Systematic analysis of alternative exon-dependent interactome remodeling reveals multitasking functions of gene regulatory factors.

Alternative splicing significantly expands biological complexity, particularly in the vertebrate nervous system. Increasing evidence indicates that developmental and tissue-dependent alternative exons often control protein-protein interactions; yet, only a minor fraction of these events have been characterized. Using affinity purification-mass spectrometry (AP-MS), we show that approximately 60% of analyzed neural-differential exons in proteins previously implicated in transcriptional regulation result in the gain or loss of interaction partners, which in some cases form unexpected links with coupled processes. Notably, a neural exon in Chtop regulates its interaction with the Prmt1 methyltransferase and DExD-Box helicases Ddx39b/a, affecting its methylation and activity in promoting RNA export. Additionally, a neural exon in Sap30bp affects interactions with RNA processing factors, modulating a critical function of Sap30bp in promoting the splicing of <100 nt "mini-introns" that control nuclear RNA levels. AP-MS is thus a powerful approach for elucidating the multifaceted functions of proteins imparted by context-dependent alternative exons.

Systematic mapping of nuclear domain-associated transcripts reveals speckles and lamina as hubs of functionally distinct retained introns.

The nucleus is highly compartmentalized through the formation of distinct classes of membraneless domains. However, the composition and function of many of these structures are not well understood. Using APEX2-mediated proximity labeling and RNA sequencing, we surveyed human transcripts associated with nuclear speckles, several additional domains, and the lamina. Remarkably, speckles and lamina are associated with distinct classes of retained introns enriched in genes that function in RNA processing, translation, and the cell cycle, among other processes. In contrast to the lamina-proximal introns, retained introns associated with speckles are relatively short, GC-rich, and enriched for functional sites of RNA-binding proteins that are concentrated in these domains. They are also highly differentially regulated across diverse cellular contexts, including the cell cycle. Thus, our study provides a resource of nuclear domain-associated transcripts and further reveals speckles and lamina as hubs of distinct populations of retained introns linked to gene regulation and cell cycle progression.

Genome-wide CRISPR-Cas9 Interrogation of Splicing Networks Reveals a Mechanism for Recognition of Autism-Misregulated Neuronal Microexons.

Alternative splicing is crucial for diverse cellular, developmental, and pathological processes. However, the full networks of factors that control individual splicing events are not known. Here, we describe a CRISPR-based strategy for the genome-wide elucidation of pathways that control splicing and apply it to microexons with important functions in nervous system development and that are commonly misregulated in autism. Approximately 200 genes associated with functionally diverse regulatory layers and enriched in genetic links to autism control neuronal microexons. Remarkably, the widely expressed RNA binding proteins Srsf11 and Rnps1 directly, preferentially, and frequently co-activate these microexons. These factors form critical interactions with the neuronal splicing regulator Srrm4 and a bi-partite intronic splicing enhancer element to promote spliceosome formation. Our study thus presents a versatile system for the identification of entire splicing regulatory pathways and further reveals a common mechanism for the definition of neuronal microexons that is disrupted in autism.

Misregulation of an Activity-Dependent Splicing Network as a Common Mechanism Underlying Autism Spectrum Disorders.

A key challenge in understanding and ultimately treating autism is to identify common molecular mechanisms underlying this genetically heterogeneous disorder. Transcriptomic profiling of autistic brains has revealed correlated misregulation of the neuronal splicing regulator nSR100/SRRM4 and its target microexon splicing program in more than one-third of analyzed individuals. To investigate whether nSR100 misregulation is causally linked to autism, we generated mutant mice with reduced levels of this protein and its target splicing program. Remarkably, these mice display multiple autistic-like features, including altered social behaviors, synaptic density, and signaling. Moreover, increased neuronal activity, which is often associated with autism, results in a rapid decrease in nSR100 and splicing of microexons that significantly overlap those misregulated in autistic brains. Collectively, our results provide evidence that misregulation of an nSR100-dependent splicing network controlled by changes in neuronal activity is causally linked to a substantial fraction of autism cases.

