Structural atlas of human primary microRNAs generated by SHAPE-MaP.

MicroRNA (miRNA) maturation is critically dependent on structural features of primary transcripts (pri-miRNAs). However, the scarcity of determined pri-miRNA structures has limited our understanding of miRNA maturation. Here, we employed selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP), a high-throughput RNA structure probing method, to unravel the secondary structures of 476 high-confidence human pri-miRNAs. Our SHAPE-based structures diverge substantially from those inferred solely from computation, particularly in the apical loop and basal segments, underlining the need for experimental data in RNA structure prediction. By comparing the structures with high-throughput processing data, we determined the optimal structural features of pri-miRNAs. The sequence determinants are influenced substantially by their structural contexts. Moreover, we identified an element termed the bulged GWG motif (bGWG) with a 3' bulge in the lower stem, which promotes processing. Our structure-function mapping better annotates the determinants of pri-miRNA processing and offers practical implications for designing small hairpin RNAs and predicting the impacts of miRNA mutations.

Emerging roles of RNA modification: m(6)A and U-tail.

Although more than 100 types of RNA modification have been described thus far, most of them were thought to be rare in mRNAs and in regulatory noncoding RNAs. Recent developments have unveiled that at least some of the modifications are considerably abundant and widely conserved. This Minireview summarizes the molecular machineries and biological functions of methylation (N6-methyladenosine, m(6)A) and uridylation (U-tail).

Genomic Clustering Facilitates Nuclear Processing of Suboptimal Pri-miRNA Loci.

Nuclear processing of most miRNAs is mediated by Microprocessor, comprised of RNase III enzyme Drosha and its cofactor DGCR8. Here, we uncover a hidden layer of Microprocessor regulation via studies of Dicer-independent mir-451, which is clustered with canonical mir-144. Although mir-451 is fully dependent on Drosha/DGCR8, its short stem and small terminal loop render it an intrinsically weak Microprocessor substrate. Thus, it must reside within a cluster for normal biogenesis, although the identity and orientation of its neighbor are flexible. We use DGCR8 tethering assays and operon structure-function assays to demonstrate that local recruitment and transfer of Microprocessor enhances suboptimal substrate processing. This principle applies more broadly since genomic analysis indicates suboptimal canonical miRNAs are enriched in operons, and we validate several of these experimentally. Proximity-based enhancement of suboptimal hairpin processing provides a rationale for genomic retention of certain miRNA operons and may explain preferential evolutionary emergence of miRNA operons.

TUT7 controls the fate of precursor microRNAs by using three different uridylation mechanisms.

Terminal uridylyl transferases (TUTs) function as integral regulators of microRNA (miRNA) biogenesis. Using biochemistry, single-molecule, and deep sequencing techniques, we here investigate the mechanism by which human TUT7 (also known as ZCCHC6) recognizes and uridylates precursor miRNAs (pre-miRNAs) in the absence of Lin28. We find that the overhang of a pre-miRNA is the key structural element that is recognized by TUT7 and its paralogues, TUT4 (ZCCHC11) and TUT2 (GLD2/PAPD4). For group II pre-miRNAs, which have a 1-nt 3' overhang, TUT7 restores the canonical end structure (2-nt 3' overhang) through mono-uridylation, thereby promoting miRNA biogenesis. For pre-miRNAs where the 3' end is further recessed into the stem (as in 3' trimmed pre-miRNAs), TUT7 generates an oligo-U tail that leads to degradation. In contrast to Lin28-stimulated oligo-uridylation, which is processive, a distributive mode is employed by TUT7 for both mono- and oligo-uridylation in the absence of Lin28. The overhang length dictates the frequency (but not duration) of the TUT7-RNA interaction, thus explaining how TUT7 differentiates pre-miRNA species with different overhangs. Our study reveals dual roles and mechanisms of uridylation in repair and removal of defective pre-miRNAs.

Cell adhesion-dependent control of microRNA decay.

Mammalian microRNAs (miRNAs) are highly stable in most cell types, and their decay mechanism remains largely unknown. Here we report that some miRNAs degrade rapidly upon the loss of cell adhesion. When cells are grown at low density or cells are detached by trypsinization or EGTA treatment, mature miR-141 is downregulated while miR-200c from a common primary transcript (pri-miR-200c∼141) remains unaffected. Blockade of transcription by Actinomycin D leads to rapid depletion of miR-141 with a half-life of <1 hr when cells are detached, indicating that the regulation occurs via RNA decay. A sequence motif (UGUCU) in the center of miR-141 is necessary for the regulation. We further find that many other miRNAs including miR-200a, miR-34a, miR-29b, miR-301a, and miR-21 are degraded upon cell splitting. Induced destruction of persistent regulatory molecules such as miRNAs may increase cellular plasticity and facilitate cellular remodeling in response to the changes in cell adhesion.

