Distinct Processing of lncRNAs Contributes to Non-conserved Functions in Stem Cells.

Long noncoding RNAs (lncRNAs) evolve more rapidly than mRNAs. Whether conserved lncRNAs undergo conserved processing, localization, and function remains unexplored. We report differing subcellular localization of lncRNAs in human and mouse embryonic stem cells (ESCs). A significantly higher fraction of lncRNAs is localized in the cytoplasm of hESCs than in mESCs. This turns out to be important for hESC pluripotency. FAST is a positionally conserved lncRNA but is not conserved in its processing and localization. In hESCs, cytoplasm-localized hFAST binds to the WD40 domain of the E3 ubiquitin ligase ß-TrCP and blocks its interaction with phosphorylated ß-catenin to prevent degradation, leading to activated WNT signaling, required for pluripotency. In contrast, mFast is nuclear retained in mESCs, and its processing is suppressed by the splicing factor PPIE, which is highly expressed in mESCs but not hESCs. These findings reveal that lncRNA processing and localization are previously under-appreciated contributors to the rapid evolution of function.

SLERT Regulates DDX21 Rings Associated with Pol I Transcription.

Dysregulated rRNA synthesis by RNA polymerase I (Pol I) is associated with uncontrolled cell proliferation. Here, we report a box H/ACA small nucleolar RNA (snoRNA)-ended long noncoding RNA (lncRNA) that enhances pre-rRNA transcription (SLERT). SLERT requires box H/ACA snoRNAs at both ends for its biogenesis and translocation to the nucleolus. Deletion of SLERT impairs pre-rRNA transcription and rRNA production, leading to decreased tumorigenesis. Mechanistically, SLERT interacts with DEAD-box RNA helicase DDX21 via a 143-nt non-snoRNA sequence. Super-resolution images reveal that DDX21 forms ring-shaped structures surrounding multiple Pol I complexes and suppresses pre-rRNA transcription. Binding by SLERT allosterically alters individual DDX21 molecules, loosens the DDX21 ring, and evicts DDX21 suppression on Pol I transcription. Together, our results reveal an important control of ribosome biogenesis by SLERT lncRNA and its regulatory role in DDX21 ring-shaped arrangements acting on Pol I complexes.

Condensates induced by transcription inhibition localize active chromatin to nucleoli.

The proper function of the genome relies on spatial organization of DNA, RNA, and proteins, but how transcription contributes to the organization is unclear. Here, we show that condensates induced by transcription inhibition (CITIs) drastically alter genome spatial organization. CITIs are formed by SFPQ, NONO, FUS, and TAF15 in nucleoli upon inhibition of RNA polymerase II (RNAPII). Mechanistically, RNAPII inhibition perturbs ribosomal RNA (rRNA) processing, releases rRNA-processing factors from nucleoli, and enables SFPQ to bind rRNA. While accumulating in CITIs, SFPQ/TAF15 remain associated with active genes and tether active chromatin to nucleoli. In the presence of DNA double-strand breaks (DSBs), the altered chromatin compartmentalization induced by RNAPII inhibition increases gene fusions in CITIs and stimulates the formation of fusion oncogenes. Thus, proper RNAPII transcription and rRNA processing prevent the altered compartmentalization of active chromatin in CITIs, suppressing the generation of gene fusions from DSBs.

Nascent Pre-rRNA Sorting via Phase Separation Drives the Assembly of Dense Fibrillar Components in the Human Nucleolus.

Fibrillar centers (FCs) and dense fibrillar components (DFCs) are essential morphologically distinct sub-regions of mammalian cell nucleoli for rDNA transcription and pre-rRNA processing. Here, we report that a human nucleolus consists of several dozen FC/DFC units, each containing 2-3 transcriptionally active rDNAs at the FC/DFC border. Pre-rRNA processing factors, such as fibrillarin (FBL), form 18-24 clusters that further assemble into the DFC surrounding the FC. Mechanistically, the 5' end of nascent 47S pre-rRNA binds co-transcriptionally to the RNA-binding domain of FBL. FBL diffuses to the DFC, where local self-association via its glycine- and arginine-rich (GAR) domain forms phase-separated clusters to immobilize FBL-interacting pre-rRNA, thus promoting directional traffic of nascent pre-rRNA while facilitating pre-rRNA processing and DFC formation. These results unveil FC/DFC ultrastructures in nucleoli and suggest a conceptual framework for considering nascent RNA sorting using multivalent interactions of their binding proteins.

