m6A modification negatively regulates translation by switching mRNA from polysome to P-body via IGF2BP3.

In the cytoplasm, mRNAs are dynamically partitioned into translating and non-translating pools, but the mechanism for this regulation has largely remained elusive. Here, we report that m6A regulates mRNA partitioning between polysome and P-body where a pool of non-translating mRNAs resides. By quantifying the m6A level of polysomal and cytoplasmic mRNAs with m6A-LAIC-seq and m6A-LC-MS/MS in HeLa cells, we observed that polysome-associated mRNAs are hypo-m6A-methylated, whereas those enriched in P-body are hyper-m6A-methylated. Downregulation of the m6A writer METTL14 enhances translation by switching originally hyper-m6A-modified mRNAs from P-body to polysome. Conversely, by proteomic analysis, we identify a specific m6A reader IGF2BP3 enriched in P-body, and via knockdown and molecular tethering assays, we demonstrate that IGF2BP3 is both necessary and sufficient to switch target mRNAs from polysome to P-body. These findings suggest a model for the dynamic regulation of mRNA partitioning between the translating and non-translating pools in an m6A-dependent manner.

A bisulfite-assisted and ligation-based qPCR amplification technology for locus-specific pseudouridine detection at base resolution.

Over 150 types of chemical modifications have been identified in RNA to date, with pseudouridine (Ψ) being one of the most prevalent modifications in RNA. Ψ plays vital roles in various biological processes, and precise, base-resolution detection methods are fundamental for deep analysis of its distribution and function. In this study, we introduced a novel base-resolution Ψ detection method named pseU-TRACE. pseU-TRACE relied on the fact that RNA containing Ψ underwent a base deletion after treatment of bisulfite (BS) during reverse transcription, which enabled efficient ligation of two probes complementary to the cDNA sequence on either side of the Ψ site and successful amplification in subsequent real-time quantitative PCR (qPCR), thereby achieving selective and accurate Ψ detection. Our method accurately and sensitively detected several known Ψ sites in 28S, 18S, 5.8S, and even mRNA. Moreover, pseU-TRACE could be employed to measure the Ψ fraction in RNA and explore the Ψ metabolism of different pseudouridine synthases (PUSs), providing valuable insights into the function of Ψ. Overall, pseU-TRACE represents a reliable, time-efficient and sensitive Ψ detection method.

Antibody-Free Fluorine-Assisted Metabolic Sequencing of RNA N4-Acetylcytidine.

N4-Acetylcytidine (ac4C) has been found to affect a variety of cellular and biological processes. For a mechanistic understanding of the roles of ac4C in biology and disease, we present an antibody-free, fluorine-assisted metabolic sequencing method to detect RNA ac4C, called "FAM-seq". We successfully applied FAM-seq to profile ac4C landscapes in human 293T, HeLa, and MDA cell lines in parallel with the reported acRIP-seq method. By comparison with the classic ac4C antibody sequencing method, we found that FAM-seq is a convenient and reliable method for transcriptome-wide mapping of ac4C. Because this method holds promise for detecting nascent RNA ac4C modifications, we further investigated the role of ac4C in regulating chemotherapy drug resistance in chronic myeloid leukemia. The results indicated that drug development or combination therapy could be enhanced by appreciating the key role of ac4C modification in cancer therapy.

The ghrelin receptor GHSR has two efficient agonists in the lobe-finned fish Latimeria chalumnae.

