Finite Element Simulation and Microstructural Analysis of Roll Forming for DP590 High-Strength Dual-Phase Steel Wheel Rims.

In this study, finite element (FE) simulation by the software Abaqus was relied on to investigate the roll forming process of a wheel rim made of an innovative dual-phase steel, i.e., DP590, after flash butt welding (FBW). In the simulation, an FE model was generated, including the design of the dies for flaring, three-roll forming, and expansion, and detailed key processing parameters based on practical production of the selected DP590. Combined with the microstructures and properties of the weld zone (WZ) and heat-affected zones (HAZs) after FBW, the distribution of stress/strain and the change in thickness of the base metal (BM), WZ and HAZs were analyzed, and compared in the important stages of roll forming. Theoretically, the variation in the microstructure and the corresponding stress-strain behaviors of the BM, WZ, and HAZs after FBW have led to the thickness reduction of DP590 that originated from softening behaviors occurring at the region of subcritical HAZs (SCHAZs), and a small amount of tempered martensite has evidently reduced the hardness and strength of the SCHAZ. Meanwhile, the distribution of stress/strain has been influenced to some extent. Further, the study includes the influence of the friction coefficient on the forming quality of the wheel rim to guarantee the simulation accuracy in practical applications. In sum, the dual-phase steel has to be carefully applied to the wheel rim, which needs to experience the processes of FBW and roll forming, focusing on the performance of SCHAZs.

Blockade of the mitochondrial DNA release ameliorates hepatic ischemia-reperfusion injury through avoiding the activation of cGAS-Sting pathway.

BACKGROUND: Liver surgery during the perioperative period often leads to a significant complication known as hepatic ischemia-reperfusion (I/R) injury. Hepatic I/R injury is linked to the innate immune response. The cGAS-STING pathway triggers the activation of innate immune through the detection of DNA within cells. Nevertheless, the precise mechanism and significance of the cGAS-STING pathway in hepatic I/R injury are yet to be investigated. METHODS: Mouse model of hepatic I/R injury was used in the C57BL/6 WT mice and the STING knockout (STING-KO) mice. In addition, purified primary hepatocytes were used to construct oxygen-glucose deprivation reperfusion (OGD-Rep) treatment models. RESULTS: Our research revealed a notable increase in mRNA and protein levels of cGAS and STING in liver during I/R injury. Interestingly, the lack of STING exhibited a safeguarding impact on hepatic I/R injury by suppressing the elevation of liver enzymes, liver cell death, and inflammation. Furthermore, pharmacological cGAS and STING inhibition recapitulated these phenomena. Macrophages play a crucial role in the activation of the cGAS-STING pathway during hepatic I/R injury. The cGAS-STING pathway experiences a significant decrease in activity and hepatic I/R injury is greatly diminished following the elimination of macrophages. Significantly, we demonstrate that the activation of the cGAS-STING pathway is primarily caused by the liberation of mitochondrial DNA (mtDNA) rather than nuclear DNA (nDNA). Moreover, the safeguarding of the liver against I/R injury is also attributed to the hindrance of mtDNA release through the utilization of inhibitors targeting mPTP and VDAC oligomerization. CONCLUSIONS: The results of our study suggest that the release of mtDNA plays a significant role in causing damage to liver by activating the cGAS-STING pathway during I/R injury. Furthermore, inhibiting the release of mtDNA can provide effective protection against hepatic I/R injury.

Niclosamide modulates phenotypic switch and inflammatory responses in human pulmonary arterial smooth muscle cells.

