Cascade Aryne Aminoarylation for Biaryl Phenol Synthesis.

We describe a transition metal-free approach to hindered 3-amino-2-aryl phenols through a cascade nucleophilic addition / Smiles-Truce rearrangement of a functionalized Kobayashi aryne precursor. Under anionic conditions, secondary alkyl amines add to the aryne intermediate to set up an aryl transfer from a neighboring sulfonate group. The use of a sulfonate, rather than the more typical sulfonamide, enables access to phenolic biaryl products that are important motifs in natural products and pharmaceuticals.

Sanger Sequencing to Determine the Full-Length Sequence of Circular RNAs.

The pre-existing theory of pre-mRNA splicing into linear mature RNA was questioned with the introduction of circular RNAs (circRNAs). Hundreds of studies using high throughput RNA-sequencing (RNA-seq) techniques and novel computational programs reported the abundant and ubiquitous expression of circRNAs originating by pre-mRNA backsplicing. CircRNAs are mostly involved in gene expression by regulating functions of interacting microRNAs (miRNAs) and RNA-binding proteins (RBPs) or translating into functional polypeptides. Although all circRNA annotation tools identify circRNAs based on the backsplice junction (BSJ) sequences, only a few identify the internal sequences of circRNAs. However, the full-length sequence of circRNAs from RNA-seq data could be error-prone due to its similarity with the counterpart linear RNA. Since circRNA function depends on the mature sequence, validation of the mature sequence is the prerequisite for their further characterization. In this chapter, we discuss the validation of circRNA BSJ sequence by RT-PCR using divergent primer followed by Sanger sequencing. Furthermore, we describe the circRNA-rolling circle amplification (circRNA-RCA; circRNA enrichment by RNase R treatment, full-length cDNA synthesis, rolling circle PCR amplification using full-length primers, and Sanger sequencing of the PCR product) to validate the mature splice sequence of circRNAs. This chapter highlights the basic guidelines for designing divergent and full-length primers for PCR amplification and Sanger sequencing to validate circRNA sequences.

Large-scale recording of neuronal activity in freely-moving mice at cellular resolution.

Current methods for recording large-scale neuronal activity from behaving mice at single-cell resolution require either fixing the mouse head under a microscope or attachment of a recording device to the animal's skull. Both of these options significantly affect the animal behavior and hence also the recorded brain activity patterns. Here, we introduce a different method to acquire snapshots of single-cell cortical activity maps from freely-moving mice using a calcium sensor called CaMPARI. CaMPARI has a unique property of irreversibly changing its color from green to red inside active neurons when illuminated with 400 nm light. We capitalize on this property to demonstrate cortex-wide activity recording without any head fixation, tethering, or attachment of a miniaturized device to the mouse's head. Multiple cortical regions were recorded while the mouse was performing a battery of behavioral and cognitive tests. We identified task-dependent activity patterns across motor and somatosensory cortices, with significant differences across sub-regions of the motor cortex and correlations across several activity patterns and task parameters. This CaMPARI-based recording method expands the capabilities of recording neuronal activity from freely-moving and behaving mice under minimally-restrictive experimental conditions and provides large-scale volumetric data that are currently not accessible otherwise.

Identification of potential proteins translated from circular RNA splice variants.

Circular RNAs (circRNAs) are covalently closed RNA molecules generated from precursor RNAs by the head-to-tail backsplicing of exons. Hundreds of studies demonstrated that circRNAs are ubiquitously expressed and regulate cellular events by modulating microRNA (miRNA) and RNA-binding protein (RBP) activities. A few circRNAs are also known to translate into functional polypeptides regulating cellular physiology. All these functions primarily depend on the full-length sequence of the circRNAs. CircRNA backsplice junction sequence is the key to identifying circRNAs and their full-length mature sequence. However, some multi-exonic circRNAs exist in different isoforms sharing identical backsplice junction sequences and are termed circRNA splice variants. Here, we analyzed the previously published HeLa cell RNA-seq datasets to identify circRNA splice variants using the de novo module of the CIRCexplorer2 circRNA annotation pipeline. A subset of circRNAs with splice variants was validated by the circRNA-rolling circle amplification (circRNA-RCA) method. Interestingly, several validated circRNAs were predicted to translate into proteins by the riboCIRC database. Furthermore, polyribosome fractionation followed by quantitative PCR confirmed the association of a subset of circRNAs with polyribosome supporting their protein-coding potential. Finally, bioinformatics analysis of proteins derived from splice variants of circCORO1C and circASPH suggested altered protein sequences and structures that could affect their physiological functions. Together, our study identified novel circRNA splice variants and their potential translation into protein isoforms which may regulate various physiological processes.