Re-evaluation of the roles of DROSHA, Export in 5, and DICER in microRNA biogenesis.

Biogenesis of canonical microRNAs (miRNAs) involves multiple steps: nuclear processing of primary miRNA (pri-miRNA) by DROSHA, nuclear export of precursor miRNA (pre-miRNA) by Export in 5 (XPO5), and cytoplasmic processing of pre-miRNA by DICER. To gain a deeper understanding of the contribution of each of these maturation steps, we deleted DROSHA, XPO5, and DICER in the same human cell line, and analyzed their effects on miRNA biogenesis. Canonical miRNA production was completely abolished in DROSHA-deleted cells, whereas we detected a few DROSHA-independent miRNAs including three previously unidentified noncanonical miRNAs (miR-7706, miR-3615, and miR-1254). In contrast to DROSHA knockout, many canonical miRNAs were still detected without DICER albeit at markedly reduced levels. In the absence of DICER, pre-miRNAs are loaded directly onto AGO and trimmed at the 3' end, yielding miRNAs from the 5' strand (5p miRNAs). Interestingly, in XPO5 knockout cells, most miRNAs are affected only modestly, suggesting that XPO5 is necessary but not critical for miRNA maturation. Our study demonstrates an essential role of DROSHA and an important contribution of DICER in the canonical miRNA pathway, and reveals that the function of XPO5 can be complemented by alternative mechanisms. Thus, this study allows us to understand differential contributions of key biogenesis factors, and provides with valuable resources for miRNA research.

RNATACs: Multispecific small molecules targeting RNA by induced proximity.

RNA-targeting small molecules (rSMs) have become an attractive modality to tackle traditionally undruggable proteins and expand the druggable space. Among many innovative concepts, RNA-targeting chimeras (RNATACs) represent a new class of multispecific, induced proximity small molecules that act by chemically bringing RNA targets into proximity with an endogenous RNA effector, such as a ribonuclease (RNase). Depending on the RNA effector, RNATACs can alter the stability, localization, translation, or splicing of the target RNA. Although still in its infancy, this new modality has the potential for broad applications in the future to treat diseases with high unmet need. In this review, we discuss potential advantages of RNATACs, recent progress in the field, and challenges to this cutting-edge technology.

Assessment of Lambda-Cyhalothrin and Spinetoram Toxicity and Their Effects on the Activities of Antioxidant Enzymes and Acetylcholinesterase in Honey Bee (Apis mellifera) Larvae.

Honeybees play a crucial role as agricultural pollinators and are frequently exposed to various pollutants, including pesticides. In this study, we aimed to evaluate the toxicity of lambda-cyhalothrin (LCY) and spinetoram (SPI) in honey bee larvae reared in vitro through single (acute) and repeated (chronic) exposure. The acute LD50 values for LCY and SPI were 0.058 (0.051-0.066) and 0.026 (0.01-0.045) µg a.i./larva, respectively. In chronic exposure, the LD50 values of LCY and SPI were 0.040 (0.033-0.046) and 0.017 (0.014-0.019) µg a.i./larva, respectively. The chronic no-observed-effect dose of LCY and SPI was 0.0125 µg a.i./larva. Adult deformation rates exceeded 30% in all LCY treatment groups, showing statistically significant differences compared to the solvent control group (SCG). Similarly, SPI-treated bees exhibited significantly more deformities than SCG. Furthermore, we examined the activities of several enzymes, namely, acetylcholinesterase (AChE), glutathione-S-transferase (GST), catalase (CAT), and superoxide dismutase (SOD), in larvae, pupae, and newly emerged bees after chronic exposure at the larval stage (honey bee larval chronic LD50, LD50/10 (1/10th of LD50), and LD50/20 (1/20th of LD50)). LCY and SPI induced significant changes in detoxification (GST), antioxidative (SOD and CAT), and signaling enzymes (AChE) during the developmental stages (larvae, pupae, and adults) of honey bees at sublethal and residue levels. Our results indicate that LCY and SPI may affect the development of honey bees and alter the activity of enzymes associated with oxidative stress, detoxification, and neurotransmission. These results highlight the potential risks that LCY and SPI may pose to the health and normal development of honey bees.

