Characterization of Xenopus laevis guanine deaminase reveals new insights for its expression and function in the embryonic kidney.

BACKGROUND: The mammalian guanine deaminase (GDA), called cypin, is important for proper neural development, by regulating dendritic arborization through modulation of microtubule (MT) dynamics. Additionally, cypin can promote MT assembly in vitro. However, it has never been tested whether cypin (or other GDA orthologs) binds to MTs or modulates MT dynamics. Here, we address these questions and characterize Xenopus laevis GDA (Gda) for the first time during embryonic development. RESULTS: We find that exogenously expressed human cypin and Gda both display a cytosolic distribution in primary embryonic cells. Furthermore, while expression of human cypin can promote MT polymerization, Xenopus Gda has no effect. Additionally, we find that the tubulin-binding collapsin response mediator protein (CRMP) homology domain is only partially conserved between cypin and Gda. This likely explains the divergence in function, as we discovered that the cypin region containing the CRMP homology and PDZ-binding domain is necessary for regulating MT dynamics. Finally, we observed that gda is strongly expressed in the kidneys during late embryonic development, although it does not appear to be critical for kidney development. CONCLUSIONS: Together, these results suggest that GDA has diverged in function between mammals and amphibians, and that mammalian GDA plays an indirect role in regulating MT dynamics. Developmental Dynamics 248:296-305, 2019. © 2019 Wiley Periodicals, Inc.

Neurofilaments form flexible bundles during neuritogenesis in culture and in mature axons in situ.

Neurofilaments (NFs) undergo cation-dependent phospho-mediated associations with each other and other cytoskeletal elements that support axonal outgrowth. Progressive NF-NF associations generate a resident, bundled population that undergoes exchange with transporting NFs. We examined the properties of bundled NFs. Bundles did not always display a fully linear profile but curved and twisted at various points along the neurite length. Bundles retracted faster than neurites and retracted bundles did not expand following extraction with Triton, indicating that they coiled passively rather than due to pressure from the cell. Bundles consisted of helically wound NFs, which may provide flexibility necessary for turning of growing axons during pathfinding. Interactions between NFs and other cytoskeletal elements may be disrupted en masse during neurite retraction or regionally during remodeling. It is suggested that bundles within long axons that cannot be fully retracted into the soma could provide maintain proximal support yet still allow more distal flexibility for remodeling and changing direction during pathfinding.

Divergent and convergent roles for kinases and phosphatases in neurofilament dynamics.

C-terminal neurofilament phosphorylation mediates cation-dependent self-association leading to neurofilament incorporation into the stationary axonal cytoskeleton. Multiple kinases phosphorylate the C-terminal domains of the heavy neurofilament subunit (NF-H), including cyclin-dependent protein kinase 5 (CDK5), mitogen-activated protein kinases (MAPKs), casein kinase 1 and 2 (CK1 and CK2) and glycogen synthase kinase 3ß (GSK3ß). The respective contributions of these kinases have been confounded because they phosphorylate multiple substrates in addition to neurofilaments and display extensive interaction. Herein, differentiated NB2a/d1 cells were transfected with constructs expressing GFP-tagged NF-H, isolated NF-H sidearms and NF-H lacking the distal-most 187 amino acids. Cultures were treated with roscovitine, PD98059, Li(+), D4476, tetrabromobenzotriazole and calyculin, which are active against CDK5, MKK1 (also known as MAP2K1), GSK3ß, CK1, CK2 and protein phosphatase 1 (PP1), respectively. Sequential phosphorylation by CDK5 and GSK3ß mediated the neurofilament-neurofilament associations. The MAPK pathway (i.e. MKK1 to ERK1/2) was found to downregulate GSK3ß, and CK1 activated PP1, both of which promoted axonal transport and restricted neurofilament-neurofilament associations to axonal neurites. The MAPK pathway and CDK5, but not CK1 and GSK3ß, inhibited neurofilament proteolysis. These findings indicate that phosphorylation of neurofilaments by the proline-directed MAPK pathway and CDK5 counterbalance the impact of phosphorylation of neurofilaments by the non-proline-directed CK1 and GSK3ß.

