Search

Changji'an Formula (CJAF) is a Chinese herbal compound, which is effective against irritable bowel syndrome with predominant diarrhea (IBS-D) in clinic. However, the molecular mechanism has not been well defined. In the current study, the potential targets and signaling pathways of CJAF against IBS-D were predicted using network pharmacology analysis. The pharmacological mechanisms of CJAF against IBS-D and the potential mechanism were validated by using an IBS-D mouse model induced by enema with trinitrobenzene-sulfonic acid (TNBS) plus with restraint stress and further intervened with CJAF. A total of 232 active compounds of CJAF were obtained, a total of 397 potential targets for the active ingredients were retrieved and a total of 219 common targets were obtained as the potential targets of CJAF against IBS-D. GO and KEGG enrichment analyses showed that multiple targets were enriched and could be experimentally validated in a mouse model of IBS-D. The mechanisms were mainly converged on the immune and inflammatory pathways, especially the NF-κB, TNF and IL-17 signaling pathway, which were closely involved in the treatment of CJAF against IBS-D. Animal experiment showed that CJAF alleviated visceral hypersensitivity and diarrhea symptom of IBS-D. CJAF also restored the histological and ultrastructure damage of IBS-D. The result of Western blot showed that CJAF upregulated colonic tight junction proteins of ZO-1, Occludin and Claudin-1. Further results demonstrated that CJAF inhibited the protein expression of NF-κB/NLRP3 inflammasome pathway targets and downregulated proinflammatory mediators of IL-1β, IL-18, TNF-α. In conclusion, CJAF could effectively reduce inflammatory response and alleviate visceral hypersensitivity as well as diarrhea symptom of IBS-D by inhibiting the NF-κB/NLRP3 signaling pathway. This study not only reveals the mechanism of CJAF against IBS-D, but also provides a novel therapeutic strategy for IBS-D.

Summary: G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibits stress granule assembly and interacts with G3BP1/2 via an ITFG motif, including residue F17, in the N protein. Prior studies examining the impact of the G3PB1-N interaction on SARS-CoV-2 replication have produced inconsistent findings, and the role of this interaction in pathogenesis is unknown. Here, we use structural and biochemical analyses to define the residues required for G3BP1-N interaction and structure-guided mutagenesis to selectively disrupt this interaction. We find that N-F17A mutation causes highly specific loss of interaction with G3BP1/2. SARS-CoV-2 N-F17A fails to inhibit stress granule assembly in cells, has decreased viral replication, and causes decreased pathology in vivo. Further mechanistic studies indicate that the N-F17-mediated G3BP1-N interaction promotes infection by limiting sequestration of viral genomic RNA (gRNA) into stress granules.

The study presents a novel meshed horizontal–vertical (H–V) geogrid, offering promising advancements in geotechnical structure performance. The study pioneers a modeling approach for H–V geogrid foundation bearing capacity with discrete element method, expanding understanding and optimizing design strategy. By analyzing the granular displacement, contact force distribution, and vertical stress distribution within the foundation system, the study examines the impact of burial depth, vertical element height, and the number of vertical elements on H–V reinforced foundations. The findings suggest that employing a burial depth equivalent to the width of the footing enhances bearing capacity compared to conventional geogrid applications, with depths set at 0.4 times the width of the footing. This enhancement is attributed to forming a deeper slip surface in H–V systems. Moreover, raising vertical elements to 0.6 times the width of the footing enhances bearing capacity with minimal increase in geogrid usage, indicating a strategic approach to reinforcement. Increasing the number of vertical elements, particularly with three pairs, significantly enhances bearing capacity by reinforcing lateral restraint on the soil and promoting stress homogenization, thereby augmenting the “deep-footing” effect. The technical analysis underscores the efficacy of H–V geogrids in bolstering the bearing capacity of reinforced foundations, which is attributed to the robust grip and interlocking mechanism facilitated by these geogrids’ vertical ribs and mesh structure, which augment lateral confinement and diminish horizontal soil displacement.