A Study on Survival Analysis Methods Using Neural Network to Prevent Cancers.

Background: Cancer is one of the main global health threats. Early personalized prediction of cancer incidence is crucial for the population at risk. This study introduces a novel cancer prediction model based on modern recurrent survival deep learning algorithms. Methods: The study includes 160,407 participants from the blood-based cohort of the Korea Cancer Prevention Research-II Biobank, which has been ongoing since 2004. Data linkages were designed to ensure anonymity, and data collection was carried out through nationwide medical examinations. Predictive performance on ten cancer sites, evaluated using the concordance index (c-index), was compared among nDeep and its multitask variation, Cox proportional hazard (PH) regression, DeepSurv, and DeepHit. Results: Our models consistently achieved a c-index of over 0.8 for all ten cancers, with a peak of 0.8922 for lung cancer. They outperformed Cox PH regression and other survival deep neural networks. Conclusion: This study presents a survival deep learning model that demonstrates the highest predictive performance on censored health dataset, to the best of our knowledge. In the future, we plan to investigate the causal relationship between explanatory variables and cancer to reduce cancer incidence and mortality.

Effects of Customized Digital Health Care Service on Metabolic Syndrome Status and Lifestyle Using a Health Care App: Clinical Trial.

BACKGROUND: Untact cultures have rapidly spread around the world as a result of the prolongation of the COVID-19 pandemic, leading to various types of research and technological developments in the fields of medicine and health care, where digital health care refers to health care services provided in a digital environment. Previous studies relating to digital health care demonstrated its effectiveness in managing chronic diseases such as hypertension and diabetes. While many studies have applied digital health care to various diseases, daily health care is needed for healthy individuals before they are diagnosed with a disease. Accordingly, research on individuals who have not been diagnosed with a disease is also necessary. OBJECTIVE: This study aimed to identify the effects of using a customized digital health care service (CDHCS) on risk factors for metabolic syndrome (MS) and lifestyle improvement. METHODS: The population consisted of 63 adults who underwent a health checkup at the National Health Insurance Service Ilsan (NHIS) Hospital in 2020. Measured variables include basic clinical indicators, MS-related variables, and lifestyle variables. All items were measured at NHIS Ilsan Hospital before the use of the CDHCS and 3 months thereafter. The CDHCS used in this study is a mobile app that analyzes the health condition of the user by identifying their risk factors and provides appropriate health care content. For comparison between before and after CDHCS use (pre-post comparison), paired t test was used for continuous variables, and a chi-square test was used for nominal variables. RESULTS: The study population included 30 (47.6%) male and 33 (52.4%) female participants, and the mean age was 47.61 (SD 13.93) years. The changes in clinical indicators before and after intervention results showed a decrease in weight, waist circumference, triglyceride, and high-density lipoprotein cholesterol and increases in systolic blood pressure and diastolic blood pressure. The distribution of the risk group increased from 32 (50.8%) to 34 (54%) and that of the MS group decreased from 18 (28.6%) to 16 (25.4%). The mean metabolic syndrome age-chronological age before the CDHCS was 2.20 years, which decreased to 1.72 years after CDHCS, showing a decrease of 0.48 years in the mean metabolic syndrome age-chronological age after the intervention. While all lifestyle variables, except alcohol consumption, showed a tendency toward improvement, the differences were not statistically significant. CONCLUSIONS: Although there was no statistical significance in the variables under study, this pilot study will provide a foundation for more accurate verification of CDHCS in future research.

10-year follow-up study on medical expenses and medical care use according to biological age: National Health Insurance Service Health Screening Cohort (NHIS-HealS 2002~2019).