Adsorption of CO32-/HCO3- on a quartz surface: cluster formation, pH effects, and mechanistic aspects.

Soluble inorganic carbon is an important component of a soil carbon pool, and its fate in soils, sediments, and underground water environments has great effects on many physiochemical and geological processes. However, the dynamical processes, behaviors and mechanism of their adsorption by soil active components, such as quartz, are still unclear. The aim of this work is to systematically address the anchoring mechanism of CO32- and HCO3- on a quartz surface at different pH values. Three pH values (pH 7.5, pH 9.5 and pH 11) and three carbonate salt concentrations (0.07, 0.14 and 0.28 M) are considered, and molecular dynamics methods are used. The results indicate that the pH value regulates the adsorption behavior of CO32- and HCO3- on the quartz surface by affecting the CO32-/HCO3- ratio and the surface charge of quartz. In general, both HCO3- and CO32- ions were able to adsorb on the quartz surface and the adsorption capacity of CO32- is higher than that of HCO3-. HCO3- ions tended to uniformly distribute in an aqueous solution and contact the quartz surface in the form of single molecules instead of clusters. In contrast, CO32- ions were mainly adsorbed as clusters which became larger as the concentration increased. Na+ ions were essential for the adsorption of HCO3- and CO32-, because some of the Na+ and CO32- ions spontaneously associated together to form clusters, promoting the clusters to be adsorbed on the quartz surface through cationic bridges. The local structures and dynamics trajectory of CO32- and HCO3- showed that the anchoring mechanism of carbonate solvates on quartz involved H-bonds and cationic bridges, which changed in relation to the concentration and pH values. However, the HCO3- ions mainly adsorbed on the quartz surface via H-bonds while the CO32- ions tended to be adsorbed through cationic bridges. These results may help in understanding the geochemical behavior of soil inorganic carbon and further the processes of the Earth's carbon chemical cycle.

Effect of perioperative application of dexmedetomidine on post-operative stress reaction, pain and prognostic adverse effects in patients undergoing gynaecological laparoscopy.

Introduction: The aim of this study was to analyse the effect of perioperative dexmedetomidine (DEX) application on stress response, post-operative pain and prognosis in patients undergoing gynaecologic laparoscopy. Patients and Methods: One hundred and sixty-eight patients admitted for gynaecologic laparoscopic surgery from May 2020 to November 2022 were included in the study. The patients were randomly divided into pre-operative DEX group (n = 56), intraoperative DEX group (n = 56) and post-operative DEX group (n = 56) according to the application of DEX in the perioperative period. The visual analogue scale (VAS), time awake, extubation time, pneumoperitoneum time, post-anaesthesia care unit (PACU) stay time and Richmond agitation-sedation scale score (RASS) were recorded. Results: Patients in both the pre-operative and intraoperative DEX groups had substantially shorter wakeup and extubation times than those in the post-operative DEX group. Patients in the pre-operative DEX group had considerably shorter wakeup and extubation times than those in the intraoperative DEX group, and their pneumoperitoneum time was significantly shorter than that of the post-operative DEX group (P < 0.001). The RASS scores of the pre-operative DEX group and intraoperative DEX group were significantly lower than those of the post-operative DEX group at 1 h, 6 h and 12 h after surgery. Meanwhile, at all time periods, the RASS scores of patients in the pre-operative DEX group were considerably lower than those in the intraoperative DEX group (P < 0.01). The VAS scores of patients in the pre-operative DEX group and intraoperative DEX group were evidently lower than those in the post-operative DEX group at 0.5 h, 2 h and 12 h postoperatively, and the VAS scores of patients in the pre-operative DEX group were markedly lower than those in the intraoperative DEX group (P < 0.001). The incidence of nausea and vomiting was significantly lower in the pre-operative DEX group than in the intraoperative DEX group and the post-operative DEX group at 0-2 h, >2-12 h and >12-24 h postoperatively (P < 0.001). The incidence of nausea and vomiting in the intraoperative DEX group was significantly lower than that in the post-operative DEX group from 0 to 2 h after surgery (P < 0.05). The incidence of adverse reactions was not significantly different amongst the three groups of patients (P > 0.05). Conclusion: Pre-operative and intraoperative application of DEX can help reduce post-operative pain and stress responses, help patients recover quickly after surgery and improve patient prognosis, especially the pre-operative application of DEX.

