Highly selective, reversible water activation by P,N-cooperativity in pyridyl-functionalized phosphinines.

Tetrapyridyl-functionalized phosphinines were prepared and structurally characterized. The donor-functionalized aromatic phosphorus heterocycles react highly selectively and even reversibly with water. Calculations reveal P,N-cooperativity for this process, with the flanking pyridyl groups serving to kinetically enhance the formal oxidative addition process of H2O to the low-coordinate phosphorus atom via H-bonding. Subsequent tautomerization forms 1,2-dihydrophosphinine derivatives, which can be quantitatively converted back to the phosphinine by applying vacuum, even at room temperature. This process can be repeated numerous times, without any sign of decomposition of the phosphinine. In the presence of CuI·SMe2, dimeric species of the type ([Cu2I2(phosphinine)]2) are formed, in which each phosphorus atom shows the less common µ2-bridging 2e--lone-pair-donation to two Cu(i)-centres. Our results demonstrate that fully unsaturated phosphorus heterocycles, containing reactive P[double bond, length as m-dash]C double bonds, are interesting candidates for the activation of E-H bonds, while the aromaticity of such compounds plays an appreciable role in the reversibility of the reaction, supported by NICS calculations.

Precision and Accuracy of Single-Cell/Nuclei RNA Sequencing Data.

Single-cell/nuclei RNA sequencing (sc/snRNA-Seq) is widely used for profiling cell-type gene expressions in biomedical research. An important but underappreciated issue is the quality of sc/snRNA-Seq data that would impact the reliability of downstream analyses. Here we evaluated the precision and accuracy in 18 sc/snRNA-Seq datasets. The precision was assessed on data from human brain studies with a total of 3,483,905 cells from 297 individuals, by utilizing technical replicates. The accuracy was evaluated with sample-matched scRNA-Seq and pooled-cell RNA-Seq data of cultured mononuclear phagocytes from four species. The results revealed low precision and accuracy at the single-cell level across all evaluated data. Cell number and RNA quality were highlighted as two key factors determining the expression precision, accuracy, and reproducibility of differential expression analysis in sc/snRNA-Seq. This study underscores the necessity of sequencing enough high-quality cells per cell type per individual, preferably in the hundreds, to mitigate noise in expression quantification.

Brain eQTLs of European, African American, and Asian ancestry improve interpretation of schizophrenia GWAS.

Research on brain expression quantitative trait loci (eQTLs) has illuminated the genetic underpinnings of schizophrenia (SCZ). Yet, the majority of these studies have been centered on European populations, leading to a constrained understanding of population diversities and disease risks. To address this gap, we examined genotype and RNA-seq data from African Americans (AA, n=158), Europeans (EUR, n=408), and East Asians (EAS, n=217). When comparing eQTLs between EUR and non-EUR populations, we observed concordant patterns of genetic regulatory effect, particularly in terms of the effect sizes of the eQTLs. However, 343,737 cis-eQTLs (representing ∼17% of all eQTLs pairs) linked to 1,276 genes (about 10% of all eGenes) and 198,769 SNPs (approximately 16% of all eSNPs) were identified only in the non-EUR populations. Over 90% of observed population differences in eQTLs could be traced back to differences in allele frequency. Furthermore, 35% of these eQTLs were notably rare (MAF < 0.05) in the EUR population. Integrating brain eQTLs with SCZ signals from diverse populations, we observed a higher disease heritability enrichment of brain eQTLs in matched populations compared to mismatched ones. Prioritization analysis identified seven new risk genes ( SFXN2 , RP11-282018.3 , CYP17A1 , VPS37B , DENR , FTCDNL1 , and NT5DC2 ), and three potential novel regulatory variants in known risk genes ( CNNM2 , C12orf65 , and MPHOSPH9 ) that were missed in the EUR dataset. Our findings underscore that increasing genetic ancestral diversity is more efficient for power improvement than merely increasing the sample size within single-ancestry eQTLs datasets. Such a strategy will not only improve our understanding of the biological underpinnings of population structures but also pave the way for the identification of novel risk genes in SCZ.

