Nicotinic acetylcholine receptors (nAChRs), prototype members of the cys‐loop ligand‐gated ion channel family, are key mediators of cholinergic transmission in the central nervous system. Despite ...their importance, technical gaps exist in our ability to dissect the function of individual subunits in the brain. To overcome these barriers, we designed CRISPR/Cas9 small guide RNA sequences (sgRNAs) for the production of loss‐of‐function alleles in mouse nAChR genes. These sgRNAs were validated in vitro via deep sequencing. We subsequently targeted candidate nAChR genes in vivo by creating herpes simplex virus (HSV) vectors delivering sgRNAs and Cas9 expression to mouse brain. The production of loss‐of‐function insertions or deletions (indels) by these ‘all‐in‐one’ HSV vectors was confirmed using brain slice patch clamp electrophysiology coupled with pharmacological analysis. Next, we developed a scheme for cell type‐specific gene editing in mouse brain. Knockin mice expressing Cas9 in a Cre‐dependent manner were validated using viral microinjections and genetic crosses to common Cre‐driver mouse lines. We subsequently confirmed functional Cas9 activity by targeting the ubiquitous neuronal protein, NeuN, using adeno‐associated virus (AAV) delivery of sgRNAs. Finally, the mouse β2 nAChR gene was successfully targeted in dopamine transporter (DAT)‐positive neurons via CRISPR/Cas9. The sgRNA sequences and viral vectors, including our scheme for Cre‐dependent gene editing, should be generally useful to the scientific research community. These tools could lead to new discoveries related to the function of nAChRs in neurotransmission and behavioral processes.
Peng et al. introduce a set of viral vectors for nicotinic acetylcholine receptor (nAChR) gene editing. Herpes simplex virus vectors allow all‐in‐one delivery of CRISPR/Cas9 elements for gene editing in any mouse strain, and adeno‐associated virus vectors allow cell type‐specific nAChR gene editing. These vectors will be useful for next‐generation molecular‐level queries into the contribution of nAChR subunits in addiction and psychiatric disorders.
Ventral tegmental area (VTA) neurons receive glutamatergic and/or GABAergic input from other local neurons within the VTA. Nicotinic acetylcholine receptor (nAChR) activity is capable of modulating ...such intra-VTA transmission, but the mechanisms are unclear. Here, we isolated monosynaptic glutamate or GABA transmission from mouse medial VTA (mVTA) to lateral VTA (latVTA) using pharmacology and optogenetics, and we studied the ability of nicotine to modulate these modes of transmission. The action of nicotine on mVTA to latVTA glutamate transmission was bidirectional; nicotine enhanced glutamate release in half of the recorded latVTA cells and inhibited release in the other half. Nicotine-mediated reduction in glutamate release was reversed by blockade of GABA
receptors. This, coupled with expression data demonstrating coexpression of vesicular glutamate transporter 2 (VGluT2) and glutamate decarboxylase 2 (Gad2) in mVTA neurons, suggests that nicotine is able to stimulate GABA corelease from mVTA VGluT2
neurons. Nicotine had an altogether different effect on mVTA to latVTA GABA release from Gad2
cells; nicotine suppressed GABA release from mVTA Gad2
terminals in nearly all cells tested. Together, these data uncover a complex system of local circuitry in the VTA that is modulated by nAChR activity. These actions of nicotine, which occurred at concentrations of nicotine found in the artificial CSF of cigarette smokers, may play a role in the adaptive response of the reward system to repeated nicotine exposure.
The immunoglobulin (Ig)-like domain containing receptor 1 (ILDR1) gene encodes angulin-2/ILDR1, a recently discovered tight junction protein, which forms tricellular tight junction (tTJ) structures ...with tricellulin and lipolysis-stimulated lipoprotein receptor (LSR) at tricellular contacts (TCs) in the inner ear. Previously reported recessive mutations within ILDR1 have been shown to cause severe to profound nonsyndromic sensorineural hearing loss (SNHL), DFNB42. Whole-exome sequencing of a Korean multiplex family segregating partial deafness identified a novel homozygous ILDR1 variant (p.P69H) within the Ig-like domain. To address the pathogenicity of p.P69H, the angulin-2/ILDR1 p.P69H variant protein, along with the previously reported pathogenic ILDR1 mutations, was expressed in angulin-1/LSR knockdown epithelial cells. Interestingly, partial mislocalization of the p.P69H variant protein and tricellulin at TCs was observed, in contrast to a severe mislocalization and complete failure of tricellulin recruitment of the other reported ILDR1 mutations. Additionally, three-dimensional protein modeling revealed that angulin-2/ILDR1 contributed to tTJ by forming a homo-trimer structure through its Ig-like domain, and the p.P69H variant was predicted to disturb homo-trimer formation. In this study, we propose a possible role of angulin-2/ILDR1 in tTJ formation in the inner ear and a wider audiologic phenotypic spectrum of DFNB42 caused by mutations within ILDR1.