Environmental enrichment and social interaction improve cognitive function and decrease reactive oxidative species in normal adult mice.

Environmental stimulation and increased social interactions stimulate cognitive performance, while decrease in these parameters can exacerbate cognitive decline as a function of illness, injury, or age. We examined the impact of environmental stimulation and social interactions on cognitive performance in healthy adult C57B1/6J mice. Mice were housed for 1 month individually or in groups of three (to prevent or allow social interaction) in either a standard environment (SE) or an enlarged cage containing nesting material and items classically utilized to stimulate exploration and activity ("enriched environment"; EE). Cognitive performance was tested by Y maze navigation and Novel Object Recognition (NOR; which compares the relative amount of time mice spent investigating a novel vs. a familiar object). Mice maintained for 1 month under isolated conditions in the SE statistically declined in performance versus baseline in the Y maze (p < 0.02; ANOVA). Performance under all other conditions did not change from baseline. Maintenance in groups in the SE statistically improved NOR (p < 0.01), whereas maintenance in isolation in the SE did not alter performance from baseline. Maintenance in the EE statistically improved performance in NOR for mice housed in groups and individually (p < 0.01). Maintenance under isolated conditions slightly increased reactive oxygen/nitrogen species (ROS/RNS) in brain. Environmental enrichment did not influence ROS/RNS. These findings indicate that environmental and social enrichment can positively influence cognitive performance in healthy adult mice, and support the notion that proactive approaches may delay age-related cognitive decline.

Differential roles of kinesin and dynein in translocation of neurofilaments into axonal neurites.

Neurofilament (NF) subunits translocate within axons as short NFs, non-filamentous punctate structures ('puncta') and diffuse material that might comprise individual subunits and/or oligomers. Transport of NFs into and along axons is mediated by the microtubule (MT) motor proteins kinesin and dynein. Despite being characterized as a retrograde motor, dynein nevertheless participates in anterograde NF transport through associating with long MTs or the actin cortex through its cargo domain; relatively shorter MTs associated with the motor domain are then propelled in an anterograde direction, along with any linked NFs. Here, we show that inhibition of dynein function, through dynamitin overexpression or intracellular delivery of anti-dynein antibody, selectively reduced delivery of GFP-tagged short NFs into the axonal hillock, with a corresponding increase in the delivery of puncta, suggesting that dynein selectively delivered short NFs into axonal neurites. Nocodazole-mediated depletion of short MTs had the same effect. By contrast, intracellular delivery of anti-kinesin antibody inhibited anterograde transport of short NFs and puncta to an equal extent. These findings suggest that anterograde axonal transport of linear NFs is more dependent upon association with translocating MTs (which are themselves translocated by dynein) than is transport of NF puncta or oligomers.

Investigation of SIS epidemics on dynamic network models with temporary link deactivation control schemes.

Mathematical modeling of epidemic diseases is increasingly being used to respond to emerging diseases. Although conditions modeled by SIS dynamics will eventually reach either a disease-free steady-state or an endemic steady state without interventions, it is desirable to eradicate the disease as quickly as possible by introducing a control scheme. Here, we investigate the control methods of epidemic models on dynamic networks with temporary link deactivation. A quick link deactivation mechanism can simulate a community effort to reduce the risk of infection by temporarily avoiding infected neighbors. Once infected individuals recover, the links between the susceptible and recovered are activated. Our study suggests that a control scheme that has been shown ineffective in controlling dynamic network models may yield effective responses for networks with certain types of link dynamics, such as the temporary link deactivation mechanisms. We observe that a faster and more effective eradication could be achieved by updating control schemes frequently.

16p12.1 Deletion Orthologs are Expressed in Motile Neural Crest Cells and are Important for Regulating Craniofacial Development in Xenopus laevis.

