Transcriptomic and Proteomic Analysis of CRISPR/Cas9-Mediated ARC-Knockout HEK293 Cells.

Arc/Arg3.1 (activity-regulated cytoskeletal-associated protein (ARC)) is a critical regulator of long-term synaptic plasticity and is involved in the pathophysiology of schizophrenia. The functions and mechanisms of human ARC action are poorly understood and worthy of further investigation. To investigate the function of the ARC gene in vitro, we generated an ARC-knockout (KO) HEK293 cell line via CRISPR/Cas9-mediated gene editing and conducted RNA sequencing and label-free LC-MS/MS analysis to identify the differentially expressed genes and proteins in isogenic ARC-KO HEK293 cells. Furthermore, we used bioluminescence resonance energy transfer (BRET) assays to detect interactions between the ARC protein and differentially expressed proteins. Genetic deletion of ARC disturbed multiple genes involved in the extracellular matrix and synaptic membrane. Seven proteins (HSPA1A, ENO1, VCP, HMGCS1, ALDH1B1, FSCN1, and HINT2) were found to be differentially expressed between ARC-KO cells and ARC wild-type cells. BRET assay results showed that ARC interacted with PSD95 and HSPA1A. Overall, we found that ARC regulates the differential expression of genes involved in the extracellular matrix, synaptic membrane, and heat shock protein family. The transcriptomic and proteomic profiles of ARC-KO HEK293 cells presented here provide new evidence for the mechanisms underlying the effects of ARC and molecular pathways involved in schizophrenia pathophysiology.

Effect of clustering on ellipsometric spectra of randomly distributed gold nanoparticles on a substrate.

We present a theoretical model for describing light scattering from randomly distributed Au nanoparticles on a substrate, including the clustering effect. By using the finite-element Green's function method and spherical harmonic basis functions, we are able to calculate the polarization-dependent reflectivity spectra of the system (modeled by randomly distributed nanoparticles coupled with clusters) efficiently and accurately. The calculated ellipsometric spectra of the system with clusters can adequately describe the experimental data for the whole frequency range. We find that the clustering effect leads to some prominent features in the low frequency range of the ellipsometric spectra, which are attributed to plasmonic resonances associated with the coupling of Au nanoparticles and clusters.

Identification of rare missense mutations in the glutamate ionotropic receptor AMPA type subunit genes in schizophrenia.

OBJECTIVE: The alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors significantly regulate the synaptic transmission and functions of various synaptic receptors. This study aimed to identify single nucleotide mutations in the glutamate receptor, ionotropic, AMPA type (GRIA) gene family, which is associated with schizophrenia. METHODS: The exon regions of four genes (GRIA1, GRIA2, GRIA3, and GRIA4) encoding glutamate ionotropic receptor AMPA type proteins were resequenced in 516 patients with schizophrenia. We analyzed the protein function of the identified rare mutants via immunoblotting. RESULTS: A total of 24 coding variants were detected in the GRIA gene family, including six missense mutations, 17 synonymous mutations, and one frameshift insertion. Notably, three ultra-rare missense mutations (GRIA1p.V182A, GRIA2p.P123Q, and GRIA4p.Y491H) were not documented in the single nucleotide polymorphism database, gnomAD genomes, and 1517 healthy controls available from Taiwan BioBank. Immunoblotting revealed GRIA4p.Y491H mutant with altered protein expressions in cultured cells compared with the wild type. CONCLUSION: Our findings suggest that, in some patients affected by schizophrenia, the GRIA gene family harbors rare functional mutations, which support rare coding variants that could contribute to the genetic architecture of this illness. The in-vitro impacts of these rare pathological mutations on the pathophysiology of schizophrenia are worthy of future investigation.

Indicators of HSV1 Infection, ECM-Receptor Interaction, and Chromatin Modulation in a Nuclear Family with Schizophrenia.

