A spatiotemporal barrier formed by Follistatin is required for left-right patterning.

How left-right (LR) asymmetry emerges in a patterning field along the anterior-posterior axis remains an unresolved problem in developmental biology. Left-biased Nodal emanating from the LR organizer propagates from posterior to anterior (PA) and establishes the LR pattern of the whole embryo. However, little is known about the regulatory mechanism of the PA spread of Nodal and its asymmetric activation in the forebrain. Here, we identify bilaterally expressed Follistatin (Fst) as a regulator blocking the propagation of the zebrafish Nodal ortholog Southpaw (Spaw) in the right lateral plate mesoderm (LPM), and restricting Spaw transmission in the left LPM to facilitate the establishment of a robust LR asymmetric Nodal patterning. In addition, Fst inhibits the Activin-Nodal signaling pathway in the forebrain thus preventing Nodal activation prior to the arrival, at a later time, of Spaw emanating from the left LPM. This contributes to the orderly propagation of asymmetric Nodal activation along the PA axis. The LR regulation function of Fst is further confirmed in chick and frog embryos. Overall, our results suggest that a robust LR patterning emerges by counteracting a Fst barrier formed along the PA axis.

Septin 7 is a centrosomal protein that ensures S phase entry and microtubule nucleation by maintaining the abundance of p150glued.

Septins play important roles in regulating development and differentiation. Septin 7 (SEPT7) is a crucial component in orchestrating the septin core complex into highly ordered filamentous structures. Here, we showed that genetic depletion of SEPT7 or treatment with forchlorfenuron (FCF; a compound known to affect septin filament assembly) led to reduced the S phase entry in cell models and zebrafish embryos. In addition to colocalizing with actin filaments, SEPT7 resided in the centrosome, and SEPT7 depletion led to aberrant mitotic spindle pole formation. This mitotic defect was rescued in SEPT7-deficient cells by wild-type SEPT7, suggesting that SEPT7 maintained mitotic spindle poles. In addition, we observed disorganized microtubule nucleation and reduced cell migration with SEPT7 depletion. Furthermore, SEPT7 formed a complex with and maintained the abundance of p150glued , the component of centriole subdistal appendages. Depletion of p150glued resulted in a phenotype reminiscent of SEPT7-deficient cells, and overexpression of p150glued reversed the defective phenotypes. Thus, SEPT7 is a centrosomal protein that maintains proper cell proliferation and microtubule array formation via maintaining the abundance of p150glued .

Sodium phenylbutyrate inhibits Schwann cell inflammation via HDAC and NFκB to promote axonal regeneration and remyelination.

BACKGROUND: Epigenetic regulation by histone deacetylases (HDACs) in Schwann cells (SCs) after injury facilitates them to undergo de- and redifferentiation processes necessary to support various stages of nerve repair. Although de-differentiation activates the synthesis and secretion of inflammatory cytokines by SCs to initiate an immune response during nerve repair, changes in either the timing or duration of prolonged inflammation mediated by SCs can affect later processes associated with repair and regeneration. Limited studies have investigated the regulatory processes through which HDACs in SCs control inflammatory cytokines to provide a favorable environment for peripheral nerve regeneration. METHODS: We employed the HDAC inhibitor (HDACi) sodium phenylbutyrate (PBA) to address this question in an in vitro RT4 SC inflammation model and an in vivo sciatic nerve transection injury model to examine the effects of HDAC inhibition on the expression of pro-inflammatory cytokines. Furthermore, we assessed the outcomes of suppression of extended inflammation on the regenerative potential of nerves by assessing axonal regeneration, remyelination, and reinnervation. RESULTS: Significant reductions in lipopolysaccharide (LPS)-induced pro-inflammatory cytokine (tumor necrosis factor-α [TNFα]) expression and secretion were observed in vitro following PBA treatment. PBA treatment also affected the transient changes in nuclear factor κB (NFκB)-p65 phosphorylation and translocation in response to LPS induction in RT4 SCs. Similarly, PBA mediated long-term suppressive effects on HDAC3 expression and activity. PBA administration resulted in marked inhibition of pro-inflammatory cytokine secretion at the site of transection injury when compared with that in the hydrogel control group at 6-week post-injury. A conducive microenvironment for axonal regrowth and remyelination was generated by increasing expression levels of protein gene product 9.5 (PGP9.5) and myelin basic protein (MBP) in regenerating nerve tissues. PBA administration increased the relative gastrocnemius muscle weight percentage and maintained the intactness of muscle bundles when compared with those in the hydrogel control group. CONCLUSIONS: Suppressing the lengthened state of inflammation using PBA treatment favors axonal regrowth and remyelination following nerve transection injury. PBA treatment also regulates pro-inflammatory cytokine expression by inhibiting the transcriptional activation of NFκB-p65 and HDAC3 in SCs in vitro.

