC and Par3 as polarity markers. We show that the levels of Ric-8A are critical during migration and affect the localization of polarity markers and the subcellular localization of GTPase activity, suggesting that Ric-8A, probably through heterotrimeric G-protein signaling, regulates cell polarity during CNC migration. Highlights • Ric-8A levels are crucial for cell polarity establishment during CNC cell migration. • Ric-8A morphant cells present active Rac1 at cell-cell contacts. • Par3 lost its localization at cell-cell contacts in Ric-8A morphant explants. • In CNC control explants, aPKC is concentrated in the cell cortex at the leading edge. [ABSTRACT FROM AUTHOR]

Abstract The frog neuromuscular junction (NMJ) has been extensively used as a model system to dissect the mechanisms involved in synapse formation, maturation, maintenance, regeneration, and function. Early NMJ synaptogenesis relies on a combination of cell-autonomous and interdependent pre/postsynaptic communication processes. Due to their transparency, comparatively easy manipulation, and remarkable regenerative abilities, frog tadpoles constitute an excellent model to study NMJ formation and regeneration. Here, we aimed to contribute new aspects on the characterization of the ontogeny of NMJ formation in Xenopus embryos and to explore the morphological changes occurring at the NMJ after spinal cord injury. Following analyses of X. tropicalis tadpoles during development we found that the early pathfinding of rostral motor axons is likely helped by previously formed postsynaptic specializations, whereas NMJ formation in recently differentiated ventral muscles in caudal segments seems to rely on presynaptic inputs. After spinal cord injury of X. laevis tadpoles our results suggest that rostral motor axon projections help caudal NMJ re-innervation before spinal cord connectivity is repaired. Highlights • In Xenopus embryonic NMJs, rostral motor axons innervate preformed AChR clusters. • Presynaptic axons drive NMJ formation in ventral muscles of new caudal segments. • After SCI, rostral motor axon collaterals help caudal NMJ re-innervation. [ABSTRACT FROM AUTHOR]

Abstract The main purpose of regenerative biology is to improve human health by exploiting cellular and molecular mechanisms favoring tissue repair. In recent years, non-mammalian vertebrates have emerged as powerful model organisms to tackle the problem of tissue regeneration. Here, we analyze the process of bone repair in metamorphosing Xenopus tropicalis tadpoles subjected to traumatic skull injury. Five days after skull perforation, a dense and highly vascularized mesenchymal is apparent over the injury site. Using an in vivo bone staining procedure based on independent pulses of Alizarin red and Calcein green, we show that the deposition of new bone matrix completely closes the wound in 15 days. The absence of cartilage implies that bone repair follows an intramembranous ossification route. Collagen second harmonic imaging reveals that while a well-organized lamellar type of bone is deposited during development, a woven type of bone is produced during the early-phase of the regeneration process. Osteoblasts lying against the regenerating bone robustly express fibrillar collagen 1a1 , SPARC and Dlx5. These analyses establish Xenopus tropicalis as a new model system to improve traumatic skull injury recovery. Highlights • A novel traumatic skull injury assay was implemented in Xenopus tropicalis tadpoles and froglets. • A dense and highly irrigated mesenchymal plug forms over the wound in 5 days. • A woven type of bone closes the lesion in 15 days, and subsequently converts into lamellar bone. • Bone repair occurs in the absence of cartilage. • Osteoblasts involved in regeneration robustly express Collagen 1a1 , SPARC and Dlx5 transcripts. [ABSTRACT FROM AUTHOR]

An instructive role for metabolism in embryonic patterning is emerging, although a role for mitochondria is poorly defined. We demonstrate that mitochondrial oxidative metabolism establishes the embryonic patterning center, the Spemann-Mangold Organizer, via hypoxia-inducible factor 1α (Hif-1α) in Xenopus. Hypoxia or decoupling ATP production from oxygen consumption expands the Organizer by activating Hif-1α. In addition, oxygen consumption is 20% higher in the Organizer than in the ventral mesoderm, indicating an elevation in mitochondrial respiration. To reconcile increased mitochondrial respiration with activation of Hif-1α, we discovered that the "free" c-subunit ring of the F 1 F o ATP synthase creates an inner mitochondrial membrane leak, which decouples ATP production from respiration at the Organizer, driving Hif-1α activation there. Overexpression of either the c-subunit or Hif-1α is sufficient to induce Organizer cell fates even when β-catenin is inhibited. We propose that mitochondrial leak metabolism could be a general mechanism for activating Hif-1α and Wnt signaling. [Display omitted] • The Spemann-Mangold Organizer has elevated oxygen consumption • A mitochondrial membrane leak activates Hif-1α in the Organizer • The mitochondrial leak is caused by the "free" c-subunit of F 1 F o ATP synthase • Hif-1α is sufficient to induce Organizer cell fates and activate Wnt reporters MacColl Garfinkel et al. define an instructive role for mitochondrial metabolism in establishing the Spemann-Mangold Organizer in Xenopus. A mitochondrial inner membrane leak leads to a local increase in oxygen consumption and activation of Hif-1α. Hif-1α, which can activate a Wnt reporter, induces Organizer gene expression. [ABSTRACT FROM AUTHOR]