Chromatin context-dependent regulation and epigenetic manipulation of prime editing.

We set out to exhaustively characterize the impact of the cis-chromatin environment on prime editing, a precise genome engineering tool. Using a highly sensitive method for mapping the genomic locations of randomly integrated reporters, we discover massive position effects, exemplified by editing efficiencies ranging from ∼0% to 94% for an identical target site and edit. Position effects on prime editing efficiency are well predicted by chromatin marks, e.g., positively by H3K79me2 and negatively by H3K9me3. Next, we developed a multiplex perturbational framework to assess the interaction of trans-acting factors with the cis-chromatin environment on editing outcomes. Applying this framework to DNA repair factors, we identify HLTF as a context-dependent repressor of prime editing. Finally, several lines of evidence suggest that active transcriptional elongation enhances prime editing. Consistent with this, we show we can robustly decrease or increase the efficiency of prime editing by preceding it with CRISPR-mediated silencing or activation, respectively.

Sequence determinant of small RNA production by DICER.

RNA silencing relies on specific and efficient processing of double-stranded RNA by Dicer, which yields microRNAs (miRNAs) and small interfering RNAs (siRNAs)1,2. However, our current knowledge of the specificity of Dicer is limited to the secondary structures of its substrates: a double-stranded RNA of approximately 22 base pairs with a 2-nucleotide 3' overhang and a terminal loop3-11. Here we found evidence pointing to an additional sequence-dependent determinant beyond these structural properties. To systematically interrogate the features of precursor miRNAs (pre-miRNAs), we carried out massively parallel assays with pre-miRNA variants and human DICER (also known as DICER1). Our analyses revealed a deeply conserved cis-acting element, termed the 'GYM motif' (paired G, paired pyrimidine and mismatched C or A), near the cleavage site. The GYM motif promotes processing at a specific position and can override the previously identified 'ruler'-like counting mechanisms from the 5' and 3' ends of pre-miRNA3-6. Consistently, integrating this motif into short hairpin RNA or Dicer-substrate siRNA potentiates RNA interference. Furthermore, we find that the C-terminal double-stranded RNA-binding domain (dsRBD) of DICER recognizes the GYM motif. Alterations in the dsRBD reduce processing and change cleavage sites in a motif-dependent fashion, affecting the miRNA repertoire in cells. In particular, the cancer-associated R1855L substitution in the dsRBD strongly impairs GYM motif recognition. This study uncovers an ancient principle of substrate recognition by metazoan Dicer and implicates its potential in the design of RNA therapeutics.

Structure of the human DICER-pre-miRNA complex in a dicing state.

Dicer has a key role in small RNA biogenesis, processing double-stranded RNAs (dsRNAs)1,2. Human DICER (hDICER, also known as DICER1) is specialized for cleaving small hairpin structures such as precursor microRNAs (pre-miRNAs) and has limited activity towards long dsRNAs-unlike its homologues in lower eukaryotes and plants, which cleave long dsRNAs. Although the mechanism by which long dsRNAs are cleaved has been well documented, our understanding of pre-miRNA processing is incomplete because structures of hDICER in a catalytic state are lacking. Here we report the cryo-electron microscopy structure of hDICER bound to pre-miRNA in a dicing state and uncover the structural basis of pre-miRNA processing. hDICER undergoes large conformational changes to attain the active state. The helicase domain becomes flexible, which allows the binding of pre-miRNA to the catalytic valley. The double-stranded RNA-binding domain relocates and anchors pre-miRNA in a specific position through both sequence-independent and sequence-specific recognition of the newly identified 'GYM motif'3. The DICER-specific PAZ helix is also reoriented to accommodate the RNA. Furthermore, our structure identifies a configuration of the 5' end of pre-miRNA inserted into a basic pocket. In this pocket, a group of arginine residues recognize the 5' terminal base (disfavouring guanine) and terminal monophosphate; this explains the specificity of hDICER and how it determines the cleavage site. We identify cancer-associated mutations in the 5' pocket residues that impair miRNA biogenesis. Our study reveals how hDICER recognizes pre-miRNAs with stringent specificity and enables a mechanistic understanding of hDICER-related diseases.

A quantitative map of human primary microRNA processing sites.