Correlation between gerotranscendence and oral health-related quality of life among elderly population in Davanagere city: A cross-sectional survey.

Aim: To assess the relationship between oral health related quality of life (OHRQoL) and gerotranscendence among elderly subjects in Davangere city. Settings and Design: Field Setting and cross-sectional survey design. Materials and Methods: Study involved a stratified sample of 400 elderly population aged 60 years and above. Data related to demographic details, systemic and oral health related factors, nutritional status, gerotranscendence level and geriatric oral health related quality of life of study participants was recorded using study proforma, Mini Nutritional Scale Assessment- Short form (MNA-SF) index, Gerotranscendence Scale Type 2 (GST2) questionnaire and GOHAI questionnaire respectively. Statistical Analysis Used: Significance level was fixed at P < 0.05. Chi-square, Pearson's/Spearman's correlation and Multiple linear regression tests were used for analysis. Results: Participants had good oral health related quality of life (mean GOHAI - 41.33±10.8) and moderate level of gerotranscendence (GST2- 19.5 ± 8.7). The gerotranscendence scores were significantly (P<0.05) negatively correlated with socioeconomic status (r = -0.19), education (r = -0.55), self-perceived oral health (r = -0.43), nutritional status (r = -0.64), GOHAI (r = -0.17), utilization of dental services (r = -0.26) and marital status (r = -0.39) and were significantly (P < 0.05) positively correlated with age (r = 0.77), systemic problems (r = 0.49), number of missing teeth (r = 0.57), self-perceived need for treatment (r = 0.24), and pan chewing (r = 0.62). Gerotranscendence was not a significant predictor of GOHAI (P = 0.43). Conclusion: Gerotranscendence was negatively correlated with oral health related quality of life among elderly population in Davanagere city.

A heterotypic assembly mechanism regulates CHIP E3 ligase activity.

CHIP (C-terminus of Hsc70-interacting protein) and its worm ortholog CHN-1 are E3 ubiquitin ligases that link the chaperone system with the ubiquitin-proteasome system (UPS). CHN-1 can cooperate with UFD-2, another E3 ligase, to accelerate ubiquitin chain formation; however, the basis for the high processivity of this E3s set has remained obscure. Here, we studied the molecular mechanism and function of the CHN-1-UFD-2 complex in Caenorhabditis elegans. Our data show that UFD-2 binding promotes the cooperation between CHN-1 and ubiquitin-conjugating E2 enzymes by stabilizing the CHN-1 U-box dimer. However, HSP70/HSP-1 chaperone outcompetes UFD-2 for CHN-1 binding, thereby promoting a shift to the autoinhibited CHN-1 state by acting on a conserved residue in its U-box domain. The interaction with UFD-2 enables CHN-1 to efficiently ubiquitylate and regulate S-adenosylhomocysteinase (AHCY-1), a key enzyme in the S-adenosylmethionine (SAM) regeneration cycle, which is essential for SAM-dependent methylation. Our results define the molecular mechanism underlying the synergistic cooperation of CHN-1 and UFD-2 in substrate ubiquitylation.

Reactivity of Nitric Oxide and Nitrosonium Ion with Copper(II/I) Schiff Base Complexes: Mechanistic Aspects of Imine C═N Bond Cleavage and Oxidation of Pyridine-2-aldehyde to Pyridine-2-carboxylic Acid.