Schizophrenia (SCZ) is a complex psychiatric disorder with high heritability; identifying risk genes is essential for deciphering the disorder's pathogenesis and developing novel treatments. Using whole-exome sequencing, we screened for mutations within protein-coding sequences in a single family of patients with SCZ. In a pathway enrichment analysis, we found multiple transmitted variant genes associated with two KEGG pathways: herpes simplex virus 1 (HSV1) infection and the extracellular matrix (ECM)-receptor interaction. When searching for rare variants, six variants, SLC6A19p.L541R, CYP2E1p.T376S, NAT10p.E811D, N4BP1p.L7V, CBX2p.S520C, and ZNF460p.K190E, segregated with SCZ. A bioinformatic analysis showed that three of these mutated genes were associated with chromatin modulation. We found that HSV1 infection, ECM-receptor interaction pathways, and epigenetic mechanisms may contribute to the pathogenesis of SCZ in certain families. The identified polygenetic risk factors from the sample family provide distinctive underlying biological mechanisms of the pathophysiology of SCZ and may be useful in clinical practice and patient care.

Ultrarare Loss-of-Function Mutations in the Genes Encoding the Ionotropic Glutamate Receptors of Kainate Subtypes Associated with Schizophrenia Disrupt the Interaction with PSD95.

Schizophrenia is a complex mental disorder with a genetic component. The GRIK gene family encodes ionotropic glutamate receptors of the kainate subtype, which are considered candidate genes for schizophrenia. We screened for rare and pathogenic mutations in the protein-coding sequences of the GRIK gene family in 516 unrelated patients with schizophrenia using the ion semiconductor sequencing method. We identified 44 protein-altered variants, and in silico analysis indicated that 36 of these mutations were rare and damaging or pathological based on putative protein function. Notably, we identified four truncating mutations, including two frameshift deletion mutations (GRIK1p.Phe24fs and GRIK1p.Thr882fs) and two nonsense mutations (GRIK2p.Arg300Ter and GRIK4p.Gln342Ter) in four unrelated patients with schizophrenia. They exhibited minor allele frequencies of less than 0.01% and were absent in 1517 healthy controls from Taiwan Biobank. Functional analysis identified these four truncating mutants as loss-of-function (LoF) mutants in HEK-293 cells. We also showed that three mutations (GRIK1p.Phe24fs, GRIK1p.Thr882fs, and GRIK2p.Arg300Ter) weakened the interaction with the PSD95 protein. The results suggest that the GRIK gene family harbors ultrarare LoF mutations in some patients with schizophrenia. The identification of proteins that interact with the kainate receptors will be essential to determine kainate receptor-mediated signaling in the brain.

Combined micro- and nano-scale surface textures for enhanced near-infrared light harvesting in silicon photovoltaics.

As silicon photovoltaics evolve towards thin-wafer technologies, efficient optical absorption for the near-infrared wavelengths has become particularly challenging. In this work, we present a solution that employs combined micro- and nano-scale surface textures to increase light harvesting in the near-infrared for crystalline silicon photovoltaics, and discuss the associated antireflection and scattering mechanisms. The surface textures are achieved by uniformly depositing a layer of indium-tin-oxide nanowhiskers on micro-grooved silicon substrates using electron-beam evaporation. The nanowhiskers facilitate optical transmission in the near-infrared by functioning as impedance matching layers with effective refractive indices gradually varying from 1 to 1.3. Materials with such unique refractive index characteristics are not readily available in nature. As a result, the solar cell with combined textures achieves over 90% external quantum efficiencies for a broad wavelength range of 460-980 nm, which is crucial to the development of advanced thin-substrate silicon solar cells.

Sequencing of the coding regions of GNBIL on chromosome 22q11.2 as a risk gene of schizophrenia.

GNB1L haploinsufficiency caused by 22q11.2 deletion syndrome may contribute to schizophrenia pathophysiology. We resequenced the protein-coding sequences of GNB1L in 553 patients with schizophrenia and 535 controls from Taiwan. Four common single-nucleotide polymorphisms showed no association with patients with schizophrenia. We identified 17 rare missense mutations, including three that were schizophrenia-associated and predicted as pathogenic (p.R57W, p.G68S, and p.R265C). Given that rare mutations with high penetrance contribute to schizophrenia, missense mutations of GNB1L might increase the risk of schizophrenia in some patients.

Optical metrology of randomly-distributed Au colloids on a multilayer film.

Spectroscopic ellipsometry (SE) measurements coupled with efficient theoretical modeling and scanning electron microscopy analysis are used in the metrology of randomly-distributed gold nanoparticles on a multilayer film. Measurements were conducted in the ultraviolet to near infrared region at several angles of incidence. To understand the size, shape, and distribution of nanoparticles, a finite-element Green's function approach considering the scattering from multiple nanoparticles was employed to calculate the ellipsometry parameters. Our calculations are in fair agreement with the ellipsometry measurements when suitable size, shape, and distribution pattern of nanoparticles are found. This demonstrates that SE could be a useful tool to the metrology of arbitrarily-distributed nanoparticles on a multilayer film.