The peptide hormone ghrelin (an agonist) and LEAP2 (an antagonist) play important functions in energy metabolism via their receptor GHSR, an A-class G protein-coupled receptor. Ghrelin, LEAP2, and GHSR are widely present from fishes to mammals. However, our recent study suggested that fish GHSRs have different binding properties to ghrelin: a GHSR from the lobe-finned fish Latimeria chalumnae (coelacanth) is efficiently activated by ghrelin, but GHSRs from the ray-finned fish Danio rerio (zebrafish) and Larimichthys crocea (large yellow croaker) have lost binding to ghrelin. Do fish GHSRs use another peptide as their agonist? In the present study we tested to two fish motilins from D. rerio and L. chalumnae because motilin is distantly related to ghrelin. In ligand binding and activation assays, the fish GHSRs from D. rerio and L. crocea displayed no detectable or very low binding to all tested motilins; however, the fish GHSR from L. chalumnae bound to its motilin with high affinity and was efficiently activated by it. Therefore, it seemed that motilin is not a ligand for GHSR in the ray-finned fish D. rerio and L. crocea, but is an efficient agonist for GHSR in the lobe-finned fish L. chalumnae, one of the closest fish relatives of tetrapods. The results of present study suggested that GHSR might have two efficient agonists, ghrelin and motilin, in ancient fishes; however, this feature might be only preserved in some extant fishes with ancient evolutionary origins.

Nanomolar range of FAM237B can activate receptor GPR83.

Our recent study confirmed that the mature neuropeptide FAM237A, also known as neurosecretory protein GL (NPGL), is an efficient agonist for GPR83. The paralog FAM237B was previously reported as a weak agonist for GPR83. In the present study, we prepared mature human FAM237B via an intein-fusion approach and demonstrated that it could cause a significant activation effect at the nanomolar range (1‒10 nM) in a NanoBiT-based ß-arrestin recruitment assay. Thus, FAM237B appears to be another endogenous agonist for GPR83 and future in vivo studies will be required to confirm this.

Ambient ultrafine particle (PM0.1): Sources, characteristics, measurements and exposure implications on human health.

The problem of ultrafine particles (UFPs; PM0.1) has been prevalent since the past decades. In addition to become easily inhaled by human respiratory system due to their ultrafine diameter (<100 nm), ambient UFPs possess various physicochemical properties which make it more toxic. These properties vary based on the emission source profile. The current development of UFPs studies is hindered by the problem of expensive instruments and the inexistence of standardized measurement method. This review provides detailed insights on ambient UFPs sources, physicochemical properties, measurements, and estimation models development. Implications on health impacts due to short-term and long-term exposure of ambient UFPs are also presented alongside the development progress of potentially low-cost UFPs sensors which can be used for future UFPs studies references. Current challenge and future outlook of ambient UFPs research are also discussed in this review. Based on the review results, ambient UFPs may originate from primary and secondary sources which include anthropogenic and natural activities. In addition to that, it is confirmed from various chemical content analysis that UFPs carry heavy metals, PAHs, BCs which are toxic in its nature. Measurement of ambient UFPs may be performed through stationary and mobile methods for environmental profiling and exposure assessment purposes. UFPs PNC estimation model (LUR) developed from measurement data could be deployed to support future epidemiological study of ambient UFPs. Low-cost sensors such as bipolar ion and ionization sensor from common smoke detector device may be further developed as affordable instrument to monitor ambient UFPs. Recent studies indicate that short-term exposure of UFPs can be associated with HRV change and increased cardiopulmonary effects. On the other hand, long-term UFPs exposure have positive association with COPD, CVD, CHF, pre-term birth, asthma, and also acute myocardial infarction cases.

The interaction between autophagy and the epithelial-mesenchymal transition mediated by NICD/ULK1 is involved in the formation of diabetic cataracts.