Abstract Covalent organic frameworks (COFs) are one class of porous materials with permanent porosity and regular channels, and have a covalent bond structure. Due to their interesting characteristics, COFs have exhibited diverse potential applications in many fields. However, some applications require the frameworks to possess high structural stability, excellent crystallinity, and suitable pore size. COFs based on β-ketoenamine and imines are prepared through the irreversible enol-to-keto tautomerization. These materials have high crystallinity and exhibit high stability in boiling water, with strong resistance to acids and bases, resulting in various possible applications. In this review, we first summarize the preparation methods for COFs based on β-ketoenamine, in the form of powders, films and foams. Then, the effects of different synthetic methods on the crystallinity and pore structure of COFs based on β-ketoenamine are analyzed and compared. The relationship between structures and different applications including fluorescence sensors, energy storage, photocatalysis, electrocatalysis, batteries and proton conduction are carefully summarized. Finally, the potential applications, large-scale industrial preparation and challenges in the future are presented. Graphical Abstract

Melanoma is the most aggressive and difficult to treat of all skin cancers. Despite advances in the treatment of melanoma, the prognosis for melanoma patients remains poor, and the recurrence rate remains high. There is substantial evidence that Chinese herbals effectively prevent and treat melanoma. The bioactive ingredient Salvianolic acid B (SAB) found in Salvia miltiorrhiza, a well-known Chinese herbal with various biological functions, exhibits inhibitory activity against various cancers. A375 and mouse B16 cell lines were used to evaluate the main targets and mechanisms of SAB in inhibiting melanoma migration. Online bioinformatics analysis, Western blotting, immunofluorescence, molecular fishing, dot blot, and molecular docking assays were carried out to clarify the potential molecular mechanism. We found that SAB prevents the migration and invasion of melanoma cells by inhibiting the epithelial–mesenchymal transition (EMT) process of melanoma cells. As well as interacting directly with the N-terminal domain of β-actin, SAB enhanced its compactness and stability, thereby inhibiting the migration of cells. Taken together, SAB could significantly suppress the migration of melanoma cells via direct binding with β-actin, suggesting that SAB could be a helpful supplement that may enhance chemotherapeutic outcomes and benefit melanoma patients.

Colon cancer is a highly malignant cancer with poor prognosis. Astragalus membranaceus (Fisch.) Bunge (Huang Qi in Chinese, HQ), a well-known Chinese herbal medicine and a popular food additive, possesses various biological functions and has been frequently used for clinical treatment of colon cancer. However, the underlying mechanism is not fully understood. Isoflavonoids, including formononetin (FMNT) and calycosin (CS), are the main bioactive ingredients isolated from HQ. Thus, this study aimed to explore the inhibitory effects and mechanism of HQ, FMNT and CS against colon cancer by using network pharmacology coupled with experimental validation and molecular docking. The network pharmacology analysis revealed that FMNT and CS exerted their anticarcinogenic actions against colon cancer by regulating multiple signaling molecules and pathways, including MAPK and PI3K-Akt signaling pathways. The experimental validation data showed that HQ, FMNT and CS significantly suppressed the viability and proliferation, and promoted the apoptosis in colon cancer Caco2 and HT-29 cells. HQ, FMNT and CS also markedly inhibited the migration of Caco2 and HT-29 cells, accompanied by a marked increase in E-cadherin expression, and a notable decrease in N-cadherin and Vimentin expression. In addition, HQ, FMNT and CS strikingly decreased the expression of ERK1/2 phosphorylation (p-ERK1/2) without marked change in total ERK1/2 expression. They also slightly downregulated the p-Akt expression without significant alteration in total Akt expression. Pearson correlation analysis showed a significant positive correlation between the inactivation of ERK1/2 signaling pathway and the HQ, FMNT and CS-induced suppression of colon cancer. The molecular docking results indicated that FMNT and CS had a strong binding affinity for the key molecules of ERK1/2 signaling pathway. Conclusively, HQ, FMNT and CS exerted good therapeutic effects against colon cancer by mainly inhibiting the ERK1/2 signaling pathway, suggesting that HQ, FMNT and CS could be useful supplements that may enhance chemotherapeutic outcomes and benefit colon cancer patients.

Worldwide, cancer is a growing epidemic that results in large social and economic burdens. Despite advances in current diagnosis and treatment, most of the prognosis in cancer patients remains poor. It is urgent to find alternative therapies with effective cancer prevention and treatment. Chinese herbal medicines (CHMs) have been increasingly used worldwide for cancer prevention and treatment due to their privileged properties. CHMs are useful in the suppression of various types of cancers through different mechanisms of action. Non-coding RNAs (ncRNAs), including microRNAs, long non-coding RNAs and circular RNAs, are closely involved in the cancer progression and development. Regulation of ncRNAs in tumor cells may be a useful pharmacological strategy for the cancer prevention and treatment. Substantial evidence exists that various phytochemicals from CHMs exert potent anticarcinogenic effects by regulating ncRNAs-related targets and signaling pathways. Herein, the purpose of this paper is to conclude the current understanding of phytochemicals from CHMs in ncRNAs-mediated cancer suppression and the molecular mechanisms. This review will help to provide beneficial clues related to the clinical use of CHMs in the cancer prevention and treatment and further promote new drug discovery against cancer.