Maturation of canonical microRNA (miRNA) is initiated by DROSHA that cleaves the primary transcript (pri-miRNA). More than 1,800 miRNA loci are annotated in humans, but it remains largely unknown whether and at which sites pri-miRNAs are cleaved by DROSHA. Here, we performed in vitro processing on a full set of human pri-miRNAs (miRBase version 21) followed by sequencing. This comprehensive profiling enabled us to classify miRNAs on the basis of DROSHA dependence and map their cleavage sites with respective processing efficiency measures. Only 758 pri-miRNAs are confidently processed by DROSHA, while the majority may be non-canonical or false entries. Analyses of the DROSHA-dependent pri-miRNAs show key cis-elements for processing. We observe widespread alternative processing and unproductive cleavage events such as "nick" or "inverse" processing. SRSF3 is a broad-acting auxiliary factor modulating alternative processing and suppressing unproductive processing. The profiling data and methods developed in this study will allow systematic analyses of miRNA regulation.

A Mechanism for microRNA Arm Switching Regulated by Uridylation.

Strand selection is a critical step in microRNA (miRNA) biogenesis. Although the dominant strand may change depending on cellular contexts, the molecular mechanism and physiological significance of such alternative strand selection (or "arm switching") remain elusive. Here we find miR-324 to be one of the strongly regulated miRNAs by arm switching and identify the terminal uridylyl transferases TUT4 and TUT7 to be the key regulators. Uridylation of pre-miR-324 by TUT4/7 re-positions DICER on the pre-miRNA and shifts the cleavage site. This alternative processing produces a duplex with a different terminus from which the 3' strand (3p) is selected instead of the 5' strand (5p). In glioblastoma, the TUT4/7 and 3p levels are upregulated, whereas the 5p level is reduced. Manipulation of the strand ratio is sufficient to impair glioblastoma cell proliferation. This study uncovers a role of uridylation as a molecular switch in alternative strand selection and implicates its therapeutic potential.

Different perceptual worlds: Parent and youth perspectives on parenting outcome trajectories from a Latino family-based program.

Discrepancies between parent and youth perceptions of their relationship are a common aspect of generational acculturation gaps influencing immigrant families. Programs designed to strengthen parenting practices among immigrant Latino families commonly address immigration stresses, including differences between parent and youth perceptions, but little is known about discrepancies in their appraisals of program effects on parenting behavior. A randomized trial was conducted examining effects on parent behavior of a program for immigrant families with youth aged 10-14, developed through community-based participatory research principles. Families (346 parents and youth) were recruited by organizations serving Latino families in a Midwestern metropolitan area and randomly assigned to the eight-session psychoeducation and skill-building program or a waitlist control. Parents and youth completed self-report measures at pre-intervention, post-intervention (4 months), and a 6-month follow-up regarding parents' expression of acceptance, efforts to solicit information about the child's experiences, and consistency of discipline, key foci of the program. Based on social cognition theory, the study focused on possible differences in parents' and youths' perceptions of change in parenting behavior. Parents in the treatment group reported pre-post improved acceptance, consistent discipline, and solicitation, whereas youth reported improvement only in parental solicitation, a pattern maintained at follow-up. In the control group, the only change was youth-reported reduction in parental acceptance. Parents' perceptions of improvement are encouraging, but overall lack of improvements from the youth perspective poses a potential problem for impact on parent-child relations. Interventions may need to target both parent and youth cognitions about behavior changes directly.

Bias-minimized quantification of microRNA reveals widespread alternative processing and 3' end modification.

MicroRNAs (miRNAs) modulate diverse biological and pathological processes via post-transcriptional gene silencing. High-throughput small RNA sequencing (sRNA-seq) has been widely adopted to investigate the functions and regulatory mechanisms of miRNAs. However, accurate quantification of miRNAs has been limited owing to the severe ligation bias in conventional sRNA-seq methods. Here, we quantify miRNAs and their variants (known as isomiRs) by an improved sRNA-seq protocol, termed AQ-seq (accurate quantification by sequencing), that utilizes adapters with terminal degenerate sequences and a high concentration of polyethylene glycol (PEG), which minimize the ligation bias during library preparation. Measurement using AQ-seq allows us to correct the previously misannotated 5' end usage and strand preference in public databases. Importantly, the analysis of 5' terminal heterogeneity reveals widespread alternative processing events which have been underestimated. We also identify highly uridylated miRNAs originating from the 3p strands, indicating regulations mediated by terminal uridylyl transferases at the pre-miRNA stage. Taken together, our study reveals the complexity of the miRNA isoform landscape, allowing us to refine miRNA annotation and to advance our understanding of miRNA regulation. Furthermore, AQ-seq can be adopted to improve other ligation-based sequencing methods including crosslinking-immunoprecipitation-sequencing (CLIP-seq) and ribosome profiling (Ribo-seq).

Design of Anticancer 2,4-Diaminopyrimidines as Novel Anoctamin 1 (ANO1) Ion Channel Blockers.