Investigation of Neuropathology after Nerve Release in Chronic Constriction Injury of Rat Sciatic Nerve.

Peripheral compressive neuropathy causes significant neuropathic pain, muscle weakness and prolong neuroinflammation. Surgical decompression remains the gold standard of treatment but the outcome is suboptimal with a high recurrence rate. From mechanical compression to chemical propagation of the local inflammatory signals, little is known about the distinct neuropathologic patterns and the genetic signatures after nerve decompression. In this study, controllable mechanical constriction forces over rat sciatic nerve induces irreversible sensorimotor dysfunction with sustained local neuroinflammation, even 4 weeks after nerve release. Significant gene upregulations are found in the dorsal root ganglia, regarding inflammatory, proapoptotic and neuropathic pain signals. Genetic profiling of neuroinflammation at the local injured nerve reveals persistent upregulation of multiple genes involving oxysterol metabolism, neuronal apoptosis, and proliferation after nerve release. Further validation of the independent roles of each signal pathway will contribute to molecular therapies for compressive neuropathy in the future.

Involvement of BIG1 and BIG2 in regulating VEGF expression and angiogenesis.

VEGF stimulates the formation of new blood vessels by inducing endothelial cell (EC) proliferation and migration. Brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein (BIG)1 and 2 accelerate the replacement of bound GDP with GTP to activate ADP-ribosylation factor (Arf)1, which regulates vesicular transport between the Golgi and plasma membrane. Although it has been reported that treating cells with BFA interferes with Arf1 activation to inhibit VEGF secretion, the role of BIG1 and BIG2 in VEGF trafficking and expression, EC migration and proliferation, and vascular development remains unknown. Here, we found that inactivation of Arf1 reduced VEGF secretion but did not affect the levels of VEGF protein. Interestingly, however, BIG1 and BIG2 knockdown significantly decreased the levels of VEGF mRNA and protein in glioblastoma U251 cells and HUVECs. Furthermore, depletion of BIG1 and BIG2 inhibited HUVEC angiogenesis by diminishing cell migration. Angioblast migration and intersegmental vessel sprouting were also impaired when the BIG2 homolog, Arf guanine nucleotide exchange factor (arfgef)2, was knocked down in zebrafish with endothelial expression of green fluorescent protein (GFP). Depletion of arfgef2 by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) also caused defects in vascular development of zebrafish embryos. Taken together, these data reveal that BIG1 and BIG2 participate in endothelial cell angiogenesis.-Lu, F.-I., Wang, Y.-T., Wang, Y.-S., Wu, C.-Y., Li, C.-C. Involvement of BIG1 and BIG2 in regulating VEGF expression and angiogenesis.

The Effect of the Chorion on Size-Dependent Acute Toxicity and Underlying Mechanisms of Amine-Modified Silver Nanoparticles in Zebrafish Embryos.

As the worldwide application of nanomaterials in commercial products increases every year, various nanoparticles from industry might present possible risks to aquatic systems and human health. Presently, there are many unknowns about the toxic effects of nanomaterials, especially because the unique physicochemical properties of nanomaterials affect functional and toxic reactions. In our research, we sought to identify the targets and mechanisms for the deleterious effects of two different sizes (~10 and ~50 nm) of amine-modified silver nanoparticles (AgNPs) in a zebrafish embryo model. Fluorescently labeled AgNPs were taken up into embryos via the chorion. The larger-sized AgNPs (LAS) were distributed throughout developing zebrafish tissues to a greater extent than small-sized AgNPs (SAS), which led to an enlarged chorion pore size. Time-course survivorship revealed dose- and particle size-responsive effects, and consequently triggered abnormal phenotypes. LAS exposure led to lysosomal activity changes and higher number of apoptotic cells distributed among the developmental organs of the zebrafish embryo. Overall, AgNPs of ~50 nm in diameter exhibited different behavior from the ~10-nm-diameter AgNPs. The specific toxic effects caused by these differences in nanoscale particle size may result from the different mechanisms, which remain to be further investigated in a follow-up study.