Copy number variants (CNVs) associated with neurodevelopmental disorders are characterized by extensive phenotypic heterogeneity. In particular, one CNV was identified in a subset of children clinically diagnosed with intellectual disabilities (ID) that results in a hemizygous deletion of multiple genes at chromosome 16p12.1. In addition to ID, individuals with this deletion display a variety of symptoms including microcephaly, seizures, cardiac defects, and growth retardation. Moreover, patients also manifest severe craniofacial abnormalities, such as micrognathia, cartilage malformation of the ears and nose, and facial asymmetries; however, the function of the genes within the 16p12.1 region have not been studied in the context of vertebrate craniofacial development. The craniofacial tissues affected in patients with this deletion all derive from the same embryonic precursor, the cranial neural crest, leading to the hypothesis that one or more of the 16p12.1 genes may be involved in regulating neural crest cell (NCC)-related processes. To examine this, we characterized the developmental role of the 16p12.1-affected gene orthologs, polr3e, mosmo, uqcrc2, and cdr2, during craniofacial morphogenesis in the vertebrate model system, Xenopus laevis. While the currently-known cellular functions of these genes are diverse, we find that they share similar expression patterns along the neural tube, pharyngeal arches, and later craniofacial structures. As these genes show co-expression in the pharyngeal arches where NCCs reside, we sought to elucidate the effect of individual gene depletion on craniofacial development and NCC migration. We find that reduction of several 16p12.1 genes significantly disrupts craniofacial and cartilage formation, pharyngeal arch migration, as well as NCC specification and motility. Thus, we have determined that some of these genes play an essential role during vertebrate craniofacial patterning by regulating specific processes during NCC development, which may be an underlying mechanism contributing to the craniofacial defects associated with the 16p12.1 deletion.

Neurofilament cross-bridging competes with kinesin-dependent association of neurofilaments with microtubules.

The phosphorylation of neurofilaments (NFs) has long been considered to regulate their axonal transport rate and in doing so to provide stability to mature axons. Axons contain a centrally situated ;bundle' of closely opposed phospho-NFs that display a high degree of NF-NF associations and phospho-epitopes, surrounded by less phosphorylated ;individual' NFs that are often associated with kinesin and microtubules (MTs). Bundled NFs transport substantially slower than the surrounding individual NFs and might represent a resident population that stabilizes axons and undergoes replacement by individual NFs. To examine this possibility, fractions enriched in bundled NFs and individual NFs were generated from mice and NB2a/d1 cells by sedimentation of cytoskeletons over a sucrose cushion. More kinesin was recovered within individual versus bundled NF fractions. Individual but not bundled NFs aligned with purified MTs under cell-free conditions. The percentage of NFs that aligned with MTs was increased by the addition of kinesin, and inhibited by anti-kinesin antibodies. Bundles dissociated following incubation with EGTA or alkaline phosphatase, generating individual NFs that retained or were depleted of phospho-epitopes, respectively. These dissociated NFs aligned with MTs at a level identical to those originally isolated as individual NFs regardless of phosphorylation state. EGTA-mediated dissociation of bundles was prevented and reversed by excess Ca(2+), whereas individual NFs did not associate in the presence of excess Ca(2+). These findings confirm that bundling competes with NF-MT association, and provide a mechanism by which C-terminal NF phosphorylation might indirectly contribute to the observed slowing in axonal transport of phospho-NFs.

An anatomic and clinical study of the suprascapular and axillary nerve blocks for shoulder arthroscopy.

HYPOTHESIS: The combination of suprascapular nerve block (SSNB) and axillary nerve block (ANB) has been reported to provide safe and effective analgesia for arthroscopic shoulder surgery. This study was designed to identify anatomic landmarks of the suprascapular nerve (SSN) and axillary nerve (AN) and to evaluate the effects of SSNB and ANB using the identified landmarks. MATERIALS AND METHODS: This study included 52 cadaveric shoulders and 30 patients in the anatomic and clinical studies, respectively. After the exact location of the SSN and AN was identified from the cadavers, the clinical study at the end of the operation and at 8, 16, 24, 32, 40, and 48 hours postoperatively was performed in 2 groups: without both SSNB and ANB (group I) and with both SSNB and ANB (group II). RESULTS: The SSN was located at a length of one-half (2/5-3/5, 88%) from the anterior tip of the acromion to the superior angle of the scapula and at a length of two-fifths (1/3-1/2, 100%) from the anterior tip of the acromion to the medial border of the spine. The AN was located at a length of three-fifths (2/5-4/5, 98%) from the acromial angle to the inferior insertion of the teres major muscle. The depth from the skin was 3.20 ± 0.58 cm for the SSN and 2.07 ± 0.45 cm for the AN. The clinical study showed that the total amount of analgesic for patient-controlled anesthesia was markedly decreased at the end of the operation and at 8 hours postoperatively in group II compared with group I. CONCLUSIONS: The SSNB and ANB were considered to provide safe and effective analgesia in terms of early postoperative pain in arthroscopic shoulder surgery.