Genetic analysis of the NR2E1 gene as a candidate gene of schizophrenia.

NR2E1 is implicated in the regulation of neurogenesis and considered as a candidate gene for schizophrenia. We resequenced all the exons of NR2E1 in 547 patients with schizophrenia and 567 controls from Taiwan. We identified five common SNPs with no association with patients with schizophrenia. Further haplotype-based association analysis showed that two haplotypes within NR2E1 were correlated with the schizophrenia risk. Four rare mutations located at untranslated regions were identified in patients with schizophrenia but not in our control sample. The present study suggests that NR2E1 is likely to play a significant role in conferring susceptibility to schizophrenia.

Differential Expression of Multiple Disease-Related Protein Groups Induced by Valproic Acid in Human SH-SY5Y Neuroblastoma Cells.

Valproic acid (VPA) is a multifunctional medication used for the treatment of epilepsy, mania associated with bipolar disorder, and migraine. The pharmacological effects of VPA involve a variety of neurotransmitter and cell signaling systems, but the molecular mechanisms underlying its clinical efficacy is to date largely unknown. In this study, we used the isobaric tags for relative and absolute quantitation shotgun proteomic analysis to screen differentially expressed proteins in VPA-treated SH-SY5Y cells. We identified changes in the expression levels of multiple proteins involved in Alzheimer's disease, Parkinson's disease, chromatin remodeling, controlling gene expression via the vitamin D receptor, ribosome biogenesis, ubiquitin-mediated proteolysis, and the mitochondrial oxidative phosphorylation and electron transport chain. Our data indicate that VPA may modulate the differential expression of proteins involved in mitochondrial function and vitamin D receptor-mediated chromatin transcriptional regulation and proteins implicated in the pathogenesis of neurodegenerative diseases.

Multiple Rare Risk Coding Variants in Postsynaptic Density-Related Genes Associated With Schizophrenia Susceptibility.

OBJECTIVE: Schizophrenia is a chronic debilitating neurobiological disorder of aberrant synaptic connectivity and synaptogenesis. Postsynaptic density (PSD)-related proteins in N-methyl-D-aspartate receptor-postsynaptic signaling complexes are crucial to regulating the synaptic transmission and functions of various synaptic receptors. This study examined the role of PSD-related genes in susceptibility to schizophrenia. METHODS: We resequenced 18 genes encoding the disks large-associated protein (DLGAP), HOMER, neuroligin (NLGN), neurexin, and SH3 and multiple ankyrin repeat domains (SHANK) protein families in 98 schizophrenic patients with family psychiatric history using semiconductor sequencing. We analyzed the protein function of the identified rare schizophrenia-associated mutants via immunoblotting and immunocytochemistry. RESULTS: We identified 50 missense heterozygous mutations in 98 schizophrenic patients with family psychiatric history, and in silico analysis revealed some as damaging or pathological to the protein function. Ten missense mutations were absent from the dbSNP database, the gnomAD (non-neuro) dataset, and 1,517 healthy controls from Taiwan BioBank. Immunoblotting revealed eight missense mutants with altered protein expressions in cultured cells compared with the wild type. CONCLUSION: Our findings suggest that PSD-related genes, especially the NLGN, SHANK, and DLGAP families, harbor rare functional mutations that might alter protein expression in some patients with schizophrenia, supporting contributing rare coding variants into the genetic architecture of schizophrenia.

Manipulative depolarization and reflectance spectra of morphologically controlled nano-pillars and nano-rods.

Depolarization of sub-mum-high Si nano-pillar/nano-rod surface reflectance with morphologically controlled anti-reflection spectrum is demonstrated. Extremely small reflectance dip of 1.5% at 400-450 nm for Si nano-pillars is extraordinary when comparing with Si nano-rods, in which the reflectance vs. L/lambda for Si nano-pillars coincides well with the graded-index multilayer based modeling spectrum. Alternatively, Si nano-rods preserve its flattened reflectance spectrum up to 1700 nm, whereas the Si nano-pillar surface reflectance monotonically increases to approach that of bulk Si. The destructive interference is only induced on Si nano-pillar surface with larger aspect-ratio > or =15 and small sidewall slope <7 to suppress surface reflectance at blue-green wavelength region. Anomalous depolarization observed from disordered Si nano-pillar/nano-rod surface reflection indicates that TM-mode incidence interacts with more bound electrons than TE-mode to preserve its effective dielectric permittivity less deviated from the bulk Si. The degraded depolarization ratio observed under TE-mode incidence which correlates well with a simplified bounded-electron resonance model is elucidated.