Excessive proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) represent key steps of pulmonary vascular remodeling, leading to the development of pulmonary arterial hypertension (PAH) and right ventricular failure. Niclosamide (NCL), an FDA-approved anthelmintic, has been shown to regulate cell proliferation, migration, invasion, and apoptosis through a variety of signaling pathways. However, its role on modulating the phenotypic switch and inflammatory responses in PASMCs remains unclear. In this study, cell proliferation assay showed that NCL inhibited PDGF-BB induced proliferation of human PASMCs in a dose-dependent manner. Western blot analysis further confirmed a notable reduction in the expression of cyclin D1 and PCNA proteins. Subsequently, flow cytometry analysis demonstrated that NCL induced an increased percentage of cells in the G1 phase while promoting apoptosis in PASMCs. Moreover, both scratch wound assay and transwell assay confirmed that NCL decreased PDGF-BB-induced migration of PASMCs. Mechanistically, western blot revealed that pretreatment of PASMCs with NCL markedly restored the protein levels of SMA, SM22, and calponin, while reducing phosphorylation of P38/STAT3 signaling in the presence of PDGF-BB. Interestingly, macrophages adhesion assay showed that NCL markedly reduced recruitment of Calcein-AM labeled RAW264.7 by TNFα-stimulated PASMCs. Western blot revealed that NCL suppressed TNFα-induced expression of both of VCAM-1 and ICAM-1 proteins. Furthermore, pretreatment of PASMCs with NCL significantly inhibited NLRP3 inflammasome activity through reducing NLRP3, AIM2, mature interleukin-1ß (IL-ß), and cleaved Caspase-1 proteins expression. Together, these results suggested versatile effects of NCL on controlling of proliferation, migration, and inflammatory responses in PASMCs through modulating different pathways, indicating that repurposing of NCL may emerge as a highly effective drug for PAH treatment.

Bio-gel nanoarchitectonics in tissue engineering.

Given the creation of materials based on nanoscale science, nanotechnology must be combined with other disciplines. This role is played by the new concept of nanoarchitectonics, the process of constructing functional materials from nanocomponents. Nanoarchitectonics may be highly compatible with applications in biological systems. Conversely, it would be meaningful to consider nanoarchitectonics research oriented toward biological applications with a focus on materials systems. Perhaps, hydrogels are promising as a model medium to realize nanoarchitectonics in biofunctional materials science. In this review, we will provide an overview of some of the defined targets, especially for tissue engineering. Specifically, we will discuss (i) hydrogel bio-inks for 3D bioprinting, (ii) dynamic hydrogels as an artificial extracellular matrix (ECM), and (iii) topographical hydrogels for tissue organization. Based on these backgrounds and conceptual evolution, the construction strategies and functions of bio-gel nanoarchitectonics in medical applications and tissue engineering will be discussed.

New Prospects Arising from Dynamically Crosslinked Polymers: Reprogramming Their Properties.

Dynamically crosslinked polymers (DCPs) have gained significant attention owing to their applications in fabricating (re)processable, recyclable, and self-healable thermosets, which hold great promise in addressing ecological issues, such as plastic pollution and resource scarcity. However, the current research predominantly focuses on redefining and/or manipulating their geometries while replicating their bulk properties. Given the inherent design flexibility of dynamic covalent networks, DCPs also exhibit a remarkable potential for various novel applications through postsynthesis reprogramming their properties. In this review, the recent advancements in strategies that enable DCPs to transform their bulk properties after synthesis are presented. The underlying mechanisms and associated material properties are overviewed mainly through three distinct strategies, namely latent catalysts, material-growth, and topology isomerizable networks. Furthermore, the mutual relationship and impact of these strategies when integrated within one material system are also discussed. Finally, the application prospects and relevant issues necessitating further investigation, along with the potential solutions are analyzed.

Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens using a dual fluorescence readthrough reporter.

Messenger RNA precursors (pre-mRNA) generally undergo 3' end processing by cleavage and polyadenylation (CPA), which is specified by a polyadenylation site (PAS) and adjacent RNA sequences and regulated by a large variety of core and auxiliary CPA factors. To date, most of the human CPA factors have been discovered through biochemical and proteomic studies. However, genetic identification of the human CPA factors has been hampered by the lack of a reliable genome-wide screening method. We describe here a dual fluorescence readthrough reporter system with a PAS inserted between two fluorescent reporters. This system enables measurement of the efficiency of 3' end processing in living cells. Using this system in combination with a human genome-wide CRISPR/Cas9 library, we conducted a screen for CPA factors. The screens identified most components of the known core CPA complexes and other known CPA factors. The screens also identified CCNK/CDK12 as a potential core CPA factor, and RPRD1B as a CPA factor that binds RNA and regulates the release of RNA polymerase II at the 3' ends of genes. Thus, this dual fluorescence reporter coupled with CRISPR/Cas9 screens reliably identifies bona fide CPA factors and provides a platform for investigating the requirements for CPA in various contexts.