Cell Cycle-Dependent Localization of Dynactin Subunit p150 glued at Centrosome.

p150(glued) is the largest subunit of dynactin protein complex, through which cargo vesicles link to the microtubule minus-end directed motor protein dynein. In addition, p150(glued) also locates in the mother centriole where it organizes the subdistal appendage. The components of appendage are dynamically regulated throughout the cell cycle stages, but it is still unclear whether the centrosomal residency of p150(glued) correlated with cell cycle progression. Here we found that p150(glued) was located in the mother centriole during G1/S stage and its centrosomal residency was independent of microtubule transportation. However, the centrosomal p150(glued) became blurred at G2/M phase and this event was not regulated by its phosphorylation. Entering into mitosis, p150(glued) was robustly enriched in the mitotic spindle nearby the spindle poles but not in the centrosome. During serum starvation (G0 stage), p150(glued) appeared at the base of primary cilium and its depletion attenuated starvation-induced primary cilium formation. We also checked its role in the maintenance of centrosome homeostasis and configuration, and found depletion of p150(glued) did not induce centrosome amplification or splitting but inhibited U2OS cell growth. G1 arrest and reduced EdU incorporation were observed in p150(glued) deficient U2OS cells. In addition, cyclin E was downregulated following p150(glued) depletion. The p53/p21 signaling was activated indicating that CDKs were inactivated. The reduced cell growth was ameliorated in the p150(glued) depleted cells when treated with p53 inhibitor. Thus, we have identified the centrosomal targeting of p150(glued) in distinct cell cycle stage and uncovered its role in controlling G1/S transition.

Identification and mechanism of regulation of the zebrafish dorsal determinant.

In vertebrates, the animal-vegetal axis is determined during oogenesis and at ovulation, the egg is radially symmetric. In anamniotes, following fertilization, a microtubule-dependent movement leads to the displacement of maternal dorsal determinants from the vegetal pole to the future dorsal side of the embryo, providing the initial breaking of radial symmetry [Weaver C, Kimelman D (2004) Development 131:3491-3499]. These dorsal determinants induce ß-catenin nuclear translocation in dorsal cells of the blastula. Previous work in amphibians has shown that secreted Wnt11/5a complexes, regulated by the Wnt antagonist Dkk-1, are required for the initiation of embryonic axis formation [Cha et al. (2009) Curr Biol 29:1573-1580]. In the current study, we determined that the vegetal maternal dorsal determinant in fish is not the Wnt11/5a complex but the canonical Wnt, Wnt8a. Translation of this mRNA and secretion of the Wnt8a protein result in a dorsal-to-ventral gradient of Wnt stimulation, extending across the entire embryo. This gradient is counterbalanced by two Wnt inhibitors, Sfrp1a and Frzb. These proteins are essential to restrict the activation of the canonical Wnt pathway to the dorsal marginal blastomeres by defining the domain where the Wnt8a activity gradient is above the threshold value necessary for triggering the canonical ß-catenin pathway. In summary, this study establishes that the zebrafish maternal dorsal determinant, Wnt8a, is required to localize the primary dorsal center, and that the extent of this domain is defined by the activity of two maternally provided Wnt antagonists, Sfrp1a and Frzb.

Rice straw-derived chitosan-enhanced plasticizers as biologically and environmentally friendly alternatives for sustainable materials.

Plasticizers like Bis(2-ethylhexyl)phthalate (DEHP) are commonly used to enhance plastic properties but pose environmental and health risks. This study successfully derived plasticizers X and Y from rice straws, demonstrating efficacy in chitosan polymer coatings. Chitosan-based polymers exhibit exceptional hardness, with a value of 300 MPa, due to their enriched structure and robust chitosan bonding. This surpasses the hardness of DEHP. Zebrafish exposure over 5 days revealed that X and Y had no significant behavioral impact, while DEHP caused noticeable toxic effects. Maternal DEHP exposure reduced placental cell growth, unlike X and Y, which had no adverse effects on uterine differentiation or placenta formation, suggesting their safety in human pregnancy. The successful development of X and Y represents a crucial step towards greener plasticizers, addressing environmental concerns and promoting safer alternatives in various industries.