No Increased Detection of Nucleic Acids of CNS-related Viruses in the Brains of Patients with Schizophrenia, Bipolar Disorder, and Autism Spectrum Disorder.

BACKGROUND AND HYPOTHESIS: Viral infections are increasingly recognized in the etiology of psychiatric disorders based on epidemiological and serological studies. Few studies have analyzed viruses directly within the brain and no comprehensive investigation of viral infection within diseased brains has been completed. This study aims to determine whether viral infection in brain tissues is a risk factor for 3 major psychiatric disorders, including schizophrenia, bipolar disorder, and autism spectrum disorder. STUDY DESIGN: This study directly evaluated the presence of viral DNA or RNA in 1569 brains of patients and controls using whole-genome sequencing and RNA sequencing data with 4 independent cohorts. The PathSeq tool was used to identify known human viruses in the genome and transcriptome of patients and controls. STUDY RESULTS: A variety of DNA and RNA viruses related to the central nervous system were detected in the brains of patients with major psychiatric disorders, including viruses belonging to Herpesviridae, Polyomaviridae, Retroviridae, Flaviviridae, Parvoviridae, and Adenoviridae. However, no consistent significant differences were found between patients and controls in terms of types and amount of virus detected at both DNA and RNA levels. CONCLUSIONS: The findings of this study do not suggest an association between viral infection in postmortem brains and major psychiatric disorders.

Neuroimmune transcriptome changes in patient brains of psychiatric and neurological disorders.

Neuroinflammation has been implicated in multiple brain disorders but the extent and the magnitude of change in immune-related genes (IRGs) across distinct brain disorders has not been directly compared. In this study, 1275 IRGs were curated and their expression changes investigated in 2467 postmortem brains of controls and patients with six major brain disorders, including schizophrenia (SCZ), bipolar disorder (BD), autism spectrum disorder (ASD), major depressive disorder (MDD), Alzheimer's disease (AD), and Parkinson's disease (PD). There were 865 IRGs present across all microarray and RNA-seq datasets. More than 60% of the IRGs had significantly altered expression in at least one of the six disorders. The differentially expressed immune-related genes (dIRGs) shared across disorders were mainly related to innate immunity. Moreover, sex, tissue, and putative cell type were systematically evaluated for immune alterations in different neuropsychiatric disorders. Co-expression networks revealed that transcripts of the neuroimmune systems interacted with neuronal-systems, both of which contribute to the pathology of brain disorders. However, only a few genes with expression changes were also identified as containing risk variants in genome-wide association studies. The transcriptome alterations at gene and network levels may clarify the immune-related pathophysiology and help to better define neuropsychiatric and neurological disorders.

Absence of coding somatic single nucleotide variants within well-known candidate genes in late-onset sporadic Alzheimer's Disease based on the analysis of multi-omics data.

Somatic mutations arise randomly or are induced by environmental factors, which may increase the risk of Alzheimer's disease (AD). Identifying somatic mutations in sporadic AD (SAD) may provide new insight of the disease. To evaluate the potential contribution of somatic single nucleotide variations (SNVs), particularly that of well-known AD-candidate genes, we investigated sequencing data sets from four platforms: whole-genome sequencing (WGS), deep whole-exome sequencing (WES) on paired brain and liver samples, RNA sequencing (RNA-seq), and single-cell whole-genome sequencing (scWGS) of brain samples from 16 AD patients and 16 non-AD individuals. We found that the average number, mean variant allele fractions (VAFs) and mutational signatures of somatic SNVs have similar distributions between AD brains and non-AD brains. We did not identify any somatic SNVs within coding regions of the APP, PSEN1, PSEN2, nor in APOE. This study shows that somatic SNVs within the coding region of AD-candidate genes are unlikely to be a common causal factor for SAD.

Drug Response-Related DNA Methylation Changes in Schizophrenia, Bipolar Disorder, and Major Depressive Disorder.