Maternal PFOS exposure in mice induces hepatic lipid accumulation and inflammation in adult female offspring: Involvement of microbiome-gut-liver axis and autophagy.

Perfluorooctane sulfonates (PFOS) are the persistent organic pollutants. In the present study, 0, 0.3, or 3-mg/kg PFOS were administered to pregnant mice from GD 11 to GD 18. The histopathology of liver and intestine, serum and hepatic lipid levels, lipid metabolism related genes, and gut microbiota were examined in adult female offspring. The results suggested that maternal PFOS exposure increased serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and induced F4/80+ macrophage infiltration in adult female offspring, in addition to the elevation of TNF-α and IL-1ß mRNA levels in low-dose and high-dose groups, respectively. Furthermore, maternal exposure to PFOS increased serum triglyceride (TG) and hepatic total cholesterol (TC) levels, which was associated with the alteration of the process of fatty acid transport and ß-oxidation, TG synthesis and transport, cholesterol synthesis and excretion in the liver. The AMPK/mTOR/autophagy signaling was also inhibited in the liver of adult female offspring. Moreover, changes in gut microbiota were also related to lipid metabolism, especially for the Desulfovibrio, Ligilactobacillus, Enterorhabdus, HT002 and Peptococcaceae_unclassified. Additionally, maternal exposure to PFOS decreased mRNA expressions of the tight junction protein and AB+ goblet cells in the colon, while increasing the overproduction of lipopolysaccharides (LPS) and F4/80+ macrophage infiltration. Collectively, maternal PFOS exposure induced liver lipid accumulation and inflammation, which strongly correlated with the disruption of the gut-liver axis and autophagy in adult female offspring, highlighting the persistent adverse effects in offspring exposed to PFOS.

Molecular machines working at interfaces: physics, chemistry, evolution and nanoarchitectonics.

As a post-nanotechnology concept, nanoarchitectonics combines nanotechnology with advanced materials science. Molecular machines made by assembling molecular units and their organizational bodies are also products of nanoarchitectonics. They can be regarded as the smallest functional materials. Originally, studies on molecular machines analyzed the average properties of objects dispersed in solution by spectroscopic methods. Researchers' playgrounds partially shifted to solid interfaces, because high-resolution observation of molecular machines is usually done on solid interfaces under high vacuum and cryogenic conditions. Additionally, to ensure the practical applicability of molecular machines, operation under ambient conditions is necessary. The latter conditions are met in dynamic interfacial environments such as the surface of water at room temperature. According to these backgrounds, this review summarizes the trends of molecular machines that continue to evolve under the concept of nanoarchitectonics in interfacial environments. Some recent examples of molecular machines in solution are briefly introduced first, which is followed by an overview of studies of molecular machines and similar supramolecular structures in various interfacial environments. The interfacial environments are classified into (i) solid interfaces, (ii) liquid interfaces, and (iii) various material and biological interfaces. Molecular machines are expanding their activities from the static environment of a solid interface to the more dynamic environment of a liquid interface. Molecular machines change their field of activity while maintaining their basic functions and induce the accumulation of individual molecular machines into macroscopic physical properties molecular machines through macroscopic mechanical motions can be employed to control molecular machines. Moreover, research on molecular machines is not limited to solid and liquid interfaces; interfaces with living organisms are also crucial.

Diverse temporal and spatial mechanisms work, partially through Stanniocalcin-1, V-ATPase and senescence, to activate the extracellular ATP-mediated drug resistance in human cancer cells.