OBJECTIVES: The world is witnessing a sharp increase in its elderly population, accelerated by longer life expectancy and lower birth rates, which in turn imposes enormous medical burden on society. Although numerous studies have predicted medical expenses based on region, gender, and chronological age (CA), any attempt has rarely been made to utilize biological age (BA)-an indicator of health and aging-to ascertain and predict factors related to medical expenses and medical care use. Thus, this study employs BA to predict factors that affect medical expenses and medical care use. MATERIALS AND METHODS: Referring to the health screening cohort database of the National Health Insurance Service (NHIS), this study targeted 276,723 adults who underwent health check-ups in 2009-2010 and kept track of the data on their medical expenses and medical care use up to 2019. The average follow-up period is 9.12 years. Twelve clinical indicators were used to measure BA, while the total annual medical expenses, total annual number of outpatient days, total annual number of days in hospital, and average annual increases in medical expenses were used as the variables for medical expenses and medical care use. For statistical analysis, this study employed Pearson correlation analysis and multiple regression analysis. RESULTS: Regression analysis of the differences between corrected biological age (cBA) and CA exhibited statistically significant increases (p<0.05) in all the variables of the total annual medical expenses, total annual number of outpatient days, total annual number of days in hospital, and average annual increases in medical expenses. CONCLUSIONS: This study quantified decreases in the variables for medical expenses and medical care use based on improved BA, thereby motivating people to become more health-conscious. In particular, this study is significant in that it is the first of its kind to predict medical expenses and medical care use through BA.

H19X-encoded microRNAs induced by IL-4 in adipocyte precursors regulate proliferation to facilitate differentiation.

Adipose tissue, an organ critical for systemic energy homeostasis, is influenced by type 2 immunity in its development and function. The type 2 cytokine interleukin (IL)-4 induces the proliferation of bipotential adipocyte precursors (APs) in white fat tissue and primes these cells for differentiation into beige adipocytes, which are specialized for thermogenesis. However, the underlying mechanisms have not yet been comprehensively examined. Here, we identified six microRNA (miRNA) genes upregulated upon IL-4 stimulation in APs, miR-322, miR-503, miR-351, miR-542, miR-450a, and miR-450b; these are encoded in the H19X locus of the genome. Their expression is positively regulated by the transcription factor Klf4, whose expression also increases upon IL-4 stimulation. These miRNAs shared a large set of target genes, of which 381 genes were downregulated in mRNA expression upon IL-4 stimulation and enriched in Wnt signaling pathways. Two genes with downregulated expression, Ccnd1 and Fzd6, were repressed by H19X-encoded miRNAs. Additionally, the Wnt signaling activator LiCl downregulated the expression of this group of miRNAs in APs, indicating that Wnt signaling-related genes and these miRNAs form a double-negative feedback regulatory loop. This miRNA/Wnt feedback regulation modulated the elevated proliferation of APs induced by IL-4 stimulation and contributed to priming them for beige adipocyte differentiation. Moreover, the aberrant expression of these miRNAs attenuates the differentiation of APs into beige adipocytes. Collectively, our results suggest that H19X-encoded miRNAs facilitate the transition of APs from proliferation to differentiation in the IL-4-mediated regulation.

Predicting the incidence of age-related diseases based on biological age: The 11-year national health examination data follow-up.

PURPOSE: As the population ages rapidly, the incidence of age-related diseases (ARDs) is also increasing fast. Predicting the incidence of ARDs is a challenge since the rates of individual aging vary, and objective assessments of the stages of aging based on chronological age (CA) may be inaccurate. Thus, in this study, we developed a biological age (BA) model based on the National Health Examination (NHE) data and analyzed the model prediction results for the incidence of 16 ARDs. METHODS: This study was based on the 2002-2019 National Health Information Databases of the National Health Insurance Service (NHIS-NHID). The data from a total of 10,002,494 subjects were selected between 2009 and 2010, and the principal component analysis (PCA) was performed to develop the BA model. The Cox-proportional hazard model was used to perform predictive analysis of the ARD incidence. RESULTS: For the unit increase in the difference between corrected biological age (cBA) and chronological age (CA), the hazard ratios (HRs) of ARDs increased significantly for both sexes (p < 0.001). In descending order, the corresponding ARDs' HRs were obesity (1.655), chronic renal failure (1.362), hypertension (1.301), hyperlipidemia (1.264), diabetes mellitus (1.261), fracture (1.119), dementia (1.163), cataract (1.116), myocardial infarction (1.097), stroke (1.169), macular degeneration (1.075), osteoarthritis (1.059), osteoporosis (1.124), Parkinson's disease (1.048), and chronic obstructive pulmonary disease (1.026). CONCLUSIONS: In this study, the incidence of 16 ARDs were analyzed based on BA. Therefore, conducting the NHIS health examination can facilitate the prevention of ARDs by estimating HRs for at least 16 diseases.

Assessment of acute and chronic toxicity of cyantraniliprole and sulfoxaflor on honey bee (Apis mellifera) larvae.