Histone Trimethylations and HDAC5 Regulate Spheroid Subpopulation and Differentiation Signaling of Human Adipose-Derived Stem Cells.

Human adipose-derived stem cells (ASCs) have shown immense potential for regenerative medicine. Our previous work demonstrated that chitosan nano-deposited surfaces induce spheroid formation and differentiation of ASCs for treating sciatic nerve injuries. However, the underlying cell fate and differentiation mechanisms of ASC-derived spheroids remain unknown. Here, we investigate the epigenetic regulation and signaling coordination of these therapeutic spheroids. During spheroid formation, we observed significant increases in histone 3 trimethylation at lysine 4 (H3K4me3), lysine 9 (H3K9me3), and lysine 27 (H3K27me3), accompanied by increased histone deacetylase (HDAC) activities and decreased histone acetyltransferase activities. Additionally, HDAC5 translocated from the cytoplasm to the nucleus, along with increased nuclear HDAC5 activities. Utilizing single-cell RNA sequencing (scRNA-seq), we analyzed the chitosan-induced ASC spheroids and discovered distinct cluster subpopulations, cell fate trajectories, differentiation traits, and signaling networks using the 10x Genomics platform, R studio/language, and the Ingenuity Pathway Analysis (IPA) tool. Specific subpopulations were identified within the spheroids that corresponded to a transient reprogramming state (Cluster 6) and the endpoint cell state (Cluster 3). H3K4me3 and H3K9me3 were discovered as key epigenetic regulators by IPA to initiate stem cell differentiation in Cluster 6 cells, and confirmed by qPCR and their respective histone methyltransferase inhibitors: SNDX-5613 (a KMT2A inhibitor for H3K4me3) and SUVi (an SUV39H1 inhibitor for H3K9me3). Moreover, H3K9me3 and HDAC5 were involved in regulating downstream signaling and neuronal markers during differentiation in Cluster 3 cells. These findings emphasize the critical role of epigenetic regulation, particularly H3K4me3, H3K9me3, and HDAC5, in shaping stem cell fate and directing lineage-specific differentiation.

Generational effects and abnormalities in craniofacial chondrogenesis in zebrafish (Danio rerio) embryos upon maternal exposure to estrogen endocrine disrupting chemicals.

Bisphenol A (BPA) and diethyl phthalate (DEP) are estrogenic endocrine disrupting chemicals (EEDCs). The present study reconfirmed that the angle of the ceratohyal cartilage (CH) in embryos were larger from maternal BPA and E2, but smaller from DEP compared to the control. However, it is still unknown whether both the BPA and DEP chemicals disrupted the action of E2 and thereby influence the estrogen signaling pathways. Additionally, it remains unclear whether they also disrupted certain related genes in the migratory pathways of neural crest cells (NCCs) in their offspring. The present data showed that nuclear estrogen receptors and membrane estrogen receptors have different disrupted profiles among female zebrafish exposed to BPA (F-BPA), and DEP (F-DEP), and external E2 (F-E2). However, certain related genes in the migratory pathways of NCCs in embryos from F-BPA and F-E2 such as the sox10, chm1, and tgfbr1a mRNA expressions showed a positive relationship compared with CH angles; the gene expressions of sox9a, smad3, and col2a1a and the CH angles of embryos exhibited an opposite relationship upon F-DEP treatments. Thus, we suggested that the genes involved in NCCs migration were potentially induced by the residual maternal DEP contents. Two sets of genes, chm1/tgfb3 and chm1/gper1, exhibited an identical profile in the ovary and its offspring at 2 h of post fertilization upon F-E2 and F-BPA treatments, respectively. We suggested that the maternal mRNA from female to embryos were transferred before the maternal-to-zygotic transition stage.

Adipose-derived stem cells modulate neuroinflammation and improve functional recovery in chronic constriction injury of the rat sciatic nerve.

