Search

Visualizing the subcellular localization of presynaptic proteins with fluorescent proteins is a powerful tool to dissect the genetic and molecular mechanisms underlying synapse formation and patterning in live animals. Here, we utilize split green and red fluorescent proteins to visualize the localization of endogenously expressed presynaptic proteins at a single neuron resolution in Caenorhabditis elegans. By using CRISPR/Cas9 genome editing, we generated a collection of C. elegans strains in which endogenously expressed presynaptic proteins (RAB-3/Rab3, SNG-1/Synaptogyrin, CLA-1/Piccolo, SYD-2/Liprin-α, UNC-10/RIM, RIMB-1/RIM-BP, and ELKS-1/ELKS) are tagged with tandem repeats of GFP11 and/or wrmScarlet11. We show that the expression of GFP1-10 and wrmScarlet1-10 under neuron-specific promoters can robustly label presynaptic proteins in different neuron types. We believe that the combination of our knock-in strains and GFP1-10 and wrmScarlet1-10 plasmids is a versatile modular system useful for neuroscientists to examine the localization of endogenous presynaptic proteins in any neuron type in C. elegans., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Purpose: To evaluate how the relationship between respiratory interval (RI) and temporal resolution (TR) impacts image quality in free-breathing abdominal MRI (FB-aMRI) using golden-angle radial sparse parallel (GRASP)., Methods: Ten healthy volunteers (25.9 ± 2.5 years, four women) underwent 2 mins free-breathing fat-suppression T1-weighted imaging using GRASP at RIs of 3 and 5s (RI 3 and RI 5 , respectively) and retrospectively reconstructed at TR of 1.8, 2.9, 4.8, and 7.7s (TR 1.8 , TR 2.9 , TR 4.8 , and TR 7.7 , respectively) in each patient. The standard deviation (SD) under the diaphragm was measured using SD maps showing the discrepancy for each horizontal section at all TRs. Two radiologists evaluated image quality (visualization of the right hepatic vein at the confluence of the inferior vena cava, posterior segment branch of portal vein, pancreas, left kidney, and artifacts) at all TRs using a 5-point scale., Results: The SD was significantly higher at TR 1.8 compared to TR 4.8 (P < 0.01) and TR 7.7 (P < 0.001), as well as at TR 2.9 compared to TR 7.7 (P < 0.01) for both RIs. The SD between TR 4.8 and TR 7.7 did not differ for both RIs. For all visual assessment metrics, the TR 1.8 scores were significantly lower than the TR 4.8 and TR 7.7 scores for both RIs. The pancreas and left kidney scores at TR 2.9 were significantly lower than those at TR 7.7 (P < 0.05) for RI 5 . Additionally, the left kidney score at TR 1.8 was lower than that at TR 2.9 (P < 0.05) for RI 3 . All scores at TR 2.9 , TR 4.8 , and TR 7.7 were similar for RI 3 , while those at TR 4.8 and TR 7.7 were similar for RI 5 ., Conclusion: Prolonging the TRs compared to RIs enhances image quality in FB-aMRI using GRASP.

The auxin-inducible degron (AID) provides reversible, spatiotemporal control for the knockdown of target proteins. Here, we present a protocol for AID-mediated protein knockdown in Caenorhabditis elegans. We describe steps for generating the knock-in mutants using two CRISPR-Cas9 genome editing techniques and preparing the auxin-containing nematode growth media (NGM) plates. We also detail AID-mediated spatiotemporal protein knockdown. For complete details on the use and execution of this protocol, please refer to Kurashina et al. (2021). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Neuronal cell fate determinants establish the identities of neurons by controlling gene expression to regulate neuronal morphology and synaptic connectivity. However, it is not understood if neuronal cell fate determinants have postmitotic functions in synapse pattern formation. Here we identify a novel role for UNC-4 homeobox protein and its corepressor UNC-37/Groucho, in tiled synaptic patterning of the cholinergic motor neurons in Caenorhabditis elegans . We show that unc-4 is not required during neurogenesis but is required in the postmitotic neurons for proper synapse patterning. In contrast, unc-37 is required in both developing and postmitotic neurons. The synaptic tiling defects of unc-4 mutants are suppressed by bar-1/β-catenin mutation, which positively regulates the expression of ceh-12/HB9 . Ectopic ceh-12 expression partly underlies the synaptic tiling defects of unc-4 and unc-37 mutants. Our results reveal a novel postmitotic role of neuronal cell fate determinants in synapse pattern formation through inhibiting the canonical Wnt signaling pathway., Competing Interests: MK, JW, JL, KL, AJ, KM No competing interests declared, (© 2021, Kurashina et al.)

Precise neuronal wiring is critical for the function of the nervous system and is ultimately determined at the level of individual synapses. Neurons integrate various intrinsic and extrinsic cues to form synapses onto their correct targets in a stereotyped manner. In the past decades, the nervous system of nematode (Caenorhabditis elegans) has provided the genetic platform to reveal the genetic and molecular mechanisms of synapse formation and specificity. In this review, we will summarize the recent discoveries in synapse formation and specificity in C. elegans.

