Mapping RNA-protein interactions with subcellular resolution using colocalization CLIP.

RNA-binding proteins (RBPs) are essential for RNA metabolism and profoundly impact health and disease. The subcellular organization of RBP interaction networks with target RNAs remains largely unexplored. Here, we develop colocalization CLIP (coCLIP), a method that combines cross-linking and immunoprecipitation (CLIP) with proximity labeling, to explore in-depth the subcellular RNA interactions of the RBP human antigen R (HuR). Using this method, we uncover HuR's dynamic and location-specific interactions with RNA, revealing alterations in sequence preferences and interactions in the nucleus, cytosol, or stress granule (SG) compartments. We uncover HuR's unique binding preferences within SGs during arsenite stress, illuminating intricate interactions that conventional methodologies cannot capture. Overall, coCLIP provides a powerful method for revealing RBP-RNA interactions based on localization and lays the foundation for an advanced understanding of RBP models that incorporate subcellular location as a critical determinant of their functions.

Mapping RNA-Protein Interactions with Subcellular Resolution Using Colocalization CLIP.

RNA binding proteins (RBPs) are essential for RNA metabolism and profoundly impact health and disease. The subcellular organization of RBP interaction networks with target RNAs remains largely unexplored. Here, we develop colocalization CLIP, a method that combines CrossLinking and ImmunoPrecipitation (CLIP) with proximity labeling, to explore in-depth the subcellular RNA interactions of the well-studied RNA-binding protein HuR. Using this method, we uncover HuR's dynamic and location-specific interactions with RNA, revealing alterations in sequence preferences and interactions in the nucleus, cytosol, or stress granule compartments. We uncover HuR's unique binding preferences within stress granules during arsenite stress, illuminating intricate interactions that conventional methodologies cannot capture. Overall, coCLIP provides a powerful method for revealing RBP:RNA interactions based on localization and lays the foundation for an advanced understanding of RBP models that incorporate subcellular location as a critical determinant of their functions.

Cellular functions of eukaryotic RNA helicases and their links to human diseases.

RNA helicases are highly conserved proteins that use nucleoside triphosphates to bind or remodel RNA, RNA-protein complexes or both. RNA helicases are classified into the DEAD-box, DEAH/RHA, Ski2-like, Upf1-like and RIG-I families, and are the largest class of enzymes active in eukaryotic RNA metabolism - virtually all aspects of gene expression and its regulation involve RNA helicases. Mutation and dysregulation of these enzymes have been linked to a multitude of diseases, including cancer and neurological disorders. In this Review, we discuss the regulation and functional mechanisms of RNA helicases and their roles in eukaryotic RNA metabolism, including in transcription regulation, pre-mRNA splicing, ribosome assembly, translation and RNA decay. We highlight intriguing models that link helicase structure, mechanisms of function (such as local strand unwinding, translocation, winching, RNA clamping and displacing RNA-binding proteins) and biological roles, including emerging connections between RNA helicases and cellular condensates formed through liquid-liquid phase separation. We also discuss associations of RNA helicases with human diseases and recent efforts towards the design of small-molecule inhibitors of these pivotal regulators of eukaryotic gene expression.

Two distinct binding modes provide the RNA-binding protein RbFox with extraordinary sequence specificity.

Specificity of RNA-binding proteins for target sequences varies considerably. Yet, it is not understood how certain few proteins achieve markedly higher sequence specificity than most others. Here we show that the RNA Recognition Motif of RbFox accomplishes extraordinary sequence specificity by employing functionally and structurally distinct binding modes. Affinity measurements of RbFox for all binding site variants reveal the existence of two distinct binding modes. The first exclusively accommodates cognate and closely related RNAs with high affinity. The second mode accommodates all other RNAs with reduced affinity by imposing large thermodynamic penalties on non-cognate sequences. NMR studies indicate marked structural differences between the two binding modes, including large conformational rearrangements distant from the RNA-binding site. Distinct binding modes by a single RNA-binding module explain extraordinary sequence selectivity and reveal an unknown layer of functional diversity, cross talk and regulation in RNA-protein interactions.