Aspect-ratio-dependent ultra-low reflection and luminescence of dry-etched Si nanopillars on Si substrate.

The Si nanopillars with high aspect ratio were fabricated by dry-etching the thin SiO(2)-covered Si substrate with a rapidly self-assembled Ni nanodot patterned mask. Aspect-ratio-dependent ultra-low reflection and anomalous luminescence of Si nanopillars are analyzed for applications in all-Si based lighting and energy transferring systems. The Si nanopillars induce an ultra-low reflectance and refractive index of 0.88% and 1.12, respectively, at 435 nm due to the air/Si mixed structure and highly roughened surface. The reflectance can be <10% with a corresponding refractive index of<1.80 between 190 and 670 nm. Lengthening the Si nanopillars from 150 +/- 15 to 230 +/- 20 nm further results in a decreasing reflectance, corresponding to a reduction in refractive index by Delta n/n = 18% in the visible and near-infrared wavelength region. After dry-etching an Si wafer into Si nanopillars, the weak blue-green luminescence with double consecutive peaks at 418-451 nm is attributed to the oxygen defect (O(2-))-induced radiation, which reveals less relevance with the ultra-low-reflective Si nanopillar surface.

Functional analyses and effect of DNA methylation on the EGR1 gene in patients with schizophrenia.

EGR1, involved in the regulation of synaptic plasticity, learning, and memory, is considered a candidate gene for schizophrenia. We resequenced the exonic regions of EGR1 in 516 patients with schizophrenia and conducted a reporter gene assay. We found two mutations including a rare mutation (c.-8C>T, rs561524195) and one common SNP (c.308-42C>T, rs11743810). The reporter gene assay showed c.-8C>T mutant did not affect promoter activity. Gene expression analyses showed that the average EGR1 mRNA and protein levels in lymphoblastoid cell lines of schizophrenia in male, but not female, were significantly higher than those in controls. We conducted in vitro DNA methylation reaction, luciferase activity assay, and pyrosequencing to assess DNA methylation of EGR1 expression underlying the pathophysiology of schizophrenia. DNA methylation of the EGR1 promoter region attenuated reporter activity, suggesting that DNA methylation regulates EGR1 expression. There were no statistically significant differences in DNA methylation levels of 17 CpG sites at the EGR1 promoter region between 64 patients with schizophrenia compared with 64 controls. These results suggest that the exonic mutations in EGR1 and DNA methylation regulating EGR1 expression might not be associated with schizophrenia. However, the gender-specific association of elevated EGR1 expression might be involved in the pathophysiology of schizophrenia.

Mutation analysis of the WNT7A gene in patients with schizophrenia.

Aberrant WNT signaling has been implicated in the pathophysiology of schizophrenia. WNT7A, a member of the WNT gene family, is considered a potential candidate of schizophrenia. All exons of WNT7A in 570 schizophrenic patients and 563 controls were sequenced, and protein functional analysis was conducted. Five common variants were identified, but none were noted to be associated with schizophrenia. Nevertheless, nine rare mutations, including one schizophrenia-specific missense mutation (c.305G > A), were discovered. However, immunoblot analysis findings revealed that the c.305G > A mutation did not affect protein expression. These results suggest that WNT7A is unlikely to be associated with susceptibility to schizophrenia.

DNA methylation analysis of the EGR3 gene in patients of schizophrenia.

DNA methylation has been implicated in the pathogenesis of schizophrenia. EGR3 is considered as a potential candidate gene for schizophrenia. We conducted in vitro DNA methylation reaction, Lucia luciferase activity assay, and pyrosequencing assay to assess the DNA methylation of the EGR3 expression underlying the pathophysiology of schizophrenia. We found that DNA methylation of the putative EGR3 regulatory regions attenuated Lucia luciferase activity. There was no difference in the DNA methylation pattern of EGR3 between in 50 schizophrenic patients and 47 controls. Our data suggest that DNA methylation regulated the expression of EGR3 might not be associated with schizophrenia.

