In cells, mRNA synthesis and decay are influenced by each other, and their balance is altered by either external or internal cues, resulting in changes in cell dynamics. We previously reported that it is important that an array of mRNAs that shape a phenotype are degraded before cellular transitions, such as cellular reprogramming and differentiation. In adipogenesis, the interaction between DDX6 and 4E-T had a definitive impact on the pathway in the processing body (PB). We screened a library of α-helix analogs with an alkaloid-like backbone to identify compounds that inhibit the binding between DDX6 and 4E-T proteins, which occurs between the α-helix of structured and internally disordered proteins. IAMC-00192 was identified as a lead compound. This compound directly inhibited the interaction between DDX6 and 4E-T. IAMC-00192 inhibited the temporal increase in PB formation that occurs during adipogenesis and epithelial-mesenchymal transition (EMT) and significantly suppressed these cellular transitions. In the EMT model, the half-life of preexisting mRNAs in PBs was extended twofold by the compound. The novel inhibitor of RNA decay not only represents a potentially useful tool to analyze in detail the pathological conditions affected by RNA decay and how it regulates the pathological state. The identification of this inhibitor may lead to the discovery of a first-in-class RNA decay inhibitor drug. Cell fate upon differentiation depends on not only gene expression programs at the transcriptional level but also posttranscriptional processes, such as alternative splicing, polyadenylation, and mechanisms altering mRNA availability, including RNA decay . RNA decThe elaborate molecular machinery involved in RNA decay has been analyzed in great detail Fig. . One of The molecular machinery of RNA quality control has been analyzed in great detail , and numIn this study, we investigated whether RNA decay inhibition could inhibit adipogenesis as a physiological process and EMT as a pathological process. The binding of DDX6 and 4E-T, a core component of RNA decay, promotes the decay process by looping single-stranded RNA structures, and we decided to search for small-molecule compounds that inhibit binding at this step. DDX6 and 4E-T are structured and unstructured  proteins, respectively, and a previous report on the interface structure indicated α-helix involvement . We hypoIt has been reported that DDX6 and 4E-T bind directly through RecA2 and the CHD region, respectively, in PBs and are involved in RNA decay . AdditioRenilla reniformis) luciferase (Rluc)-overexpressing HEK293FT cells, and luciferase activity was observed in the cells using Rluc as an internal control. Either RecA2 of DDX6 or CHD of 4E-T suppressed Fluc activity in genetically modified HEK293FT cells   Fig. 2A2A. DDX6 on and Koff constant rate more specifically, we performed regeneration-free kinetics using the WAVE system  to evaluate its function as an inhibitor  using the WAVE system (Creoptix AG). The proteins required for this WAVE system assay were designed as HA-tagged DDX6 and 4E-T Fig. , force-etem Fig. . First, tor Fig. . To corrtor Fig.  right cotor Fig.  left.FigCebpa and Fabp4 levels were lower in the cells treated with IAMC-00192 at all time points. Cebpb and Cebpd, which are transiently expressed in the early stage of adipogenic differentiation, were maintained by IAMC-00192. Reduced Pparg expression was noted in cells treated with IAMC-00192 alone for 8 days. Next, we evaluated factors associated with EMT that change with adipogenic differentiation and found that Zeb1, Snai1, and Twist1 genes were all highly elevated from Day 3 to Day 8 in cells treated with IAMC-00192, indicating that several key transcripts for adipogenesis were maintained even after the external cue. These results suggest that the inhibitor of DDX6 and the 4E-T interaction can modulate RNA decay and intervene in adipogenesis.We previously reported that RNA decay in PBs is indispensable for the induction of adipogenic differentiation . When DDGSTK1, ELF3, CDK1, and DNPH1, were found in the PB fraction with Tb2 -based methods . Direct The effective in vitro IAMC-00192 concentration discovered in this study is 10 μM, and it is still a lead compound that needs to be optimized. Using this compound as a foothold, we will need to establish its efficacy at subμM concentrations and investigate its off-target and precise allosteric effects. In addition, it is necessary to reproduce the in vitro effects in vivo as a step toward clinical use. In the postgenomic era, post-transcriptional modification has been shown to be involved in a wide variety of biological reactions, but the development of compounds to modify these processes has not progressed as expected. The discovery of compounds that inhibit RNA decay, which is one of the intracellular PPIs and is regulated by and involved in a large number of proteins through LLPS, opens up the possibility of developing various drugs that target RNA decay and provides a strategy for targeting EMT and ectopic adipogenesis. This is particularly important given the lack of treatments is currently available to treat these conditions.3 per well) seeded with Nluc_DDX6 and Cluc_4E-T plasmid (20 ng per well) and Lipofectamine 3000 (Thermo Fisher Scientific Incorporated) in White/96-well Clear Flat Bottom TC-treated Culture Microplate . Briefly, the plasmid and lipofectamine 3000 reacted in Opti-MEM for 10 min at room temperature, mixed with trypsin-treated cells, and seeded onto 96-well plates. Eighteen hours after seeding, the cells were incubated for 48 h with exposure to the small molecule compound. Eighteen hours after seeding, the cells were further incubated with small molecular inhibitors, and 48 h later, these cells were assayed for luciferase levels using the Dual-luciferase reporter assay system (Promega Corporation) and Tecan Infinite F200 plate reader  was used for measurement.HEK293FT Rluc cells  and the cells were harvested at the indicated time points using TRIzol reagent and Direct-zol RNA Miniprep Kits. Determine the mRNA decay rate by nonlinear regression curve fitting (one phase decay) using GraphPad Prism  .t-tests for bar graphs using GraphPad Prism. P < 0.05 was considered to indicate significance.The results are expressed as the means ± Standard Deviation (SD). The statistical significance of differences among groups was evaluated using parametric unpaired Supplementary informationSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary Table 1Supplementary Table 2Data Set


Ultimately, the involvement of the RNA modifications and their writers, erasers, and readers in human diseases and cancer is also discussed.Although a plethora of DNA modifications have been extensively investigated in the last decade, recent breakthroughs in molecular biology, including high throughput sequencing techniques, have enabled the identification of post-transcriptional marks that decorate RNAs; hence, epitranscriptomics has arisen. This recent scientific field aims to decode the regulatory layer of the transcriptome and set the ground for the detection of modifications in ribose nucleotides. Until now, more than 170 RNA modifications have been reported in diverse types of RNA that contribute to various biological processes, such as RNA biogenesis, stability, and transcriptional and translational accuracy. However, dysfunctions in the RNA-modifying enzymes that regulate their dynamic level can lead to human diseases and cancer. The present review aims to highlight the epitranscriptomic landscape in human RNAs and match the catalytic proteins with the deposition or deletion of a specific mark. In the current review, the most abundant RNA modifications, such as N6-methyladenosine (m Ever since the central dogma of molecular biology was proposed by Francis Crick, our perception of the flow of genetic information has been dramatically broadened. Over the years, the flux of information has revealed numerous processing steps that ensure proper gene expression and has highlighted the high complexity of the human genome, paving the way for deciphering the hidden aspects of life . MoreoveFor decades, it was known that RNA molecules were decorated with various chemical modifications, but it was only after the revolution in molecular biology and the advent of RNA sequencing techniques that the emerging field of epitranscriptomics spawned. Until now, more than hundreds of multiple RNA chemical modifications have been reported, whereas only a few of them have been systematically studied  Figure . These pRNA modifications are implicated in the regulation of several facets of RNA processing, thus possessing an indispensable role for the generation of functional RNA molecules ,8. These6A), N5-methylcytosine (m5C), pseudouridine (Ψ) and inosine (I) are the most abundant RNA modifications in eukaryotic cells and their roles have been extensively investigated. Additionally, 7-methylguanosine (m7G), N1-methyladenosine (m1A), 3-methylcytidine (m3C), queuosine (Q), uridine (U) and ribose methylation (2′-O-Me or Nm) are RNA-related chemical marks that also fall under the scope of modern epitranscriptomics research  can affect the Watson–Crick base-pairing, leading to either the misincorporation of nucleotides in the cDNA synthesis procedure or a blockage in the reverse transcriptase .Adenine, the most heavily modified RNA nucleotide, can be altered in different ways; this includes, for example, methylations on the nitrogen atoms of the adenine base generating m6A was discovered, it is still the center of attention; many studies aim to investigate this type of RNA modification and its functionality, since it constitutes the most prevalent internal RNA change both in yeast and higher eukaryotes [6A sites [6A peaks in human RNAs, while their bioinformatics analysis supported that ~95% of the m6A are located at mRNAs, whereas <2% of the m6A sites are found in ncRNAs [6A is in abundance on mRNAs, highlighting its involvement in multiple biological processes, whereas its presence in rRNAs affects translation. It should be noted that m6A methylation has also been detected in other RNA species, such as microRNAs (miRNAs) and circRNAs, contributing to their biogenesis and maturation; meanwhile, in lncRNAs m6A, bases are responsible for mediating transcriptional repression, thus affecting their functionality . Additional proteins, such as the Wilms tumor 1-associated protein (WTAP), the Vir-like m6A methyltransferase-associated (VIRMA) protein, the RNA binding motif protein 15/15B (RBM15/15B), and the Zinc-finger CCCH-type-containing 13 (ZC3H13) protein, are implicated in the formation and regulation of the m6A methyltransferase complex [6A formation in both mRNAs and lncRNAs [However, these enzymes form a multi-subunit complex that mediates the generation of m complex ,21. For  complex ,23,24. F lncRNAs ,26.6A levels in circRNAs [6A “writer” of the 18S rRNA, which is stabilized by TRMT112, whereas ZCCHC4 acts on the 28S rRNA , and the Human AlkB homolog H5 (ALKBH5) protein [6A alterations, including members of the YT521-B homology domain-containing proteins (YTHDF1-3 and YTHDC1-2), insulin-like growth factor 2 (IGF2), mRNA-binding proteins (IGF2BP1-3), Fragile X messenger ribonucleoprotein 1 (FMR1), Leucine-rich PPR motif-containing protein (LRPPRC), heterogeneous nuclear ribonucleoprotein (HNRNP) protein family members (HNRNPC and HNRNPA2B1) and the eukaryotic initiation factor 3 (eIF3) [On the other hand, only two demethylases have emerged as the universal molecular “erasers” of m protein ,34; mean3 (eIF3) ,11,13,356A in multiple RNA types manifests its involvement in the regulation of almost all phases of RNA metabolism, including RNA structure, localization, stability, and shelf life [6A sites have been reported: one at position A1832 in 18S rRNA, as well as one at position A4220 in 28S rRNA ; however, it is particularly enriched in 3′-UTRs around the termination codons. In particular, approximately 50% of the m6A sites are