Icetexane diterpenoids as Cav3.2 T-type calcium channel inhibitors from Salvia prattii and analgesic effect of their Semi-synthesized derivatives.

Ten new icetexane diterpenoids, salpratins E-N (1-10) and a known analogue (11) were characterized from Salvia prattii Hemsl. Structurally, 1 is the first 19(4 â†’ 3)-abeo-icetexane diterpenoid featuring with a 6/7/6 ring system. The structures of isolated compounds were determined by comprehensive analyses of spectroscopic data, ECD calculation, and single-crystal X-ray diffraction. Biological studies initially revealed that 1, 7, 10, and 11 are notable Cav3.2 T-type Ca2+ channel (TTCC) inhibitors with IC50 values of 2.9, 5.1, 2.3, and 3.2 µM, respectively. Five icetexane related derivatives (13-17) were synthesized from an abietane type precursor, (+)-carnosic acid (12), for the purpose of overcoming the poor water solubility of aforementioned active compounds and further investigating diverse diterpenes with valuable activity. Among them, 13 and 14 showed potent inhibitions on Cav3.2, having IC50 values of 6.7 and 2.4 µM, respectively. Significantly, they exhibited dose-dependent (1, 3, and 10 mg/kg) and comparable analgesic effects as that of Z944, a TTCCs inhibitor under clinical trial for pain management, in the mouse acetic acid writhing test. These findings further enrich structural diversity and bioactivity of Salvia diterpenoids, as well as provide promising structural templates for the development of Cav3.2 analgesics.

Sliding mode control with self-adaptive parameters of a 5-DOF hybrid robot.

Due to the advantages of high stiffness, high precision, high load capacity and large workspace, hybrid robots are applicable to drilling and milling of complicated components with large sizes, for instance car panels. However, the difficulty in establishing an exact dynamic model and external disturbances affect the high accuracy control directly, which will decrease the machining accuracy and thereby affect the machining quality and efficiency of the system. Sliding mode control is an effective approach for high-order nonlinear dynamic systems since that it is very insensitive to disturbances and parameter variations. However, chattering may exist in traditional sliding mode control with fixed parameters, which results from a constant approaching speed. Besides, the approaching speed will affect the chattering strength directly. To solve these problems, a modified sliding mode controller with self-adaptive parameters is proposed to enhance the trajectory-tracking performance of a 5-degree-of-freedom hybrid robot. Firstly, the kinematic model of the robot is established. Then adopting the principle of virtual work, a rigid dynamic model of the robot is built. Based on the built dynamic model, a modified sliding mode control method is developed, of which the approaching speed is dependent on the system state. Finally, the sliding mode controller with self-adaptive parameters is created for a hybrid robot. The proposed sliding mode controller can achieve a rapid approaching speed and suppress chattering simultaneously. Simulation results demonstrate that the proposed modified sliding mode controller can achieve a comparatively accurate and smooth trajectory, which owns good robustness to external disturbances.

Quantitative Risk Assessment of an Oil-Gas-Hydrogen-Electricity Integrated Energy Station in China.

This paper presents a quantitative risk assessment (QRA) study of an oil-gas-hydrogen-electricity integrated energy station in China. A comprehensive assessment of the station was made in terms of consequences and risks, respectively. Consequence analysis shows that the severity of hazardous chemical leakage accidents in the station is in the order of natural gas, hydrogen, gasoline, and diesel fuel, especially in the liquefied natural gas (LNG) and compressed natural gas (CNG) storage tank areas, which may cause major accidents in the event of leakage. The results of the risk assessment showed that the individual and societal risks of the integrated oil-gas-hydrogen-electricity energy station exceeded the risk acceptance criteria. Moreover, the catastrophic rupture of LNG and CNG storage tanks is the main cause of the unacceptable risk. Additional mitigation measures for them, mainly including the installation of emergency manual shut-off valves, the installation of combustible gas detection and alarm devices, and pressure relief protection devices, can effectively reduce the risk to an acceptable level.