Introduction: Compressive neuropathy, a common chronic traumatic injury of peripheral nerves, leads to variable impairment in sensory and motor function. Clinical symptoms persist in a significant portion of patients despite decompression, with muscle atrophy and persistent neuropathic pain affecting 10%-25% of cases. Excessive inflammation and immune cell infiltration in the injured nerve hinder axon regeneration and functional recovery. Although adipose-derived stem cells (ASCs) have demonstrated neural regeneration and immunomodulatory potential, their specific effects on compressive neuropathy are still unclear. Methods: We conducted modified CCI models on adult male Sprague-Dawley rats to induce irreversible neuropathic pain and muscle atrophy in the sciatic nerve. Intraneural ASC injection and nerve decompression were performed. Behavioral analysis, muscle examination, electrophysiological evaluation, and immunofluorescent examination of the injured nerve and associated DRG were conducted to explore axon regeneration, neuroinflammation, and the modulation of inflammatory gene expression. Transplanted ASCs were tracked to investigate potential beneficial mechanisms on the local nerve and DRG. Results: Persistent neuropathic pain was induced by chronic constriction of the rat sciatic nerve. Local ASC treatment has demonstrated robust beneficial outcomes, including the alleviation of mechanical allodynia, improvement of gait, regeneration of muscle fibers, and electrophysiological recovery. In addition, locally transplanted ASCs facilitated axon remyelination, alleviated neuroinflammation, and reduced inflammatory cell infiltration of the injured nerve and associated dorsal root ganglion (DRG). Trafficking of the transplanted ASC preserved viability and phenotype less than 7 days but contributed to robust immunomodulatory regulation of inflammatory gene expression in both the injured nerve and DRG. Discussion: Locally transplanted ASC on compressed nerve improve sensory and motor recoveries from irreversible chronic constriction injury of rat sciatic nerve via alleviation of both local and remote neuroinflammation, suggesting the promising role of adjuvant ASC therapies for clinical compressive neuropathy.

Therapeutic potential of nanoceria pretreatment in preventing the development of urological chronic pelvic pain syndrome: Immunomodulation via reactive oxygen species scavenging and SerpinB2 downregulation.

Urological chronic pelvic pain syndrome (UCPPS) manifests as pelvic pain with frequent urination and has a 10% prevalence rate without effective therapy. Nanoceria (cerium oxide nanoparticles [CNPs]) were synthesized in this study to achieve potential long-term pain relief, using a commonly used UCPPS mouse model with cyclophosphamide-induced cystitis. Transcriptome sequencing analysis revealed that serpin family B member 2 (SerpinB2) was the most upregulated marker in mouse bladder, and SerpinB2 was downregulated with CNP pretreatment. The transcriptome sequencing analysis results agreed with quantitative polymerase chain reaction and western blot analysis results for the expression of related mRNAs and proteins. Analysis of Gene Expression Omnibus (GEO) datasets revealed that SerpinB2 was a differentially upregulated gene in human UCPPS. In vitro SerpinB2 knockdown downregulated proinflammatory chemokine expression (chemokine receptor CXCR3 and C-X-C motif chemokine ligand 10) upon treatment with 4-hydroperoxycyclophosphamide. In conclusion, CNP pretreatment may prevent the development of UCPPS, and reactive oxygen species (ROS) scavenging and SerpinB2 downregulation may modulate the immune response in UCPPS.

Identification of Novel Vascular Genes Downstream of Islet2 and Nr2f1b Transcription Factors.

The genetic regulation of vascular development is not elucidated completely. We previously characterized the transcription factors Islet2 (Isl2) and Nr2f1b as being critical for vascular growth. In this study, we further performed combinatorial microarrays to identify genes that are potentially regulated by these factors. We verified the changed expression of several targets in isl2/nr2f1b morphants. Those genes expressed in vessels during embryogenesis suggested their functions in vascular development. We selectively assayed a potential target follistatin a (fsta). Follistatin is known to inhibit BMP, and BMP signaling has been shown to be important for angiogenesis. However, the fsta's role in vascular development has not been well studied. Here, we showed the vascular defects in ISV growth and CVP patterning while overexpressing fsta in the embryo, which mimics the phenotype of isl2/nr2f1b morphants. The vascular abnormalities are likely caused by defects in migration and proliferation. We further observed the altered expression of vessel markers consistent with the vascular defects in (fli:fsta) embryos. We showed that the knockdown of fsta can rescue the vascular defects in (fli:fsta) fish, suggesting the functional specificity of fsta. Moreover, the decreased expression of fsta rescues abnormal vessel growth in isl2 and nr2f1b morphants, indicating that fsta functions downstream of isl2/nr2f1b. Lastly, we showed that Isl2/Nr2f1b control vascular development, via Fsta-BMP signaling in part. Collectively, our microarray data identify many interesting genes regulated by isl2/nr2f1b, which likely function in the vasculature. Our research provides useful information on the genetic control of vascular development.