Introduction: Resistance to drug therapies is associated with a large majority of cancer-related deaths. ATP-binding cassette (ABC) transporter-mediated drug efflux, epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs), glutathione (GSH), senescence, and vacuole-type ATPase (V-ATPase) all contribute to the resistance. We recently showed that extracellular ATP (eATP) induces and regulates EMT, CSC formation, and ABC transporters in human cancer cells and tumors. eATP also consistently upregulates Stanniocalcin-1 (STC1), a gene that significantly contributes to EMT, CSC formation, and tumor growth. We also found that eATP enhances drug resistance in cancer cells through eATP internalization mediated by macropinocytosis, leading to an elevation of intracellular ATP (iATP) levels, induction of EMT, and CSC formation. However, these factors have never been systematically investigated in the context of eATP-induced drug resistance. Methods: In this study, we hypothesized that eATP increases drug resistance via inducing ABC efflux, EMT, CSCs, STC1, and their accompanied processes such as GSH reducing activity, senescence, and V-ATPase. RNA sequencing, metabolomics, gene knockdown and knockout, and functional assays were performed to investigate these pathways and processes. Results and discussion: Our study results showed that, in multiple human cancer lines, eATP induced genes involved in drug resistance, elevated ABC transporters' efflux activity of anticancer drugs; generated transcriptomic and metabolic profiles representing a drug resistant state; upregulated activities of GSH, senescence, and V-ATPase to promote drug resistance. Collectively, these newly found players shed light on the mechanisms of eATP-induced as well as STC1- and V-ATPase-mediated drug resistance and offer potential novel targets for combating drug resistance in cancers.

Layer-by-layer designer nanoarchitectonics for physical and chemical communications in functional materials.

Nanoarchitectonics, as a post-nanotechnology concept, constructs functional materials and structures using nanounits of atoms, molecules, and nanomaterials as materials. With the concept of nanoarchitectonics, asymmetric structures, and hierarchical organization, rather than mere assembly and organization of structures, can be produced, where rational physical and chemical communications will lead to the development of more advanced functional materials. Layer-by-layer assembly can be a powerful tool for this purpose, as exemplified in this feature paper. This feature article explores the possibility of constructing advanced functional systems based on recent examples of layer-by-layer assembly. We will illustrate both the development of more basic methods and more advanced nanoarchitectonics systems aiming towards practical applications. Specifically, the following sections will provide examples of (i) advancement in basics and methods, (ii) physico-chemical aspects and applications, (iii) bio-chemical aspects and applications, and (iv) bio-medical applications. It can be concluded that materials nanoarchitectonics based on layer-by-layer assembly is a useful method for assembling asymmetric structures and hierarchical organization, and is a powerful technique for developing functions through physical and chemical communication.

Nucleation During Storage Impeded Supersaturation in the Dissolution Process of Amorphous Celecoxib.

The aqueous solubility of active pharmaceutical ingredients (APIs) is one of the most critical factors in determining the absorption of orally administered drugs. Amorphization of API may offer better drug absorption than the crystalline state owing to enhanced solubility. However, if crystal nuclei are formed during storage, they may develop into crystals upon contact with water, thus limiting the dissolution advantage. In an earlier study, we found that the nuclei of amorphous celecoxib (CEL) could be formed at freezing temperatures (FT) without further crystal growth. Following this finding, we compared the dissolution performances of amorphous CEL annealed at room temperature (RT, 25 °C) or FT (-20 °C). We found that only the RT-annealed CEL could achieve a supersaturated state effectively during the dissolution process, which could be explained by the fast conversion of the FT-annealed amorphous CEL to a crystalline state owing to the presence of nuclei. Investigation of the residual solids revealed that supersaturation could be maintained for a while after the appearance of the crystals, which could be explained by heterogeneous nucleation and competition between the dissolution of amorphous parts and crystallization. In addition, a new crystalline form of CEL was observed during dissolution.

Physical Simulation and Numerical Simulation of Flash Butt Welding for Innovative Dual Phase Steel DP590: A Comparative Study.