Ferroptosis, which has attracted much attention, is a new form of regulated cell death that is closely associated with the progression of various diseases. Ferroptosis is dependent upon intracellular iron and reactive oxygen species and is morphologically, biochemically, and genetically distinct from other nonapoptotic forms of cell death. Cumulative evidence shows that ferroptosis plays a pivotal role in the development and progression of tumorigenesis. Ferroptosis induction has become a potential effective strategy in cancer prevention and treatment in the clinic. Thus, it is worth identifying the active chemical compounds that induce ferroptosis for cancer suppression. Chinese herbal medicines (CHMs), which contain a host of bioactive phytochemicals that could target many signaling pathways, have been increasingly used worldwide due to their effects against various diseases. Compelling evidence has demonstrated that many phytochemicals from CHMs exert potent anticarcinogenic effects and have been widely used for the clinical prevention and treatment of a variety of cancers. Furthermore, growing evidence suggests that phytochemicals could exhibit potent inhibitory effects against different cancers by inducing ferroptosis through the regulation of a large number of ferroptosis-related signaling pathways and targets. Herein, we summarized the current understanding of phytochemicals from CHMs in ferroptosis-mediated cancer prevention and treatment and the molecular mechanisms. We also concluded with potential applications of emerging ferroptosis-related strategies against cancer. This study may expand our current knowledge about the mechanisms of phytochemicals from CHMs as anticancer agents, which could be used clinically to enhance the effect of future ferroptosis-inducing CHM therapies against cancers.

The integrated braking control system (IBC) has become one of the most popular brake-by-wire (BBW) solutions due to its compactness and versatility. Accurate monitoring of wheel cylinder pressure in real time is the basis for brake pressure control, and pressure estimation is a low-cost and reliable method. However, the IBC is an electromechanical hydraulic coupling system that has significant nonlinear behaviors; moreover, vehicle dynamics also have a critical impact on the accuracy of pressure estimation. To solve this problem, this paper proposes a novel adaptive extended Kalman filter (EKF) approach that combines a hydraulic model and a single-wheel model. This novel strategy has better estimation than the hydraulic model when the pressure is accurately estimated by the single-wheel model, while when the single-wheel model is not accurate, the EKF degrades to the hydraulic model. Finally, vehicle experimental data under high- and low-mu braking are collected. The pressure estimation error of the EKF is within 0.4 MPa in the low-mu road and 2 MPa in the high-mu road. It is proven that the proposed pressure estimation strategy is highly effective.

Abstract Background Horizontal gene transfer (HGT), which is affected by environmental pollution and climate change, promotes genetic communication, changing bacterial pathogenicity and drug resistance. However, few studies have been conducted on the effect of HGT on the high pathogenicity and drug resistance of the opportunistic pathogen Vibrio harveyi. Results V. harveyi 345 that was multidrug resistant and infected Epinephelus oanceolutus was isolated from a diseased organism in Shenzhen, Southern China, an important and contaminated aquaculture area. Analysis of the entire genome sequence predicted 5678 genes including 487 virulence genes contributing to bacterial pathogenesis and 25 antibiotic-resistance genes (ARGs) contributing to antimicrobial resistance. Five ARGs (tetm, tetb, qnrs, dfra17, and sul2) and one virulence gene (CU052_28670) on the pAQU-type plasmid p345–185, provided direct evidence for HGT. Comparative genome analysis of 31 V. harveyi strains indicated that 217 genes and 7 gene families, including a class C beta-lactamase gene, a virulence-associated protein D gene, and an OmpA family protein gene were specific to strain V. harveyi 345. These genes could contribute to HGT or be horizontally transferred from other bacteria to enhance the virulence or antibiotic resistance of 345. Mobile genetic elements in 71 genomic islands encoding virulence factors for three type III secretion proteins and 13 type VI secretion system proteins, and two incomplete prophage sequences were detected that could be HGT transfer tools. Evaluation of the complete genome of V. harveyi 345 and comparative genomics indicated genomic exchange, especially exchange of pathogenic genes and drug-resistance genes by HGT contributing to pathogenicity and drug resistance. Climate change and continued environmental deterioration are expected to accelerate the HGT of V. harveyi, increasing its pathogenicity and drug resistance. Conclusion This study provides timely information for further analysis of V. harveyi pathogenesis and antimicrobial resistance and developing pollution control measurements for coastal areas.