located at the last exon of the transcripts [6A modifications have been observed near exonic splice junctions in the CDS region and have been shown to affect the alternative splicing of human precursor mRNAs. More precisely, the mechanism involves the binding of the nuclear reader YTHDC1 to m6A sites, which recruits multiple splicing factors, including the SRSF3 that enhances exon inclusion and regulates the mRNA splicing by directly guiding the spliceosome in the appropriate splice sites [Recently, multiple studies have focused on the functional role of mnscripts . In addice sites ,39.6A. Specifically, m6A hypermethylated sites facilitate the transportation of the mRNA into the cytosol by their binding to the nuclear protein YTHDC1, which delivers the molecule to nuclear export proteins [6A methylation regulates protein synthesis through a plethora of enzymes that cooperate to increase translation efficiency [6A into the UTRs and binds to eIF3 to form the mRNA loop and promote translation, whereas YTHDF1 and YTHDF3 can also enhance translation through the YTHDF-eIF3 pathway [6A readers is also necessary for maintaining mRNA stability, while m6A sites regulate secondary structures [6A levels directly affect the mRNA’s fate [The nuclear export of mature transcripts is catalyzed by writers, erasers, and readers of mproteins . Furtherficiency ,42. METT pathway ,44. Notaructures . AssociaA’s fate .1A constitutes an additional methylation of adenine and the m1A RNA-modifying proteins regulate its dynamic abundancy in both ncRNAs and mRNAs. Most m1A sites have been identified at specific positions in tRNAs, around the GC-rich regions, , as well as in HGGAGRA motifs  of the lncRNAs [1A modification is present in each transcript within the GUUCNANNC sequence, which is mainly located in the 5′ UTRs and/or in the first splice site [6A, the addition of a methyl group into the first atom of the base is catalyzed by TRMT10 and the TRMT6/TRMT61 complex, which are members of the tRNA methyltransferase protein family (TRMT); meanwhile, ALKBH1 and ALKBH3 are the key enzymes that erase this type of modification  is characterized as a factor involved in the RNase P/MRP-mediated endoribonucleolytic cleavage of m6A, a recent report supports that HRSP12 functions as a reader protein for m1A [M lncRNAs . In the ice site ,49. In cfication . Notably for m1A .1A methylations may play a regulatory or stabilizing role in modified RNAs. As for the tRNA molecules, m1A in position 58 stabilizes the T-loop structure. Especially in tRNAiMet, the m1A58 increases translation by activating polysomes [1A erasers decreases the translation levels, while in the case of ALKBH3 overexpression, tRNA fragments (tRFs) can be formed through tRNA cleavage [1A participates in the formation of the 60S subunit of the 80S complex [1A alterations in 5′ UTRs of the mRNAs enhance translation efficiency, m1A in the CDS prevents protein synthesis [1A YTHDF2 and YTHDF3 proteins mediate the mRNA destabilization [To continue, molysomes . On the cleavage . In rRNA complex . Althougynthesis . Finallylization ,57.In humans, the most common RNA editing procedure that results in the substitution of a specific nucleotide is adenine-to-inosine (A-to-I); in mRNA, this is catalyzed by adenosine deaminase enzymes, namely ADARs, that act on double-stranded RNAs (dsRNAs). The ADAR family includes three proteins, which are encoded by three ADAR genes . The typ34 of tRNA is located at the wobble position and has been related to the codon recognition mechanism [Recent RNA-seq studies have confirmed a great number of A-to-I RNA editing sites in human mRNAs. Interestingly, the majority of inosine residues are located at UTRs and intronic regions; meanwhile, approximately 1000 editing sites have been found in protein-coding regions, indicating that A-to-I editing has two distinct and critical roles in RNAs ,64,65. Fechanism ,63. Take5C, m3C methylations, C-to-U RNA editing and the synthesis of pseudouridine and dihydrouridine participate in various biological processes; thus, these are at the center of epitranscriptome research.Apart from adenine, a great number of post-transcriptional marks have also been found in cytosine, among which m5C. This type of modification has been found in diverse types of RNAs. Two distinct methyltransferase groups have been confirmed to incorporate m5C in RNAs: the NOP2/Sun RNA methyltransferase (NSUN) family that includes seven proteins and the DNMT2 [5C eraser, whereas TET2 and TET3, MBD2/4, ALKBH1 and ALKBH6 are potential m5C demethylases; however, none of them have been confirmed [5C recognition proteins [The most dominant methylation of C in RNAs is the one occurring in the fifth nitrogen atom of the C, namely mhe DNMT2 . On the onfirmed ,70,71,72proteins ,75,76,775C is present at multiple positions in human tRNAs, including C38, C48 and C72, which are modified by NSUN2, NSUN6 and DNMT2  converts the ribobase C to U in mRNA editing sites . AlthougThe Ψ modification is the well-studied derivative of U and is prevalent in all classes of RNAs. In humans, Ψ is mainly formed by members of the pseudouridine synthase (PUS) family, PUS1-PUS10, while its readers and erasers remain uncertain. Ψ writers can be divided into two separate groups: the guide RNA-dependent synthases that include the small ribonucleoproteins H/ACA sRNPs, and the guide-independent PUS enzymes. Briefly, the guide-dependent process is catalyzed by a two-step reaction that requires a complementary RNA to guide the enzyme in the target region and a protein that forms the modification . On the rRNAs are also subjected to pseudouridylation through Dyskerin Pseudouridine Synthase 1 (DKC1), an alternative enzyme that incorporates Ψ in 28S rRNAs . NotablyIn mRNAs, Ψ is added co-transcriptionally by PUS1, PUS7, and RPUSD4 enzymes, and plays a critical role in the alternative pre-mRNA processing steps that affect gene expression ,106,107.Besides Ψ, C can be edited into U to synthesize D, a highly conserved alteration found in great abundancy at specific positions in the D-loop of tRNAs . DihydroEven though modern epitranscriptomics is mainly focused on modifications occurring in adenine and cytosine, several guanine-based alterations are vital for the fate of RNAs and are thus worth mentioning.7G mark is widely known due to its presence in tRNAs originating from multiple organisms, it is also associated with the eukaryotic 5′ capping of the mRNA [7G in tRNAs, whereas WBSCR22 and TRMT112 proteins act synergistically to add m7G on the 18S rRNA [7G addition to the mRNA cap [Although the mthe mRNA ,118. Dif18S rRNA ,120,121.18S rRNA . On the mRNA cap .7G in tRNAs, the formation of m7G46 promotes the mRNA translation and increases the tRNA stability [7G1639 in the human 18S rRNA, this modification is not necessary for the biogenesis of the 40S ribosomal subunit [7G in the 5′ cap of the mRNA enable its involvement in vital biological pathways, including RNA maturation, nuclear export and cap-dependent translation [As for the physiological role of mtability . It shou subunit . Moreovenslation .1G at tRNA position 37; meanwhile, in case of mitochondrial tRNAs, TRMT5 demonstrates a notably lower activity and is replaced by TRMT10C, an additional tRNA methyltransferase that introduces the m1G9 mt-tRNA modification [1G9 methyltransferase TRMT10C can form multiprotein complexes with RG9MTD1 and SDR5C1, in order to catalyze the process of G methylation [1G in tRNAs is vital for proper tRNA folding and its tertiary structure, and can prevent frameshifting during protein production [1G writers have not been reported, the incorporation of a single m1G mark into the mRNA sequence destabilizes the translation machinery, leading to reduced levels of the generated protein [In human RNAs, the addition of a methyl group into the first atom of the ribose ring of G is mediated by various writers that act independently. More precisely, tRNA methyltransferase 5 (TRMT5) is the enzyme that catalyzes the incorporation of mfication ,124. Furhylation ,126. Accoduction . Moreove protein .34 on tRNAs protects tRNAs from ribonuclease degradation and affects the translation accuracy [Q represents a tRNA-specific hyper-modified guanosine nucleoside derived from G, which forms a plethora of derivatives, including galactosyl-queuosine , mannosyl-queuosine (ManQ), and glutamyl-queuosine (GluQ) ,130. It accuracy ,132.Although chemical modifications mainly occur in adenine, cytosine and guanine, few studies have reported the existence of post-transcriptional marks in U.5U), which constitutes a common modification both in tRNAs and rRNAs. However, high-throughput sequencing studies have also revealed the existence of m5U in mRNAs. Although TRMT2A and TRMT2B are the m5U-catalyzing protein enzymes that add the methyl group into human tRNAs, rRNAs and mRNAs, neither erasers nor readers have been identified yet [5U regions have been extensively studied in human tRNAs and are found at position m5U54 in the T-loop, but also in the mitochondrial tRNAs [5U54 mark can cause tRNA’s degradation and the generation of tRFs [In RNAs, the methylation of the fifth nitrogen atom of the U ring creates the 5-methyluridine , which recognizes the C32 and N34 regions in the anticodon loop, and the creation of Cm32 and Nm34 influences the translation . AdditioSer . In rRNASer . Nm methSer ,150.In mRNAs, Nm is present in the UTRs and more precisely in the 5′ mRNA termini, whereas in CDS regions, the AGUA motif has been found to harbor this type of modification . The 5′ Over the years, systematic efforts to catalog the repertoire of modifications that are embroidered on RNA molecules in different tissues and diverse pathophysiological conditions has illuminated the correlation between epitranscriptome deregulation and disease development .More specifically, mutations, as well as irregular expression patterns in numerous RNA modification enzymes, have been linked with defects in the epitranscriptome and, subsequently, with several human diseases, including cancer and neurological and cardiovascular disorders; thus, research has been propelled towards the elucidation of the enigmatic molecular mechanisms driving these pathological conditions .6A writer METTL3 has been found to be overexpressed in acute myeloid leukemia (AML) cell lines; it is considered to be accountable for the increased m6A methylation profiles and the translational activation of the MYC proto-oncogene, phosphatase and tensin homolog (PTEN), and the BCL2 apoptosis regulator mRNA transcripts, thus sustaining cell survival and proliferation [6A eraser FTO contributes to the bypassing of growth suppressors by demethylating; this reduces the stability and negatively regulates the retinoic acid receptor alpha (RARA), the ankyrin repeat and SOCS box-containing 2 (ASB2) mRNAs, hampering cell differentiation and promoting leukemogenesis [First and foremost, a wide array of RNA modifiers have been associated with guiding normal cells towards the acquisition of traits distinctive of cancer cells, widely known as the “hallmarks of cancer” . For insferation . On the ogenesis .6A readers YTHDF1 and YTHDF2 in hindering the efficiency of immune responses against tumor antigens, and assisting leukemia stem cells to evade apoptosis, respectively. Specifically, YTHDF1 recognizes the m6A signature and enhances the translation of the lysosomal proteases’ mRNAs found in dendritic cells, which, in turn, degrade the engulfed antigens, inhibiting their cross-presentation and suppressing the induction of CD8+ T cell responses [6A-methylated target transcripts, such as the tumor necrosis factor (TNF) receptor superfamily member 1B (TNFRSF1B) mRNAs, and promotes their degradation, affecting the TNF apoptotic signaling pathway [Notably, recent studies have also underlined the involvement of the mesponses . On the  pathway .6A is not the only epitranscriptomic mark that can promote malignancy. In the cytoplasm, Lin-28 homolog A (LIN28A) normally regulates the expression of let-7 miRNAs, the recruitment of writer TUT4. In turn, TUT4 adds uridines at the 3′ end of the precursor miRNAs (pre-let7), thus inhibiting their cleavage by Dicer and promoting their degradation [7G writer METTL1 interferes with the methylation and maturation of the same miRNA family, resulting in the enhanced migration of cancer cells [Despite being the most frequently encountered RNA modification, mradation . In brearadation . Moreoveradation ; meanwhier cells . 