Appropriate customized auditory rehabilitation for hearing impaired subjects requires prediction of residual hearing and progression of hearing loss. Mutations in
TMPRSS3
encoding a transmembrane ...serine protease were reported to be associated with two different autosomal recessive nonsyndromic hearing loss (arNSHL) phenotypes, DFNB8 and DFNB10, in terms of residual hearing that may mandate different rehabilitation. We aimed to reveal the genetic contribution of
TMPRSS3
mutations among Korean populations and to correlate the clinical phenotype with
TMPRSS3
genotypes. Fifty families that segregated arNSHL and have visited our clinic recently for 2 years were recruited for
TMPRSS3 screening
. Novel
TMPRSS3
variants detected in our cohort were modeled using a predicted three-dimensional (3D) structure of the serine protease domain. The prevalence reached up to 11.2 % (3/27) among subjects with either prelingual hearing loss but retaining some degree of language development or with postlingual ski-slope hearing loss. We also found that a p.A306T allele is a founder allele in this population. Based upon the 3D modeling, we were able to correlate significant retention of residual low-frequency hearing and slower progression of its loss to this novel variant p.T248M that was predicted to have milder pathogenicity. A yeast-based protease assay confirmed a mild pathogenic potential of the p.T248M variant and a tight correlation between the protease activity and the residual hearing. Preservation of this low-frequency hearing should be of utmost importance when considering auditory rehabilitation. Our results significantly narrow down the candidate population for
TMPRSS3
sequencing for more efficient genetic diagnosis. More importantly, genotype–phenotype correlation of this gene observed in our cohort suggests that
TMPRSS3
can be an appropriate candidate for personalized and customized auditory rehabilitation.
Key message
The prevalence of
TMPRSS3
mutations among Korean postlingual hearing loss is 8.3 %.
The p.A306T variant of
TMPRSS3
is the common founder allele in Koreans.
A novel variant, p.T248M of
TMPRSS3
, was predicted to have milder pathogenicity.
There was a genotype–phenotype correlation of this gene in Koreans.
Our data support implication of this gene for personalized rehabilitation.
The aim of this study was to understand the prevalence and molecular genetic etiology of incomplete partition type III (IP type III) anomaly in Koreans. We also attempted to verify the prevalence of ...genomic deletions in the DFNX2 locus and to look for association between inheritance patterns and mutation type in East Asian IP type III subjects.
Retrospective case review.
Tertiary referral center.
Subjects with IP type III anomaly and their biological mothers.
Sanger sequencing, array-comparative genomic hybridization (aCGH), and PCR were performed. We also analyzed the type and inheritance of the causative genetic abnormality in East Asian DFNX2 patients.
Mutation type and occurrence.
We identified IP type III in 10 (4.8%) of 206 patients with an inner ear abnormality. We confirmed an etiologic homogeneity, DFNX2, of the IP type III in this Korean population. Two (20%) of the 10 DFNX2 carried a large genomic deletion affecting POU3F4, as proved by aCGH. PCR confirmed that the 2 deletions occurred de novo. Genetic alteration occurred de novo in 29.4% (5/17) of all reported Korean IP type III cases. From this study and literature review, we observed a striking difference of de novo occurrence rate (75% versus 12.5%, p = 0.032) between large genomic deletions and point mutations in East Asian population.
Our data suggest that different POU3F4 mutations might show different recurrence rate in siblings of the IP type III families, especially in East Asian population. Genetic counseling should be provided accordingly.
Recent studies suggest that transcript isoforms significantly overlap (approximately 60%) between brain tissue and Epstein–Barr virus-transformed lymphoblastoid cell lines (LCLs). Interestingly, 14 ...cohesion-related genes with variants that cause Cornelia de Lange Syndrome (CdLS) are highly expressed in the brain and LCLs. In this context, we first performed RNA sequencing of LCLs from 22 solved (with pathogenic variants) and 19 unsolved (with no confirmed variants) CdLS cases. Next, an RNA sequencing pipeline was developed using solved cases with two different methods: short variant analysis (for single-nucleotide and indel variants) and aberrant splicing detection analysis. Then, 19 unsolved cases were subsequently applied to our pipeline, and four pathogenic variants in NIPBL (one inframe deletion and three intronic variants) were newly identified. Two of three intronic variants were located at Alu elements in deep-intronic regions, creating cryptic exons. RNA sequencing with LCLs was useful for identifying hidden variants in exome-negative cases.
•RNA sequencing using LCLs was performed in Cornelia de Lange syndrome patients.•Using our pipeline, we could newly identify four pathogenic NIPBL variants.•Two deep-intronic variants both located at Alu elements were found.•RNA sequencing was effective in finding hidden variants in exome-negative patients.