Abstract Background Lung cancer remains the most common cause of cancer-related deaths, with a high incidence and mortality in both sexes worldwide. Chemoprevention has been the most effective strategy for lung cancer prevention. Thus, exploring novel and effective candidate agents with low toxicity for chemoprevention is essential and urgent. Houttuynia cordata Thunb. (Saururaceae) (H. cordata), which is a widely used herbal medicine and is also popularly consumed as a healthy vegetable, exhibits anti-inflammatory, antioxidant and antitumor activity. However, the chemopreventive effect of H. cordata against benzo(a)pyrene (B[a]P)-initiated lung tumorigenesis and the underlying mechanism remain unclear. Methods A B[a]P-stimulated lung adenocarcinoma animal model in A/J mice in vivo and a normal lung cell model (BEAS.2B) in vitro were established to investigate the chemopreventive effects of H. cordata and its bioactive compound 2-undecanone against lung tumorigenesis and to clarify the underlying mechanisms. Results H. cordata and 2-undecanone significantly suppressed B[a]P-induced lung tumorigenesis without causing obvious systemic toxicity in mice in vivo. Moreover, H. cordata and 2-undecanone effectively decreased B[a]P-induced intracellular reactive oxygen species (ROS) overproduction and further notably protected BEAS.2B cells from B[a]P-induced DNA damage and inflammation by significantly inhibiting phosphorylated H2A.X overexpression and interleukin-1β secretion. In addition, H. cordata and 2-undecanone markedly activated the Nrf2 pathway to induce the expression of the antioxidative enzymes heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO-1). Nrf2 silencing by transfection with Nrf2 siRNA markedly decreased the expression of HO-1 and NQO-1 to diminish the reductions in B[a]P-induced ROS overproduction, DNA damage and inflammation mediated by H. cordata and 2-undecanone. Conclusions H. cordata and 2-undecanone could effectively activate the Nrf2-HO-1/NQO-1 signaling pathway to counteract intracellular ROS generation, thereby attenuating DNA damage and inflammation induced by B[a]P stimulation and playing a role in the chemoprevention of B[a]P-induced lung tumorigenesis. These findings provide new insight into the pharmacological action of H. cordata and indicate that H. cordata is a novel candidate agent for the chemoprevention of lung cancer.

Formononetin (FMNT) is a major bioactive compound from Astragalus membranaceus (Fisch.) Bunge, and has been widely used to treat conditions related to vascular insufficiency. However, the molecular mechanism for the therapeutic effect has not been well defined. This study aimed to investigate the effect and mechanism of FMNT on endothelial function. The potential targets and signaling pathways of FMNT in the setting of ischemia were predicted using network pharmacology analysis. Human umbilical vein endothelial cells (HUVECs) were used for the in vitro studies and C57BL/6 mice were used for in vivo experiments. The results of the network pharmacology analysis showed that multiple signaling molecules including MAPK and PI3K-Akt pathways could be involved in the pharmacological actions of FMNT against ischemic diseases. The experimental validation data showed that FMNT significantly promoted the growth, proliferation, migration and tube formation of HUVECs in association with activation of endothelial nitric oxide synthase (eNOS) and promotion of intracellular nitric oxide (NO) production. FMNT also markedly activated Erk1/2 and Akt signaling in HUVECs. The enhanced endothelial function by FMNT was abolished when the cells were pre-treated with eNOS inhibitor. FMNT-induced eNOS/NO activation, endothelial function and angiogenesis was also effectively attenuated when Erk1/2 or Akt signaling pathway was inhibited. In addition, FMNT significantly promoted wound healing in C57BL/6 mice associated with activation of Erk1/2 and Akt signaling. Enhanced wound healing by FMNT in mice was prevented when eNOS-, Erk1/2, or Akt-medicated signaling was inhibited. Moreover, when Akt signaling was inhibited in HUVECs, FMNT was still able to activate Erk1/2 signaling without promotion of endothelial function. Similarly, FMNT could activate Akt signaling with no change in endothelial function when Erk1/2 signaling was attenuated in HUVECs. Conclusively, the present study demonstrated that FMNT significantly enhanced endothelial function and promoted angiogenesis in vitro and in vivo through activating Erk1/2- and Akt-mediated eNOS/NO signaling pathway. The data also suggested that simultaneous activation of Erk1/2 and Akt signaling was required for FMNT-induced promotion of endothelial function. Results from the present study might provide support and evidence for the application of FMNT during the clinical treatment of conditions related to vascular insufficiency.