Pharmacotherapy is the most common treatment for schizophrenia (SCZ), bipolar disorder (BD), and major depressive disorder (MDD). Pharmacogenetic studies have achieved results with limited clinical utility. DNA methylation (DNAm), an epigenetic modification, has been proposed to be involved in both the pathology and drug treatment of these disorders. Emerging data indicates that DNAm could be used as a predictor of drug response for psychiatric disorders. In this study, we performed a systematic review to evaluate the reproducibility of published changes of drug response-related DNAm in SCZ, BD and MDD. A total of 37 publications were included. Since the studies involved patients of different treatment stages, we partitioned them into three groups based on their primary focuses: (1) medication-induced DNAm changes (n = 8); (2) the relationship between DNAm and clinical improvement (n = 24); and (3) comparison of DNAm status across different medications (n = 14). We found that only BDNF was consistent with the DNAm changes detected in four independent studies for MDD. It was positively correlated with clinical improvement in MDD. To develop better predictive DNAm factors for drug response, we also discussed future research strategies, including experimental, analytical procedures and statistical criteria. Our review shows promising possibilities for using BDNF DNAm as a predictor of antidepressant treatment response for MDD, while more pharmacoepigenetic studies are needed for treatments of various diseases. Future research should take advantage of a system-wide analysis with a strict and standard analytical procedure.

Sex-differential DNA methylation and associated regulation networks in human brain implicated in the sex-biased risks of psychiatric disorders.

Many psychiatric disorders are characterized by a strong sex difference, but the mechanisms behind sex-bias are not fully understood. DNA methylation plays important roles in regulating gene expression, ultimately impacting sexually different characteristics of the human brain. Most previous literature focused on DNA methylation alone without considering the regulatory network and its contribution to sex-bias of psychiatric disorders. Since DNA methylation acts in a complex regulatory network to connect genetic and environmental factors with high-order brain functions, we investigated the regulatory networks associated with different DNA methylation and assessed their contribution to the risks of psychiatric disorders. We compiled data from 1408 postmortem brain samples in 3 collections to identify sex-differentially methylated positions (DMPs) and regions (DMRs). We identified and replicated thousands of DMPs and DMRs. The DMR genes were enriched in neuronal related pathways. We extended the regulatory networks related to sex-differential methylation and psychiatric disorders by integrating methylation quantitative trait loci (meQTLs), gene expression, and protein-protein interaction data. We observed significant enrichment of sex-associated genes in psychiatric disorder-associated gene sets. We prioritized 2080 genes that were sex-biased and associated with psychiatric disorders, such as NRXN1, NRXN2, NRXN3, FDE4A, and SHANK2. These genes are enriched in synapse-related pathways and signaling pathways, suggesting that sex-differential genes of these neuronal pathways may cause the sex-bias of psychiatric disorders.

Transcription factor POU3F2 regulates TRIM8 expression contributing to cellular functions implicated in schizophrenia.

Schizophrenia (SCZ) is a neuropsychiatric disorder with aberrant expression of multiple genes. However, identifying its exact causal genes remains a considerable challenge. The brain-specific transcription factor POU3F2 (POU domain, class 3, transcription factor 2) has been recognized as a risk factor for SCZ, but our understanding of its target genes and pathogenic mechanisms are still limited. Here we report that POU3F2 regulates 42 SCZ-related genes in knockdown and RNA-sequencing experiments of human neural progenitor cells (NPCs). Among those SCZ-related genes, TRIM8 (Tripartite motif containing 8) is located in SCZ-associated genetic locus and is aberrantly expressed in patients with SCZ. Luciferase reporter and electrophoretic mobility shift assays (EMSA) showed that POU3F2 induces TRIM8 expression by binding to the SCZ-associated SNP (single nucleotide polymorphism) rs5011218, which affects POU3F2-binding efficiency at the promoter region of TRIM8. We investigated the cellular functions of POU3F2 and TRIM8 as they co-regulate several pathways related to neural development and synaptic function. Knocking down either POU3F2 or TRIM8 promoted the proliferation of NPCs, inhibited their neuronal differentiation, and impaired the excitatory synaptic transmission of NPC-derived neurons. These results indicate that POU3F2 regulates TRIM8 expression through the SCZ-associated SNP rs5011218, and both genes may be involved in the etiology of SCZ by regulating neural development and synaptic function.