High-efficiency leaching process for selective leaching of lithium from spent lithium iron phosphate.

With the arrival of the scrapping wave of lithium iron phosphate (LiFePO4) batteries, a green and effective solution for recycling these waste batteries is urgently required. Reasonable recycling of spent LiFePO4 (SLFP) batteries is critical for resource recovery and environmental preservation. In this study, mild and efficient, highly selective leaching of lithium from spent lithium iron phosphate was achieved using potassium pyrosulfate (K2S2O7) and hydrogen peroxide (H2O2) as leaching agents. The leaching rates of lithium and iron were 99.83 % and 0.34 %, respectively, at the optimal leaching conditions of 4 vol% 30 wt% H2O2, 0.08 mol/L K2S2O7, 25℃, 5 min, and a solid-liquid ratio of 20 g/L. Meanwhile, the mechanism of the leaching process was explored by thermodynamic, XRD, XPS, FTIR, and SEM analyses. The leaching solution was concentrated and purified, with the addition of potassium carbonate (K2CO3) to convert lithium into lithium carbonate (Li2CO3). A small amount of sulfuric acid (H2SO4) is added to the saline wastewater after precipitation, which can be converted into a leaching agent for recycling after heat treatment. This study provides a sustainable green process for the recovery of lithium iron phosphate and a new idea for resource recovery.

Recruitment of the m6A/m6Am demethylase FTO to target RNAs by the telomeric zinc finger protein ZBTB48.

BACKGROUND: N6-methyladenosine (m6A), the most abundant internal modification on eukaryotic mRNA, and N6, 2'-O-dimethyladenosine (m6Am), are epitranscriptomic marks that function in multiple aspects of posttranscriptional regulation. Fat mass and obesity-associated protein (FTO) can remove both m6A and m6Am; however, little is known about how FTO achieves its substrate selectivity. RESULTS: Here, we demonstrate that ZBTB48, a C2H2-zinc finger protein that functions in telomere maintenance, associates with FTO and binds both mRNA and the telomere-associated regulatory RNA TERRA to regulate the functional interactions of FTO with target transcripts. Specifically, depletion of ZBTB48 affects targeting of FTO to sites of m6A/m6Am modification, changes cellular m6A/m6Am levels and, consequently, alters decay rates of target RNAs. ZBTB48 ablation also accelerates growth of HCT-116 colorectal cancer cells and modulates FTO-dependent regulation of Metastasis-associated protein 1 (MTA1) transcripts by controlling the binding to MTA1 mRNA of the m6A reader IGF2BP2. CONCLUSIONS: Our findings thus uncover a previously unknown mechanism of posttranscriptional regulation in which ZBTB48 co-ordinates RNA-binding of the m6A/m6Am demethylase FTO to control expression of its target RNAs.

High-throughput sensitive screening of small molecule modulators of microexon alternative splicing using dual Nano and Firefly luciferase reporters.

Disruption of alternative splicing frequently causes or contributes to human diseases and disorders. Consequently, there is a need for efficient and sensitive reporter assays capable of screening chemical libraries for compounds with efficacy in modulating important splicing events. Here, we describe a screening workflow employing dual Nano and Firefly luciferase alternative splicing reporters that affords efficient, sensitive, and linear detection of small molecule responses. Applying this system to a screen of ~95,000 small molecules identified compounds that stimulate or repress the splicing of neuronal microexons, a class of alternative exons often disrupted in autism and activated in neuroendocrine cancers. One of these compounds rescues the splicing of several analyzed microexons in the cerebral cortex of an autism mouse model haploinsufficient for Srrm4, a major activator of brain microexons. We thus describe a broadly applicable high-throughput screening system for identifying candidate splicing therapeutics, and a resource of small molecule modulators of microexons with potential for further development in correcting aberrant splicing patterns linked to human disorders and disease.

Pathogenesis diagnosis of a pediatric patient suffering from multi-organ abscesses.