Circular intronic long noncoding RNAs.

We describe the identification and characterization of circular intronic long noncoding RNAs in human cells, which accumulate owing to a failure in debranching. The formation of such circular intronic RNAs (ciRNAs) can be recapitulated using expression vectors, and their processing depends on a consensus motif containing a 7 nt GU-rich element near the 5' splice site and an 11 nt C-rich element close to the branchpoint site. In addition, we show that ciRNAs are abundant in the nucleus and have little enrichment for microRNA target sites. Importantly, knockdown of ciRNAs led to the reduced expression of their parent genes. One abundant such RNA, ci-ankrd52, largely accumulates to its sites of transcription, associates with elongation Pol II machinery, and acts as a positive regulator of Pol II transcription. This study thus suggests a cis-regulatory role of noncoding intronic transcripts on their parent coding genes.

Processing and roles of snoRNA-ended long noncoding RNAs.

Small nucleolar RNAs (snoRNAs) are a family of conserved nuclear RNAs that function in the modification of small nuclear RNAs (snRNAs) or ribosomal RNAs (rRNAs), or participate in the processing of rRNAs during ribosome subunit maturation. Eukaryotic DNA transcription and RNA processing produce many long noncoding RNA (lncRNA) species. Although most lncRNAs are processed like typical mRNAs to be 5' capped and 3' polyadenylated, other types of lncRNAs are stabilized from primary Pol II transcripts by alternative mechanisms. One way to generate stable lncRNAs is to co-operate with snoRNA processing to produce snoRNA-ended lncRNAs (sno-lncRNAs) and 5' snoRNA-ended and 3'-polyadenylated lncRNAs (SPAs). Rather than silently accumulating in the nucleus, some sno-lncRNAs and SPAs are involved in the regulation of pre-rRNA transcription and alternative splicing of pre-mRNAs. Here we provide a mini-review to discuss the biogenesis and functions of these unusually processed lncRNAs.

Research progress of long noncoding RNA in China.

RNA is essential for all kingdoms of life and exerts important functions beyond transferring genetic information from DNA to protein. With the advent of the state-of-the-art deep sequencing technology, a large portion of noncoding transcripts in eukaryotic genomes has been broadly identified. Among them, long noncoding RNAs (lncRNAs) have been emerged as a new class of RNA molecules that have regulatory potential in a variety of physiological and pathological processes. Here we summarize recent research progresses that have been made by scientists in China on lncRNAs, including their biogenesis, functional implication and the underlying mechanism of action at the current stage. © 2016 IUBMB Life, 68(11):887-893, 2016.

DisP-seq reveals the genome-wide functional organization of DNA-associated disordered proteins.

Intrinsically disordered regions (IDRs) in DNA-associated proteins are known to influence gene regulation, but their distribution and cooperative functions in genome-wide regulatory programs remain poorly understood. Here we describe DisP-seq (disordered protein precipitation followed by DNA sequencing), an antibody-independent chemical precipitation assay that can simultaneously map endogenous DNA-associated disordered proteins genome-wide through a combination of biotinylated isoxazole precipitation and next-generation sequencing. DisP-seq profiles are composed of thousands of peaks that are associated with diverse chromatin states, are enriched for disordered transcription factors (TFs) and are often arranged in large lineage-specific clusters with high local concentrations of disordered proteins and different combinations of histone modifications linked to regulatory potential. We use DisP-seq to analyze cancer cells and reveal how disordered protein-associated islands enable IDR-dependent mechanisms that control the binding and function of disordered TFs, including oncogene-dependent sequestration of TFs through long-range interactions and the reactivation of differentiation pathways upon loss of oncogenic stimuli in Ewing sarcoma.

Controllable concentric electric field line distribution for simultaneous separation of DNA.