Pyrimidine is a privileged scaffold in many synthetic compounds exhibiting diverse pharmacological activities, and is used for therapeutic applications in a broad spectrum of human diseases. In this study, we prepared a small set of pyrimidine libraries based on the structure of two hit compounds that were identified through the screening of an in-house library in order to identify an inhibitor of anoctamin 1 (ANO1). ANO1 is amplified in various types of human malignant tumors, such as head and neck, parathyroid, and gastrointestinal stromal tumors, as well as in breast, lung, and prostate cancers. After initial screening and further structure optimization, we identified Aa3 as a dose-dependent ANO1 blocker. This compound exhibited more potent anti-cancer activity in the NCI-H460 cell line, expressing high levels of ANO1 compared with that in A549 cells that express low levels of ANO1. Our results open a new direction for the development of small-molecule ANO1 blockers composed of a pyrimidine scaffold and a nitrogen-containing heterocyclic moiety, with drug-like properties.

A Double-Edged Sword: Dual-Identity Centrality and the Health of Asian American Sexual Minority Individuals.

Asian American sexual minority individuals (AASMI) face the challenge of navigating two identities as both a sexual minority and a racial minority. However, the research base examining AASMI dual-identities remains underdeveloped. In the current study, we investigated the link between AASMI dual-identity centrality and their health and wellbeing using a subsample of 303 AASMI drawn from the Social Justice Sexuality Project dataset (n = 4,953). We conducted structural equation modeling (SEM) to test our hypothesized model and used bootstrap analysis to test the indirect effects therein. Findings indicated strong support for our model, which theorized that dual-identity centrality among AASMI can act as a "double-edged sword" on their health, whereby the process worsens their health through increased discomfort in their racial/ethnic community while simultaneously improving it through increased outness. Implications for future research and new avenues of intervention efforts at improving AASMI health are discussed.

Chromatin context-dependent regulation and epigenetic manipulation of prime editing.

Prime editing is a powerful means of introducing precise changes to specific locations in mammalian genomes. However, the widely varying efficiency of prime editing across target sites of interest has limited its adoption in the context of both basic research and clinical settings. Here, we set out to exhaustively characterize the impact of the cis- chromatin environment on prime editing efficiency. Using a newly developed and highly sensitive method for mapping the genomic locations of a randomly integrated "sensor", we identify specific epigenetic features that strongly correlate with the highly variable efficiency of prime editing across different genomic locations. Next, to assess the interaction of trans -acting factors with the cis -chromatin environment, we develop and apply a pooled genetic screening approach with which the impact of knocking down various DNA repair factors on prime editing efficiency can be stratified by cis -chromatin context. Finally, we demonstrate that we can dramatically modulate the efficiency of prime editing through epigenome editing, i.e. altering chromatin state in a locus-specific manner in order to increase or decrease the efficiency of prime editing at a target site. Looking forward, we envision that the insights and tools described here will broaden the range of both basic research and therapeutic contexts in which prime editing is useful.

Simulation Modeling of Reduced Glycosylation Effects on Potassium Channels of Mouse Cardiomyocytes.

Dilated cardiomyopathy (DCM) is the third most common cause of heart failure and the primary reason for heart transplantation; upward of 70% of DCM cases are considered idiopathic. Our in-vitro experiments showed that reduced hybrid/complex N-glycosylation in mouse cardiomyocytes is linked with DCM. Further, we observed direct effects of reduced N-glycosylation on Kv gating. However, it is difficult to rigorously determine the effects of glycosylation on Kv activity, because there are multiple Kv isoforms in cardiomyocytes contributing to the cardiac excitation. Due to complex functions of Kv isoforms, only the sum of K+ currents (IKsum) can be recorded experimentally and decomposed later using exponential fitting to estimate component currents, such as IKto, IKslow, and IKss. However, such estimation cannot adequately describe glycosylation effects and Kv mechanisms. Here, we propose a framework of simulation modeling of Kv kinetics in mouse ventricular myocytes and model calibration using the in-vitro data under normal and reduced glycosylation conditions through ablation of the Mgat1 gene (i.e., Mgat1KO). Calibrated models facilitate the prediction of Kv characteristics at different voltages that are not directly observed in the in-vitro experiments. A model calibration procedure is developed based on the genetic algorithm. Experimental results show that, in the Mgat1KO group, both IKto and IKslow densities are shown to be significantly reduced and the rate of IKslow inactivation is much slower. The proposed approach has strong potential to couple simulation models with experimental data for gaining a better understanding of glycosylation effects on Kv kinetics.