Hepatic fibrosis (HF) represents the excessive wound healing where an excess amount of connective tissues is formed within the liver, finally resulting in cirrhosis or even hepatocellular carcinoma (HCC). Therefore, it is significant to discover the efficient agents and components to treat HF, thus restraining the further progression of hepatopathy. Astragalus membranaceus (Fisch.) Bunge [also called Astragali Radix (AR)] is a famous herb in traditional Chinese medicine (TCM), which possesses a variety of biological activities and exerts good therapeutic effects in the treatment of HF. Flavonoids account for the major active ingredients related to the AR pharmacological effects. Total AR flavonoids have been proved to exert inhibitory effects on hepatic fibrosis. This study aimed to further undertake network pharmacology analysis coupled with experimental validation and molecular docking to investigate the effects and mechanism of multiple flavonoid components from AR against liver fibrosis. The results of the network pharmacology analysis showed that the flavonoids from AR exerted their pharmacological effects against liver fibrosis by modulating multiple targets and pathways. The experimental validation data showed that the flavonoids from AR were able to suppress transforming growth factor beta 1 (TGF-β1)-mediated activation of hepatic stellate cells (HSCs) and reduce extracellular matrix deposition in HSC-T6 cells via regulating the nuclear factor kappa B (NF-κB) signal transduction pathway. The results of the molecular docking study further showed that the flavonoids had a strong binding affinity for IκB kinase (IKKβ) after docking into the crystal structure. The above results indicated that, flavonoids possibly exerted the anti-inflammatory effect on treating HF by mediating inflammatory signaling pathways. The potential mechanism of these flavonoids against liver fibrosis may be related to suppression of the NF-κB pathway through effective inhibition of IKKβ. This study not only provides a scientific basis for clarifying the effects and mechanism of AR flavonoids against liver fibrosis but also suggests a novel promising therapeutic strategy for the treatment of liver fibrosis.

A resource allocation algorithm was proposed for downlink C-RAN secure communication system.This algorithm jointly optimized the parameters of base station (BS) mode,quantized noise and beamforming under the constraints of SINR requirements of Internet of things information receiver,eavesdropped receiver and fronthaul link capacity.Such a design was a non-convex optimization problem,and a step-by step optimization algorithm was proposed to decompose the original optimization problem efficiently.Semi-definite technique and function smoothing approach were adopted,and the local optimization solution of the original problem was obtained by the iterative different of convex method.Bi-section approach was further proposed to determine BS mode,and the transmitting power of the BSs were finally optimized.Simulation results indicate that the proposed joint optimization algorithm achieves better performance than group sparse optimization algorithm and baseline algorithm,and it has approximate optimal performance as the exhaustive search algorithm.

Small non-coding RNAs (sRNAs) are important modulators of gene expression and are involved in the pathogenesis and survival of prokaryotes. However, few studies have been conducted with Vibrio alginolyticus, which limits our ability to probe the regulation of virulence and environmental adaptation by sRNAs in this opportunistic pathogen. In this study, the sRNA candidate srvg23535 was identified in V. alginolyticus ZJ-T. The precise transcript end, secondary structure, and sequence conservation were determined. A srvg23535 null mutant was constructed and characterized by using Phenotype MicroArray (PM) technology. In silico target prediction was conducted by IntaRNA and TargetRNA2. Subsequently, a 107 nt transcript was validated with a sigma70 promoter at the 5′ end and a Rho-independent terminator at the 3′ end. The sRNA srvg23535 had four stem-loop structures and was conserved among Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio splendidus. Deletion of srvg23535 in V. alginolyticus ZJ-T led to a weaker utilization of D-mannose, D-melibiose, lactulose, and inosine as carbon sources but stronger utilization of L-cysteine as nitrogen source. Moreover, the srvg2353 mutant showed stronger resistance to osmotic stress but weaker resistance to pH stress. Additionally, a total of 22 common targets were identified and several were related to the observed phenotype of the mutant. This study indicated that the novel sRNA, srvg23535, is conserved and restricted to Vibrio spp., affecting the utilization of several carbon and nitrogen sources and the response to osmotic and pH stress. These results extend our understanding of sRNA regulation in V. alginolyticus and provide a significant resource for the further study of the precise target mRNAs of srvg23535, which may provide targets for antibacterial therapeutic or attenuated vaccines against Vibrio spp.