Argonaute-CLIP delineates versatile, functional RNAi networks in Aedes aegypti, a major vector of human viruses.

Argonaute (AGO) proteins bind small RNAs to silence complementary RNA transcripts, and they are central to RNA interference (RNAi). RNAi is critical for regulation of gene expression and antiviral defense in Aedes aegypti mosquitoes, which transmit Zika, chikungunya, dengue, and yellow fever viruses. In mosquitoes, AGO1 mediates miRNA interactions, while AGO2 mediates siRNA interactions. We applied AGO-crosslinking immunoprecipitation (AGO-CLIP) for both AGO1 and AGO2, and we developed a universal software package for CLIP analysis (CLIPflexR), identifying 230 small RNAs and 5,447 small RNA targets that comprise a comprehensive RNAi network map in mosquitoes. RNAi network maps predicted expression levels of small RNA targets in specific tissues. Additionally, this resource identified unexpected, context-dependent AGO2 target preferences, including endogenous viral elements and 3'UTRs. Finally, contrary to current thinking, mosquito AGO2 repressed imperfect targets. These findings expand our understanding of small RNA networks and have broad implications for the study of antiviral RNAi.

A selectable, plasmid-based system to generate CRISPR/Cas9 gene edited and knock-in mosquito cell lines.

Aedes (Ae.) aegypti and Ae. albopictus mosquitoes transmit arthropod-borne diseases around the globe, causing ~ 700,000 deaths each year. Genetic mutants are valuable tools to interrogate both fundamental vector biology and mosquito host factors important for viral infection. However, very few genetic mutants have been described in mosquitoes in comparison to model organisms. The relative ease of applying CRISPR/Cas9-based gene editing has transformed genome engineering and has rapidly increased the number of available gene mutants in mosquitoes. Yet, in vivo studies may not be practical for screening large sets of mutants or possible for laboratories that lack insectaries. Thus, it would be useful to adapt CRISPR/Cas9 systems to common mosquito cell lines. In this study, we generated and characterized a mosquito optimized, plasmid-based CRISPR/Cas9 system for use in U4.4 (Ae. albopictus) and Aag2 (Ae. aegypti) cell lines. We demonstrated highly efficient editing of the AGO1 locus and isolated U4.4 and Aag2 cell lines with reduced AGO1 expression. Further, we used homology-directed repair to establish knock-in Aag2 cell lines with a 3xFLAG-tag at the N-terminus of endogenous AGO1. These experimentally verified plasmids are versatile, cost-effective, and efficiently edit immune competent mosquito cell lines that are widely used in arbovirus studies.

Attachment security and striatal functional connectivity in typically developing children.

Attachment security is formed through interactions with a main caregiver during the first three years of life and reflects inter-individual differences in mental representations for the relationship. The striatum is known to be a key structure to initiate attachment behaviours and maintain attachment relationships as well as to modulate reward-related processing as part of the approach module in current neurobiological models of human attachment. Although findings have suggested critical roles of the striatum in inter-individual differences in attachment, most studies were based on a wide variety of tasks and very few have investigated these associations in intrinsic brain connectivity in typically developing children. In the present study, using resting-state functional magnetic resonance imaging, we examined the striatal functional connectivity according to children's attachment security in 68 nine-year-olds (Secure attachment = 39, Insecure attachment = 29, mean age/SD = 9.62/0.69). Children with secure attachment demonstrated increased functional connectivity in the tempro-limbic region, compared to children with insecure attachment. In addition, the child-reported attachment security scores were negatively associated with the caudate-prefrontal connectivity, but positively with the putamen-visual area connectivity. These data demonstrate that inter-individual differences in attachment can be captured in striatal functional connectivity organization in the typical brain.

TMEM41B Is a Pan-flavivirus Host Factor.

Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection, we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results, we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms present at nearly 20% in East Asian populations reduce flavivirus infection. Based on our mechanistic studies, we propose that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication.