Four Schiff base ligands of the general formulas [6-(R)-2-pyridyl-N-(2'-methylthiophenyl)methylenimine] (RL1) and 6-p-chlorophenyl-2-pyridyl-N-(2'-phenylthiophenyl)methylenimine (RL2), where R = H, Me, p-ClPh, and their bis-ligand copper(II) and copper(I) complexes, 1-4 and 1'-4', respectively, were synthesized and characterized. The reactivities of 1-4 with nitric oxide (NO) gas and of 1'-4' with solid NOBF4 (NO+) were examined in dry acetonitrile in the presence and absence of water (H2O). The results revealed that, in the absence of H2O, complexes 1-4 (or 1'-4') reacts with NO (or NOBF4), leading to imine C═N bond cleavage of both (or one) Schiff base(s) that generates 2 (or 1) equiv of 2-(methyl/phenyl)thiobenzenediazonium perchlorates (5/6) and the corresponding picolaldehyde (RPial) via a copper nitrosyl of a {CuNO}10-type intermediate. In the presence of H2O, the in situ formed RPial get oxidized to the corresponding picolinic acid (RPicH) via an in situ formed LCuIOH intermediate (LCuI + HO-NO → LCuIOH + NO+; L = RL1/RL2/RPic- and νO-H of CuIOH = 3650 cm-1) and subsequently produces, with the aid of NO+ oxidant, the picolinate-ligated copper(II) complexes (i) [(HPic)2Cu] (7), [(MePic)4Cu3(NO3)2]n·H2O (8·H2O), or [(ClPhPic)2Cu] (9) when NO reacts with 1-4 or (ii) [(RPic)CuII(RL1/RL2)]+ when NO+ reacts with 1'-4'. The CuII to CuI reduction of [(RPic)CuII(RL1/RL2)]+ is essential for C═N cleavage of the remaining RL1/RL2 Schiff base; excess NO can do it. The X-ray structures (1, 1', 3', 5, 7, and 8) and spectroscopic results revealed the role of CuII/I, NO, NO+, and H2O, shedding light on the mechanism of C═N bond cleavage and the oxidation of pyridine-2-aldehyde to pyridine-2-carboxylic acid. The reaction of 1 with 15NO revealed that the terminal N of the N2+ group of 5 originates from 15NO [ν14N14N- = 2248 cm-1 and ν15N14N- = 2212 cm-1].

Childhood police encounters, social isolation and epigenetic age acceleration among older U.S. adults.

OBJECTIVES: This study examined associations of childhood police encounters with biological age acceleration in later life, and their mediation by subjective or objective social isolation. METHODS: Data were from the Health and Retirement Study, nationally representative of older U.S. adults. Age acceleration was proxied through newly available epigenetic measures. Doubly robust estimation was used to establish baseline linkages, and heterogenous treatment effect models to examine variations in effects by one's increasing propensity for early police encounters. Mediation analysis was through a recently developed regression-with-residuals approach for structural nested mean models. RESULTS: Childhood police encounters was prospectively associated with age acceleration. Those with such early experiences also reported more loneliness and isolation from their community, although their ties to family and friends seemed stronger. Associations did not significantly decline with increasing propensity for such childhood experiences. Treatment effects on age acceleration seemed partly mediated by loneliness and by community isolation. DISCUSSION: Findings add to the growing evidence on the "long arm of childhood," and highlight public health implications of policy-driven social exposures.

Enhanced detection sensitivity of neuronal activity patterns using CaMPARI1 vs. CaMPARI2.