Organotrialkoxysilanes (OTAS) containing bulky UV chromophores were polymerized under basic conditions to prepare insoluble spherical polysilsesquioxane (PSQ) particles potentially useful as UV ...blocking ingredients in sunscreens. The OTAS used in this study were 3-aminopropyltrimethoxysilyl-
p
-methoxycinnamamide (APTMSMCA), 3-aminopropyltriethoxysilyl-
p
-ethoxycinnamamide (APTESMCA), 3-aminopropyltrimethoxysilyl-
p
-
N,N
′-dimethylaminobezamide (APTMSDMABA), and 3-aminopropyltriethoxysilyl-
p-N,N
′-dimethylaminobenzamide (APTESDMABA). Mixtures of insoluble spherical particles, soluble solids, and insoluble monolithic solids were produced in various yields depending on the reaction conditions and the nature of the chromophore. The molar extinction coefficients of PSQ particles were 1/2 to 1/4 of those of corresponding unreacted chromophores and OTAS. The
λ
max
and extinction coefficients of the PSQ particles varied according to their sizes.
In vitro
sun protection factor (SPF) values of petrolatum containing 10 wt% of the particles were between 10 and 21. The photostability of polyAPTMSMCA and polyAPTMSDMABA particles were significantly poorer than those of APTMSMCA and APTMSDMABA. Copolymerization of APTMSDMABA and methyltrimethoxysilane (MTMS) significantly improved the photostability. The particles dispersed in water and silicon oil showed photomechanical effects under UV light that were most likely caused by heat released during the relaxation process.
Background
In this study, the authors sought to describe the cytologic features of primary gynecologic germ cell tumors and carcinomas exhibiting germ cell differentiation because little information ...currently exists.
Methods
An institutional database search was performed to identify histologically confirmed gynecologic germ cell tumors and carcinomas with germ cell tumor differentiation. Available cytologic material was reviewed by three observers, and morphologic features were recorded in addition to patient age at original diagnosis, primary tumor site, site(s) from which the examined cytologic material was obtained, and the type of examined cytologic preparations.
Results
In total, 15 cytologic specimens from 12 women (aged 19–82 years) were identified and included touch preparations of core biopsies from various sites (n = 6), fine‐needle biopsies (n = 2), pelvic washings (n = 1), ascitic fluids (n = 4), pelvic cyst fluid (n = 1), and endometrial aspirate (n = 1). Of the 12 patients, seven had primary gynecologic germ cell tumors, four had gynecologic (ovarian and endometrial) tumors exhibiting somatic yolk sac tumor‐like differentiation, and the remaining patient had an intestinal‐type adenocarcinoma arising within an ovarian teratoma. There was morphologic overlap among many of the cases, although cytoplasmic vacuolation/granular cytoplasm was seen in 75% of primary yolk sac tumors or carcinomas with yolk sac tumor differentiation, and dense/squamoid cytoplasm was seen in 100% of teratomatous elements that were sampled.
Conclusions
Germ cell tumors and somatic neoplasms exhibiting germ cell tumor differentiation occurring in adult women share some cytologic features and may be difficult to distinguish from one another, although some tumor types showed characteristic cytomorphologic findings.
Primary gynecologic germ cell tumors and gynecologic carcinomas exhibiting germ cell tumor differentiation occurring in adult women shared overlapping cytologic features. Cytoplasmic vacuolation/granular cytoplasm was seen in the majority of samples involved by primary yolk sac tumor or carcinoma with yolk sac tumor differentiation (six of eight tumors, 75%) and dense/squamoid cytoplasm was seen in both cases with teratomatous elements (two of two tumors, 100%).
Two of the most critical factors for the survival of glioblastoma (GBM) patients are precision diagnosis and the tracking of treatment progress. At the moment, various sophisticated and specific ...diagnostic procedures are being used, but there are relatively few simple diagnosis methods. This work introduces a sensing probe based on a turn-on type fluorescence response that can measure the cysteine (Cys) level, which is recognized as a new biomarker of GBM, in human-derived cells and within on-site human clinical biopsy samples. The Cys-initiated chemical reactions of the probe cause a significant fluorescence response with high selectivity, high sensitivity, a fast response time, and a two-photon excitable excitation pathway, which allows the imaging of GBM in both mouse models and human tissue samples. The probe can distinguish the GBM cells and disease sites in clinical samples from individual patients. Besides, the probe has no short or long-term toxicity and immune response. The present findings hold promise for application of the probe to a relatively simple and straightforward following of GBM at clinical sites.
A fluorescent molecular probe for the identification of glioblastoma is developed. The probe allows the tracing of the cysteine (Cys) level, which is recognized as a new biomarker of GBM.
By approaching Asian and Latin American relations not only as a research object but as an epistemic framework, this article sheds light on the ways in which the figure of the “West,” even in its ...apparent absence, continues to govern and structure periphery-to-periphery encounters. It contends that in order to forge the project of the Global South, the unequal power relations, cacophonies, and contradictions that characterize Asian and Latin American relations must be examined. The article's use of Asia–Latin America as method enables new perspectives on epistemic violence and the politics of knowledge as well as a fundamental reassessment of the workings of culture, race, economy, globality, geopolitics, and biopolitics.
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