BACKGROUND: Cataracts are the leading cause of blindness and a common ocular complication of diabetes. The epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) and altered autophagic activity occur during the development of diabetic cataracts. The disturbed interaction of autophagy with EMT in LECs stimulated by high glucose levels may participate in cataract formation. METHODS: A rat diabetic cataract model induced by streptozotocin (STZ) and human lens epithelial cells (HLE-B3) stimulated with a high glucose concentration were employed in the study. These models were treated with rapamycin (an inhibitor of mammalian target of rapamycin (mTOR)), and N-(N-[3,5-difluorophenacetyl]-1-alanyl)-S-phenylglycine t-butyl ester (DAPT, an inhibitor of γ-secretase) alone or in combination. Lens opacity was observed and photographed under a slit-lamp microscope. Histological changes in paraffin sections of lenses were detected under a light microscope after hematoxylin and eosin staining. Alterations of autophagosomes in LECs were counted and evaluated under a transmission electron microscope. The expression levels of proteins involved in the EMT, autophagy, and the signaling pathways in LECs were measured using Western blotting and immunofluorescence staining. Cell migration was determined by performing transwell and scratch wound assays. Coimmunoprecipitation (Co-IP) was performed to verify protein-protein interactions. Proteins were overexpressed in transfected cells to confirm their roles in the signaling pathways of interest. RESULTS: In LECs, a high glucose concentration induces the EMT by activating Jagged1/Notch1/Notch intracellular domain (NICD)/Snail signaling and inhibits autophagy through the AKT/mTOR/unc 51-like kinase 1 (ULK1) signaling pathway in vivo and in vitro, resulting in diabetic cataracts. Enhanced autophagic activity induced by rapamycin suppressed the EMT by inducing Notch1 degradation by SQSTM1/p62 and microtubule-associated protein light chain 3 (LC3) in LECs, while inhibition of the Notch signaling pathway with DAPT not only prevented the EMT but also activated autophagy by decreasing the levels of NICD, which bound to ULK1, phosphorylated it, and then inhibited the initiation of autophagy. CONCLUSIONS: We describe a new interaction of autophagy and the EMT involving NICD/ULK1 signaling, which mediates crosstalk between these two important events in the formation of diabetic cataracts. Activating autophagy and suppressing the EMT mutually promote each other, revealing a potential target and strategy for the prevention of diabetic cataracts.

Chemical methods and advanced sequencing technologies for deciphering mRNA modifications.

RNA modification, like other epigenetic modifications such as DNA modification and histone modification, is an emerging player in the field of the posttranscriptional regulation of gene expression. More than 160 kinds of RNA modifications have been identified, and they are widely distributed in different types of RNA. Recently, researchers have increasingly used advanced technologies to study modified nucleic acids in order to elucidate their biological functions and expand the understanding of the central laws of epigenetics. In this tutorial review, we comprehensively outline current advanced techniques for decoding RNA modifications, highlighting some of the bottlenecks in existing approaches as well as new opportunities that may lead to innovations. With this review, we expect to provide chemistry and biology students and researchers with ideas for solving some challenging problems, such as how to simultaneously detect multiple types of modifications within the same system. Moreover, some low-coverage modifications that may act as 'candidates' in important transcriptional processes need to be further explored. These novel approaches have the potential to lay a foundation for understanding the nuanced complexities of the biological functions of RNA modification.

Exploring physical activity behavior in middle-aged Taiwanese women based on the theory of planned behavior.

The study aimed to apply the theory of planned behavior (TPB) to understand middle-aged women's behavior of physical activity (PA). We recruited 185 women between 45 and 64 years (mean: 53.2 ± 5.6) for this cross-sectional study. Participants complete demographic data and an exercise behavior questionnaire including attitudes, subjective norms, perceived behavioral control (PBC), and intention. Means, frequencies, and t-tests were used. To test TPB, we used structural equation modeling. Fit indices for this model demonstrate a good fit: chi-square and degree of freedom ratio X2/df = 2.14 (p = .34), goodness of fit =.97, comparative fit index =.99, root mean square error of approximation =.019, and Akaike information criterion = 28.14. Significant positive correlations between subjective norms and intention (ß=.18, p < .05) and between PBC and intention (ß=.48, p < .01). Women believed that close family and friends promoted their intention to perform PA. Increased PBC would positively enhance their intention. Findings showed that PBC was the strongest predictor. Enhancing women's PBC over their PA can improve their intention. Future researchers are encouraged to examine the barriers to and benefits of improving PBC so that a useful and effective intervention can be designed to promote PA.

A review on vitrification technologies of hazardous waste.