Calcium-modulated photoactivatable ratiometric integrator (CaMPARI) is a calcium ion (Ca2+)- and light-dependent genetically encoded fluorescent activity integrator that can capture snapshots of neuronal activity through an irreversible process known as photoconversion. This unique property was previously used to label neurons based upon their tuning properties in order to map synaptic connectivity and to record large-scale neuronal activity in freely moving mice without attaching any mechanical device to them. The latest version of CaMPARI (CaMPARI2) was engineered to enhance the contrast generated by photoconverting the green protein to the activity-dependent red form and to reduce the Ca2+-independent photoconversion rate compared to the first generation of CaMPARI (CaMPARI1). However, here we show that this optimization process also resulted in reduced photoconversion efficiency of active neurons in the mouse cortex and hippocampus. Through side-by-side comparison of the two CaMPARI sensors under several experimental conditions, we show that CaMPARI1 exhibits a substantially higher red-to-green ratio in active cells than CaMPARI2. In addition, we show that CaMPARI1 also functions as a more sensitive traditional Ca2+ sensor than CaMPARI2 by producing larger activity-driven dynamic fluorescence changes in the observed neurons. Therefore, we conclude that during the optimization process of CaMPARI2, some of the sensor's characteristics were not predicted properly by in vitro screening assays, and therefore in vivo screening and validation steps should be included in future optimization attempts to increase the predictability of screening pipelines.

Validation of Circular RNAs by PCR.

High-throughput RNA-sequencing (RNA-seq) technologies combined with novel bioinformatic algorithms discovered a large class of covalently closed single-stranded RNA molecules called circular RNAs (circRNAs ). Although RNA-seq has identified more than a million circRNAs, only a handful of them is validated with other techniques, including northern blotting, gel-trap electrophoresis, exonuclease treatment assays, and polymerase chain reaction (PCR). Reverse transcription (RT) of total RNA followed by PCR amplification is the most widely used technique for validating circRNAs identified in RNA-seq. RT-PCR is a highly reproducible, sensitive, and quantitative method for the detection and quantitation of circRNAs. This chapter details the basic guidelines for designing suitable primers for PCR amplification and validation of circRNAs .

Secularism, family ties and loneliness: A multilevel longitudinal study of ten European societies.

OBJECTIVES: Mass media suggest rising political and religious concern about secularism-induced decline of the family. Implications for loneliness remain unexamined. The current study filled this gap. METHODS: Data were from 10 national probability samples in the Survey of Health, Ageing and Retirement in Europe. Multilevel longitudinal models tested linkages of societal secularism with loneliness, their mediation by specific family relationships, and the role of this cultural dimension in weakening associations of family ties with loneliness. Both weighted Maximum Likelihood and unweighted Bayesian analyses were conducted, separately for each gender. RESULTS: Societal secularism was not positively linked to either gender's loneliness. Associations with family ties were inconsistent, with only men's average partnered status lower in more secular settings. Nor did any positive indirect effects emerge. Moderation results were also inconsistent, with secularism only weakening linkages of some family dimensions with loneliness. Bayesian estimates were generally nonsignificant. DISCUSSION: Societal secularism may not be a risk factor for loneliness or for weak family ties. Results stand at odds with religious and political rhetoric on secularism-induced decline of the family, and its individual and societal consequences.

Religious attendance and global cognitive function: A fixed-effects cross-lagged panel modeling study of older U.S. adults.

OBJECTIVES: Evidence linking religious attendance to better cognitive function is based on flawed study designs. No population representative longitudinal studies on the topic have taken both unobserved confounding and reverse causation into account. Recently developed fixed-effects cross-lagged panel modeling (FE-CLPM) offers simultaneous traction on these issues. It also allows parsing of long-from short-run effects. Using FE-CLPM and ten-year data from the Health and Retirement Study-a national probability sample of U.S. adults over age 50-this study began to fill the gaps above. METHODS: Gender-specific FE-CLPM models were used to examine bidirectional and within-person linkages of religious attendance with global cognitive function. Granger-Sims "causality" tests further examined short-run effects in both directions. Impulse response analysis was used to explore time patterns in these linkages. RESULTS: At least among women, religious attendance had negative short-term prospective linkages with global cognitive function. Over successive time points, these associations increased in strength among both genders. Feedback effects-of cognitive status on religious attendance-were found in both women's and men's models, but had a gender-specific pattern. DISCUSSION: Results contradict a large literature positing cognitive benefits of religiosity. Instead, they lend support to a recent "neural resource depletion" model-especially among women. Overall, findings illustrate the "dark side" of religious engagement, which studies increasingly present as a social determinant with "outcome wide" positive effects on multiple health dimensions.