5C aberrant methylation of the oncogenic transcripts of the heparin binding growth factor (HDGF) gene, have been shown to augment mRNA stability and correspond with poor cancer prognosis [1A demethylation of tRNA molecules, participate in the production of tRNA-derived small RNAs (tDRs) and contribute to an enhanced cancer cell proliferation [As far as the Ψ writer DKC1 is concerned, mutations in this gene have been linked with the reduced pseudouridylation of rRNA molecules, and subsequently with the modified translation of several cancer-associated transcripts, such as the vascular endothelial growth factor (VEGF) and the tumor suppressor p53 mRNAs ,180,181.rognosis . Finallyferation . 6A signature could also be implicated in numerous neurological diseases [6A machinery have been detected, underlining the potentially critical role of defective methylation in the establishment and progression of these diseases [6A eraser ALKBH5 and the m6A reader YTHDC2 have been shown to participate in MDD and autism spectrum disorder, respectively [Given its critical role in governing brain development and functionality, it is not surprising that perturbations in the mdiseases . So far,diseases . Specifidiseases ,185,186;diseases ,188,189,diseases ,191 and diseases . Furtherectively ,194.As for the remaining epitranscriptomic marks, alterations in the NSUN2 gene have been identified as a cause of developing autosomal-recessive intellectual disability (ID)  and Dubo6A are responsible for the glucose metabolism by regulating the β-cells in the pancreas and urging liver gluconeogenesis, thus affecting the progression of type 2 diabetes. Both METTL3 and METTL14 are downregulated in patients with type 2 diabetes, whereas FTO demonstrates an increased expression pattern, which enhances the expression of forkhead box O1 (FoxO1), glucose-6-phosphatase (G6PC), and diacylglycerol acyltransferase 2 (DGAT2) enzymes; this finally leads to hyperglycemia, due to insulin secretion dysregulation. According to additional studies, an m6A-dependent pathway, in which FTO and METTL3 possess significant roles, controls lipid production and switches on/off both adipogenesis and obesity. Last but not least, RNA modifications are the key regulators in multiple cardiovascular diseases, including adipogenesis, obesity, type 2 diabetes, different types of atherosclerosis and limb ischemia . In part6A writers, readers and erasers can also promote and/or inhibit chronic inflammation and lipid deposition, which form the atherosclerotic plaques. For instance, the overexpression of METTL14 in endothelial cells is responsible for monocyte aggregation, via affecting either the m6A levels of FoxO1 mRNA or the binding affinity of the FoxO1 protein, resulting in the progression of atherosclerosis. The dysfunction of endothelial cells is also induced by the m5C RNA modification levels. Notably, NSUN2 can promote the translation of ICAM-1, which increases the adhesion of leukocytes to the surface of endothelial cells. On the contrary, limb ischemia is connected to other RNA modifications, such as A-to-I, Ψ and m7G. Studies support that A-to-I editing and Nm can alter the targets of the tumor suppressor microRNA, miR487b, and hence promote angiogenesis; meanwhile, the increased levels of the Ψ writers, RPUSD3 and RPUSD4, induce mitochondrial protein synthesis, leading to the defective assembly of OXPHOS [7G modification in tRNAs is involved in vascular development due to the significant role of METTL1 in neovascularization [7G and the downregulation of METTL1 affect the pluripotency of the human-induced pluripotent stem cells, enable their differentiation to endothelial progenitor cells, and thus promote post-ischemic angiogenesis [In the case of atherosclerosis, the expression levels of mf OXPHOS . Recent rization . Notablyogenesis .In summary, the rise of epitranscriptomics has led to the identification of a wide spectrum of RNA modifications that exist in many RNA classes. Each modification is located at specific regions in RNAs and possesses specific roles that are often vital to cellular responses. A variety of enzymes are implicated in the mechanisms that incorporate or erase each modification, thus underlining the complexity of the eukaryotic transcriptomes. The dysfunction of the catalytic enzymes that are responsible for the post-transcriptional marks into the RNAs can lead to human diseases, such as cancer. Modern epitranscriptomics aims to decipher the molecular mechanisms that generate the RNA modifications and decode the involvement of RNA marks in cellular homeostasis. Undoubtedly, breakthroughs in molecular biology, including RNA-sequencing techniques, will enhance our efforts to unveil the mysteries of the features of RNA molecules.


These methods, such as dimethyl sulfate (DMS) mutational profiling with high-throughput sequencing (DMS-MaPseq), couple small chemical-mediated RNA modifications with next-generation sequencing. DMS-MaPseq, a powerful method for genome-wide and target-specific RNA secondary structure profiling, has been applied in yeast, mammals, Drosophila, and Arabidopsis thaliana, but not in crops. Here, we used DMS-MaPseq to conduct a target-specific and genome-wide profile of in vivo RNA secondary structure in rice (Oryza sativa). The DMS treatment conditions were optimized for rice leaf and root tissues. To increase the sequencing depth and coverage of low-abundance transcripts in genome-wide DMS-MaPseq, we used streptavidin-biotin depletion to reduce the abundance of highly expressed chloroplast transcripts during library construction. The resulting target-specific and genome-wide rice DMS-MaPseq data were of high quality and reproducibility. Furthermore, we used DMS-MaPseq to profile the in vivo RNA secondary structure of an OsmiR399 target region located at 5′UTR of OsPHO2, which participates in rice phosphate homeostasis. An unfolded RNA structure downstream of miRNA target site was observed in predicted in vivo RNA secondary structure, reminiscence of the TAM (Target Adjacent nucleotide Motif) involved in mRNA structure-mediated regulation in miRNA cleavage. Our study optimized DMS-MaPseq for probing in vivo RNA secondary structure in rice, facilitating the study of RNA structure-mediated regulations in crops.RNA transcripts form various secondary and tertiary structures that have a wide range of regulatory functions. Several methods have been developed to profile  In living cells, RNA folding is dynamic and largely dependent on the cellular context. Growing evidence has shown that in vivo RNA structure has critical functions and plays important regulatory roles in numerous biological processes, such as precursor messenger RNA (mRNA) processing, RNA stability, RNA trafficking, translation and phase separation in prokaryotes and eukaryotes  biogenesis, miRNA-mediated RNA silencing, mRNA long-distance transport, RNA N6-methyladenosine (m6A) modification, plant development, ambient stress responses, and other processes carbodiimide (in vivo RNA secondary structure probing due to its high reactivity and strong ability to penetrate cells (1-methyladenosine (m1A) and N3-methylcytidine  was developed . Insteade TGIRT; . TGIRT mf indels . The higf indels . Furtherf indels . Recentlf indels  and DRACf indels , were deArabidopsis thaliana. It demonstrated that CHR2 remodels the secondary structure of pri-miRNAs to impede miRNA biogenesis  homeostasis. Altogether, the optimized DMS-MaPseq for rice could facilitate the study of RNA structure-mediated biological functions in crops.In this study, we optimized DMS-MaPseq for profiling the Oryza sativa ssp. Japonica Nipponbare) seedlings were grown in a hydroponic nutrient system at 30°C under a 12 h light-12 h dark cycle. Three-week-old seedlings were harvested for DMS treatment.Rice (2). Then, 200, 300, 400, 500, or 600 μl of DMS  was added to the reaction buffer to give a final concentration of 1, 1.5, 2, 2.5, and 3%, respectively. For DMS treatment control, leaves of 3-week-old Arabidopsis grown on the soils were collected and treated with 1% DMS. For the no DMS control, the same volume of DEPC-treated water was added into the reaction buffer. DMS treatment was performed for 15 min at 30°C with shaking at 250 rpm or under vacuum (approximately 12 psi) at room temperature without shaking. After the DMS treatment, 6 ml of β-mercaptoethanol  was added to a final concentration of 23% and incubated under vacuum for 5 min to quench the DMS reaction. Then, the samples were washed three times with DEPC-treated water, frozen in liquid nitrogen, and ground to fine powder.DMS treatment was performed as described , 2019. TThe powdered rice materials (0.1 g) were mixed with 1 ml of Trizol reagent  and total RNA was extracted following the manufacturer’s protocol. Denatured agarose gel electrophoresis was used to validate the integrity of total RNA.2O. The solution was heated to 75°C for 3 min and put on ice for at least 1 min. Then, 4 μl of 5× First Strand buffer , 1 μl of 0.1 M DTT, 1 μl of 10 mM dNTPs and 1 μl of SUPERase-In RNase Inhibitor  were added. The mixture was heated at 35°C for 15 min, and 1 μl of SuperScript III reverse transcriptase  was added. The reaction was incubated at 55°C for 1 h, then inactivated by heating at 70°C for 15 min. Following phenol-chloroform extraction and ethanol precipitation, the cDNA was size-fractionated on a 10% urea-polyacrylamide gel. Next, the cDNA was transferred to a positively charged nylon membrane  via a semi-dry blotter (Bio-rad). Immobilized cDNA was detected following the procedure of the chemiluminescent nucleic acid detection module kit  and the signal was collected with iBright1500 (Thermo Fisher).The DMS treatment was validated by a primer extension assay as previously described , 2019 wi2O and 1 μl of TGIRT-III  were added to the solution. After incubation at room temperature for 30 min, 2 μl of 10 mM dNTPs was added and reverse transcription was conducted at 60°C for 2.5 h. Then, 2 μl of 2.5 M NaOH was added to stop the reaction and decay the RNA. The mixture was incubated at 95°C for 3 min and neutralized by adding HCl. Next, the cDNA was purified with RNAClean XP beads (Beckman). Then, the targets were amplified with KOD-FX hot-start DNA polymerase (Toyobo) using gene-specific primers . The reaction mixture including 250 ng of purified DNA templates, 1 μl of 100 mM ATP, 1 μl of 100 mM GTP, 1 μl of 100 mM CTP, 0.4 μl of 100 mM UTP, 0.8 μl of 50 mM biotin-16-UTP (Lucigen), 1.5 μl of T7 RNA Polymerase Mix (NEB) and 1 μl of SUPERase-In RNase inhibitor (Thermo Fisher) was incubated at 37°C overnight. Then, TURBO DNase (Thermo Fisher) was added to digest the DNA template at 37°C for 1 h. Finally, RNA probes were purified by RNAClean XP beads (Beckman) and mixed with a final concentration of 160 ng/μl anti-PSBA probes, 70 ng/μl anti-RBCL probes, 15 ng/μl anti-PSAB probes, 15 ng/μl anti-PSAA probes, 10 ng/μl anti-PSBC probes, 10 ng/μl anti-PSBB probes, and 10 ng/μl anti-PSBD probes.Chloroplast genes were amplified using listed primers containing T7 promoter sequence  and clon2O. Then, 0.75 μl of random primer and 0.75 μl of 10 mM dNTP were added to 10 μl of RNA. The mixture was heated to 65°C for 5 min and incubated on ice for at least 1 min. cDNA was synthesized by adding 4 μl of 5× First-strand buffer, 0.75 μl of 0.1 M DTT, 0.75 μl of SUPERase-In RNase inhibitor (Thermo Fisher) and 0.5 μl of TGIRT (InGex), and 2.5 μl of RNase-free H2O. The reverse transcription was performed at 25°C for 10 min, then 42°C for 30 min, then 60°C for 1.5 h. Next, 2 μl of 2.5 M NaOH