Interplay between ER Ca2+ Binding Proteins, STIM1 and STIM2, Is Required for Store-Operated Ca2+ Entry.

Store-operated calcium entry (SOCE), a fundamentally important homeostatic and Ca2+ signaling pathway in many types of cells, is activated by the direct interaction of stromal interaction molecule 1 (STIM1), an endoplasmic reticulum (ER) Ca2+-binding protein, with Ca2+-selective Orai1 channels localized in the plasma membrane. While much is known about the regulation of SOCE by STIM1, the role of stromal interaction molecule 2 (STIM2) in SOCE remains incompletely understood. Here, using clustered regularly interspaced short palindromic repeats -CRISPR associated protein 9 (CRISPR-Cas9) genomic editing and molecular imaging, we investigated the function of STIM2 in NIH 3T3 fibroblast and αT3 cell SOCE. We found that deletion of Stim2 expression reduced SOCE by more than 90% in NIH 3T3 cells. STIM1 expression levels were unaffected in the Stim2 null cells. However, quantitative confocal fluorescence imaging demonstrated that in the absence of Stim2 expression, STIM1 did not translocate or form punctae in plasma membrane-associated ER membrane (PAM) junctions following ER Ca2+ store depletion. Fluorescence resonance energy transfer (FRET) imaging of intact, living cells revealed that the formation of STIM1 and Orai1 complexes in PAM nanodomains was significantly reduced in the Stim2 knockout cells. Our findings indicate that STIM2 plays an essential role in regulating SOCE in NIH 3T3 and αT3 cells and suggests that dynamic interplay between STIM1 and STIM2 induced by ER Ca2+ store discharge is necessary for STIM1 translocation, its interaction with Orai1, and activation of SOCE.

Stromal Interaction Molecule 1 (STIM1) Regulates ATP-sensitive Potassium (KATP) and Store-operated Ca2+ Channels in MIN6 ß-Cells.

Stromal interaction molecule 1 (STIM1) regulates store-operated Ca2+ entry (SOCE) and other ion channels either as an endoplasmic reticulum Ca2+-sensing protein or when present in the plasma membrane. However, the role of STIM1 in insulin-secreting ß-cells is unresolved. We report that lowering expression of STIM1, the gene that encodes STIM1, in insulin-secreting MIN6 ß-cells with RNA interference inhibits SOCE and ATP-sensitive K+ (KATP) channel activation. The effects of STIM1 knockdown were reversed by transduction of MIN6 cells with an adenovirus gene shuttle vector that expressed human STIM1 Immunoprecipitation studies revealed that STIM1 binds to nucleotide binding fold-1 (NBF1) of the sulfonylurea receptor 1 (SUR1) subunit of the KATP channel. Binding of STIM1 to SUR1 was enhanced by poly-lysine. Our data indicate that SOCE and KATP channel activity are regulated by STIM1. This suggests that STIM1 is a multifunctional signaling effector that participates in the control of membrane excitability and Ca2+ signaling events in ß-cells.

Resveratrol Interferes with Fura-2 Intracellular Calcium Measurements.

Resveratrol, a naturally occurring polyphenol found in some fruits and especially in grapes, has been reported to provide diverse health benefits. Resveratrol's mechanism of action is the subject of many investigations, and some studies using the ratiometric calcium indicator Fura-2 suggest that it modulates cellular calcium responses. In the current study, contradictory cellular calcium responses to resveratrol applied at concentrations exceeding 10 µM were observed during in vitro imaging studies depending on the calcium indicator used, with Fura-2 indicating an increase in intracellular calcium while Fluo-4 and the calcium biosensor YC3.60 indicated no response. When cells loaded with Fura-2 were treated with 100 µM resveratrol, excitation at 340 nm resulted in a large intensity increase at 510 nm, but the expected concurrent decline with 380 nm excitation was not observed. Pre-treatment of cells with the calcium chelator BAPTA-AM did not prevent a rise in the 340/380 ratio when resveratrol was present, but it did prevent an increase in 340/380 when ATP was applied, suggesting that the resveratrol response was an artifact. Cautious data interpretation is recommended from imaging experiments using Fura-2 concurrently with resveratrol in calcium imaging experiments.