An approach for the controllable separation and concentration of nucleic acid using a circular nonuniform electric field was proposed and developed. Using six different lengths of DNA molecules as standard samples, the distribution of the gradient electric field was increased from the outer circular electrode to the inner rod-shaped electrode, contributing to the migration of DNA molecules at a velocity gradient towards the region with the strongest inner electric field. The DNA molecules were arranged in a distribution of concentric circles that aligned with the distribution of concentric equipotential lines. The concentration of DNA multiplied with the alternation of radius. As a result, this platform allowed simultaneous DNA separation, achieving a resolution range of 1.17-3.03 through an extended electrophoresis time, resulting in enhanced concentration factors of 1.08-6.27. Moreover, the manipulation of the relative height of the inner and outer electrodes enabled precise control over the distribution and the deflection degree of electric field lines, leading to accurate control over DNA deflection.

Overexpression of PtoMYB99 diminishes poplar tolerance to osmotic stress by suppressing ABA and JA biosynthesis.

Drought poses a serious challenge to sustained plant growth and crop yields in the context of global climate change. Drought tolerance in poplars and their underlying mechanisms still remain largely unknown. In this article, we investigated the overexpression of PtoMYB99 - both a drought and abscisic acid (ABA) induced gene constraining drought tolerance in poplars (as compared with wild type poplars). First, we found that PtoMYB99-OE lines exhibited increased stomatal opening and conductance, higher transpiration and photosynthetic rates, as well as reduced levels of ABA and jasmonic acid (JA). Second, PtoMYB99-OE lines accumulated more reactive oxygen species (ROS), including H2O2 and O2-, as well as malonaldehyde (MDA), proline, and soluble sugar under osmotic stress; conversely, the activity of antioxidant enzymes (SOD, POD, and CAT), was weakened in the PtoMYB99-OE lines. Third, the expression of ABA biosynthetic genes, PtoNCED3.1 and PtoNCED3.2, as well as JA biosynthetic genes, PtoOPR3.1 and PtoOPR3.2, was significantly reduced in the PtoMYB99-OE lines under both normal conditions and osmotic stress. Based on our results, we conclude that the overexpression of PtoMYB99 compromises tolerance to osmotic stress in poplar. These findings contribute to the understanding of the role of the MYB genes in drought stress and the biosynthesis of ABA and JA.

Simultaneous separation and analysis of multiple doxorubicin hydrochloride liposomes forms in serum by circular nonuniform electric field gel electrophoresis.

BACKGROUND: Liposomal formulations have traditionally been considered the most therapeutically effective drug delivery systems (DDS). However, their pharmacokinetics study and efficacy assessment are still challenging given size heterogeneity and unknown forms in vivo. The pharmacodynamic evaluation that solely analyzes total drug concentration is unfit for the liposomal formulation study. Hence, it is crucial to develop effective strategies for the separation and analysis of different forms of liposomal formulations in order to contribute to the study of pharmacokinetic profiles associated with both liposome-incorporated and non-liposomal drugs. (84) RESULTS: A laboratory-built circular nonuniform electric field gel electrophoresis (CNEFGE) system was developed in this study for simultaneous separation and analysis of various forms of doxorubicin hydrochloride (DOX•HCl) liposomes. Liposomes were effectively fractionized based on their size and higher concentration in situ in the concentration zone, obtaining liposome recovery >95 % and a 3.04 concentration factor. It was found that the technique could be used to evaluate not only the size distribution of liposomes but also the drug loading capacity related to size. The charge-to-size-based separation mechanism has also allowed the simultaneous separation of liposome-entrapped drugs, protein-bound drugs, and free drugs in various forms, and the technique has been successfully employed in serum. Moreover, the quantification analysis of liposomes incubated with serum for 72 h showed that the proportion of the ratio of DOX•HCl in liposome-entrapped drugs, protein-bound drugs, and free drugs is approximately 97:2:1. (143) SIGNIFICANCE: Using the separation principle of gel electrophoresis and the electrification characteristics of drug carriers, this study developed and implemented an efficient approach for the simultaneous separation and concentration of multiple forms of drug liposomes in vivo. This approach offers a wide range of applications in the pharmacokinetics, efficacy, and safety evaluation of drug carriers and liposomes. (56).