In this study, the microstructure and performance of newly designed dual-phase steel (DP590) after joining by flash butt welding (FBW) for vehicle wheel rims was analysed and compared by two simulations, i.e., physical simulation and numerical simulation, due to the high acceptance of these two methodologies. Physical simulation is regarded as a thermal-mechanical solution conducted by the Gleeble 3500 simulator and which can distribute the heat-affected zone (HAZ) of the obtained weld joint into four typical HAZs. These are coarse-grained HAZ, fine-grained HAZ, inter-critical HAZ and sub-critical HAZ. A combination of ferrite and tempered martensite leads to the softening behaviour at the sub-critical HAZ of DP590, which is verified to be the weakest area, and influences the final performance due to ~9% reduction of hardness and tensile strength. The numerical simulation, relying on finite element method (FEM) analysis, can distinguish the temperature distribution, which helps us to understand the relationship between the temperature distribution and real microstructure/performance. Based on this study, the combination of physical and numerical simulations can be used to optimise the flash butt welding parameters (flash and butt processes) from the points of temperature distribution (varied areas), microstructure and performance, which are guidelines for the investigation of flash butt welding for innovative materials.

Manipulating the Dynamic Adaptivity of a Fluid Interface to Maintain the Multipotency of Mesenchymal Stromal Cells.

The native extracellular matrix is highly dynamic with continuous mutual feedback between cells being responsible for many important cell function regulators. However, establishing bidirectional interaction between complex adaptive microenvironments and cells remains elusive. Herein an adaptive biomaterial based on lysozyme monolayers self-assembled at a perfluorocarbon FC40-water interface is reported. The dynamic adaptivity of interfacially assembled protein nanosheets is modulated independently of bulk mechanical properties by covalent crosslinking. This provides a scenario to establish bidirectional interactions of cells with liquid interfaces of varying dynamic adaptivity. This is found that growth and multipotency of human mesenchymal stromal cells (hMSCs) are enhanced at the highly adaptive fluid interface. The multipotency retention of hMSCs is mediated by low cell contractility and metabolomic activity involving the continuous mutual feedback between the cells and materials. Consequently, an understanding of the cells' response to dynamic adaptivity has substantial implications for regenerative medicine and tissue engineering.

The HIF-1α/miR-26a-5p/PFKFB3/ULK1/2 axis regulates vascular remodeling in hypoxia-induced pulmonary hypertension by modulation of autophagy.

Pulmonary arterial hypertension (PAH) is a progressive and life-threatening disease characterized by pulmonary vascular remodeling, which may cause right heart failure and even death. Accumulated evidence confirmed that microRNA-26 family play critical roles in cardiovascular disease; however, their function in PAH remains largely unknown. Here, we investigated the expression of miR-26 family in plasma from PAH patients using quantitative RT-PCR, and identified miR-26a-5p as the most downregulated member, which was also decreased in hypoxia-induced pulmonary arterial smooth muscle cell (PASMC) autophagy models and lung tissues of PAH patients. Furthermore, chromatin immunoprecipitation (ChIP) analysis and luciferase reporter assays revealed that hypoxia-inducible factor 1α (HIF-1α) specifically interacted with the promoter of miR-26a-5p and inhibited its expression in PASMCs. Tandem mRFP-GFP-LC3B fluorescence microscopy demonstrated that miR-26a-5p inhibited hypoxia-induced PAMSC autophagy, characterized by reduced formation of autophagosomes and autolysosomes. In addition, results showed that miR-26a-5p overexpression potently inhibited PASMC proliferation and migration, as determined by cell counting kit-8, EdU staining, wound-healing, and transwell assays. Mechanistically, PFKFB3, ULK1, and ULK2 were direct targets of miR-26a-5p, as determined by dual-luciferase reporter gene assays and western blots. Meanwhile, PFKFB3 could further enhance the phosphorylation level of ULK1 and promote autophagy in PASMCs. Moreover, intratracheal administration of adeno-miR-26a-5p markedly alleviated right ventricular hypertrophy and pulmonary vascular remodeling in hypoxia-induced PAH rat models in vivo. Taken together, the HIF-1α/miR-26a-5p/PFKFB3/ULK1/2 axis plays critical roles in the regulation of hypoxia-induced PASMC autophagy and proliferation. MiR-26a-5p may represent as an attractive biomarker for the diagnosis and treatment of PAH.

Coelenterazine-Type Bioluminescence-Induced Optical Probes for Sensing and Controlling Biological Processes.