Vitrification technology provides a solution for the issue of safe disposal of hazardous waste containing harmful chemical composition and organic pollutants. This review discusses application of vitrification technologies to treat hazardous waste including, asbestos, fly ash, electronic sludge, nuclear waste, medical waste and radioactive waste. Vitrification processes via Joule heating, microwave heating, plasma technology, electric arc furnaces and incinerators are compared herein. Stabilization of hazardous waste can be achieved by vitrification with the addition of flux agents/additives. Furthermore, crystalline structures, containing the silicate-glass network, are formed as a result of vitrification, depending on the type of flux agents/additives used. In addition, the concentration of heavy metals can be degraded in the final residue and leaching resistance can be achieved. Moreover, energy consumption, pollution prevention and the foreground of the practical application of vitrification are discussed. Vitrification with the advantage of encapsulating pollutants from the hazardous waste is proven to be a promising approach for hazardous waste treatment.

Sequencing 5-Formyluracil in Genomic DNA at Single-Base Resolution.

Albeit with low content, 5-formyluracil has been an important modification in genomic DNA. 5-formyluracil was found to be widely distributed among living bodies. Due to the equilibrium of keto-enol form, 5-formyluracil could be base-paired with guanine, thus inducing mutations in DNA. The highly reactive aldehyde group of 5-formyluracil could also cross-link with proteins nearby, preventing gene replication and expression. In certain cancerous tissues, the content of 5-formyluracil was found to be higher than the normal tissues adjacent to the tumor, and 5-formyluracil might be an important potential epigenetic mark. Nevertheless, the lack of a higher resolution sequencing technique has hampered the studies of 5-formyluracil. We adjusted the base-pairing of 5-formyluracil during the PCR amplification by changing the pH. Hence, we adopted the Alkaline Modulated 5-formyluracil Sequencing (AMfU-Seq), a single-base resolution analysis method, to profile 5-formyluracil at the genome scale. We analyzed the distribution of 5-formyluracil in the human thyroid carcinoma cells using AMfU-Seq. This technique can be used in the future investigations of 5-formyluracil.

Highly sensitive detection of 6mA at single-base resolution based on A-C mismatch.

We first demonstrated that 6mA can be selectively recognized based on the selective ligation reaction of DNA ligase toward A-C mismatch and 6mA-C mismatch. This method, when further combined with amplification using RCA, achieved highly sensitive identification of 6mA in dsDNA at single-base resolution.

Fluorescence assay based on the thioflavin T-induced conformation switch of G-quadruplexes for TET1 detection.

Ten-eleven translocation (TET) dioxygenase is of great significance in cytosine demethylation and in the control of cell differentiation and transformation. Herein, a fluorescence method has been developed for the highly sensitive detection of TET1, a member of the TET dioxygenase family. Based on the ThT-induced specific conformation of the G-quadruplex structure, the fluorescence signal decreased linearly with increasing TET1 concentration. The method shows potential clinical applications in the screening of TET protein inhibitors for anticancer drug discovery.

End-labeling-based electrochemical strategy for detection of adenine methylation in nucleic acid by differential pulse voltammetry.

A promising electrochemical strategy for assay of N6-methyladenosine (m6A)/N6-methyladenine (6mA) in RNA/DNA is proposed. The key of this strategy is the end-labeling of nucleic acid, which makes it possible to detect methylation level in unknown sequence. Firstly, the end of m6A-RNA or 6mA-DNA was labeled with sulfhydryl group through T4 polynucleotide kinase (T4 PNK) and then directly assembled on a gold nanoparticle-modified glassy carbon electrode (AuNPs/GCE). Secondly, methylation sites in RNA/DNA were specifically recognized by anti-m6A-antibody, and then, horseradish peroxidase-labeled goat anti-rabbit IgG (HRP-IgG) was further conjugated on the antibody. Thirdly, HRP-IgG catalyzed the hydroquinone oxidation reaction to generate amplified current signal which correlates with the amount of m6A/6mA in nucleic acid. This method showed a wide linear range from 0.0001 to 10 nM for m6A-RNA, 0.001 to 100 nM for 6mA-dsDNA, and 0.0001 to 10 nM for 6mA-ssDNA. The method was successfully applied to detection of m6A/6mA in RNA/DNA from HeLa cells and E. coli cells and validation of the decrease of m6A-RNA in HeLa cells after treatment with FTO protein.

