CELSR1 variants are associated with partial epilepsy of childhood.

CELSR1 gene, encoding cadherin EGF LAG seven-pass G-type receptor 1, is mainly expressed in neural stem cells during the embryonic period. It plays an important role in neurodevelopment. However, the relationship between CELSR1 and disease of the central nervous system has not been defined. In this study, we performed trios-based whole-exome sequencing in a cohort of 356 unrelated cases with partial epilepsy without acquired causes and identified CELSR1 variants in six unrelated cases. The variants included one de novo heterozygous nonsense variant, one de novo heterozygous missense variant, and four compound heterozygous missense variants that had one variant was located in the extracellular region and the other in the cytoplasm. The patients with biallelic variants presented severe epileptic phenotypes, whereas those with heterozygous variants were associated with a mild epileptic phenotype of benign epilepsy with centrotemporal spikes (BECTS). These variants had no or low allele frequency in the gnomAD database. The frequencies of the CELSR1 variants in this cohort were significantly higher than those in the control populations. The evidence from ClinGen Clinical-Validity Framework suggested a strong association between CELSR1 variants and epilepsy. These findings provide evidence that CELSR1 is potentially a candidate pathogenic gene of partial epilepsy of childhood.

Polymersomes from dual responsive block copolymers: drug encapsulation by heating and acid-triggered release.

A series of well-defined thermoresponsive diblock copolymers (PEO45-b-PtNEAn, n=22, 44, 63, 91, 172) were prepared by the atom transfer radical polymerization of trans-N-(2-ethoxy-1,3-dioxan-5-yl) acrylamide (tNEA) using a poly(ethylene oxide) (PEO45) macroinitiator. All copolymers are water-soluble at low temperature, but upon quickly heating to 37 °C, laser light scattering (LLS) and transmission electron microscopy (TEM) characterizations indicate that these copolymers self-assemble into aggregates with different morphologies depending on the chain length of PtNEA and the polymer concentration; the morphologies gradually evolved from spherical solid nanoparticles to a polymersome as the degree of polymerization ("n") of PtNEA block increased from 22 to 172, with the formation of clusters with rod-like structure at the intermediate PtNEA length. Both the spherical nanoparticle and the polymersome are stable at physiological pH but susceptible to the mildly acidic medium. Acid-triggered hydrolysis behaviors of the aggregates were investigated by LLS, Nile red fluorescence, TEM, and (1)H NMR spectroscopy. The results revealed that the spherical nanoparticles formed from PEO45-b-PtNEA44 dissociated faster than the polymersomes of PEO45-b-PtNEA172, and both aggregates showed an enhanced hydrolysis under acidic conditions. Both the spherical nanoparticle and polymersome are able to efficiently load the hydrophobic doxorubicin (DOX), and water-soluble fluorescein isothiocyanate-lysozyme (FITC-Lys) can be conveniently encapsulated into the polymersome without using any organic solvent. Moreover, FITC-Lys and DOX could be coloaded in the polymersome. The drugs loaded either in the polymersome or in the spherical nanoparticle could be released by acid triggering. Finally, the DOX-loaded assemblies display concentration-dependent cytotoxicity to HepG2 cells, while the copolymers themselves are nontoxic.

Efficient strategies based on behavioral and electrophysiological methods for epilepsy-related gene screening in the Drosophila model.

Introduction: With the advent of trio-based whole-exome sequencing, the identification of epilepsy candidate genes has become easier, resulting in a large number of potential genes that need to be validated in a whole-organism context. However, conducting animal experiments systematically and efficiently remains a challenge due to their laborious and time-consuming nature. This study aims to develop optimized strategies for validating epilepsy candidate genes using the Drosophila model. Methods: This study incorporate behavior, morphology, and electrophysiology for genetic manipulation and phenotypic examination. We utilized the Gal4/UAS system in combination with RNAi techniques to generate loss-of-function models. We performed a range of behavioral tests, including two previously unreported seizure phenotypes, to evaluate the seizure behavior of mutant and wild-type flies. We used Gal4/UAS-mGFP flies to observe the morphological alterations in the brain under a confocal microscope. We also implemented patch-clamp recordings, including a novel electrophysiological method for studying synapse function and improved methods for recording action potential currents and spontaneous EPSCs on targeted neurons. Results: We applied different techniques or methods mentioned above to investigate four epilepsy-associated genes, namely Tango14, Klp3A, Cac, and Sbf, based on their genotype-phenotype correlation. Our findings showcase the feasibility and efficiency of our screening system for confirming epilepsy candidate genes in the Drosophila model. Discussion: This efficient screening system holds the potential to significantly accelerate and optimize the process of identifying epilepsy candidate genes, particularly in conjunction with trio-based whole-exome sequencing.

Reduction-degradable linear cationic polymers as gene carriers prepared by Cu(I)-catalyzed azide-alkyne cycloaddition.