Bioluminescence-based probes have long been used to quantify and visualize biological processes in vitro and in vivo. Over the past years, we have witnessed the trend of bioluminescence-driven optogenetic systems. Typically, bioluminescence emitted from coelenterazine-type luciferin-luciferase reactions activate light-sensitive proteins, which induce downstream events. The development of coelenterazine-type bioluminescence-induced photosensory domain-based probes has been applied in the imaging, sensing, and control of cellular activities, signaling pathways, and synthetic genetic circuits in vitro and in vivo. This strategy can not only shed light on the mechanisms of diseases, but also promote interrelated therapy development. Here, this review provides an overview of these optical probes for sensing and controlling biological processes, highlights their applications and optimizations, and discusses the possible future directions.

An emerging master inducer and regulator for epithelial-mesenchymal transition and tumor metastasis: extracellular and intracellular ATP and its molecular functions and therapeutic potential.

Despite the rapid development of therapeutic strategies in cancer treatment, metastasis remains the major cause of cancer-related death and scientific challenge. Epithelial-Mesenchymal Transition (EMT) plays a crucial role in cancer invasion and progression, a process by which tumor cells lose cell-cell adhesion and acquire increased invasiveness and metastatic activity. Recent work has uncovered some crucial roles of extracellular adenosine 5'- triphosphate (eATP), a major component of the tumor microenvironment (TME), in promoting tumor growth and metastasis. Intratumoral extracellular ATP (eATP), at levels of 100-700 µM, is 103-104 times higher than in normal tissues. In the current literature, eATP's function in promoting metastasis has been relatively poorly understood as compared with intracellular ATP (iATP). Recent evidence has shown that cancer cells internalize eATP via macropinocytosis in vitro and in vivo, promoting cell growth and survival, drug resistance, and metastasis. Furthermore, ATP acts as a messenger molecule that activates P2 purinergic receptors expressed on both tumor and host cells, stimulating downstream signaling pathways to enhance the invasive and metastatic properties of tumor cells. Here, we review recent progress in understanding eATP's role in each step of the metastatic cascade, including initiating invasion, inducing EMT, overcoming anoikis, facilitating intravasation, circulation, and extravasation, and eventually establishing metastatic colonization. Collectively, these studies reveal eATP's important functions in many steps of metastasis and identify new opportunities for developing more effective therapeutic strategies to target ATP-associated processes in cancer.

Cancer Stem Cell Formation Induced and Regulated by Extracellular ATP and Stanniocalcin-1 in Human Lung Cancer Cells and Tumors.

Cancer stem cells (CSCs) are closely associated with metastasis and epithelial mesenchymal transition (EMT). We previously reported that extracellular ATP (eATP) induces and regulates EMT in cancer cells. We recently found that the gene stanniocalcin 1 (STC1) is significantly upregulated by eATP in human non-small lung cancer (NSCLC) A549 cells; however, the relationships among eATP, CSCs, and STC1 were largely unknown. In this study, we performed gene knockdown and knockout, and a wide variety of functional assays to determine if and how eATP and STC1 induce CSCs in NSCLC A549 and H1299 cells. Our data show that, in both cultured cells and tumors, eATP increased the number of CSCs in the cancer cell population and upregulated CSC-related genes and protein markers. STC1 deletion led to drastically slower cell and tumor growth, reduced intracellular ATP levels and CSC markers, and metabolically shifted STC1-deficient cells from an energetic state to a quiescent state. These findings indicate that eATP induces and regulates CSCs at transcriptional, translational, and metabolic levels, and these activities are mediated through STC1 via mitochondria-associated ATP synthesis. These novel findings offer insights into eATP-induced CSCs and identify new targets for inhibiting CSCs.

Molecule-to-Material-to-Bio Nanoarchitectonics with Biomedical Fullerene Nanoparticles.