Systemic Lupus Erythematosus and Lung Involvement: A Comprehensive Review.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with multiorgan manifestations, including pleuropulmonary involvement (20-90%). The precise mechanism of pleuropulmonary involvement in SLE is not well-understood; however, systemic type 1 interferons, circulating immune complexes, and neutrophils seem to play essential roles. There are eight types of pleuropulmonary involvement: lupus pleuritis, pleural effusion, acute lupus pneumonitis, shrinking lung syndrome, interstitial lung disease, diffuse alveolar hemorrhage (DAH), pulmonary arterial hypertension, and pulmonary embolism. DAH has a high mortality rate (68-75%). The diagnostic tools for pleuropulmonary involvement in SLE include chest X-ray (CXR), computed tomography (CT), pulmonary function tests (PFT), bronchoalveolar lavage, biopsy, technetium-99m hexamethylprophylene amine oxime perfusion scan, and (18)F-fluorodeoxyglucose positron emission tomography. An approach for detecting pleuropulmonary involvement in SLE includes high-resolution CT, CXR, and PFT. Little is known about specific therapies for pleuropulmonary involvement in SLE. However, immunosuppressive therapies such as corticosteroids and cyclophosphamide are generally used. Rituximab has also been successfully used in three of the eight pleuropulmonary involvement forms: lupus pleuritis, acute lupus pneumonitis, and shrinking lung syndrome. Pleuropulmonary manifestations are part of the clinical criteria for SLE diagnosis. However, no review article has focused on the involvement of pleuropulmonary disease in SLE. Therefore, this article summarizes the literature on the epidemiology, pathogenesis, diagnosis, and management of pleuropulmonary involvement in SLE.

Racism, stress and health in Asian Americans: A structural equation analysis of mediation and social support group differences.

The buffering effect of social support on the negative effects of racism exposure on health outcomes has been mixed in prior studies regarding Asian Americans. Based on the stress-coping framework and using structural equation modelling (SEM) methods, we tested a theoretical model portraying simultaneous mediational paths from racism exposure to general physical and mental health through racism-related stress. Bootstrap analysis was used to test the indirect effects present in the model. Additionally, multi-group SEM analysis was conducted to investigate the moderation effect of social support from family and friends on the paths in the model. The sample consisted of 310 Asian American adults who completed an online survey. The results from the two-step SEM analysis and bootstrap analysis supported the theoretical model-racism exposure can simultaneously have a negative indirect effect on Asian Americans' physical and mental health via racism-related stress. Multi-group SEM analysis showed that there were no differences in model path coefficients based on having varying levels of social support from friends or family.

"Race was something we didn't talk about": Racial Socialization in Asian American Families.

OBJECTIVE: Our goal was to explore racial socialization practices in Asian American families during a time of heightened racial tension. BACKGROUND: Asian Americans hold a complex racial position in the United States, made even more complicated by an increase in public protests regarding socioracial injustices in the United States experienced by racial minority groups. Discussions about race and ethnicity occur within Asian American families but often focus on cultural heritage rather than awareness of discrimination and the historical roots of racism. METHOD: Our study used an inductive-deductive thematic analysis to collect data from 12 Asian American young adults. Semistructured interviews queried participants' experiences with racial socialization in their nuclear families and their own racial identity. FINDINGS: Qualitative analysis revealed the following themes: (a) Participants received limited messages regarding racial issues, (b) participants engaged in "bottom-up" racial socialization and taught their parents about race, and (c) participants felt left out of society's racial dialogue. CONCLUSION: During this time of heightened racial tension, Asian American young adults struggle to find their place, despite wanting to participate in community building. IMPLICATIONS: Without strong Asian American racial socialization practices in families, young adults must educate themselves and initiate racial meaning-making in their families.

Statistical Analysis of County-Level Contributing Factors to Opioid-Related Overdose Deaths in the United States.

This paper examines county-level characteristic factors contributing to opioid-related overdose deaths in the United States. We categorized factors into three groups: demographic, socio-economic, and health care environmental group. These features were used as predictors to model the overdose deaths from all types of opioids including prescription (e.g., oxycodone and hydrocodone) and illicit opioids (e.g., heroin and fentanyl) to investigate general trend, as well as separate models for heroin and fentanyl. Multilevel mixed-effect regression was adopted to adequately model grouping effect across counties.

Micro-bubble flow simulation of dissolved air flotation process for water treatment using computational fluid dynamics technique.