Tau interferes with axonal neurite stabilization and cytoskeletal composition independently of its ability to associate with microtubules.

Tau impacts overall axonal transport particularly when overexpressed by interfering with translocation of kinesin along microtubules (MTs) and/or as a cargo of kinesin by outcompeting other kinesin cargo. To discern between which of these mechanisms was more robust during axonal outgrowth, we overexpressed phosphomimetic (E18; which is incapable of MT binding), phospho-null (A18) or wild-type (WT) full-length human tau conjugated to EGFP, the latter two of which bind MTs. Expression of WT and A18 displayed increased acetylated MTs and resistance to colchicine, while expression of E18 did not, indicating that E18 did not contribute to MT stabilization. Expression of all tau constructs reduced overall levels of neurofilaments (NFs) within axonal neurites, and distribution of NFs along neurite lengths. Since NFs are another prominent cargo of kinesin during axonal neurite outgrowth, this finding is consistent with WT, A18 and E18 inhibiting NF transport to the same extent by competing as cargo of kinesin. These findings indicate that tau can impair axonal transport independently of association with MTs in growing axonal neurites.

Corrigendum: Wolf-Hirschhorn Syndrome-Associated Genes Are Enriched in Motile Neural Crest Cells and Affect Craniofacial Development in Xenopus laevis.

[This corrects the article DOI: 10.3389/fphys.2019.00431.].

Exonic point mutations of human tau enhance its toxicity and cause characteristic changes in neuronal morphology, tau distribution and tau phosphorylation in the lamprey cellular model of tauopathy.

Exonic mutations in the gene coding for human tau cause familial neurofibrillary degenerative diseases (tauopathies) which exhibit mutation-specific characteristics. It is thus unclear whether such mutations have similar effects on tau structure and function in vivo and if they act via similar cytopathological mechanisms in vulnerable neuron types. We have previously shown that overexpressing wild type human tau isoforms in identified giant neurons (ABCs) of the lamprey CNS results in characteristic, stereotyped cytopathological changes in these cells over several weeks. Here, we use this model to compare the cytopathological consequences of expressing wild type and exonic mutant tau isoforms (P301L, G272V, V337M, and R406W) at a high level of resolution. We show that each of the four exonic htau mutations tested accelerate degeneration in ABCs when compared to their WT parent isoforms, and that the patterns of human tau distribution, phosphorylation and cytopathology, while similar, vary characteristically from one another among both WT and mutant isoforms in a single identified neuron in situ. Our results therefore suggest that at least some of the differences between the effects of these mutations in humans are due to cell autonomous, mutation specific differences in the cytopathological mechanism of tau-induced neurodegeneration.

The Influence of Tourists' Experience of Quality of Street Foods on Destination's Image, Life Satisfaction, and Word of Mouth: The Moderating Impact of Food Neophobia.

Due to growing food-related tourism, there is increasing interest about street foods worldwide, including South Korea. Many types of food-related experiences have been considered as one of the significant elements to develop positive perceptions about a destination, and street food has been recognized as a critical clue for encouraging tourists to a destination. Previous scholars mentioned street food as a public health risk element as well as a significant factor to attract tourists' attention. Therefore, this study aims to find out how experiential quality of street foods is related to the destination image, life satisfaction, and word of mouth as perceived by tourists in night markets of South Korea. Data was collected from 325 foreigners who visited night markets and have experienced street foods in Korea. This study demonstrates the results of the influence of quality of street foods on tourist experience, on destination image, on life satisfaction, and on word of mouth in Korea. In addition, the result shows a moderating impact of food neophobia on the formulated relationships. There are statistically significant differences between groups with high neophobia perception and low neophobia perception of street foods. Based on the results of this study, we propose not only academic implications for future studies, but also managerial implications for food enterprises and food tourism organizers related to street food.