Insulin secretion and Ca2+ dynamics in ß-cells are regulated by PERK (EIF2AK3) in concert with calcineurin.

Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) (EIF2AK3) is essential for normal development and function of the insulin-secreting ß-cell. Although genetic ablation of PERK in ß-cells results in permanent neonatal diabetes in humans and mice, the underlying mechanisms remain unclear. Here, we used a newly developed and highly specific inhibitor of PERK to determine the immediate effects of acute ablation of PERK activity. We found that inhibition of PERK in human and rodent ß-cells causes a rapid inhibition of secretagogue-stimulated subcellular Ca(2+) signaling and insulin secretion. These dysfunctions stem from alterations in store-operated Ca(2+) entry and sarcoplasmic endoplasmic reticulum Ca(2+)-ATPase activity. We also found that PERK regulates calcineurin, and pharmacological inhibition of calcineurin results in similar defects on stimulus-secretion coupling. Our findings suggest that interplay between calcineurin and PERK regulates ß-cell Ca(2+) signaling and insulin secretion, and that loss of this interaction may have profound implications in insulin secretion defects associated with diabetes.

Serum- and glucocorticoid-induced protein kinase 1 (SGK1) is regulated by store-operated Ca2+ entry and mediates cytoprotection against necrotic cell death.

Serum and glucocorticoid-regulated kinase 1 (SGK1) encodes a phosphatidylinositol 3-kinase-dependent serine/threonine kinase that is rapidly induced in response to cellular stressors and is an important cell survival signal. Previous studies have suggested that an increase in cytoplasmic Ca(2+) concentration ([Ca(2+)]c) is required for increased SGK1 expression, but the subcellular source of Ca(2+) regulating SGK1 transcription remains uncertain. Activation of endoplasmic reticulum stress (ERS) with thapsigargin (TG) increased SGK1 mRNA and protein expression in MDA-MB-231 cells. Intracellular Ca(2+) imaging revealed that store-operated Ca(2+) entry played a prominent role in SGK1 induction by TG. Neither ERS nor release of Ca(2+) from the ER was sufficient to activate SGK1. Prolonged elevation of intracellular Ca(2+) levels, however, triggered cell death with a much greater proportion of the cells undergoing necrosis rather than apoptosis. A relative increase in the percentage of cells undergoing necrosis was observed in cells expressing a short hairpin RNA targeted to the SGK1 gene. Necrotic cell death evoked by cytoplasmic Ca(2+) overloading was associated with persistent hyperpolarization of the inner mitochondrial membrane and a modest increase in calpain activation, but did not involve detectable caspase 3 or caspase 7 activation. The effects of cytoplasmic Ca(2+) overloading on mitochondrial membrane potential were significantly reduced in cells expressing SGK1 compared with SGK1-depleted cells. Our findings indicate that store-operated Ca(2+) entry regulates SGK1 expression in epithelial cells and suggest that SGK1-dependent cytoprotective signaling involves effects on maintaining mitochondrial function.

Gating of connexin 43 gap junctions by a cytoplasmic loop calmodulin binding domain.