BACKGROUND: Recently, cyantraniliprole (CYA) and sulfoxaflor (SUL) have been considered as alternatives to neonicotinoid insecticides. In this study, we evaluated the acute and chronic toxicities of CYA and SUL on honey bee (Apis mellifera L.) larvae reared in vitro. RESULTS: In the acute toxicity test, the following test doses were used to determine the median lethal dose (LD50 ): CYA 0.007, 0.014, 0.028, 0.056 and 0.112 µg larva-1 ; SUL 2.5, 5, 10, 20 and 40 µg larva-1 . In the chronic toxicity test, the following test doses were used to determine the LD50 : CYA 0.00512, 0.0128, 0.032, 0.08 and 0.2 µg larva-1 ; SUL 0.0625, 0.125, 0.25, 0.5 and 1.0 µg larva-1 . The acute LD50 values of CYA and SUL were 0.047 and 11.404 µg larva-1 , respectively. Larvae acutely exposed to SUL had significantly lower body weight than controls, but those exposed to CYA showed no difference. The no observed adverse effect level (NOAEL) and LD50 values of the chronic toxicity tests for each insecticide were 0.00512 and 0.064 µg larva-1 for CYA, and 0.0625 µg larva-1 and 0.212 µg larva-1 for SUL, respectively. Larvae chronically exposed to SUL emerged as bees with deformed wings, reaching adult deformation rates of over 50%; however, CYA had no effect on adult deformation. CONCLUSION: Exposure to CYA increased larval mortality but did not cause any adult deformation, whereas SUL exposure increased pupal mortality and caused wing deformation in newly emerged bees. Our study may be useful for the assessment of pesticide toxicity by providing valuable findings on the effects of these insecticides on honey bee larvae. © 2022 Society of Chemical Industry.

Horizontal Honey-Bee Larvae Rearing Plates Can Increase the Deformation Rate of Newly Emerged Adult Honey Bees.

Rearing honey bee larvae in vitro is an ideal method to study honey bee larval diseases or the toxicity of pesticides on honey bee larvae under standardized conditions. However, recent studies reported that a horizontal position may cause the deformation of emerged bees. Accordingly, the purpose of this study was to evaluate the emergence and deformation rates of honey bee (Apis mellifera ligustica) larvae reared in horizontal and vertical positions. The study was conducted under the same laboratory conditions with three experimental groups, non-capped or capped horizontal plates and capped vertical plates. However, our results demonstrated that the exhibited adult deformation rates of the horizontal plates were significantly higher (27.8% and 26.1%) than those of the vertical plates (11.9%). In particular, the most common symptoms were deformed wings and an abnormal abdomen in the horizontal plates. Additionally, adults reared on horizontal plates were substantially smaller (10.88 and 10.82 mm) than those on vertical plates (11.55 mm). Considering these conclusions, we suggest that a vertical rearing method is more suitable when considering the deformation rates of the control groups to verify the sublethal effects of pesticides on honey bees.

The Association between Taking Dietary Supplements and Healthy Habits among Korean Adults: Results from the Fifth Korea National Health and Nutritional Examination Survey (2010-2012).

BACKGROUND: Recently, the number of people interested in health in South Korea has increased, and the rate of dietary supplement use is rising. Researchers have hypothesized that the rate of practicing healthy habits is higher among those who use dietary supplements than those who do not. Therefore, this study aimed to discover the association between taking dietary supplements and practicing various healthy habits in the Korean, adult population. METHODS: The sample included 15,789 adults over 19 years old who participated in the fifth Korea National Health and Nutrition Examination Survey. The user group was defined as those taking dietary supplements for more than 2 weeks during the previous year or once during the past month. Measures for the seven healthy habits were based on those included in the Alameda study and were analyzed accounting for the complex sampling design. RESULTS: The rate of taking dietary supplements was significantly higher in women, middle aged participants, urban residents, those with a higher income, those with a higher education level, and nonsmokers as well as among women with a moderate subjective health status, women who limited their alcohol content, and women with dyslipidemia. In the adjusted analysis, the rate of performing three of the 'Alameda 7' habits-eating breakfast regularly, restricting snacking, and limiting drinking-was higher in the female dietary supplement user group than in the other groups. Women practiced more healthy habits and had a higher dietary supplement intake rate than men. CONCLUSION: We found that taking dietary supplements in Korean adults is highly associated with demographic and social factors. Taking dietary supplements had a relationship with dietary habits, and there was no significant association between dietary supplement and other healthy habits. Thus in the health clinic, we suggest that taking dietary supplements complements a patient's healthy habits, with the exception of dietary habits, for health promotion.