Oxidized sodium alginate hydrogel-mouse nerve growth factor sustained release system promotes repair of peripheral nerve injury.

In recent years, mouse nerve growth factor (mNGF) has emerged as an important biological regulator to repair peripheral nerve injury, but its systemic application is restricted by low efficiency and large dosage requirement. These limitations prompted us to search for biomaterials that can be locally loaded. Oxidized sodium alginate hydrogel (OSA) exhibits good biocompatibility and physicochemical properties, and can be loaded with drugs to construct a sustained-release system that can act locally on nerve injury. Here, we constructed a sustained-release system of OSA-mouse nerve growth factor (mNGF), and investigated the loading and release of the drug through Fourier transform infrared spectroscopy and drug release curves. In vitro and in vivo experiments showed that OSA-mNGF significantly promoted the biological activities of RSC-96 cells and facilitated the recovery from sciatic nerve crush injury in rats. This observation may be attributed to the additive effect of OSA on promoting Schwann cell biological activities or its synergistic effect of cross-activating phosphoinositide 3-kinase (PI3K) through extracellular signal regulated kinase (ERK) signaling. Although the specific mechanism of OSA action needs to be explored in the future, the current results provide a valuable preliminary research basis for the clinical application of the OSA-mNGF sustained-release system for nerve repair.

Capillary suction under unsaturated condition drives strong specific affinity of ions at the surface of clay mineral.

Mineral-solution interface is of great importance in many soil and geochemical processes as well as industrial applications. Most relevant studies were based on saturated condition and given the corresponding theory, model, and mechanism. However, soils are usually in the non-saturation with different capillary suction. Our study presents substantially different scenery for ions interacting with mineral surface under unsaturated condition using molecular dynamics method. Under partially hydrated state, both cations (Ca2+) and anions (Cl-) can be adsorbed as outer-sphere complexes at the montmorillonite surface, and the number significantly increased with the increase of unsaturated degree. Ions preferred to interact with clay mineral instead of water molecules under unsaturated state, and the mobility of both cations and anions substantially decreased with the increase of capillary suction as reflected by the diffusion coefficient analysis. Potential of mean force calculations further clearly revealed that the adsorption strength of both Ca2+ and Cl- increased with capillary suction. Such an increase was more obvious for Cl- compared to Ca2+, despite the adsorption strength of Cl- was much weaker than Ca2+ at a certain capillary suction. Therefore, it is the capillary suction under unsaturated condition that drives the strong specific affinity of ions at the surface of clay mineral, which was tightly related to the steric effect of confined water film, the destruction of EDL structure, and the cation-anion pair interaction. This suggests that our common understanding of mineral-solution interaction should be largely improved.

Genetic liability between COVID-19 and pre-eclampsia/eclampsia: a Mendelian randomization study.

OBJECTIVE: The aim of this study was to investigate the possible causal relationship between COVID-19 and the risk of pre-eclampsia/eclampsia using a Mendelian randomized (MR) design. METHODS: We estimated their genetic correlations and then performed two-sample Mendelian randomization analyses using pooled statistics from the COVID-19 susceptibility/hospitalization genome-wide association study and the pre-eclampsia/eclampsia datasets. The main analyses were performed using the inverse variance weighting method, supplemented by the weighted median method and the MR-Egger method. RESULTS: We identified a significant and positive genetic correlation between COVID-19 susceptibility and pre-eclampsia/eclampsia [OR = 1.23 (1.01-1.51), p = 0.043]. Meanwhile, hospitalization of COVID-19 was significantly associated with a higher risk of pre-eclampsia/eclampsia [OR = 1.15 (1.02-1.30), p = 0.024]. Consistently, hospitalization of COVID-19 were nominally associated with higher risk of pre-eclampsia [OR = 1.14, (1.01-1.30), p = 0.040]. The results were robust under all sensitivity analyses. CONCLUSION: These results suggest that COVID-19 may increase the risk of pre-eclampsia/eclampsia. Future development of preventive or therapeutic interventions should emphasize this to mitigate the complications of COVID-19. [Figure: see text].