was added to stop the reaction and decaying RNA. The expression of chloroplast RNAs were measured by RT-PCR. The intensity of PCR products was quantified by ImageJ. The primers used for RT-PCR were listed in Total RNA was extracted from DMS-treated rice leaves and was treated with TURBO DNase (Thermo Fisher). One microgram of DNase-treated total RNA was mixed with 0, 0.25 or 0.5 μl anti-chloroplast RNA Probe mixture in a 20 μl hybridization reaction . The mixture was put in a thermocycler at 68°C for 5 min, then ramped down by −0.1°C/s to 22°C, and finally held at 22°C for 5 min. Then, 100 μl of Dynabeads MyOne Streptavidin C1  was washed and re-suspended in 40 μl of 2× binding and washing buffer according to the manufacturer’s manual. Biotinylated probe-target hybrids were immobilized by streptavidin beads twice. The depleted RNA solution was precipitated by ethanol and re-suspended in 10 μl of RNase-free HGenome-wide DMS-MaPseq was performed as described  with somData analysis for target-specific or genome-wide DMS-MaPseq was performed as described . BrieflyBased on the DMS mutation signal, the secondary structures were modeled by RNAStructure .3 DMS siGraphs with dot plots  were drawn using GraphPad Prism 8The GEO accession number of the DMS-MaPseq data in this study is GSE197245.in vivo. However, rice absorbs a lot of silicon from the soil and deposits it in the leaves, stem, and husks to form silica bodies (v/v) and different incubation conditions . We also treated 3-week-old Arabidopsis leaves as a reference. As seen in the Arabidopsis sample, total RNAs of DMS-treated rice samples were slightly degraded while untreated RNA was intact  and incubation conditions (shaking or vacuum for 15 min). Consistent with the leaf results, total RNAs of root samples were modified under our DMS treatment conditions  in untreated and DMS-treated leaf samples. The primer extension assay was modified from a previously published protocol , with soncations , implyinncations . Of notencations .in vivo RNA. Higher DMS concentration treatment results in more DMS modifications on RNA, but also causes greater RNA decay that deteriorates the quality of DMS-MaPseq library  among the 1, 2, and 2.5% DMS-treated samples . We alsoin vivo RNA secondary structure, we mapped our DMS mutation signals of 1% DMS treated leaf sample to the evolutionally conserved 18S rRNA secondary structure  by TopHat with 10% mismatch tolerance  with known RNA structure. The predicted RNA secondary structure exhibited a four-way junction, highly consistent with the reference structure , facilitates miRNA cleavage , while Arabidopsis roots contain only one cortical-cell layer (Triticum aestivum) and maize (Zea mays).There are some hurdles for RNA structure study in crops. First, the complexity of the cell wall and presence of multiple cell layers hinder penetration of small chemicals into cells to react with RNA. For instance, rice deposits silica bodies on the leaf and stem surface to resist pathogen infection and abiotic stress , which all layer . Multiplll layer . Our DMSin vivo RNA secondary structure from different isoforms  can be found below: National Center for Biotechnology Information (NCBI) BioProject database under accession number GSE197245.ZW conceived and designed research. QJ, SH, and GW conducted experiments. QJ and LZ performed data analysis. ZW and QJ wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by grants from the National Key Research and Development Program of China (2021YFF1000402) and the National Natural Science Foundation of China (32170262).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Arctium lappa on MC3T3-E1 osteoblast differentiation. In this study, after treatment with different concentrations of Arctiin, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to estimate the expression of osteogenesis markers. Then, the activity of alkaline phosphatase (ALP) was detected by an ALP assay kit and calcium nodules staining was evaluated by alizarin red staining (ARS). Additionally, the regulatory effects of Arctiin on cyclin D1 (Ccnd1) was assessed by measurement of protein expression. Subsequently, the functions of Ccnd1 silencing on the osteogenic differentiation was examined in Arctiin-treated MC3T3-E1 cells. Results indicated that Arctiin dose-dependently upregulated the expression of runt-related transcription factor 2 (RUNX2), collagen type 1 (COL1A1), osteocalcin (OCN) and osteopontin (OPN). Elevated ALP activity and calcification degree was prominently observed in the Arctiin-treated groups. Moreover, Ccnd1 expression was notably enhanced after Arctiin intervention. Importantly, Ccnd1-knockdown abrogated the impacts of Arctiin on osteogenic differentiation of MC3T3-E1. To conclude, findings in this study suggested that Arctiin could regulate MC3T3-E1 osteoblast differentiation via up-regulating Ccnd1, supporting that Arctiin might be a therapeutic target for osteoporosis.Osteoporosis is a systemic disorder of bone metabolism. This study aimed to investigate the impacts and possible mechanisms of Arctiin, a lignin isolated from   Osteoporosis is a frequent disease in bone characterized by bone weakening, low bone mineral density and bone mass, which contributes to a decrease in bone strength and ultimately leads to increased risk of fracture . The higArctium lappa L [Osteogenic differentiation is deemed as a pivotal determinant in bone regeneration. Accumulating literature has supported that abnormal bone remodeling and osteoblastic bone formation are responsible for the initiation and development of osteoporosis . It is c lappa L . A consi lappa L . Emergin lappa L . Osteopo lappa L ,11. TherThis paper was intended to clarify the roles of Arctiin in the capacity of mouse pre-osteoblast MC3T3-E1 to differentiate into osteoblast, and further explored the regulatory effects of Arctiin on cyclin D1 (Ccnd1) expression. These results might provide novel insight into anti-osteoporosis effect of Arctiin.2. For osteoblastic differentiation experiments, to induce osteogenic differentiation, cells were cultivated in α-MEM supplemented with 10% FBS, 20 mM beta-glycerophosphate and 100 mg/mL ascorbic acid  for a longest period of 14 days [Murine preosteoblast MC3T3-E1 cells acquired from Chinese Academy of Sciences  were maintained in Minimum Essential Medium α  medium followed by the addition of 10% fetal bovine serum  under the condition of 37°C and 5% CO 14 days . Differe 14 days . Arctiinhttp://stitch.embl.de/) was used to predict the possible interactions among Arctiin and proteins using Mus musculus as an organism. The protein had higher score and related to the progression of osteoporosis was selected to perform the further experiments.The STITCH website (3 cells/well) osteoblasts plated into 96-well plates at 37°C for 48 h. Then, a microplate reader  was applied to record the absorbance at 450 nm 4 h later after CCK-8 solution (10 μL) was jointed to each well.The cell viability assay was conducted using a cell counting kit-8 kit . Various concentrations of Arctiin  were utilized to treat MC3T3-E1 (2 × 105 cells/well) inoculated in 24-well plates were exposed to osteogenic induction medium for 2 weeks. The obtained lysates by adioimmunoprecipitation (RIPA) buffer (Sigma-Aldrich) were centrifugated at 200 × g for 5 min. A microplate reader  was to calculate ALP activity using the optical density (OD) at 405.ALP activity was appraised by an ALP assay kit  according to the standard protocol. Briefly, MC3T3-E1 cells (1 × 105 cells per well) and osteogenesis was determined at 14 days in osteogenic medium with or without Arctiin. After fixation in 4% paraformaldehyde for 30 min, cells were washed by PBS. Then, cells were stained by 1% ARS solution (Sigma-Aldrich) for 15 min and a light microscope  was employed to photograph stained cells. To quantify mineralization, calcium deposits were desorbed using 10% cetylpyridinium chloride  for 30 min, and the absorbance at 562 nm was measured in a plate reader .ARS assay was to examine mineralization nodule formation . MC3T3-EShort hairpin RNA (shRNA) targeting Ccnd1  and its negative control (shNC) were purchased from GenePharma . MC3T3-E1 cells grown on 6-well plates were transfected with shCcnd1 or shNC by means of lipofectamine 2000 reagent  in accordance with the manufacturer’s guidelines. At 48 h after post-transfection, the transfection efficiency was tested.The concentration of isolated proteins by RIPA buffer (Sigma-Aldrich) was quantified by a bicinchoninic acid protein assay kit . Following, 40 μg of proteins per lane were applied to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and loaded onto polyvinylidene fluoride membrane . A total of 5% fat-free milk solution was used to block these blots. The blots were incubated with specific primary antibodies overnight at 4°C, followed by incubation of horseradish peroxide (HRP)-conjugated secondary antibody at room temperature for 1.5 h. Immune complexes were recognized with an enhanced chemiluminescence reagent (EMD Millipore). Image Lab™ Software  was employed to evaluate the band density. The relative protein expression was calculated based on glyceraldehyde-phosphate dehydrogenase (GAPDH) as the loading control. The primary antibodies used in this study included anti-runt-related transcription factor 2 , anti-collagen type 1 , anti-osteocalcin , anti-osteopontin , anti-Ccnd1  and anti-GAPDH .−ΔΔCq method [The isolation of total RNA was conducted by TRIzol® reagent . Then, the complementary DNA (cDNA) was prepared using RT reactions with a first Strand cDNA Synthesis Kit . An ABI 7500 Thermocycler  was introduced to qPCR implementation. The primers used in this study were provided by Sangon Biotech. Fold changes of gene expression were determined based on the 2q method . GAPDH wAll experiments were repeated independently in triplicate. Values are presented as mean ± standard deviation and analysis was performed using GraphPad Prism 6.0 . Differences among groups were estimated with the aid of one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. A value of p < 0.05 was considered as a statistically significant result.As a lignin bioactive compound, Arctiin has been reported to block osteoclastogenesis and bone resorption via the suppression of RANKL-induced reactive oxygen species . HoweverOsteogenic differentiation is deemed as a pivotal determinant in bone regeneration, which is considered as an indispensable therapeutic strategy for osteoporosis ,16. Aftehttp://stitch.embl.de/) was used to detect the possible targets of Arctiin. It was found that Arctiin could bind to Ccnd1 and had higher score  was used to predict the possible targets of Arctiin. It was found that Arctiin could bind to Ccnd1. Ccnd1, the abbreviation of cyclin D1 (Mus musculus), is located on chromosome 7 and encoded by mouse Ccnd1 gene. A previous study reported that Ccnd1 was targeted by Let-7b to modulate osteoblast differentiation in mouse MC3T3-E1 cells [Figuring out the mechanisms underlying osteoblast differentiation and calcification is critical to improve the therapy for osteoporosis. To elucidate the potential mechanism of Arctiin functioning in the regulation of MC3T3-E1 cell differentiation, the STITCH website (E1 cells . MiR-23bE1 cells . Ccnd1 eE1 cells . MoreoveE1 cells . In thisTaken together, this is the first study to show that the impacts of Arctiin on MC3T3-E1 osteoblast differentiation. Mechanically, we validated that Arctiin promoted MC3T3-E1 cells’ osteogenic differentiation ability by targeting Ccnd1. Our findings provide experimental supports for the application of Arctiin in improving new bone formation and treating pathological conditions of bone loss. As Ccnd1 is a target of various pathways, the next experiments will investigate the effects on these pathways by Arctiin. Additionally, we will make the animal experiment in the future study to enhance the reliability of experimental results and provide theoretical basis for clinical application.Click here for additional data file.