Water-soluble nonionic polymers of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), and polyvinylpyrrolidone (PVP) were examined to develop the dispersion of graphene in an aqueous surfactant solution. Sodium dodecylbenzenesulfonate was used as an anionic surfactant to disperse graphene in an aqueous solution and to give negative charge on it. The dispersion of graphene was monitored through the electropherograms in affinity capillary electrophoresis; a broad peak for the dispersed graphene and shot signals for the aggregated one. When PEG was added in the separation buffer as an affinity reagent, the number of the shot signals in the electropherogram was reduced; PEG can develop the dispersion of graphene in an aqueous surfactant solution. The dispersion was also developed with PVP or PVA. The effective electrophoretic mobility of the dispersed graphene was reduced by using the polymer as an affinity reagent. The result suggested that the anionic surfactant on the graphene surface was competitively substituted with the nonionic polymer. The degree of the decrease in the effective electrophoretic mobility was larger with PEG with a high-molecular mass. The broad peak of the dispersed graphene got narrower by the addition of PEG, and the number of theoretical plates was improved.

During development, neurons form synapses with their fate-determined targets. While we begin to elucidate the mechanisms by which extracellular ligand-receptor interactions enhance synapse specificity by inhibiting synaptogenesis, our knowledge about their intracellular mechanisms remains limited. Here we show that Rap2 GTPase ( rap-2 ) and its effector, TNIK ( mig-15 ), act genetically downstream of Plexin ( plx-1 ) to restrict presynaptic assembly and to form tiled synaptic innervation in C. elegans . Both constitutively GTP- and GDP-forms of rap-2 mutants exhibit synaptic tiling defects as plx-1 mutants, suggesting that cycling of the RAP-2 nucleotide state is critical for synapse inhibition. Consistently, PLX-1 suppresses local RAP-2 activity. Excessive ectopic synapse formation in mig-15 mutants causes a severe synaptic tiling defect. Conversely, overexpression of mig-15 strongly inhibited synapse formation, suggesting that mig-15 is a negative regulator of synapse formation. These results reveal that subcellular regulation of small GTPase activity by Plexin shapes proper synapse patterning in vivo., Competing Interests: XC, AS, AH, MK, EF, NW, BM, HM, KM No competing interests declared, (© 2018, Chen et al.)

The acid-base equilibrium of hexamethylenetetramine (hexamine) was analyzed with its effective electrophoretic mobility by capillary zone electrophoresis. Although hexamine is degradable in a weakly acidic aqueous solution, and the degraded products of ammonia and formaldehyde can be formed, the effective electrophoretic mobility of hexamine was measured in the pH range between 2.8 and 6.9. An acid-base dissociation equilibrium of the protonated hexamine was analyzed based on the mobility change, and an acid dissociation constant of pK a = 4.93 ± 0.01 (mean ± standard error, ionic strength: 0.020 mol dm -3 ) was determined. The monoprotic acid-base equilibrium of hexamine was confirmed through comparisons of its electrophoretic mobility with the N-ethylquinolinium ion and with the monocationic N-ethyl derivative of hexamine, as well as a slope analysis of the dissociation equilibrium.

After UV-irradiation of a TiO(2) shell on Pt anode was cut off, anodic current was observed continuously for some time because long-lived MV(+)* produced from MV(2+) embedded inside the shell, as a result of the electron transfer from the conduction band of the irradiated TiO(2) shell, diffused slowly and discharged to the shell thus realizing electron accumulation inside the TiO(2) shell.

Reactions of quinquedentate Schiff base ligands with Mn and Cu ions afforded icosa- and hexadecanuclear mixed-metal clusters in which dinuclear CuII complexes trapped oxo-bridged [MnIII8MnIV4O12] and [MnIII6O6] cores, respectively. Maximum entropy method analysis for synchrotron X-ray diffraction data was used to determine the oxidation states of the Mn ions.

A ruthenacyclopentatriene polymer 2 was synthesized by metallacycling polymerization (MCP). Polymer 2 with Mn = 3400 and Mw = 5800 consisted of identical ruthenacyclopentatriene units. The UV-vis spectra of 2 showed a shift of the pi-pi* band to a longer wavelength due to the enlargement of the pi-conjugation. Polymer 2 underwent reversible reduction due to the ruthenacycle moiety at -1.01 V versus ferrocenium/ferrocene. The EPR spectrum of the reduced form of 2 in frozen THF indicated ferromagnetic interaction of spins at ruthenacyclopentatriene units.

Cytological features of a malignant spindle-cell variant and a benign tubular variant of adenomyoepithelioma were examined to improve diagnosis of this tumor. Fine-needle aspiration cytology, of both a malignant and a benign case, characterized cellular and cohesive cell clusters composed of epithelial and myoepithelial cells. The smears of the malignant case were rich in spindle-shaped myoepithelial cells, admixed with a few epithelial cells. In about a fourth of the clusters, atypical cells with enlarged hyperchromatic nuclei and prominent nucleoli comprised more than 20% of cells. The smears of the benign case were composed of tubular epithelial cells surrounding one to several layers of myoepithelial cells with clear cytoplasm. Mild atypia was occasionally noticed. A review of the literature showed that a cytological diagnosis of malignancy is not warranted, if nuclear atypia is not generally severe. Focal severe atypia is not definitively indicative of benignity or malignancy., (Copyright 2002 Wiley-Liss, Inc.)

To determine the area of surgical resection and the prognosis in patients with intrahepatic bile duct carcinoma, we studied the mode of spread of this cancer, using 27 autopsy and 4 lobectomized cases. In hilar carcinoma, there was lymphatic invasion to the extrahepatic bile duct before invation to the hepatic lobules. Scirrhous cancer spread more distantly toward the peripheral areas of the liver than non-scirrhous cancer. The occurrence of extrahepatic metastasis might involve the following factors; depth of infiltration, extent of venous invasion, intrahepatic metastasis, size of the main tumor, and number of hepatic segments invaded by the cancer.