Raw Pinelliae Rhizoma (RPR) is a representative toxic herb that is widely used for eliminating phlegm or treating cough and vomiting. Given its irritant toxicity, its processed products, including Pinelliae Rhizoma Praeparatum (PRP) and Pinelliae Rhizoma Praeparatum cum Zingibere et Alumine (PRPZA), are more commonly applied and administered concomitantly with other chemical drugs, such as cough medications. This study aimed to investigate the effects of RPR, PRP, and PRPZA on CYP3A activity. Testosterone (Tes) and buspirone (BP) were used as specific probe substrates ex vivo and in vivo, respectively. CYP3A activity was determined by the metabolite formation ratios from the substrates. Ex vivo results show that the metabolite formation ratios from Tes significantly decreased, indicating that RPR, PRP, and PRPZA could inhibit CYP3A activity in rats. CYP3A protein and mRNA levels were determined to explore the underlying mechanism. These levels showed marked and consistent down-regulation with CYP3A activity. A significant decrease in metabolite formation ratios from BP was also found in PRPZA group in vivo, implying that PRPZA could inhibit CYP3A activity. Conclusively, co-administration of PR with other CYP3A-metabolizing drugs may cause drug–drug interactions. Clinical use of PR-related formulae should be monitored carefully to avoid adverse interactions.

Astragali radix (AR) is one of the most widely used traditional Chinese herbal medicines. Modern pharmacological studies and clinical practices indicate that AR possesses various biological functions, including potent immunomodulation, antioxidant, anti-inflammation and antitumor activities. To date, more than 200 chemical constituents have been isolated and identified from AR. Among them, isoflavonoids, saponins and polysaccharides are the three main types of beneficial compounds responsible for its pharmacological activities and therapeutic efficacy. After ingestion of AR, the metabolism and biotransformation of the bioactive compounds were extensive in vivo. The isoflavonoids and saponins and their metabolites are the major type of constituents absorbed in plasma. The bioavailability barrier (BB), which is mainly composed of efflux transporters and conjugating enzymes, is expected to have a significant impact on the bioavailability of AR. This review summarizes studies on the phytochemistry, pharmacology and pharmacokinetics on AR. Additionally, the use of AR as a personalized medicine based on the BB is also discussed, which may provide beneficial information to achieve a better and more accurate therapeutic response of AR in clinical practice.

Seven new triterpenoid saponins (1–7), together with three known ones (8–10), were isolated from Ilex pubescens. Elucidation of their structures was performed based on high-resolution electrospray ionisation mass spectrometry (HR-ESI-MS), infrared spectra (IR), and nuclear magnetic resonance (NMR) spectroscopic data. The anti-inflammatory activity of the isolates toward lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages was investigated. The results demonstrated that compounds 3, 5, and 6 inhibited the expression of inducible nitric oxide synthase (iNOS) protein in comparison with LPS stimulation in RAW264.7 cells.

Nuclear speckles are nuclear membraneless organelles in higher eukaryotic cells playing a vital role in gene expression. Using an in situ reverse transcription-based sequencing method, we study nuclear speckle-associated human transcripts. Our data indicate the existence of three gene groups whose transcripts demonstrate different speckle localization properties: stably enriched in nuclear speckles, transiently enriched in speckles at the pre-messenger RNA stage, and not enriched. We find that stably enriched transcripts contain inefficiently excised introns and that disruption of nuclear speckles specifically affects splicing of speckle-enriched transcripts. We further reveal RNA sequence features contributing to transcript speckle localization, indicating a tight interplay between transcript speckle enrichment, genome organization, and splicing efficiency. Collectively, our data highlight a role of nuclear speckles in both co-and posttranscriptional splicing regulation. Last, we show that genes with stably enriched transcripts are over-represented among genes with heat shock-up-regulated intron retention, hinting at a connection between speckle localization and cellular stress response. [ABSTRACT FROM AUTHOR]