Opsariichthys bidens have not been determined. In this study, the expression patterns and potential functions of PHB in spermatogenesis in O. bidens were characterized using histological microscopic observations, PCR cloning, real-time quantitative PCR (qPCR), Western blotting (WB) and immunofluorescence (IF). The full-length cDNA of Ob-phb was 1500 bp encoding 271 amino acids. A sequence alignment demonstrated that the PHB protein is conserved among different animals. qPCR revealed that phb mRNA is widely distributed in O. bidens and highly expressed in the testes at stages IV and V. WB revealed that Ob-PHB is located in the mitochondria of testes. IF revealed the colocalization of PHB signals and mitochondria. Signals were detected around nuclei in spermatogonia and spermatocytes, gradually moving to the tail region during spermiogenesis, and finally aggregating in the midpiece. These results indicate that Ob-PHB was expressed in the mitochondria during spermatogenesis. In addition, this study proposed Ob-PHB may participate in the degradation of mitochondria and cell differentiation during spermatogenesis.Spermatogenesis is the intricate and coordinated process by which spermatogonia develop into haploid differentiated spermatozoa. Mitochondria are essential for spermatogenesis, and prohibitin (PHB) is closely associated with mitochondrial structure and function during spermatogenesis. Although PHB has been implicated in spermatogenesis in some taxa, its roles in  Spermatogenesis in teleosts is a complex and dynamic developmental process involving a series of physiological and morphological changes in germ cells . This prSaccharomyces cerevisiae, PHB mutations alter the morphology of mitochondria but do not lead to cell death [Drosophila at the larval stage [Prohibitins (PHBs) are highly conserved proteins and widely distributed in bacteria , yeast , plants ll death ,22, wheral stage . It has al stage , maintaial stage ,25, regual stage  and promal stage .Bubalus bubalis is correlated with low sperm motility [Caenorhabditis elegans causes abnormal spermatogenesis [Boleophthalmus pectinirostris and silver pomfret Pampus argenteus, PHB is expressed at all stages of spermatogenesis and maintains the normal morphology and function of mitochondria [PHB is involved in spermatogenesis and has various biological functions. For instance, decreased PHB expression in motility ; a lack ogenesis . In humaogenesis . The rologenesis ,31,32. Iogenesis ,31,32. Ichondria ,34. TherOpsariichthys bidens  is an important commercial fish in China and aquaculture production is increasing [O. bidens, research on the mechanisms underlying reproduction in the species has attracted substantial attention. To date, cytological features of spermatogenesis in O. bidens have been studied by Tang et al. (2020) [O. bidens begins with the proliferation of spermatogonia, and after meiotic divisions of spermatocytes, haploid spermatids differentiate into flagellated sperm via morphological changes during spermiogenesis [O. bidens. Based on the roles of PHB in reproduction and evolutionary conservation, we speculate that PHB plays an important role in O. bidens spermatogenesis.creasing . To deve. (2020) . Spermatogenesis . HoweverO. bidens. PHB may play a role in maintaining the integrity and function of mitochondria and mediate mitochondrial degradation during fish spermatogenesis, laying the foundation for the elucidation of the mechanism of spermatogenesis in O. bidens.In the present study, we observed the histological characteristics of testes and spermatogenic cells and explored the expression, subcellular localization and possible functions of PHB in O. bidens by histological analyzation. In stage II of testis development, testes were mainly composed of spermatogonia and included low amounts of primary spermatocytes. The seminiferous tubules were formed of some small tubule lumens. The spermatogonia were the main germ cells and were developed in the cysts and scattered in seminiferous tubules , 816 bp open reading frame (ORF) and 618 bp 3’ UTR (Ob-PHB was approximately 29.6 kDa and its isoelectric point was 5.16. The Ob-PHB protein contains 271 amino acids (aa) and its predicted secondary structure is shown in Ob-PHB protein was predicted  applied in WB was demonstrated a. WB resOb-PHB protein acting as an antigen. IF was used to evaluate the expression and distribution of Ob-PHB during spermatogenesis. Ob-PHB signals were consistently co-localized with mitochondrial signals. In the spermatogonia and spermatocytes, Ob-PHB and mitochondrial signals were distributed around the nuclei  can be used for IF a. In IF,Daniorerio were roughly classified into eight phases according to time period  [Tor brevifilis [Acrossocheilus fasciatus [O. Bidens, a species of Cypriniformes, is divided into four successive stages: stages I–V, stage I, and stage VI are not observed in O. bidens. In stage I, spermatogonia constantly proliferate and this can be referred to as the proliferating period. In stage III, the testis is dominated by spermatocytes in meiosis, which can also be r meferred to as the meiosis period, as described by Huszno et al. (2012) [O. bidens testis development provides evidence for the identification of the reproductive cycle of O. bidens.Testis development of the zebrafish e cycle) . Accordievifilis  and the asciatus  were divasciatus . Similar. (2012) . In stag. (2012) . Above aD. rerio [Ophidion SP. and Bryconops affinis, spermatogenic cysts burst when spermatids occurs [O. bidens belong to the cystic type, which is consistent with the report by Tang et al. (2020) [With testis development, spermatogenesis was spontaneously activated. Spermatogenesis in teleosts are classified into two types: cystic and semi-cystic . In the D. rerio . In the D. rerio ,41. For s occurs ,41. Base. (2020) .P. argenteus and the mudskipper B. pectinirostris, mitochondria gradually shift to the area where flagellum formation occurs [O. bidens  experiments. In addition, nine tissues, including the liver, testes, kidney, brain, gill, muscle, heart, intestine, and spleen were obtained, frozen in liquid nitrogen and then stored at −80 °C until subsequent RNA and protein extraction.From September 2020 to June 2021, O. bidens and to observe the stages of testis development and the cytological characteristics of spermatogenesis and, finally, images were obtained.Paraffin sectioning and hematoxylin-eosin staining was performed to observe the stages of testis development and the cytological characteristics of spermatogenesis. In brief, the gonad fixed with Bouin’s solution dehydrated by an ethanol concentration gradient, permeabilized with xylene, and embedded in paraffin. Then, samples were cut into 7 μm sections and stained with hematoxylin-eosin. Following paraffin sectioning completed, a Nikon NI-U light microscope  was used to select the male O. Bidens. RNA extraction was performed as previously described [® RT Reagent Kit . Correspondingly, the 5’ and 3’ RACE cDNAs were synthesized by reverse transcription using the Smart RACE cDNA Amplification Kit  with RNA extracted before as the template.RNA was extracted from testes in escribed . First, Ob-phb transcript obtained from the National Center for Biotechnology Information , we designed primers using Primer Premier version 5.0  ; 27 cycles of 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 50 s; and 72 °C for 10 min for a final extension. Then, purpose straps were separated by performing gel electrophoresis and recycled using an Agarose gel Recovery Kit II . Then, the purified cDNA product was inserted into the pMD19-T vector  to form recombinant plasmids, which were transferred into competent DH5α cells  and sent to GENEWIZ company for sequencing.Based on the Ob-phb cDNA fragment was obtained, specific primers for 5’ and 3’ RACE were designed  with the neighbor-joining method. PHB from the following species (and accession numbers) included in the analysis were as follows: H. sapiens (AAB21614.1), B. taurus (NP_001029744.1), M. musculus (NP_032857.1), G. gallus (NP_001171206.1), A. platyrhynchos (XP_027301386.1), C. orientalis (AJF36071.1), X. tropicalis (NP_989038.1), P. Promelas (XP_039543669.1), D. rerio (NP_958454.1), S. salar (XP_014059527.1), O. tankahkeei (AEI91930.1), O. bimaculoides (XP_014779632.1), E. sinensis (ADM64319.1), P. clarkii (AGU02225.1) and S. cerevisiae (AAA53144.1). The isoelectric point and the molecular weight were predicted using ExPASy ProtParam . COILS  was used to predict the coiled-coil domain in PHB, and TMpred  was used to predict the transmembrane domain. CD-search  was used to search conserved domains (SPFH domain) in PHB. In addition, the tertiary structure of the PHB protein was predicted by the online tool I-Tasser .Vector NTI 11.5  was used to determine the similarity between the PHB amino acid sequences of −ΔΔCT method and expressed as the mean ± SD . The one-way analysis of variance (ANOVA) with SPSS v20.0  was used to determine significant differences. p < 0.05 was considered a statistically significant difference.We extracted mRNA from nine tissues of three male O. bidens (testes at stage IV) and testes of twelve male O. bidens . Then, cDNA was synthetized using a PrimeScript® RT Reagent Kit  for qPCR. We performed qPCR to evaluate phb mRNA expression levels in all of the tissues examined and stage II–V testes of O. bidens as described by Lin et al. . AccordiThe samples used for WB were the same as described in qPCR. Protein extraction assay was performed based on Zhang’s research . First, p < 0.05.WB assay was used to detect the expression pattern of Ob-PHB protein. WB experiments were performed according to Zhang’s research . In the Ob-PHB protein was detected in the mitochondria and cytoplasm by WB. Rabbit anti-SDHA and rabbit anti-β-tubulin antibodies were purchased from Beyotime .Mitochondria and cytoplasm were extracted from stage IV testes with the tissue Mitochondria Isolation Kit , according to the manufacturer’ instructions. In the WB experiment, the mitochondrial protein succinate dehydrogenase (SDHA) and cytoplasmic protein β-tubulin were used to confirm the separation between the mitochondria and cytoplasm. O. bidens testes were fixed in 4% PFA-PBS overnight. Following washing in PBS and after being dehydrated in 0.5 M sucrose-PBS at 4 °C, the testis was embedded in an optimum cutting temperature (O.C.T.) compound and stored at −80 °C. Then, samples were sliced into 5 μm frozen sections.Frozen sectioning was performed as follows: IF assay was conducted according to Zhang’s research . For IF,O. bidens and explored the structure, distribution and functions of mitochondrial protein PHB during spermatogenesis. As determined by qPCR and WB, PHB was expressed in different organs and during different stages of testis development. IF showed that PHB and mitochondria are co-localized during spermatogenesis. These results indicated that PHB plays different roles in different stages of spermatogenesis. It may not only affect the structure, function, and degradation of mitochondria but also participate in the differentiation of spermatogenic cells. However, the exact mechanism by which PHB participates in spermatogenesis in O. bidens is still unknown and should be explored in further research.In this study, we analyzed the histological characteristics of testes and germ cells in