A pediatric patient suffered from renal and hepatic abscesses (through hepatorenal space) and recovered by antibiotics and other therapies. By clinical analysis, the multi-organ abscesses might be caused by bloodstream-disseminated infection. In order to identify the pathogen, we collected kidney biopsy tissue, swabs, and plasma samples, and used metagenomics next-generation sequencing (mNGS) and some traditional methods. The results revealed that polymicrobial especially anaerobic bacteremia (Bacteroides fragilis, et al.) contributed to the abscess formation. What is more, systematic human adenovirus C (HAdV-C) infection was shown, and the virus was isolated. The titer of HAdV-2 neutralizing antibodies was 1/4 in the plasma after symptoms onset. Although the exact mechanism of HAdV-2 infection in multiple abscess formation has not been clarified, the case of multi-organ abscesses in the context of polymicrobial especially anaerobic bacteremia and HAdV infection in healthy children is infrequent.

Profiling of SARS-CoV-2 neutralizing antibody-associated antigenic peptides signature using proteome microarray.

The profile of antibodies against antigenic epitopes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during neutralizing antibody (NAb) decay has not been clarified. Using a SARS-CoV-2 proteome microarray that contained viral antigenic peptides, we analyzed the characteristics of the humoral response in patients with coronavirus disease 19 (COVID-19) in a longitudinal study. A total of 89 patients were recruited, and 226 plasma samples were serially collected in 2020. In the antigenic peptide microarray, the level of immunoglobulin G (IgG) antibodies against peptides within the S2 subunit (S-82) and a conserved gene region in variants of interest, open reading frame protein 10 (ORF10-3), were closely associated with the presence of SARS-CoV-2 NAbs. In an independent evaluation cohort of 232 plasma samples collected from 116 COVID-19 cases in 2020, S82-IgG titers were higher in NAbs-positive samples (p = 0.002) than in NAbs-negative samples using enzyme-linked immunosorbent assay. We further collected 66 plasma samples from another cohort infected by Omicron BA.1 virus in 2022. Compared with the samples with lower S82-IgG titers, NAb titers were significantly higher in the samples with higher S82-IgG titers (p = 0.04). Our findings provide insights into the understanding of the decay-associated signatures of SARS-CoV-2 NAbs.

Banxia Xiexin decoction alleviates AS co-depression disease by regulating the gut microbiome-lipid metabolic axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Banxia Xiexin decoction (BXD) is a classic Chinese herbal formulation consisting of 7 herbs including Pinelliae Rhizoma, Scutellariae Radix, Zingiberis Rhizoma, Ginseng Radix, Glycyrrhizae Radix, Coptidis Rhizoma, and Jujubae Fructus, which can exert effects on lowering lipids and alleviating depressive mood disorders via affecting gastrointestinal tract. AIM OF THE STUDY: The pathogenesis of atherosclerosis (AS) co-depression disease has not been well studied, and the current clinical treatment strategies are not satisfactory. As a result, it is critical to find novel methods of treatment. Based on the hypothesis that the gut microbiome may promote the development of AS co-depression disease by regulating host lipid metabolism, this study sought to evaluate the effectiveness and action mechanism of BXD in regulation of the gut microbiome via an intervention in AS co-depression mice. MATERIALS AND METHODS: To determine the primary constituents of BXD, UPLC-Q/TOF-MS analysis was carried out. Sixteen C56BL/6 mice were fed normal chow as a control group; 64 ApoE-/- mice were randomized into four groups (model group and three treatment groups) and fed high-fat chow combined with daily bind stimulation for sixteen weeks to develop the AS co-depression mouse model and were administered saline or low, medium or high concentrations of BXD during the experimental modeling period. The antidepressant efficacy of BXD was examined by weighing, a sucrose preference test, an open field test, and a tail suspension experiment. The effectiveness of BXD as an anti-AS treatment was evaluated by means of biochemical indices, the HE staining method, and the Oil red O staining method. The impacts of BXD on the gut microbiome structure and brain (hippocampus and prefrontal cortex tissue) lipids in mice with the AS co-depression model were examined by 16S rDNA sequencing combined with lipidomics analysis. RESULTS: The main components of BXD include baicalin, berberine, ginsenoside Rb1, and 18 other substances. BXD could improve depression-like behavioral characteristics and AS-related indices in AS co-depression mice; BXD could regulate the abundance of some flora (phylum level: reduced abundance of Proteobacteria and Deferribacteres; genus level: reduced abundance of Clostridium_IV, Helicobacter, and Pseudoflavonifractor, Acetatifactor, Oscillibacter, which were significantly different). The lipidomics analysis showed that the differential lipids between the model and gavaged high-dose BXD (BXH) groups were enriched in glycerophospholipid metabolism, and lysophosphatidylcholine (LPC(20:3)(rep)(rep)) in the hippocampus and LPC(20:4)(rep) in the prefrontal cortex both showed downregulation in BXH. The correlation analysis illustrated that the screened differential lipids were mainly linked to Deferribacteres and Actinobacteria. CONCLUSION: BXD may exert an anti-AS co-depression therapeutic effect by modulating the abundance of some flora and thus intervening in peripheral lipid and brain lipid metabolism (via downregulation of LPC levels).