Cellular-resolution monitoring of ischemic stroke pathologies in the rat cortex.

Stroke is a leading cause of disability in the Western world. Current post-stroke rehabilitation treatments are only effective in approximately half of the patients. Therefore, there is a pressing clinical need for developing new rehabilitation approaches for enhancing the recovery process, which requires the use of appropriate animal models. Here, we demonstrate the use of nonlinear microscopy of calcium sensors in the rat brain to study the effects of ischemic stroke injury on cortical activity patterns. We longitudinally recorded from thousands of neurons labeled with a genetically-encoded calcium indicator before and after an ischemic stroke injury in the primary motor cortex. We show that this injury has an effect on the activity patterns of neurons not only in the motor and somatosensory cortices, but also in the more distant visual cortex, and that these changes include modified firing rates and kinetics of neuronal activity patterns in response to a sensory stimulus. Changes in neuronal population activity provided animal-specific, circuit-level information on the post-stroke cortical reorganization process, which may be essential for evaluating the efficacy of new approaches for enhancing the recovery process.

Antisense Oligo Pulldown of Circular RNA for Downstream Analysis.

Circular RNAs (circRNAs) are a large family of noncoding RNA molecules that have emerged as novel regulators of gene expression by sequestering microRNAs (miRNAs) and RNA-binding proteins (RBPs). Several computational tools have been developed to predict circRNA interaction with target miRNAs and RBPs with a view to studying their potential effect on downstream target genes and cellular physiology. Biochemical assays, including reporter assays, AGO2 pulldown, ribonucleoprotein pulldown, and biotin-labeled RNA pulldown, are used to capture the association of miRNAs and RBPs with circRNAs. Only a few studies have used circRNA pulldown assays to capture the associated miRNAs and RBPs under physiological conditions. In this detailed protocol, the circRNA of interest (e.g., circHipk2) was captured using a biotin-labeled antisense oligo (ASO) targeting the circHipk2 backsplice junction sequence followed by pulldown with streptavidin-conjugated magnetic beads. The specific enrichment of circRNA was analyzed using reverse transcription quantitative PCR (RT-qPCR). Furthermore, the ASO pulldown assay can be coupled to miRNA RT-qPCR and western blotting analysis to confirm the association of miRNAs and RBPs predicted to interact with the target circRNA. In summary, the specific pulldown of circRNA using this quick and easy method makes it a useful tool for identifying and validating circRNA interaction with specific miRNAs and RBPs.

Iron-catalyzed carboarylation of alkynes via activation of π-activated alcohols: rapid synthesis of substituted benzofused six-membered heterocycles.

An Fe(OTf)3-catalysed carboarylation of alkynes is reported for the straightforward synthesis of densely substituted 1,2-dihydroquinolines from N-propargyl anilides and π-activated alcohols. The reaction provides a new method for the synthesis of highly substituted benzofused six-membered heterocycles by the formation of two carbon-carbon bonds and one ring in a single step. The power of the methodology was further extended to the synthesis of substituted chromene and thiochromene derivatives in high yields. In addition, substituted quinoline derivatives were also achieved in a single step in the presence of FeCl3 through detosylation/aromatisation. A number of control experiments have been performed and a plausible mechanism has also been proposed to explain the formation of the products.

The relational genomics of cognitive function: A longitudinal study.