N-methylmesoporphyrin IX (NMM) was used as a fluorescent indicator. The sensor is excellent in analyzing folded RNA targets and differentiating between closely related sequences due to the multicomponent character of the target-interrogating probe. Response to unamplified samples of total bacterial RNA from Mycobacterium tuberculosis complex or Escherichia coli was observed with excellent selectivity within 30 min under isothermal conditions at 50°C in a one-tube one-step assay. Several bacterial species can be detected in multiplex by utilizing biosensors with the template strands encoding different light-up aptamers. The isothermal one-tube-one-step format of the assay and the possibility to monitor the signal visually makes it amenable to use in a point-of-care scenario.We report on a single-tube biosensor for real-time detection of bacterial pathogens with multiplex capabilities. The biosensor consists of two DNA probes, which bind to the complementary fragment of a bacterial RNA to form a three-way junction (3WJ) nucleic acid structure. One of the probes encodes a fluorescent light-up RNA aptamer under T7 promoter. It allows for generation of multiple aptamer copies due to elongation and transcription of the 3WJ structure in the presence of the complementary target. The aptamer coordinates and thereby enhances fluorescence of a cognate fluorogenic dye, allowing for fluorescent detection of the RNA target. Multiple aptamer copies can be produced from a single target-dependent 3WJ structure allowing for amplification and visual observation of the signal. The limit of detection depended on the assay time and was found to be 1.7 nM or 0.6 nM for 30-min or 60-min assay, respectively, when  Detection of bacterial pathogens is of significance for disease diagnostics and surveillance, as well for monitoring food and water samples . This imAn alternative approach to NAATs is to amplify a signal rather than the nucleic acid target, for example, with the help of enzymes . Some ofN-methylmesoporphyrin IX (NMM)  comprising etiological agents of tuberculosis  , as fluo species . Ribosom species , which mfections , and Mycrculosis . The assBsm DNA polymerase, ultra-low range DNA ladder, RiboRuler High Range RNA ladder, Middlebrook 7H9 with OADC, Luria-Bertani (LB) broth, and Ambion Turbo DNA-free DNase kit were obtained from ThermoFisher Scientific . Hi-T7 RNA polymerase, Hi-T7 RNA polymerase buffer, ribonucleotide triphosphate (rNTP) mix, and deoxyribonucleotide (dNTP) mixes were purchased from New England BioLabs . GelRed® nucleic acid gel stain was from Biotium . N-methylmesoporphyrin IX (NMM) and malachite green were purchased from Sigma-Aldrich . Dithiothreitol (DTT) and ammonium chloride were purchased from Acros Organics . All other reagents were purchased from Fischer Scientific.Oligonucleotides were custom made by Integrated DNA Technologies, Inc.  and were used without purification. Non-DEPC treated DNase/RNase-free water was from Boston Bio Products . M. bovis BCG, M. simiae, or M. abscessus were grown to log phase (OD600 = 0.5–0.8) in 75 cm2 tissue culture flasks in 7H9-OADC medium. Pelleted bacilli were lysed in 65°C Trizol using a BeadBeater and 0.1 mm silicon beads. Total RNA was isolated from Trizol lysates by chloroform extraction and Qiagen RNeasy column purification. DNA contamination was removed using an Ambion Turbo DNA-free DNAse kit according to manufacture protocol. Following DNAse treatment, the concentration of RNA was measured using a NanoDrop UV-Vis spectrophotometer .Total RNA from mycobacterial species was isolated following the previously published protocol . In brieE. coli (strain DH10B), the cells were grown an OD600 of 1 in LB medium, pelleted by centrifugation and processed using a Monarch Total RNA Purification kit from New England Biolabs  according to the vendor-recommended protocol. The isolated RNA was analyzed by 0.8% agarose gel electrophoresis. The concentration of 16S rRNA in the total RNA preparation was determined by comparing the intensity of the corresponding band in the agarose gel with the intensity of the band containing a 1500-nt marker of the RiboRuler High Range RNA ladder.To isolate total RNA from Bsm DNA polymerase, 2.5 U/μL Hi-T7 RNA polymerase, 0.125 mM dNTPs, 2.5 mM rNTPs, 1 × Hi-T7 RNA polymerase buffer, 5 mM NH4Cl, 5 mM DTT, 20 mM MgCl2, 2 μM NMM and/or malachite green (MG) were incubated in the absence or presence of synthetic DNA target (0–100 nM) at 50°C for 30 min or 60 min. The signal was measured using a Cary Eclipse Fluorescence Spectrophotometer  using a 3-mm cuvette. For NMM, the signal was measured at 608 nm upon excitation at 399 nm, with the excitation/emission slits of 5/10 nm. For MG, the signal was measured at 650 nm upon excitation at 617 nm, with the excitation/emission slits of 10/10 nm. In both cases, PMT was set to “medium” (600 V). The data was processed in Microsoft Excel and/or OriginLab© 2018b graphing software. Tube images were acquired using a smartphone camera upon exciting the samples’ fluorescence using a Spectroline™ Slimline™ UV Transilluminator.Samples (50 μL) containing EP (0.1 μM), TP (0.1 μM), 0.1 U/μL 2. The gels were run at a constant voltage of 80 V in 1 × TBE buffer at 50°C. Denaturing PAAG electrophoresis was performed using 12% PAAG supplemented with 7M urea in 1 × TBE buffer at 7 V/cm, 50°C. The gels were stained with 1 × GelRed DNA staining dye, and the gel images were acquired using a BioRad GelDoc XR + Molecular Imager coupled with ImageLab software.Polyacrylamide gel (PAAG) electrophoresis under native conditions was performed using 10% PAAG supplemented with 10 mM MgCl© 2018b graphing software. Linear dynamic range corresponds to the range of target concentrations, at which the signal is directly proportional to the concentration. The LOD was calculated using a 3 σ/S rule  containing EP (0.1 μM), TP (0.1 μM), 0.1 U/μL Bsm DNA polymerase, 2.5 U/μL Hi-T7 RNA polymerase, 0.125 mM dNTPs, 2.5 mM rNTPs, 1 × Hi-T7 RNA polymerase buffer, 5 mM NH4Cl, 5 mM DTT, 20 mM MgCl2, and 2 μM NMM were incubated in the absence or presence of total bacterial RNA (17 or 30 ng/μL) at 50°C for 30 min. The signal was measured at 608 nm upon excitation at 399 nm using a Cary Eclipse Fluorescence Spectrophotometer  with a 3-mm cuvette. The data from three independent trials was processed in Microsoft Excel.To verify that the assay can be used for the detection of a native RNA sample, total bacterial RNA from either Bsm DNA polymerase into an extended 3WJ structure (ext-3WJ) (The aptamer-expressing probe assay relies on the formation of a 3WJ structure consisting of a nucleic acid target of interest and two strands—template probe (TP) and extension probe (EP) , step 1.ext-3WJ) , step 2.ext-3WJ) , step 3.Each aptamer molecule folds into an active dye-binding conformation and binds its cognate dye, thereby enhancing the dye’s fluorescence , step 4.E. coli (NCBI accession number NC_010473.1) or from M. tuberculosis (NCBI accession number NR_102810.2). Ribosomal RNAs are known to exhibit stable secondary and tertiary interactions that would challenge efficiency of conventional hybridization probes  linker connecting the target-binding and the EP-binding fragments of TP helps to decrease the background signal  without significantly affecting the target-induced NMM fluorescence  containing ∼10 nM 16S rRNA  triggered the signal of a similar intensity as in the presence of 10 nM synthetic MTC target (M. simiae (Msim) and M. abscessus (Mabs)—did not exceed the fluorescence intensity corresponding to LOD  to form a 3WJ complex, as well as gel electrophoretic analysis of the complex formation and fluorescent response of the system to EC or total E. coli RNA, are shown in In some applications, it may be important to detect several pathogens simultaneously. Therefore, we combined two EP/TP sets—MTC-MGA-TP/MTC-EP yielding MGA in response to MTC, and EC-G4-TPeg/EC-EP , which rM. tuberculosis complex and non-tuberculous mycobacteria. In this case, the affinity of the 3WJ probe to the target was maintained by the length of the TP strand. Previously, we have shown that even if one component of the split probe is shortened to allow for discrimination of point mutations, the length of the target-recognizing fragment of another probe’s component controls the overall affinity to keep the signal high  than a conventional 37°C transcription optimum. Elevated temperature contributes to greater accessibility of the target for probe’s binding.In the aptamer-encoding 3WJ assay reported here, generation of the signalling sequence of an RNA light-up aptamer depends on the action of two enzymes—It is possible to limit the use of enzymes for the light-up aptamer-encoding 3WJ technology to only RNA polymerase. When this paper was in preparation, a similar system was reported, in which DNA polymerase to produce the promotor sequence was avoided due to the use of a split T7 promoter . In thisWe have developed a one-tube-one-step real-time label-free signal amplification molecular assay, which can detect the presence of bacterial rRNA isothermally within as little as 30 min in a mixture with other RNA sequences as part of total bacterial RNA. The target’s presence is monitored as increase in the fluorescence emission of a fluorogenic dye bound to a corresponded light-up RNA aptamer encoded by a strand of the 3WJ probe utilized by the assay. Modular design of the probe allows easy adaptation of the assay to a new pathogen, and for closely related species it is possible to re-use a more expensive TP strand and only tailor the unmodified EP strand to be species-specific. Several pathogens in one and the same sample can be simultaneously detected in a multiplex format if the target-specific probes express different aptamer sequences that light-up fluorogens with emission maxima at different wavelengths. The assay can be employed for the real-time detection of bacterial or viral pathogens.