Depletion of securin increases arsenite-induced chromosome instability and apoptosis via a p53-independent pathway.

Arsenic is a pathologic factor of cardiovascular diseases and cancers; nevertheless, it also acts as an anticancer agent effective on acute promyelocytic leukemia and multiple myeloma. Securin, a proposed proto-oncogene, regulates cell proliferation and tumorigenesis. However, roles of securin on the arsenic-induced cell cycle arrest and apoptosis remain unknown. In this study, the effects of sodium arsenite on the expression of securin in two tissue types of cell lines, the vascular endothelial and colorectal epithelial cells, were investigated. Arsenite (8-16 microM, 24 h) increased the cytotoxicity, apoptosis, and growth inhibition in both endothelial and epithelial cells. The levels of phospho-CDC2 (threonine-161), CDC2, and cyclin B1 proteins were decreased, and the G2/M fractions were increased by arsenite. Concomitantly, arsenite markedly diminished the securin protein expression and induced the abnormal sister chromatid separation. The depletion of securin proteins increased the induction of mitotic arrest, aberrant chromosome segregation, and apoptosis after arsenite treatment. p53, a tumor suppressor protein, balances the cell survival and apoptosis. Arsenite raised the levels of phospho-p53 (serine-15) and p53 (DO-1) proteins in both the securin-wild-type and -null cells. The p53-functional cells were more susceptible than the p53-mutational cells to arsenite on the cytotoxicity and apoptosis. Besides, arsenite decreased the levels of securin proteins to a similar degree in both the p53-functional and -mutational cells. Together, it is the first time to demonstrate that the inhibition of securin expression induced by arsenite increases the chromosomal instability and apoptosis via a p53-independent pathway.

Screening for Mutations in the TBX1 Gene on Chromosome 22q11.2 in Schizophrenia.

A higher-than-expected frequency of schizophrenia in patients with 22q11.2 deletion syndrome suggests that chromosome 22q11.2 harbors the responsive genes related to the pathophysiology of schizophrenia. The TBX1 gene, which maps to the region on chromosome 22q11.2, plays a vital role in neuronal functions. Haploinsufficiency of the TBX1 gene is associated with schizophrenia endophenotype. This study aimed to investigate whether the TBX1 gene is associated with schizophrenia. We searched for mutations in the TBX1 gene in 652 patients with schizophrenia and 567 control subjects using a re-sequencing method and conducted a reporter gene assay. We identified six SNPs and 25 rare mutations with no association with schizophrenia from Taiwan. Notably, we identified two rare schizophrenia-specific mutations (c.-123G>C and c.-11delC) located at 5' UTR of the TBX1 gene. The reporter gene assay showed that c.-123C significantly decreased promoter activity, while c.-11delC increased promoter activity compared with the wild-type. Our findings suggest that the TBX1 gene is unlikely a major susceptible gene for schizophrenia in an ethnic Chinese population for Taiwan, but a few rare mutations in the TBX1 gene may contribute to the pathogenesis of schizophrenia in some patients.

Rare mutations and hypermethylation of the ARC gene associated with schizophrenia.

Activity-regulated cytoskeleton-associated protein (ARC), which interacts with the N-methyl-d-aspartate receptor (NMDAR) complex, is a critical effector molecule downstream of multiple neuronal signaling pathways. Dysregulation of the ARC/NMDAR complex can disrupt learning, memory, and normal brain functions. This study examined the role of ARC in susceptibility to schizophrenia. We used a resequencing strategy to identify the variants of ARC in 1078 subjects, including patients with schizophrenia and normal controls. We identified 16 known SNPs and 27 rare mutations. SNP-based analysis showed no association of ARC with schizophrenia. In addition, the rare mutations did not increase the burden in patients compared with controls. However, one patient-specific allele in the putative ARC promoter region and seven patient-specific mutants in ARC exon regions significantly reduced the reporter gene activity compared with ARC wild-type. Methylation of a putative ARC promoter attenuated reporter activity in vitro, suggesting that ARC expression is regulated by DNA methylation. Pyrosequencing revealed eight hypermethylated CpG sites in the putative ARC promoter region in 64 schizophrenic patients compared with 63 controls. Taken together, our results suggest that both rare variants and epigenetic regulation of ARC contribute to the pathogenesis of schizophrenia in some patients.