OBJECTIVES: Research in social genetics indicates a person's genome may influence outcomes of those in close relationships. Implications for cognitive function remain unexplored. The current study examined such "metagenomic" patterns among older U.S. couples. METHODS: Data were from married or cohabiting couples in the 2006-2016 waves of the Health and Retirement Study, nationally representative of U.S. adults over 50. Measures included cognitive function as well as separate polygenic scores for cognition and for educational attainment. Analysis was through parallel process latent growth models. RESULTS: Consistent with a recent "genetic externalities" conception, one partner's polygenic score for educational attainment was linked to the other's baseline levels of cognitive function. Contrary to relational moderation speculations, neither a partner's genetic scores nor educational attainment altered individual-level genetic influences. DISCUSSION: Findings add to the growing evidence that transpersonal genetic influences in one's proximal context have substantively important implications. Research is needed on the role of non-partnership ties in channeling such effects. Implications for life course theory are discussed.

Transpersonal Genetic Effects Among Older U.S. Couples: A Longitudinal Study.

BACKGROUND: Emerging social genetics research suggests one's genes may influence not just one's own outcomes but also those of close social alters. Health implications, particularly in late life, remain underexplored. Using combined genetic and survey data, this study examined such transpersonal genetic associations among older U.S. couples. METHOD: Data were from married or cohabiting couples in the 2006-2016 waves of the Health and Retirement Study, nationally representative of U.S. adults over 50. Measures included a polygenic score for educational attainment, and self-rated health. Analysis was through parallel process latent growth models. RESULTS: Women's and men's genetic scores for education had transpersonal linkages with their partner's health. Such associations were solely with life-course variations and not late-life change in outcomes. Moreover, they were indirect, mediated by educational attainment itself. Evidence also emerged for individual-level genetic effects mediated by the partner's education. DISCUSSION: In addition to the subject-specific linkages emphasized in extant genetics literature, relational contexts involve multiple transpersonal genetic associations. These appear to have consequences for a partner's and one's own health. Life-course theory indicates that a person is never not embedded in such contexts, suggesting that these patterns may be widespread. Research is needed on their implications for the life-course and gene-environment correlation literature.

Genes-in-Dyads: A Study of Relationship Quality.

OBJECTIVES: Using dyadic genetic information on older couples, this study queried associations of a polygenic score for well-being with one's own as well as a partner's relationship experiences. METHOD: Data were from the 2010 wave of the U.S. Health and Retirement Study. Analysis was through structural equation modeling. RESULTS: Especially among women, the genetic score was associated with individuals' own relationship experiences. Genetic externalities-linkages of one's genes with a partner's experiences-were also observed. No significant gender variations emerged. DISCUSSION: Contrary to conceptions implicit in much of existing genetics literature-which focuses on individuals' own gene-trait associations-the interpersonal environments most crucial to life course and health outcomes are shaped by the genes of all involved actors. Genetic externalities are a central component. Implications for the life course and gene-environment literatures are discussed.

Is Loneliness Adaptive? A Dynamic Panel Model Study of Older U.S. Adults.

OBJECTIVES: Recent evolutionary psychological theory proposes that loneliness is an adaptive mechanism, designed to trigger maintenance and repair of social ties. No population representative analyses have probed loneliness effects on sociality. The present study addressed this gap. METHOD: Data were from the 2006, 2010, 2014, and 2018 waves of the Health and Retirement Study, nationally representative of U.S. adults over age 50. Recently developed cross-lagged models with fixed effects were used to test prospective within-person associations of loneliness with specific dimensions of sociality, taking into account reverse causality as well as all time-invariant confounders with stable effects. Both gender-combined and -specific analyses were conducted. RESULTS: Loneliness did not consistently predict overall sociality: sparse linkages were found only among women. The same null pattern held with family ties. Non-family ties, in contrast, were associated with prior loneliness, but in a gender-specific way. Loneliness positively predicted women's social interactions with friends, but seemed linked to withdrawal from these relationships among men. There were indications that lonely men instead used religious attendance as a social outlet. DISCUSSION: Loneliness seems to induce domain- and gender-specific sociality responses. Findings suggest implications for evolutionary models of sociality as well as for psychosocial and physical health. Pending replication in independent samples, inferences remain tentative.