Wolf-Hirschhorn Syndrome-Associated Genes Are Enriched in Motile Neural Crest Cells and Affect Craniofacial Development in Xenopus laevis.

Wolf-Hirschhorn Syndrome (WHS) is a human developmental disorder arising from a hemizygous perturbation, typically a microdeletion, on the short arm of chromosome four. In addition to pronounced intellectual disability, seizures, and delayed growth, WHS presents with a characteristic facial dysmorphism and varying prevalence of microcephaly, micrognathia, cartilage malformation in the ear and nose, and facial asymmetries. These affected craniofacial tissues all derive from a shared embryonic precursor, the cranial neural crest (CNC), inviting the hypothesis that one or more WHS-affected genes may be critical regulators of neural crest development or migration. To explore this, we characterized expression of multiple genes within or immediately proximal to defined WHS critical regions, across the span of craniofacial development in the vertebrate model system Xenopus laevis. This subset of genes, whsc1, whsc2, letm1, and tacc3, are diverse in their currently-elucidated cellular functions; yet we find that their expression demonstrates shared tissue-specific enrichment within the anterior neural tube, migratory neural crest, and later craniofacial structures. We examine the ramifications of this by characterizing craniofacial development and neural crest migration following individual gene depletion. We observe that several WHS-associated genes significantly impact facial patterning, cartilage formation, neural crest motility in vivo and in vitro, and can separately contribute to forebrain scaling. Thus, we have determined that numerous genes within and surrounding the defined WHS critical regions potently impact craniofacial patterning, suggesting their role in WHS presentation may stem from essential functions during neural crest-derived tissue formation.

A multiple star WDM-PON using a band splitting WDM filter.

We propose a multiple star wavelength division multiplexing-passive optical network (WDM-PON) to serve several subscriber groups located at a widely distributed area. The architecture based on a band splitting WDM (BSWDM) filter separates upstream and downstream bands to several sub-bands and assign them to different subscriber groups. As a result, it enables to use a single type AWG for second stage splitting points. Thus, it provides color-free outside plant and simplifies management issues. The proposed architecture also provides pay as you grow feature.

The glutamate-rich region of the larger lamprey neurofilament sidearm is essential for proper neurofilament architecture.

The carboxyl terminal "tail" domains of the heavy and middle molecular weight mammalian neurofilament (NF) proteins regulate inter-NF spacing and formation of organized networks. The C-terminal region of the larger of the two lamprey NF subunits (NF-180) resembles these mammalian proteins in that it consists of a proximal glutamate-rich region and a distal region containing multiple phosphorylation sites. To investigate the role of these two sidearm domains in the organization of lamprey NFs, we generated plasmids lacking the glutamate-rich domain, the domain containing multiple phosphorylation sites, or both, and examined the impact of the resultant mutant proteins on the endogenous NF network in differentiated NB21/d1 neuroblastoma cells. We present evidence that, like mammalian NFs, the glutamate-rich region of NF-180 sidearm plays a critical role in NF architecture.

Assembly and turnover of neurofilaments in growing axonal neurites.