TMEM41B is a pan-flavivirus host factor.

Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms (SNPs) present at nearly twenty percent in East Asian populations reduce flavivirus infection. Based on our mechanistic studies we hypothesize that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication. HIGHLIGHTS: TMEM41B and VMP1 are required for both autophagy and flavivirus infection, however, autophagy is not required for flavivirus infection.TMEM41B associates with viral proteins and likely facilitates membrane remodeling to establish viral RNA replication complexes.TMEM41B single nucleotide polymorphisms (SNPs) present at nearly twenty percent in East Asian populations reduce flavivirus infection.TMEM41B-deficient cells display an exaggerated innate immune response upon high multiplicity flavivirus infection.

The moderating effect of Korean preschoolers' receptive and expressive language skills on the link between Korean PA and English PA.

The purpose of the current study was to examine whether oral language skills moderate the effect of Korean phonological awareness (PA) on English PA for Korean preschoolers in the initial stage of learning English as a second language. The study participants comprised 81 five- to six-year-old Korean preschoolers attending Korean-medium preschools in South Korea. The findings demonstrated that Korean PA was significantly associated with English PA. In addition, Korean receptive and expressive language skills had moderating effects on the relationship between Korean PA and English PA, respectively. This study is discussed not only in terms of cross-language PA transfer in processing two phonologically and orthographically different languages but also in light of the importance of native language skills interacting with native PA in the second-language PA development of preschool children in the initial stage of learning a second language.

Gaming-addicted teens identify more with their cyber-self than their own self: Neural evidence.

According to existing neuroimaging studies of social cognition, individuals use knowledge about themselves to infer the mental states of others and to mentalize in a different way when the other is perceived to be similar versus dissimilar to oneself. In this study, we examined whether adolescent boys make mental state inferences for their online game characters and whether adolescents who were diagnosed as addicted to the internet game perceived their personal game character to be similar to themselves. Twelve internet-addicted adolescents and fifteen adolescents without addiction reported whether short phrases described themselves, a well-known historical person, or their own game character while undergoing a functional magnetic resonance imaging (fMRI). Different patterns of activity emerged for adolescents with internet game addiction compared to healthy adolescents when they were thinking about themselves, another person, and their game characters. Specifically, when addicted adolescents were thinking about their own game characters, more global and significant medial prefrontal (MPFC) and anterior cingulate (ACC) activations were observed, than even when compared to thinking about themselves. The ACC activation was correlated with the symptom severity. The activation patterns demonstrated that addicted adolescents were most attached to their game characters and equated their game characters to human.

The neural correlates of attachment security in typically developing children.

This study investigated neural correlates of children's attachment security using functional magnetic resonance imaging. Fifty-one boys' attachment styles (age mean = 9.5 years, SD = 0.61) were assessed with the Separation Anxiety Test (SAT). We created an fMRI version of the SAT to activate children's attachment system in fMRI environment and contrasted two conditions in which children were instructed to infer the specific feeling of the boy in the picture or to identify objects or physical activities. In the final fMRI analysis (N = 21), attachment security could be detected at the neural level corresponding to the behavioural differences in the attachment interview. Securely attached children showed greater activation in the frontal, limbic and basal ganglia area which included the dorsolateral prefrontal cortex, amygdala, cingulate cortex and striatum, compared to other children who had lower quality of attachment. These regions have a key role in socio-emotional information processing and also represent a brain network related to approach and avoidance motivation in humans. Especially the striatum, strongly linked to reward processing underpinning social approach and avoidance motivation, showed the largest effects in these differences and also positively correlated with emotional openness scores in SAT. This suggests that the quality of attachment configures the approach and avoidance motivational system in our brain mediated by the striatum.

A selective involvement of putamen functional connectivity in youth with internet gaming disorder.