Fabrication and characterization of l-ascorbyl palmitate and phospholipid-based hybrid liposomes and their impacts on the stability of loaded hydrophobic polyphenols.

The aim of this study was to evaluate the influence of l-ascorbyl palmitate (LAP) as an additive to liposome formulations by self-assembling with soy lecithin to form hybrid liposomes, in order to enhance the physical stability and bioactivator-loaded retention ratio of the LAP incorporated liposomes (LAP-LP). The addition of LAP significantly increased its surface negative charge and strong hydrophobic interactions occurred between the hydrophobic tails of LAP and phospholipids resulting in more compactly ordered, rigid and hydrophobic phospholipid bilayers as indicated by surface tension, fluorescence probes and DSC. These changes enhanced the stability of hydrophobic polyphenol loaded LAP-LP during storage. Particularly, after four weeks storage at 37 °C for naringenin loaded liposomes, the retention ratio of pure liposome decreased dramatically to 12.5 %, while the LAP-LP remained above 74.5 %. This study opens up the potential for the LAP-LP to be developed as a food-grade multifunctional formulation for encapsulating and delivering bioactivators.

Highly connected 3D chromatin networks established by an oncogenic fusion protein shape tumor cell identity.

Cell fate transitions observed in embryonic development involve changes in three-dimensional genomic organization that provide proper lineage specification. Whether similar events occur within tumor cells and contribute to cancer evolution remains largely unexplored. We modeled this process in the pediatric cancer Ewing sarcoma and investigated high-resolution looping and large-scale nuclear conformation changes associated with the oncogenic fusion protein EWS-FLI1. We show that chromatin interactions in tumor cells are dominated by highly connected looping hubs centered on EWS-FLI1 binding sites, which directly control the activity of linked enhancers and promoters to establish oncogenic expression programs. Conversely, EWS-FLI1 depletion led to the disassembly of these looping networks and a widespread nuclear reorganization through the establishment of new looping patterns and large-scale compartment configuration matching those observed in mesenchymal stem cells, a candidate Ewing sarcoma progenitor. Our data demonstrate that major architectural features of nuclear organization in cancer cells can depend on single oncogenes and are readily reversed to reestablish latent differentiation programs.

EWSR1-ATF1 dependent 3D connectivity regulates oncogenic and differentiation programs in Clear Cell Sarcoma.

Oncogenic fusion proteins generated by chromosomal translocations play major roles in cancer. Among them, fusions between EWSR1 and transcription factors generate oncogenes with powerful chromatin regulatory activities, capable of establishing complex gene expression programs in permissive precursor cells. Here we define the epigenetic and 3D connectivity landscape of Clear Cell Sarcoma, an aggressive cancer driven by the EWSR1-ATF1 fusion gene. We find that EWSR1-ATF1 displays a distinct DNA binding pattern that requires the EWSR1 domain and promotes ATF1 retargeting to new distal sites, leading to chromatin activation and the establishment of a 3D network that controls oncogenic and differentiation signatures observed in primary CCS tumors. Conversely, EWSR1-ATF1 depletion results in a marked reconfiguration of 3D connectivity, including the emergence of regulatory circuits that promote neural crest-related developmental programs. Taken together, our study elucidates the epigenetic mechanisms utilized by EWSR1-ATF1 to establish regulatory networks in CCS, and points to precursor cells in the neural crest lineage as candidate cells of origin for these tumors.

Injectable Hydrogel Based on Modified Gelatin and Sodium Alginate for Soft-Tissue Adhesive.

To assist or replace the traditional suture techniques for wound closure, soft-tissue adhesives with excellent adhesion strength and favorable biocompatibility are of great significance in biomedical applications. In this study, an injectable hydrogel tissue adhesive containing adipic acid dihydrazide-modified gelatin (Gel-ADH) and oxidized sodium alginate (OSA) was developed. It was found that this tissue adhesive possessed a uniform structure, appropriate swelling ratio, good injectability, and excellent hemocompatibility and cytocompatibility. The adhesion capacity of the developed adhesive with optimized component and concentration was stronger than that of the commercial adhesive Porcine Fibrin Sealant Kit. All these results suggested that the developed hydrogel was a promising candidate for a soft-tissue adhesive.