Neurofilaments (NFs) are thought to provide stability to the axon. We examined NF dynamics within axonal neurites of NB2a/d1 neuroblastoma by transient transfection with green fluorescent protein-tagged NF-heavy (GFP-H) under the control of a tetracycline-inducible promoter. Immunofluorescent and biochemical analyses demonstrated that GFP-H expressed early during neurite outgrowth associated with a population of centrally-situated, highly-phosphorylated crosslinked NFs along the length of axonal neurites ('bundled NFs'). By contrast, GFP-H expressed after considerable neurite outgrowth displayed markedly reduced association with bundled NFs and was instead more evenly distributed throughout the axon. This differential localization was maintained for up to 2 weeks in culture. Once considerable neurite outgrowth had progressed, GFP that had previously associated with the NF bundle during early expression was irreversibly depleted by photobleaching. Cessation of expression allowed monitoring of NF turnover. GFP-H associated bundled NFs underwent slower decay than GFP-H associated with surrounding, less-phosphorylated NFs. Notably, GFP associated with bundled NFs underwent similar decay rates within the core and edges of this bundle. These results are consistent with previous demonstration of a resident NF population within axonal neurites, but suggest that this population is more dynamic than previously considered.

The origin and migration of the dissolved sulfate from precipitation in Seoul, Korea.

This study incorporated stable isotope analyses with chemical analyses to determine the origin and migration of sulfur sources in East Asia, and these findings were compared with our decadal research from 2000 to 2001 and 2002 to 2003. The multiple sulfur isotope composition (32S, 33S and 34S) of the dissolved sulfate in precipitation was first measured from 2011 to 2013 in Seoul, South Korea. The δ34Snss values were -1.1‰ to 7.9‰ (avg. 3.6‰), strongly suggesting that sulfur derived from the combustion of Chinese coal is the predominant source of sulfate in the Seoul region. Low NO3/SO42- ratios in the precipitation samples indicated an insignificant effect of sulfur from vehicle exhaust. The seasonal variation of δ34Snss values appears to be caused by increasing biogenic sulfur activity during the spring and summer seasons. The some Δ33S values (0.13‰-0.16‰) measured in the three samples were sufficiently small; thus, whether these values can be attributed to mass-independent fractionation remains unclear. Measuring the Δ33S anomalies in dissolved sulfate provides valuable insights for identifying the sources of sulfur transferred from the stratosphere to the troposphere and upper troposphere.

A self-restorable architecture for bidirectional wavelength-division-multiplexed passive optical network with colorless ONUs.

We propose and experimentally demonstrate a novel protection scheme for wavelength-division-multiplexed passive optical network (WDM-PON) employing colorless optical transceivers. The proposed network employs 2 x N arrayed waveguide grating (AWG) to utilize its routing characteristics. The colorless operation is achieved by using wavelength-locked Fabry-Perot laser diodes (FP-LDs) injected with spectrum-sliced amplified spontaneous emission (ASE) light. The experimental results show that the restoration can be achieved within 8 ms against the feeder fiber fault and the power penalty introduced by the restoration process is negligible.

Robot-Embodied Neuronal Networks as an Interactive Model of Learning.

BACKGROUND AND OBJECTIVE: The reductionist approach of neuronal cell culture has been useful for analyses of synaptic signaling. Murine cortical neurons in culture spontaneously form an ex vivo network capable of transmitting complex signals, and have been useful for analyses of several fundamental aspects of neuronal development hitherto difficult to clarify in situ. However, these networks lack the ability to receive and respond to sensory input from the environment as do neurons in vivo. Establishment of these networks in culture chambers containing multi-electrode arrays allows recording of synaptic activity as well as stimulation. METHOD: This article describes the embodiment of ex vivo neuronal networks neurons in a closed-loop cybernetic system, consisting of digitized video signals as sensory input and a robot arm as motor output. RESULTS: In this system, the neuronal network essentially functions as a simple central nervous system. This embodied network displays the ability to track a target in a naturalistic environment. These findings underscore that ex vivo neuronal networks can respond to sensory input and direct motor output. CONCLUSION: These analyses may contribute to optimization of neuronal-computer interfaces for perceptive and locomotive prosthetic applications. Ex vivo networks display critical alterations in signal patterns following treatment with subcytotoxic concentrations of amyloid-beta. Future studies including comparison of tracking accuracy of embodied networks prepared from mice harboring key mutations with those from normal mice, accompanied with exposure to Abeta and/or other neurotoxins, may provide a useful model system for monitoring subtle impairment of neuronal function as well as normal and abnormal development.