High-throughput transcriptomics has revolutionised the field of transcriptome research by offering a cost-effective and powerful screening tool. Standard bulk RNA sequencing (RNA-Seq) enables characterisation of the average expression profiles for individual samples and facilitates identification of the molecular functions associated with genes differentially expressed across conditions. RNA-Seq can also be applied to disentangle splicing variants and discover novel transcripts, thus contributing to a comprehensive understanding of the transcriptome landscape. A closely related technique, single-cell RNA-Seq, has enabled the study of cell-type-specific gene expressions in hundreds to thousands of cells, aiding the exploration of cell heterogeneity. Nowadays, bulk RNA-Seq and single-cell RNA-Seq serve as complementary tools to advance and accelerate the development of transcriptome-based resources. This Collection illustrates how the current global research community makes use of these techniques to address a broad range of questions in life sciences. It demonstrates the usefulness and popularity of high-throughput transcriptomics and presents the best practices and potential issues for the benefit of future end-users. High-throughput transcriptomics has accelerated our understanding of the variety and complexity of transcriptomes of different tissue types and individual cells. The transcriptome refers to the full range of RNA molecules expressed by a cell, tissue, or organism in a certain physiological condition or at a specific stage of development. The study of the transcriptome is essential to (1) measure the changing levels of expression of each gene under different conditions; (2) characterise transcriptional variants and all possible splicing patterns; (3) identify non-coding RNA (ncRNA) species and study their role in different biological processes. This Collection brings together contributions from scientists addressing diverse biological and medical issues across a broad spectrum of species and cell systems using a variety of transcriptome sequencing techniques.Since its inception, bulk RNA-sequencing (RNA-Seq) has been the method of choice for studying tissue-level transcriptome changes and has brought a qualitative and quantitative improvement to the transcriptome analysis due to its unlimited dynamic range. Mean expression levels of RNAs isolated from a tissue are detected based on the number of reads mapped to each gene/transcript, and differences in gene expression profiles between samples can then be derived. Hence, the objective of gene/transcript quantification is to identify changes that occur under different experimental conditions, in disease states, and in response to medical treatments, thereby providing a snapshot of the underlying molecular processes taking place.. investigated changes in gene expression of human dermal fibroblasts following red light phototherapy and identified several genes as therapeutic targets in the treatment of skin fibrosis1. Among these, the metalloproteinase-1 (MMP1) gene, involved in extracellular matrix organization and collagen degradation, was found as one of the critical mediators of fibrotic disease. Showcasing a different application, Sánchez et al. clearly showed that transcriptomic changes in the arcuate nucleus and the remaining hypothalamic tissue of heifer calves were associated with enhanced nutrition in early life. Their data can be exploited to design new nutritional regimens capable to promote earlier onset of puberty2. RNA-Seq has also been used to examine transcriptional changes in plants; Rathore et al. performed a time-series eco-transcriptomics study to monitor temporal changes in the transcriptome of an evergreen shrub in response to changing environmental conditions in natural alpine environments. The authors found that the transcriptome reprogramming of the leaves is greater towards winters and decline during spring and that the de-acclimation period was the transcriptionally most active phase3. Finally, RNA-Seq can be successfully combined with computational methods; Yap et al. developed a deep learning classifier based on convolutional neural networks that can predict tissue classification from Genotype-Tissue Expression (GTEx) RNA-Seq data, thereby providing more information on tissue-specific gene expression than traditional differential gene expression analysis4.For example, in a study published as part of the Collection, Austin et al. reported the differential splicing of CAMK2G in two mouse models and revealed the relationship between CAMK2G and hypoxia inducible factor 1 in the pathogenesis of heart diseases5. Estill et al. analysed the transcriptome of the nucleus accumbens in the brains of mice treated with cocaine, and found many novel transcripts and splicing sites, revealing information useful in our understanding of addiction and reward6.Aside from quantifying transcript expression profiles, random fragmentation used in bulk RNA-Seq library preparation has allowed scientists to detect new features in a transcriptome, such as novel transcripts, RNA isoforms, and gene fusion transcripts. In a paper included in this Collection, Williams et alAlthough every single cell in an organism carries the same genome content, different cell types can use different portions of the same genome by expressing different subsets of all possible genes. Bulk RNA-Seq does not consider the heterogeneity of a sample and the individual complexity of each cell, so new methods have been refined to enable investigation of the transcriptome at a single-cell level.Single cell RNA-Seq (scRNA-Seq) has emerged as a breakthrough technology for dissecting tissue heterogeneity, identity, fate, and function by capturing transcriptome profiles from individual cells. Coupled with sophisticated downstream bioinformatic analysis, scRNA-Seq enables investigations of gene expressions in cells. Additionally, scRNA-Seq allows researchers to identify previously unknown cell populations, highlighting the diversity of the molecular processes affecting each individual cell or cell type and revealing differences at the cellular level that are generally obscured by bulk sampling.. described a promising and flexible droplet‐based microfluidics system capable of capturing approximately 50,000 single cells in one run7. The authors then used the system to distinguish human cell populations at different phases of the cell cycle and to study transcriptional regulatory networks, thereby pointing out several transcription factors with little or no prior association with distinct cell cycle phases. To address the problems of technical variations between cell subsets introduced by the tissue dissociation procedure, Uniken Venema et al. developed a one-step collagenase dissociation protocol suitable for scRNA-Seq and demonstrated its use on cryopreserved gut mucosal biopsies. The protocol showed many advantages, i.e. reduction of times, costs and procedures, prevention of batch effects, high level of reproducibility, and experimental flexibility, over the gold standard two-step collagenase dissociation and three-step protease dissociation protocols8.Many instruments have been developed with the aim of facilitating the use of scRNA-Seq and the analysis of relevant data. In this Collection, Davey et alMost RNA molecules in a transcriptome fall into a broad class known as non-coding RNAs, which have attracted the attention of researchers through their involvement in numerous biological processes. With unprecedented resolution and scale, variations of the RNA-Seq method allow for the identification of a growing number of ncRNAs, and investigation of their potential functions.. proposed a new method to examine the expression and activity of microRNAs (miRNAs), one of the subtypes of ncRNA, at the single-cell level9. As single-cell miRNA sequencing is not yet widespread practice, miRNA activity scores were derived indirectly from the expression profiles of all target mRNAs. The method, based on the enrichment of miRNA binding sites, was then applied to a human and a mouse scRNA-Seq data set, and returned activity measures of several miRNA candidates coherent with literature data. Finally, Su et al. studied the positive regulatory role of circular RNAs (circRNAs), another type of ncRNA, in the osteogenic differentiation of mesenchymal stromal cells of the human umbilical cord10. The authors identified over 5000 circRNAs, about one percent of which were differentially expressed. Among these, circ‐CTTN was found to play a key role during osteogenic differentiation.Nielsen et alTaken together, this Collection encompasses a broad range of studies using high-throughput transcriptomics alongside other molecular techniques in different research domains, with aims ranging from improving experimental approaches, to developing bioinformatic methods, to applications in real-world scientific questions. This Collection showcases some of the recent developments in transcriptomics studies that we hope will provide guidelines for researchers with an interest in the use of transcriptomics, or other omics techniques, in future studies.