A dissolved air flotation (DAF) system is one of the water treatment processes that purifies contaminants through a buoyancy effect by attaching the moiety of micro-bubbles on their free surface. Since the DAF system was first used in the drinking water treatment in the 1960s, it has been recognized as an effective treatment for the water purification process. Most previous works laid great emphasis on the internal flow behaviors of fluid to improve the purification efficiency of the DAF system. Nevertheless, the practical implementation with a pilot plant indeed revealed some technical incompleteness for the DAF system. To circumvent for the technical incompleteness, numerical simulation based on computational fluid dynamics (CFD) has been carried out to understand the in-depth knowledge on internal flow phenomena in the DAF system. However, the standard k-ε turbulence model has been conventionally used in the most studies without any proper consideration process. Accordingly, the objectives of this study were to investigate the major effects on the internal flow behaviors for an efficient numerical simulation of DAF when a different turbulence model and micro-bubble parameters are used. As a result, the present study found that the standard k-ε model would be not proper for the internal flow simulation of the DAF process and a careful consideration would be required for a more accurate prediction. In addition, the present study examined a desirable internal flow pattern with various operating conditions of the micro-bubble. Consequently, the main findings of this study are expected to provide realistic information to related researchers for designing the DAF system with the optimal operating parameters.

Oscillation control algorithms for resonant sensors with applications to vibratory gyroscopes.

We present two oscillation control algorithms for resonant sensors such as vibratory gyroscopes. One control algorithm tracks the resonant frequency of the resonator and the other algorithm tunes it to the specified resonant frequency by altering the resonator dynamics. Both algorithms maintain the specified amplitude of oscillations. The stability of each of the control systems is analyzed using the averaging method, and quantitative guidelines are given for selecting the control gains needed to achieve stability. The effects of displacement measurement noise on the accuracy of tracking and estimation of the resonant frequency are also analyzed. The proposed control algorithms are applied to two important problems in a vibratory gyroscope. The first is the leading-following resonator problem in the drive axis of MEMS dual-mass vibratory gyroscope where there is no mechanical linkage between the two proof-masses and the second is the on-line modal frequency matching problem in a general vibratory gyroscope. Simulation results demonstrate that the proposed control algorithms are effective. They ensure the proof-masses to oscillate in an anti-phase manner with the same resonant frequency and oscillation amplitude in a dual-mass gyroscope, and two modal frequencies to match in a general vibratory gyroscope.

Stress-activated miR-204 governs senescent phenotypes of chondrocytes to promote osteoarthritis development.

A progressive loss of cartilage matrix leads to the development of osteoarthritis (OA). Matrix homeostasis is disturbed in OA cartilage as the result of reduced production of cartilage-specific matrix and increased secretion of catabolic mediators by chondrocytes. Chondrocyte senescence is a crucial cellular event contributing to such imbalance in matrix metabolism during OA development. Here, we identify miR-204 as a markedly up-regulated microRNA in OA cartilage. miR-204 is induced by transcription factors GATA4 and NF-κB in response to senescence signals. Up-regulated miR-204 simultaneously targets multiple components of the sulfated proteoglycan (PG) biosynthesis pathway, effectively shutting down PG anabolism. Ectopic expression of miR-204 in joints triggers spontaneous cartilage loss and OA development, whereas miR-204 inhibition ameliorates experimental OA, with concomitant recovery of PG synthesis and suppression of inflammatory senescence-associated secretory phenotype (SASP) factors in cartilage. Collectively, we unravel a stress-activated senescence pathway that underlies disrupted matrix homeostasis in OA cartilage.

Functional roles of glutamic acid E143 and E705 residues in the N-terminus and transmembrane domain 7 of Anoctamin 1 in calcium and noxious heat sensing.

Anoctamin 1 (ANO1) is an anion channel that is activated by changes in cytosolic Ca2＋ concentration and noxious heat. Although the critical roles of ANO1 have been elucidated in various cell types, the control of its gating mechanisms by Ca2＋ and heat remain more elusive. To investigate critical amino acid residues for modulation of Ca2＋ and heat sensing, we constructed a randomized mutant library for ANO1. Among 695 random mutants, reduced Ca2＋ sensitivity was observed in two mutants (mutant 84 and 87). Consequently, the E143A mutant showed reduced sensitivity to Ca2＋ but not to high temperatures, whereas the E705V mutant exhibited reduced sensitivity to both Ca2＋ and noxious heat. These results suggest that the glutamic acids (E) at 143 and 705 residues in ANO1 are critical for modulation of Ca2＋ and/or heat responses. Furthermore, these findings help to provide a better understanding of the Ca2＋-mediated activation and heat-sensing mechanism of ANO1. [BMB Reports 2018; 51(5): 236-241].