Autologous Platelet-Rich Growth Factor Reduces M1 Macrophages and Modulates Inflammatory Microenvironments to Promote Sciatic Nerve Regeneration.

The failure of peripheral nerve regeneration is often associated with the inability to generate a permissive molecular and cellular microenvironment for nerve repair. Autologous therapies, such as platelet-rich plasma (PRP) or its derivative platelet-rich growth factors (PRGF), may improve peripheral nerve regeneration via unknown mechanistic roles and actions in macrophage polarization. In the current study, we hypothesize that excessive and prolonged inflammation might result in the failure of pro-inflammatory M1 macrophage transit to anti-inflammatory M2 macrophages in large nerve defects. PRGF was used in vitro at the time the unpolarized macrophages (M0) macrophages were induced to M1 macrophages to observe if PRGF altered the secretion of cytokines and resulted in a phenotypic change. PRGF was also employed in the nerve conduit of a rat sciatic nerve transection model to identify alterations in macrophages that might influence excessive inflammation and nerve regeneration. PRGF administration reduced the mRNA expression of tumor necrosis factor-α (TNFα), interleukin-1ß (IL-1ß), and IL-6 in M0 macrophages. Increased CD206 substantiated the shift of pro-inflammatory cytokines to the M2 regenerative macrophage. Administration of PRGF in the nerve conduit after rat sciatic nerve transection promoted nerve regeneration by improving nerve gross morphology and its targeted gastrocnemius muscle mass. The regenerative markers were increased for regrown axons (protein gene product, PGP9.5), Schwann cells (S100ß), and myelin basic protein (MBP) after 6 weeks of injury. The decreased expression of TNFα, IL-1ß, IL-6, and CD68+ M1 macrophages indicated that the inflammatory microenvironments were reduced in the PRGF-treated nerve tissue. The increase in RECA-positive cells suggested the PRGF also promoted angiogenesis during nerve regeneration. Taken together, these results indicate the potential role and clinical implication of autologous PRGF in regulating inflammatory microenvironments via macrophage polarization after nerve transection.

Repairing TALEN-mediated double-strand break by microhomology-mediated recombination in tobacco plastids generates abundant subgenomic DNA.

Transcription activator-like effector nuclease (TALEN) technology has been widely used to edit nuclear genomes in plants but rarely for editing organellar genomes. In addition, ciprofloxacin, commonly used to cause the double-strand break of organellar DNA for studying the repair mechanism in plants, confers no organellar selectivity and site-specificity. To demonstrate the feasibility of TALEN-mediated chloroplast DNA editing and to use it for studying the repair mechanism in plastids, we developed a TALEN-mediated editing technology fused with chloroplast transit peptide (cpTALEN) to site-specifically edit the rpoB gene via Agrobacteria-mediated transformation of tobacco leaf. Transgenic plants showed various degrees of chlorotic phenotype. Repairing damaged plastid DNA resulted in point mutation, large deletion and small inversion surrounding the rpoB gene by homologous recombination and/or microhomology-mediated recombination. In an albino line, microhomology-mediated recombination via a pair of 12-bp direct repeats between rpoC2 and ycf2 genes generated the chimeric ycf2-rpoC2 subgenome, with the level about 3- to 5-fold higher for subgenomic DNA than ycf2. Additionally, the expression of chimeric ycf2-rpoC2 transcripts versus ycf2 mRNA agreed well with the level of corresponding DNA. The ycf2-rpoC2 subgenomic DNA might independently and preferentially replicate in plastids.

Genetic compensation of γ CaMKII, an evolutionarily conserved gene.

CaMKII is a Ca2+/CaM-dependent protein kinase encoded by a family of conserved genes found throughout all metazoan species and expressed from fertilization into adulthood. One of these genes, camk2g1, is particularly important during early development as determined by pharmacologic, dominant negative and antisense morpholino approaches in zebrafish. Four other teleost fish species (cavefish, medaka, stickleback, and tilapia), exhibit sequence conservation of camk2g1 and duplication of the same CaMKII genes. A homozygous mutant of camk2g1 was generated in zebrafish using TALEN technology but yielded none of the phenotypic alterations seen using all other approaches and was reproductively viable. However, these camk2g1 mutant embryos showed a 4-fold over-expression of its paralog camk2g2. None of the other camk2 genes showed such transcriptional elevation, in fact, some of these genes were suppressed to 10% of wild type levels. In contrast, G0 camk2g1 CRISPR/Cas9 embryos recapitulated nearly all of the altered phenotypes observed in camk2g1 morphants, including renal, aural and ciliary defects. These findings validate the importance of this gene family during early zebrafish development and provide evidence for gene-specific transcriptional cross-talk consistent with genetic compensation.