Linear reduction-degradable cationic polymers with different secondary amine densities (S2 and S3) and their nonreducible counterparts (C2 and C3) were synthesized by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) step-growth polymerization of the dialkyne-oligoamine monomers and the diazide monomers. These polymers were studied with a goal of developing a set of new gene carriers. The buffering capacity and DNA binding ability of these polymers were evaluated by acid-base titration, gel retardation, and ethidium bromide (EB) exclusion assay. The polymers with lower amine density exhibit a weaker DNA-binding ability but a stronger buffering capacity in the range of pH 5.1 and 7.4. Particle size and zeta-potential measurements demonstrate that the polymers with higher amine density condense pDNA to form polyplexes with smaller sizes, while the disulfide bond in the backbone shows a negative effect on the condensing capability of the polymers, resulting in the formation of polyplexes with large size and nearly neutral surface. The reduction-sensitive polyplexes formed by polymer S2 or S3 can be disrupted by dithiothreitol (DTT) to release free DNA, which has been proven by the combination of gel retardation, EB exclusion assay, particles sizing, and zeta potential measurements. Cell viability measurements by MTT assay demonstrate that the reduction-degradable polymers (S2 and S3) have little cytotoxicity while the nonreducible polymers (C2 and C3) show obvious cytotoxicity, in particular, at high N/P ratios. In vitro transfection efficiencies of these polymers were evaluated using EGFP and luciferase plasmids as the reporter genes. Polymers S3 and S2 show much higher efficiencies than the nonreducible polymers C3 and C2 in the absence of 10% serum; unexpectedly, the lowest transfection efficiency has been observed for polymer S3 in the presence of serum.

Acid-Sensitive Polypseudorotaxanes Based on Ortho Ester-Modified Cyclodextrin and Pluronic F-127.

We demonstrate a new type of acid-sensitive amphiphilic polypseudorotaxanes (PPRs) formed via inclusion complexation between Pluronic F127 and the hydrophobic ß-cyclodextrin (CD) derivative in alcoholic solvents. The 6-OH ortho ester-substituted hydrophobic ß-CD derivative (EMD-CD) was prepared by "click" reaction of ß-CD with 2-ethylidene-4-methyl-1,3-dioxalane under mild conditions. The water-insoluble EMD-CD (host) is capable of forming PPRs with F127 (guest) in ethanol or methanol but not in water, which is confirmed by 1H NMR, wide-angle X-ray diffraction, small-angle X-ray scattering, and the time-dependent threading kinetics. Depending on the host/guest ratio, the PPRs self-assembled into sheet-like structure or vesicular nanoparticles with different sizes in water. These PPR assemblies were stable at pH 8.4 but quickly dissociated into biocompatible products in neutral or in acidic buffers due to the hydrolysis of the ortho ester groups. Good biocompatibility, ease of fabrication, and extremely pH-sensitive character make the PPRs promising carriers for anticancer drug delivery. Moreover, the present work provides an alternative method for the preparation of PPRs composed of water-insoluble CD derivatives.

Oxidation-Accelerated Hydrolysis of the Ortho Ester-Containing Acid-Labile Polymers.

We report a versatile method to tune the hydrolysis of the ortho ester-containing block copolymers by covalently incorporating oxidation-sensitive phenylboronic ester units. A series of block copolymers which contain a polyethylene glycol (PEG) block and a hydrophobic segment composed of different amounts of pendent ortho ester and phenylboronic ester groups were synthesized. These copolymers can self-assemble into narrowly dispersed micelle-like nanoparticles in phosphate buffer. The kinetics of phenylboronic ester oxidation and ortho ester hydrolysis in the nanoparticles were studied at different pH and H2O2 concentration. The results indicated that the phenylboronic ester oxidation rate was faster than the ortho ester hydrolysis rate at neutral pH, and both processes were accelerated with increasing H2O2 concentration. Nanoparticles which are extremely sensitive to the biorelevant concentration of H2O2 (50 µM) at pH 7.4 were obtained, suggesting great promise for inflammation-specific drug delivery.

Multi-responsive nanogels containing motifs of ortho ester, oligo(ethylene glycol) and disulfide linkage as carriers of hydrophobic anti-cancer drugs.

A family of multi-responsive nanogels with different compositions and crosslinking degrees have been prepared by the miniemulsion copolymerization of monomethyl oligo(ethylene glycol) acrylate (OEGA) and an ortho ester-containing acrylic monomer, 2-(5,5-dimethyl-1,3-dioxan-2-yloxy) ethyl acrylate (DMDEA), with bis(2-acryloyloxyethyl) disulfide (BADS) as a crosslinker. These nanogels are thermoresponsive and labile in the weakly acidic or reductive environments. The thermoresponsive behaviors, acid-triggered hydrolysis, and reduction-induced degradation of these nanogels were studied by means of dynamic light scattering (DLS), transmission electron microscopy (TEM) and atomic force microscopy (AFM). The results indicate that the volume phase transition temperature (VPTT), thermally induced deswelling ratio, and acid-triggered swelling ratio of the nanogels are closely relevant to their compositions and crosslinking degrees. Although these nanogels could be reductively disrupted by dithiothreitol (DTT), single polymer chains with sizes smaller than 20 nm were not detected by DLS. This is probably due to the existence of some unbreakable linkages formed by chain transfer to the disulfide bond during the radical polymerization. These nanogels are capable of encapsulating hydrophobic compounds. The loading capability of the nanogels for Nile Red (NR), paclitaxel (PTX), and doxorubicin (DOX), and the release behaviors of the drug-loaded nanogels were investigated by UV-vis spectrometry and HPLC. As expected, drug release can be greatly accelerated by a cooperative effect of both acid-triggered hydrolysis and DTT-induced degradation. Finally, the PTX-loaded nanogels exhibit a concentration-dependent toxicity to MCF-7 cells while the intact unloaded nanogels are non-toxic, thereby they may be used as potential carriers for hydrophobic anticancer drugs.