Application of plasma technology for treating e-waste: A review.

This review details the current information on e-waste treatment using plasma technology. The current status of e-waste treatment via plasma technology from the scientific literature is presented herein, namely, moist paste battery, galvanic sludge, resin, printed circuit board, and semiconductor industries. The concept of plasma technology, classification of e-waste, contaminants of e-waste (metals, metalloids, and VOCs), and vitrification of the final product are presented herein. This review paper focuses on fusing flux agents to vitrify e-waste. Furthermore, this paper covers laboratory-scale investigations, plasma technology benefits, and reuse of material from plasma post-treatment. The use of plasma technology combined with flux agents could be recommended to eliminate contaminants from e-waste. Materials from plasma post-treatment may also be applied in environmental reuse applications.

Selective Chemical Labeling and Sequencing of 5-Carboxylcytosine in DNA at Single-Base Resolution.

5-Carboxylcytosine (5caC) plays a vital role in the dynamics of DNA demethylation, and sequencing of its sites will help us dig out more biological functions of 5caC. Herein, we present a novel chemical method to efficiently label 5caC distinguished from other bases in DNA. Combined with bisulfite sequencing, 5caC sites can be located at single-base resolution, and the efficiency of 5caC labeling is 92% based on the Sanger sequencing data. Furthermore, dot blot assays have confirmed that 5caC-containing DNA isolated from HeLa cells was successfully labeled using our method. We expect that our strategy can be further applied to selectively tagging other carboxyl-modified bases and mapping their sites in RNA.

M2 macrophages promote vasculogenesis during retinal neovascularization by regulating bone marrow-derived cells via SDF-1/VEGF.

Macrophages promote vasculogenesis during retinal neovascularization (RNV) by increasing the recruitment and differentiation of bone marrow-derived cells (BMCs). Different subtypes of macrophages (M1 and M2 macrophages) are associated with RNV. However, the mechanism underlying the regulation of BMCs by different macrophage subtypes during RNV remains unclear. In the present study, we investigated the role and mechanism of action of different macrophage subtypes that regulate BMCs during the development of RNV. The retinal avascular area and neovascularization (NV) tuft area in M2 macrophage group in vivo were the largest compared to those in the control phosphate buffer saline (PBS), unpolarized-M0, and M1 macrophage groups. The number of recruited green fluorescent protein (GFP)-positive BMCs and the degree of differentiation of BMCs into CD31-positive endothelial cells (ECs) and alpha-smooth muscle actin (α-SMA)-positive smooth muscle cells (SMCs) were higher in the M2 macrophage group than in the other groups. M2-conditional medium (M2-CM) affected the in vitro migration and activation of bone marrow mesenchymal stem cells (BMSCs, a subset of BMCs) more than M1-CM. The expression of stromal cell-derived factor-1 (SDF-1) and vascular endothelial growth factor (VEGF) in M2 macrophages and BMSCs cultured with M2-CM was also higher than that in M1 macrophages and BMSCs cultured with M1-CM. Migration of BMSCs was reduced after inhibiting the SDF-1 signaling pathway. Our results indicate that M2 macrophages may express significantly higher levels of SDF-1 and VEGF than M1 macrophages, thus regulating the recruitment and differentiation of BMCs and further aggravating vasculogenesis during RNV.

Detection and Application of 5-Formylcytosine and 5-Formyluracil in DNA.