Comparison of Calcium Balancing Strategies During Hypothermic Acclimation of Tilapia (Oreochromis mossambicus) and Goldfish (Carassius auratus).

The body temperatures of teleost species fluctuate following changes in the aquatic environment. As such, decreased water temperature lowers the rates of biochemical reactions and affects many physiological processes, including active transport-dependent ion absorption. Previous studies have focused on the impacts of low temperature on the plasma ion concentrations or membrane transporters in fishes. However, very few in vivo or organism-level studies have been performed to more thoroughly elucidate the process of acclimation to low temperatures. In the present study, we compared the strategies for cold acclimation between stenothermic tilapia and eurythermic goldfish. Whole-body calcium content was more prominently diminished in tilapia than in goldfish after long-term cold exposure. This difference can be attributed to alterations in the transportation parameters for Ca2+ influx, i.e., maximum velocity (Vmax ) and binding affinity (1/Km ). There was also a significant difference in the regulation of Ca2+ efflux between the two fishes. Transcript levels for Ca2+ related transporters, including the Na+/Ca2+ exchanger and epithelial Ca2+ channel, were similarly regulated in both fishes. However, upregulation of plasma membrane Ca2+ATPase expression was more pronounced in goldfish than in tilapia. In addition, enhanced Na+/K+-ATPase abundance, which provides the major driving force for ion absorption, was only detected in tilapia, while upregulated Na+/K+-ATPase activity was only detected in goldfish. Based on the results of the present study, we have found that goldfish and tilapia differentially regulate gill epithelial plasma membrane Ca2+-ATPase (PMCA) expression and Na+/K+-ATPase activity in response to cold environments. These regulatory differences are potentially linked to more effective regulation of Ca2+ influx kinetics and better maintenance of whole body calcium content in goldfish than in tilapia.

Biopolymer Green Lubricant for Sustainable Manufacturing.

We report on the preparation of a biopolymer thin film by hydroxypropyl methylcellulose (HPMC), which can be used as a dry green lubricant in sustainable manufacturing. The thin films were characterized through scanning electron microscopy, energy-dispersive spectroscopy, and Raman spectroscopy; the films showed desirable levels of thickness, controllability, and uniformity. Tribology tests also showed desirable tribological and antiwear behaviors, caused by the formation of transfer layers. Zebrafish embryo toxicity studies showed that HPMC has excellent solubility and biocompatibility, which may show outstanding potential for applications as a green lubricant. The results of the present study show that these techniques for biopolymer HPMC provide an ecologically responsible and convenient method for preparing functional thin films, which is particularly applicable to sustainable manufacturing.

Tissue-specific derepression of TCF/LEF controls the activity of the Wnt/ß-catenin pathway.

Upon stimulation by Wnt ligands, the canonical Wnt/ß-catenin signalling pathway results in the stabilization of ß-catenin and its translocation into the nucleus to form transcriptionally active complexes with sequence-specific DNA-binding T-cell factor/lymphoid enhancer factor (TCF/LEF) family proteins. In the absence of nuclear ß-catenin, TCF proteins act as transcriptional repressors by binding to Groucho/Transducin-Like Enhancer of split (TLE) proteins that function as co-repressors by interacting with histone deacetylases whose activity leads to the generation of transcriptionally silent chromatin. Here we show that the transcription factor Ladybird homeobox 2 (Lbx2) positively controls the Wnt/ß-catenin signalling pathway in the posterior lateral and ventral mesoderm of the zebrafish embryo at the gastrula stage, by directly interfering with the binding of Groucho/TLE to TCF, thereby preventing formation of transcription repressor complexes. These findings reveal a novel level of regulation of the canonical Wnt/ß-catenin signalling pathway occurring in the nucleus and involving tissue-specific derepression of TCF by Lbx2.