Brain cortico-striatal circuits have consistently been implicated in the pathology of addiction related disorders. We applied a reliable seed-based analysis of the resting-state brain activity to comprehensively delineate the subdivisions of striatal functional connectivity implicated in internet gaming disorder. Among twelve right-handed male adolescents with internet gaming disorder and 11 right-handed and gender-matched healthy controls, we examined group differences in the functional connectivity of dorsal and ventral subdivisions of the caudate nucleus and putamen, as well as the association of these connectivity indices with behavioral measures of internet use. Adolescents with internet gaming disorder showed significantly reduced dorsal putamen functional connectivity with the posterior insula-parietal operculum. More time spent playing online games predicted significantly greater functional connectivity between the dorsal putamen and bilateral primary somatosensory cortices in adolescents with internet gaming disorder, and significantly lower functional connectivity between the dorsal putamen and bilateral sensorimotor cortices in healthy controls. The dorsal putamen functional connectivity was significantly and specifically different in adolescents with internet gaming disorder. The findings suggest a possible biomarker of internet gaming disorder.

Decreased thalamic volumes in adolescent inhalant users from Korea and Australia.

OBJECTIVES: Research investigating the impact of inhalant misuse on brain structure suggests abnormalities in subcortical regions. We investigated the association between inhalant misuse and subcortical brain volumes in adolescents. METHODS: Based on a collaborative dataset from South Korea (inhalant users: N = 15, mean age = 16.7, SD = 1.1; controls: N = 15, mean age = 15.4, SD = 1.2) and Australia (inhalant users: N = 7, mean age = 18.2, SD = 1.4; controls: N = 7, mean age = 18.9, SD = 2.6), the volumes of caudate nucleus, putamen, pallidum, amygdala, hippocampus, and thalamus were estimated in adolescent inhalant users and healthy adolescents using FreeSurfer. RESULTS: The results revealed a significantly decreased right thalamic volume in adolescent inhalant users (P = 0.042), along with a trend-level decrease in left thalamic volume (P = 0.061). A negative correlation (r = -0.544; P = 0.036) between thalamic volume and severity of inhalant use (i.e., reduced volumes associated with greater use) was identified among Korean participants. CONCLUSIONS: These findings suggest that compared with other subcortical structures, the thalamus is particularly sensitive to damage following chronic inhalant exposure during adolescence.

Decreased functional brain connectivity in adolescents with internet addiction.

BACKGROUND: Internet addiction has become increasingly recognized as a mental disorder, though its neurobiological basis is unknown. This study used functional neuroimaging to investigate whole-brain functional connectivity in adolescents diagnosed with internet addiction. Based on neurobiological changes seen in other addiction related disorders, it was predicted that connectivity disruptions in adolescents with internet addiction would be most prominent in cortico-striatal circuitry. METHODS: Participants were 12 adolescents diagnosed with internet addiction and 11 healthy comparison subjects. Resting-state functional magnetic resonance images were acquired, and group differences in brain functional connectivity were analyzed using the network-based statistic. We also analyzed network topology, testing for between-group differences in key graph-based network measures. RESULTS: Adolescents with internet addiction showed reduced functional connectivity spanning a distributed network. The majority of impaired connections involved cortico-subcortical circuits (∼24% with prefrontal and ∼27% with parietal cortex). Bilateral putamen was the most extensively involved subcortical brain region. No between-group difference was observed in network topological measures, including the clustering coefficient, characteristic path length, or the small-worldness ratio. CONCLUSIONS: Internet addiction is associated with a widespread and significant decrease of functional connectivity in cortico-striatal circuits, in the absence of global changes in brain functional network topology.

Reduced orbitofrontal cortical thickness in male adolescents with internet addiction.

BACKGROUND: The orbitofrontal cortex (OFC) has consistently been implicated in the pathology of both drug and behavioral addictions. However, no study to date has examined OFC thickness in internet addiction. In the current study, we investigated the existence of differences in cortical thickness of the OFC in adolescents with internet addiction. On the basis of recently proposed theoretical models of addiction, we predicted a reduction of thickness in the OFC of internet addicted individuals. FINDINGS: Participants were 15 male adolescents diagnosed as having internet addiction and 15 male healthy comparison subjects. Brain magnetic resonance images were acquired on a 3T MRI and group differences in cortical thickness were analyzed using FreeSurfer. Our results confirmed that male adolescents with internet addiction have significantly decreased cortical thickness in the right lateral OFC (p<0.05). CONCLUSION: This finding supports the view that the OFC alterations in adolescents with internet addiction reflect a shared neurobiological marker of addiction-related disorders in general.