Nucleic acids contain a variety of different base modifications, such as decoration at the fifth position of cytosine, which is one of the most important epigenetic modifications. Nucleic acid epigenetics mediate a wide variety of biological processes, including embryonic development and gene regulation, genomic imprinting, differentiation, and X-chromosome inactivation. Furthermore, the modification level can be aberrantly expressed in distinct sets of tissue that can indicate different tumor onsets and canceration. Thus, the analysis of modified nucleobases may contribute to the understanding of epigenetic modification-related biological processes and the correlation of modified nucleobase patterns with disease states for clinical diagnosis and treatment. In addition to 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine are found in organisms at a low content but are nevertheless extremely important chemical modifications, and 5-hydroxyuracil and 5-formyluracil compounds are also present. 5-Formyluracil is found in bacteriophages, prokaryotes, and mammalian cells. The 5-formyluracil content is higher in certain cancer tissues than in the normal tissues adjacent to the tumor. The content of 5-formyluracil in different cell tissues may have cell type specificity. With the continuous use of chemical tools, new detection technologies have greatly advanced the research on natural pyrimidine modifications. These modifications dynamically regulate the gene expression in eukaryotes and prokaryotes and provide mechanistic insights into the occurrence of diseases. Natural pyrimidine modifications act not only as intermediates for DNA demethylation or oxidative damage products but also as modulators of gene expression. Therefore, the development of more effective chemical tools will help us better understand the dynamic changes of natural pyrimidine modifications in vivo. In this Account, we summarize the recent advanced techniques for the detection of 5-formylpyrimidine (5-formylcytosine and 5-formyluracil) and highlight their great potential as biomarkers in biomedical applications. Focusing on the great urgency for the detection of epigenetic modifications, our group developed a series of methods for the qualitative and quantitative analysis of 5-formylpyrimidine in the past few years, aiming at facilitating the accurate detection and mapping of these epigenetic modifications. By the construction of probes, 5-formylpyrimidine can be selectively labeled. Using mass spectrometry, the epigenetic modifications can be quantified. Upon treatment under specific conditions, 5-formylcytosine can be recognized at single-base resolution. With this Account, we anticipate providing chemical and biological researchers with some insight to unlock the complex mechanism involved in 5-formylpyrimidine-related biological processes and stimulate more collaborative research interests from the different fields of materials, biological, medicine, and chemistry to promote the translational research of epigenetics in tumor diagnosis and treatment.

Economic and environmental evaluation of flux agents in the vitrification of resin waste: A SWOT analysis.

Flux agents play an important role in the pyrolysis treatment of vitrifying hazardous wastes. Among these is plasma jets, a cost-less flux agent derived from shell powder which can be used to create vitrification. It is a promising option to be applied in the vitrification of elements and to remove the VOCs of hazardous waste, namely, resin from PCB scrap in an atmospheric-pressure microwave plasma reactor. In this study, a laboratory scale experiment was conducted. The experiment was performed in the pyrolysis of resin which was added with flux agents. The economic evaluation of the flux agents, and the circular economy concept of the final residue derived from the plasma pyrolysis was then analyzed post treatment. To test the strength and weakness of the experiment, the SWOT analysis was performed. The outcome helped in the understanding of the cost-less flux agent used in the pyrolysis treatment of hazardous waste. Results showed that fusing shell powder in resin was better for improving the removal efficiency of VOCs, such as benzene and toluene as well as toxic metals than compared to other flux agents such as limestone and quartz sand. Moreover, the final residue of resin was found to fulfil the concept of circular economy where it could be reused as an absorbent of methyl blue, thereby indicating good absorption performance, from 1 ppm-100 ppm. The twelve strategies that were derived from the SWOT analysis could be used as information outlining the current internal and external condition for the development and application of shell powder. Shell powder, as a cost-less flux agent, has the potential for enhancing waste management and circular economy when used in the pyrolysis treatment of future hazardous wastes.

Ligation-Based qPCR-Amplification Assay for Radiolabel-Free Detection of ATP and NAD+ with High Selectivity and Sensitivity.

We have developed a new sensing system based on quantitative real-time polymerase chain reaction assay (qPCR) to detect adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD+) with high sensitivity and selectivity. T4 DNA ligase can catalyze the ligation of two short oligonucleotides (DNA1 and DNA2), which complement a template (cDNA), in the presence of its cofactor, ATP, resulting in increased template concentration and decreased Ct values in qPCR assays. Similarly, the Escherichia coli DNA ligase is also able to catalyze the ligation of DNA1 and DNA2 upon the addition of NAD+. Moreover, this approach has potential for detecting other important cofactors in related systems. Therefore, as a convenient and sensitive strategy, the method may light new beacons and find broad application in biological fields.