Nanoarchitectonics integrates nanotechnology with various other fields, with the goal of creating functional material systems from nanoscale units such as atoms, molecules, and nanomaterials. The concept bears strong similarities to the processes and functions seen in biological systems. Therefore, it is natural for materials designed through nanoarchitectonics to truly shine in bio-related applications. In this review, we present an overview of recent work exemplifying how nanoarchitectonics relates to biology and how it is being applied in biomedical research. First, we present nanoscale interactions being studied in basic biology and how they parallel nanoarchitectonics concepts. Then, we overview the state-of-the-art in biomedical applications pursuant to the nanoarchitectonics framework. On this basis, we take a deep dive into a particular building-block material frequently seen in nanoarchitectonics approaches: fullerene. We take a closer look at recent research on fullerene nanoparticles, paying special attention to biomedical applications in biosensing, gene delivery, and radical scavenging. With these subjects, we aim to illustrate the power of nanomaterials and biomimetic nanoarchitectonics when applied to bio-related applications, and we offer some considerations for future perspectives.

Regulation of alternative polyadenylation by the C2H2-zinc-finger protein Sp1.

Alternative polyadenylation (APA) enhances gene regulatory potential by increasing the diversity of mRNA transcripts. 3' UTR shortening through APA correlates with enhanced cellular proliferation and is a widespread phenomenon in tumor cells. Here, we show that the ubiquitously expressed transcription factor Sp1 binds RNA in vivo and is a common repressor of distal poly(A) site usage. RNA sequencing identified 2,344 genes (36% of the total mapped mRNA transcripts) with lengthened 3' UTRs upon Sp1 depletion. Sp1 preferentially binds the 3' UTRs of such lengthened transcripts and inhibits cleavage at distal sites by interacting with the subunits of the core cleavage and polyadenylation (CPA) machinery. The 3' UTR lengths of Sp1 target genes in breast cancer patient RNA-seq data correlate with Sp1 expression levels, implicating Sp1-mediated APA regulation in modulating tumorigenic properties. Taken together, our findings provide insights into the mechanism for dynamic APA regulation by unraveling a previously unknown function of the DNA-binding transcription factor Sp1.

From Transcriptomics, Metabolomics to Functional Studies: Extracellular ATP Induces TGF-ß-Like Epithelial Mesenchymal Transition in Lung Cancer Cells.

We and others previously showed that extracellular ATP (eATP) is implicated in epithelial mesenchymal transition (EMT). However, the mechanisms by which eATP induces EMT and ATP's relationship to TGF-ß, a well-known EMT inducer, are largely unclear. Also, eATP-induced EMT has never been studied at transcriptomic and metabolomics levels. Based on our previous studies, we hypothesized that eATP acts as a specific inducer and regulator of EMT at all levels in cancer cells. RNAseq and metabolomics analyses were performed on human non-small cell lung cancer (NSCLC) A549 cells treated with either eATP or TGF-ß. Bio-functional assays, such as invasion, intracellular ATP, cell proliferation, cytoskeleton remodeling, and others were conducted in NSCLC A549 and H1299 cells to validate changes observed from RNAseq and metabolomics studies. In the RNAseq study, eATP significantly enriched expressions of genes involved in EMT similarly to TGF-ß after 2 and 6 hours of treatment. Samples treated with eATP for 2 hours share 131 upregulated EMT genes with those of TGF-ß treated samples, and 42 genes at 6 hours treatment. Eleven genes, with known or unknown functions in EMT, are significantly upregulated by both inducers at both time points, have been identified. BLOC1S6, one of the 11 genes, was selected for further study. eATP induced numerous EMT-related changes in metabolic pathways, including cytoskeleton rearrangement, glycolysis, glutaminolysis, ROS, and individual metabolic changes similar to those induced by TGF-ß. Functional bioassays verified the findings from RNAseq and metabolomics that eATP EMT-like changes in A549 and H1299 cells similarly to TGF-ß. BLOC1S6 was found to be implicated in EMT. In these studies, eATP-induced EMT, at all levels examined, is similar but non-identical to that induced by TGF-ß, and functions in such a way that exogenous addition of TGF-ß is unnecessary for the induction. The study of BLOC1S6 further verified its potential roles in EMT and the RNAseq analysis results. All these strongly indicate that eATP is a multi-functional and multi-locational inducer and regulator of EMT, changing our thinking on how EMT is induced and regulated and pointing to new directions for inhibiting EMT in cancer.