Calmodulin (CaM) binding sites were recently identified on the cytoplasmic loop (CL) of at least three α-subfamily connexins (Cx43, Cx44, Cx50), while Cx40 does not have this putative CaM binding domain. The purpose of this study was to examine the functional relevance of the putative Cx43 CaM binding site on the Ca(2+)-dependent regulation of gap junction proteins formed by Cx43 and Cx40. Dual whole cell patch-clamp experiments were performed on stable murine Neuro-2a cells expressing Cx43 or Cx40. Addition of ionomycin to increase external Ca(2+) influx reduced Cx43 gap junction conductance (G(j)) by 95%, while increasing cytosolic Ca(2+) concentration threefold. By contrast, Cx40 G(j) declined by <20%. The Ca(2+)-induced decline in Cx43 G(j) was prevented by pretreatment with calmidazolium or reversed by the addition of 10 mM EGTA to Ca(2+)-free extracellular solution, if Ca(2+) chelation was commenced before complete uncoupling, after which g(j) was only 60% recoverable. The Cx43 CL(136-158) mimetic peptide, but not the scrambled control peptide, or Ca(2+)/CaM-dependent kinase II 290-309 inhibitory peptide also prevented the Ca(2+)/CaM-dependent decline of Cx43 G(j). Cx43 gap junction channel open probability decreased to zero without reductions in the current amplitudes during external Ca(2+)/ionomycin perfusion. We conclude that Cx43 gap junctions are gated closed by a Ca(2+)/CaM-dependent mechanism involving the carboxyl-terminal quarter of the connexin CL domain. This study provides the first evidence of intrinsic differences in the Ca(2+) regulatory properties of Cx43 and Cx40.

Development of AFLP and RAPD markers linked to a locus associated with twisted growth in corkscrew willow (Salix matsudana 'Tortuosa').

Salix matsudana Koidz. cultivar 'Tortuosa' (corkscrew willow) is characterized by extensive stem bending and curling of leaves. To investigate the genetic basis of this trait, controlled crosses were made between a corkscrew female (S. matsudana 'Tortuosa') and a straight-stemmed, wild-type male (Salix alba L. Clone 99010). Seventy-seven seedlings from this family (ID 99270) were grown in the field for phenotypic observation. Among the progeny, 39 had straight stems and leaves and 38 had bent stems and curled leaves, suggesting that a dominant allele at a single locus controls this phenotype. As a first step in characterizing the locus, we searched for amplified fragment length polymorphism (AFLP) and randomly amplified polymorphic DNA (RAPD) markers linked to the tortuosa allele using bulked segregant analysis. Samples of DNA from 10 corkscrew individuals were combined to produce a corkscrew pool, and DNA from 10 straight progeny was combined to make a wild-type pool. Sixty-four AFLP primer combinations and 640 RAPD primers were screened to identify marker bands amplified from the corkscrew parent and progeny pool, but not from the wild-type parent or progeny pool. An AFLP marker and a RAPD marker linked to and flanking the tortuosa locus were placed on a preliminary linkage map constructed based on segregation among the 77 progeny. Sectioning and analysis of shoot tips revealed that the corkscrew phenotype is associated with vascular cell collapse, smaller cell size in regions near the cambium and less developed phloem fibers than in wild-type progeny. Identification of a gene associated with this trait could lead to greater understanding of the control of normal stem development in woody plants.

Collection and storage of pollen from Salix (Salicaceae).

Genetic improvement of willows through traditional breeding can be facilitated by pollen collection and storage so that female flower receptivity need not be synchronized with pollen shed for breeding. Two experiments were completed to test the effectiveness of various organic solvents for willow pollen collection. In the first experiment, seven pollen collection treatments and an untreated control were tested with two willow clones. The other experiment tested three treatments that showed promise in the initial experiment and an untreated control with eight willow clones. Toluene and carbon tetrachloride were effective for pollen extraction, with average pollen germination percentages that were >15%, but both chemicals reduced pollen viability by 10-20% compared with an untreated control based on in vitro germination tests. Pollen extracted with carbon tetrachloride or toluene was successfully used in controlled pollination, and >100 new families were produced with this technique. Pollen viability remained high after 18 mo of storage at -20°C. Based on our results, toluene is the preferred solvent for future willow pollen extractions because it is as effective as carbon tetrachloride, is not a known carcinogen, and is less expensive.