Rapid Golden Gate assembly of exons from genomic DNA for protein expression in Escherichia coli and Pichia pastoris.

The development of a quick, single-step cloning system for generation of multiexon gene expression constructs is presented. The system allows efficient and cost-effective assembly of multiple exons of interest genes into different expression plasmids in both Escherichia coli and Pichia pastoris. The high cloning efficiency and low cost of the system make it ideal for a novel workflow for the assembly of intron-bearing genes for expression in two different expression hosts.

Discovery of novel coffee diterpenoids with inhibitions on Cav3.1 low voltage-gated Ca2+ channel.

Seven new (1-4, 6-8) diterpenoids with rare skeletons and seven known ones (9, 12, 17, 18 and 23-25) were isolated from roasted beans of Coffea arabica L. Together with previously obtained diterpenoids, a total of 26 molecules (1-25, 4a) were evaluated their activities on Cav3.1 low voltage-gated Ca2+ channel. Compounds 1, 3, 6, 7, 12, 13, 17, 19 and 24 exhibited noticeable Cav3.1 inhibitions (41.2%-96.1%) at 10 µM. The IC50 values of 1, 6, 7, 12, 13, 17 and 24 are 2.9, 2.3, 0.68, 14.8, 11.6, 6.1 and 6.8 µM, respectively. The ring moiety at C-18 and C-19, and esterification of OH-17 with long-chain fatty acids seem important for their activities. Further studies indicated that 1 and cafestol may act on different binding sits with the Cav3.1 blocker Z944, which is in clinical trial. Significantly, the present study initially shows that coffee diterpenoids are potential natural resources for Cav3.1 inhibitors.

Coupling analysis of a 5-degree-of-freedom hybrid manipulator based on a global index.

Parallel kinematic machines have been applied in aerospace and automotive manufacturing due to their potentials in high speed and high accuracy. However, there exists coupling in parallel kinematic machines, which makes dynamic analysis, rigidity enhancement, and control very complicated. In this article, coupling characteristics of a 5-degree-of-freedom (5-dof) hybrid manipulator are analyzed based on a local index and a global index. First, velocity analysis as well as acceleration analysis of the robot is conducted to provide essential information for dynamic modeling. Then the dynamic model is built based on the principle of virtual work. Whereas the mass matrix is off-diagonal, a local coupling index as well as a global index is defined, based on which coupling characteristics of the robot are analyzed. Results show that distributions of coupling indices are symmetric due to its structural features. And dimensional parameters, structural parameters, as well as mass parameters have a large influence on the system's coupling characteristics. Research conducted in the article is of great help in optimal design and control. Meanwhile, the method proposed in the article can be applied to other types of parallel kinematic machines or hybrid manipulators.