As a crucial component of urban green space (UGS), urban parks have been found to be closely associated with the health of urban residents. Drawing on a large-scale survey, “International First-class Harmonious and Livable Capital”, in Beijing conducted in 2018, this paper examines the impact of subjective and objective characteristics of UGS on residents' self-rated health (SRH) by using a binary logistic regression model. The results indicate that the overall SRH status of urban residents in Beijing is relatively good, with 73.8% of the respondents reporting good SRH. The perceived quality of UGS and objectively measured accessibility to UGS are positively associated with residents' SRH, but the subjective indicator of UGS has a greater impact on SRH than the objective indicator of UGS. In terms of influencing mechanisms, social interaction and air quality perception were the two major mediators of UGS that affected residents' SRH. The heterogeneity analysis suggests that objective accessibility to different types of urban parks has mixed effects on residents' SRH. Access to high-quality parks is positively associated with residents' SRH, whereas access to common parks has a negative impact on residents' SRH. Our findings provide important policy implications for optimizing urban park design and improving the quality of urban park provision according to human needs in the Beijing Metropolitan Area. Urbanization is advancing rapidly worldwide, with more than 60% of the global population living in cities. However, rapid, extensive urbanization in some developing countries such as China has been accompanied by serious environmental pollution and a high incidence of chronic diseases , 2. SincThe factors that influence individuals' health in the research community comprise both intrinsic and extrinsic factors. Intrinsic factors are some physiological factors and behaviors, for example, daily health behaviors and genetic factors that are potential determinants affecting individuals' health. For instance, health behaviors such as individual dietary habits and physical activity may directly affect human health . Health Urban green space (UGS) is green infrastructure, namely, for example, urban parks, urban forests, public green spaces, school playgrounds, public rest areas, city squares, and vacant lots , among wUGS provides a wide variety of social, economic, and environmental benefits , 21. ExiResearch has provided evidence of the UGS-health association. However, most of the health-related literature has measured UGS only from an objective perspective, such as the quantity and accessibility of UGS . Widely With the popularity of individual-centered urban development, the relationship between individuals' perceptions of UGS and their health has received increasing attention, and the concept of perceived accessibility has been widely used. Perceived accessibility generally refers to an individual's subjective perception of and satisfaction with the accessibility to public facilities in the physical environment , indicatIn filling these research gaps, exploring the association between UGS and residents' SRH using both subjective and objective perspectives is necessary. As an influential component of UGS, urban parks have a close relationship with residents' daily lives and health. Thus, exploring the impact and influence mechanisms of urban parks on residents' health to promote the construction of UGS as well as sustainable and healthy urban development is important. Drawing on a large-scale questionnaire survey, “International First-class Harmonious and Livable Capital”, conducted in Beijing in 2018, this study combined both objective accessibility indicators and subjective perceptions of UGS to examine the relationship between UGS and urban residents' SRH in Beijing using a binary logistic regression model while controlling for residents' socioeconomic attributes.More specifically, the objectives of this study are to (1) compare the impact intensity of both objectively and subjectively measured indicators of UGS in influencing residents' SRH; (2) explore the mediating paths through which UGS affects the SRH of urban residents; and (3) examine the heterogeneous effects of different types of urban parks on residents' SRH. Our findings provide policy insights into optimizing the allocation of UGS and improving human health through improved UGS provision.As shown in The questionnaire survey “International First-class Harmonious and Livable Capital” was conducted in the urban areas in Beijing in April 2018. It was performed by the Institute of Geographic Sciences and Natural Resource Research of the Chinese Academy of Sciences and covered 184 streets within the sixth ring road. Surveyed respondents were randomly selected from the streets with support from the local community council, and a face-to-face questionnaire survey was conducted with trained investigators. Because our survey covered many streets, it is representative of all types of urban residents' perceived living environment quality and SRH in Beijing. The spatial distribution of the study area and the surveyed respondents are shown in The survey covered seven dimensions of living environment quality  and respondents' socioeconomic characteristics such as age, gender, marital status, educational attainment, annual household income, car ownership, household registration, and occupation type. The survey also included residents' SRH and perceived quality of the park near the residential area. At the same time, in order to explore the mediating role of social interaction and air quality in the process of park affecting residents' health, we also investigated respondents' satisfaction with these two items.Our survey also collected the geographic coordinates of respondents' residences, with the support of a location-based service, which can be applied to accurately identify respondents' accessibility to UGS. The total number of questionnaires obtained in our survey was 10,651. After removing questionnaires with missing data, we finally have 10,011 effective surveys, with an effective rate of 93.99%.SRH is a widely used indicator of public health because of its easy availability in large-scale surveys  and its In this study, UGS indicators comprised objective and subjective aspects . BecauseIn this study, social interaction and air quality were used as mediator variables, both of which were measured through respondents' perceived evaluations of living environment quality. In terms of social interaction, respondents were asked, “How satisfied are you with your social interactions in the community?” As for air quality, respondents were asked, “How satisfied are you with the air quality around your residential area?” For both survey questions, response items such as “strongly dissatisfied”, “dissatisfied”, “fair”, “satisfied”, and “very satisfied” were assigned a score from 1 to 5, respectively.The control variables in this study were the socioeconomic characteristics of the respondents, such as age, gender, marital status, educational attainment, annual household income, car ownership, household registration, and occupation type. The distance to the city center from the respondents' residence was also controlled, which can reflect residents' residential location. In this study, a binary logistic regression model was used to examine the effects of UGS on urban residents' SRH in Beijing. First, the subjective indicator  was included in the model as the key explanatory variable (Model 1a). Second, the objective indicator of UGS, measured by accessibility (the distance to the nearest urban park from the respondent's residence), was included as the key explanatory variable in the model (Model 1b). Finally, both accessibility to UGS and residents' perceptions of UGS were included in the model (Model 1c), to test the joint effect of the subjective and objective measures of UGS on individuals' SRH.hukou. The logistic regression model equation is as follows:The control variables were the distance to the city center and repondents' socioeconomic characteristics, such as gender, age, marital status, educational attainment, occupation type, total annual household income, car ownership, and xi and βi are UGS variables and the corresponding regression coefficients; xj and βj are control variables and the corresponding regression coefficients.where P denotes Y = 1, the probability of occurrence of respondents having good SRH; 1-P denotes Y = 0, the probability of occurrence of respondents with poor SRH; α is the intercept; A binary logistic regression model was used to examine the effect of UGS on participants' SRH. In order to compare the effects of influencing factors, all explanatory variables were standardized before being introduced into the model. The results of the multicollinearity test showed that the variance inflation factor (VIF) values of all independent variables were below 5, indicating that multicollinearity is not a problem. The results of the regression model are shown in In addition, the results of Model 1c show that some socioeconomic attributes are also associated with respondents' SRH. Middle-aged and elderly residents had lower odds of reporting good SRH than residents aged under 20 years. Respondents with a graduate education or above had a poorer SRH than residents with a below middle school education, with a 31.7% lower odds ratio reporting good SRH. Residents with a higher annual household income had better SRH than the reference group with annual household income below 30,000 CNY, with the odds ratio ranging from 1.565 in the 300,000–500,000 CNY group to 2.835 in the 500,000–1,000,000 CNY group. Regarding household registration, respondents with a Beijing hukou had worse SRH than non-hukou residents, with a 31.4% decrease in the odds of residents reporting good SRH.To explore the specific influencing mechanisms of the effects of UGS on respondents' SRH, social interaction and air quality were added as mediating variables in the model. The results are shown in Model 2c included two mediating variables, social interaction and air quality, in the regression analysis based on the baseline model. The results showed that social interaction and air quality were significantly and positively associated with residents' SRH, with regression coefficients of 0.060 and 0.196, respectively. The mediating analysis results reveal that social interaction and air quality are important mediator variables for the UGS effect on respondents' SRH, and UGS affects respondents' SRH directly or indirectly by increasing their satisfaction with social interaction and air quality.Following the high-quality parks rating standard issued in Beijing, local authorities have differentiated urban parks into high-quality parks and common parks based on a set of criteria since 2002, such as planning and construction, greening and maintenance, supporting facilities, and order maintenance. Specifically, high-quality parks must fulfill the following criteria: (1) a park green space cover rate of over 70%; (2) a satisfaction ratio among the visitors that exceeds 90%; (3) the loess in the park is not open to air; (4) neat, standardized signage; two-star or higher toilets; (5) no advertising umbrellas or other facilities that hinder the landscape; (6) no stagnant water, dirt, spitting, or cigarette butts; no vending stalls within 50 m of the park entrance; (7) good order in the park; (8) no major complaints or safety accidents. High-quality parks generally provide healthier and more liveable environmental elements than common parks.To examine the heterogeneous effects of types of urban parks on respondents' SRH, we differentiated urban parks into high-quality and common parks for further analysis. This study explored the relationship between UGS and respondents' SRH using data collected in a large-scale survey in Beijing: the International First-class Harmonious and Liveable Capital. Our study contributes to the literature on UGS and health in at least three aspects. First, we combine the objective and subjective characteristics of UGS in the research framework. Second, mediating mechanisms such as social interaction and air quality between the UGS and residents' health are examined. Finally, our study adds new empirical evidence on the health impacts of UGS in developing countries with rapid urbanization, using Beijing as a case study.Our study found that the mean value of respondents' SRH in Beijing was 3.93 and 73.8% of residents reported good SRH, indicating that the overall SRH of urban residents in Beijing was good. This finding is similar to that in many studies : BeijingThe binary logistic regression model results indicated that the perceived quality of UGS was positively associated with respondents' SRH. This finding occurs because residents' perceived quality or satisfaction with urban parks originates from their experience , and resIn line with the literature , 47, ourThe results of the heterogeneity analysis revealed that the effects of high-quality and common parks on respondents' SRH differed. Among them, accessibility to UGS for high-quality parks was positively correlated with respondents' SRH, indicating that respondents with a longer distance to the nearest high-quality park tend to report a good SRH. This result may be observed because high-quality parks generally occupy a large area, which, to a certain extent, compresses the space for other public service facilities, leading to other types of healthy living needs of residents not being fulfilled and, in turn, affecting respondents' SRH . By contThe findings of our study provide important policy implications for improving UGS construction. Firstly, because the perceived accessibility of UGS plays a significant role in promoting residents' self-rated health, the planning and design of urban parks should pay attention to residents' perception of use and regularly collect residents' feedback on their satisfaction with urban parks. In addition, the objective distance of UGS also significantly affects the self-rated health of residents. Landscape planning departments should give priority to increasing urban parks with appropriate scale and high accessibility according to the distribution of residential areas and the urban road network system without crowding out other public service facilities.Second, social interaction and air quality are important ways in which UGS affects SRH of respondents. Therefore, when designing urban parks, landscape design departments should not only provide good internal environment design and comfortable and pleasant green landscape to purify the air, but also create a comfortable atmosphere and provide convenient and diverse activity facilities to promote residents' social activities.Finally, in view of the difference in the impact of high-quality parks and common parks on the SRH of respondents, the government departments in Beijing should take corresponding optimization measures for different types of parks. Specifically, the focus of optimisation for high-quality parks is to improve their quality, promote the transformation of high-quality parks into high-quality spaces, and fulfill the demands of some residents for high-end UGS. For common parks, the spatial quality and micro design of UGS in parks should be improved, based on not occupying land for other types of public service facilities. The area of park UGS should be appropriately expanded to increase fitness facilities and public spaces to fulfill the diversified needs of different groups with different social attribute.This study has several limitations. First, this study used cross-sectional data, which cannot fully reveal the causal relationship and changing linkage between UGS and SRH among respondents. Second, the specific mediating mechanisms of UGS on respondents' SRH in our study only considered their perceived social interaction and air quality. Other potential mediating mechanisms  were not explored and should also be included in further research. Finally, the accessibility to UGS in this study was measured only by the shortest road network distance without consideration of actual traffic speed in different grades of road segments and individuals' transport modes. Further research should focus on the impact of travel time accessibility to the UGS on residents' SRH. In addition, the areas of the original urban parks were aggregated into points according to their geometric gravity center, which could also cause some measurement bias in accessibility when encountering urban parks with a large area.Unlike developed western countries that have explored the health benefits of green space under the background of low population density, empirical evidence of green space and human health in developing countries with high population density is still relatively limited, and existing studies tend to focus only on the objective attributes of green space. Drawing on a large-scale survey and the spatial distribution of UGS in Beijing, this study quantified the association between UGS and residents' SRH by using a binary logistic regression model. It focused on the role of objectively measured accessibility to UGS in influencing residents' SRH, examined the relationship between perceived UGS quality and residents' SRH, and comprehensively measured the health benefits of UGS exposure levels from both the subjective and objective perspectives to mitigate the limitations of current research on the association between UGS and residents' health. In our study case, UGS is found to be associated with urban residents' SRH in Beijing. More specifically, both the perceived quality of UGS and objectively measured accessibility to UGS are positively related to residents' SRH, but the perceived quality of UGS has a much greater effect on residents' SRH. In addition, we identified that social interaction and air quality are important mediating paths through which UGS affects residents' SRH. Moreover, the effects of the types of urban parks on residents' SRH differed. Among these differences, accessibility to UGS for high-quality parks is positively associated with residents' SRH, and accessibility to UGS for common parks is negatively related to residents' SRH. Overall, this empirical evidence provides novel insights into optimizing green space,which could help guide planners and decision-makers to promote green space development for public health.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.DZ, BZ, and WZ: conceptualization. DZ: writing and review. QZ: writing—original. M-PK: review and editing. JL: software. BZ and QZ: data analysis. WZ: investigation. All authors contributed to the article and approved the submitted version.This work was supported by the Humanities and Social Sciences Research Program of the Ministry of Education in China (20YJCZH221), National Natural Science Foundation of China (42001120), Fundamental Research Funds for the Provincial Universities of Zhejiang (GB202103004).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.