Study on time-based variation of blood circulation index, pulse wave energy, and RAI of healthy adult men after different eating times.

BACKGROUND: The purpose of this study is to examine the effects of different eating times on blood circulation index, pulse wave energy (E), and radial augmentation index (RAI) of healthy adult men. METHODS: Blood circulation index, E, and RAI were measured using a three-dimensional (3-D) pulse imaging system (3-D MAC) at before, right after, 30 minutes after, 1 hour after, and 2 hours after eating. RESULTS: In the blood circulation index, heart rate (HR), estimated cardiac output (ECO), and estimated cardiac output index (ECI) increased significantly right after eating compared to before eating. By contrast, estimated circulation resistance (ECR) and estimated circulation resistance index (ECRI) decreased significantly right after eating compared to before eating. E had a tendency to increase right after eating compared to before eating and decrease gradually at every experimental point (left and right Chon, Kwan, and Cheok). RAI had a tendency to decrease right after eating compared to before eating and increase gradually at every experimental point. CONCLUSIONS: Different eating times can bring about changes on blood circulation index, E, and RAI. These changes show a certain tendency and coincide with the physiological factors that eating causes a rise of HR, an increase of systolic cardiac pump performance, and a reduction of peripheral vascular resistance.

Gelatin-layered and multi-sized porous ß-tricalcium phosphate for tissue engineering scaffold.

The multi-sized porous ß-tricalcium phosphate scaffolds were fabricated by freeze drying followed by slurry coating using a multi-sized porous sponge as a template. Then, gelatin was dip coated on the multi-sized porous ß-tricalcium phosphate scaffolds under vacuum. The mechanical and biological properties of the fabricated scaffolds were evaluated and compared to the uniformly sized porous scaffolds and scaffolds that were not coated by gelatin. The compressive strength was tested by a universal testing machine, and the cell viability and differentiation behavior were measured using a cell counting kit and alkaline phosphatase activity using the MC3T3-E1 cells. In comparison, the gelatin-coated multi-sized porous ß-tricalcium phosphate scaffold showed enhanced compressive strength. After 14 days, the multi-sized pores were shown to affect cell differentiation, and gelatin coatings were shown to affect the cell viability and differentiation. The results of this study demonstrated that the multi-sized porous ß-tricalcium phosphate scaffold coated by gelatin enhanced the mechanical and biological strengths.

The Patterns and Risk Factors of Smoking Relapse among People Successful in Smoking Cessation at the Smoking Cessation Clinics of Public Health Centers

PURPOSE: The purpose of this study was to investigate the patterns of smoking relapse and to identify risk factors related to smoking relapse among those successful in smoking cessation at the smoking cessation clinics of public health centers. METHODS: Data were collected from 1,705 six-month quitters and analyzed by the Kaplan-Meier analysis and the Cox proportional hazard model. RESULTS: The rate of smoking relapse was 38.2% in 1 year, 44.4% in 2 years, and 47.8% in 5 years. The vast majority of relapse (62.3%) occurred within the first six months after quitting. The risk factors related to smoking relapse were age (HR 1.964: 95% CI 1.545, to 2.497), nicotine dependence (HR 1.293: 95% CI 1.087 to 1.539), problem drinking (HR 1.497: 95% CI 1.116 to 2.008), behavioral therapy type (HR 1.398: 95% CI 1.193 to 1.638), and nicotine replacement therapy type (HR 1.363: 95% CI 1.077 to 1.724). CONCLUSION: For reducing smoking relapse, it is necessary to develop a smoking relapse prevention program for the first six months after quitting and to strengthen behavioral therapy in the course of smoking cessations clinics.