MiR-146a reduces fibrosis after glaucoma filtration surgery in rats.

PURPOSE: To explore the impact of microRNA 146a (miR-146a) and the underlying mechanisms in profibrotic changes following glaucoma filtering surgery (GFS) in rats and stimulation by transforming growth factor (TGF)-ß1 in rat Tenon's capsule fibroblasts. METHODS: Cultured rat Tenon's capsule fibroblasts were treated with TGF-ß1 and analyzed with microarrays for mRNA profiling to validate miR-146a as the target. The Tenon's capsule fibroblasts were then respectively treated with lentivirus-mediated transfection of miR-146a mimic or inhibitor following TGF-ß1 stimulation in vitro, while GFS was performed in rat eyes with respective intraoperative administration of miR-146a, mitomycin C (MMC), or 5-fluorouracil (5-FU) in vivo. Profibrotic genes expression levels (fibronectin, collagen Iα, NF-KB, IL-1ß, TNF-α, SMAD4, and α-smooth muscle actin) were determined through qPCR, Western blotting, immunofluorescence staining and/or histochemical analysis in vitro and in vivo. SMAD4 targeting siRNA was further used to treat the fibroblasts in combination with miR-146a intervention to confirm its role in underlying mechanisms. RESULTS: Upregulation of miR-146a reduced the proliferation rate and profibrotic changes of rat Tenon's capsule fibroblasts induced by TGF-ß1 in vitro, and mitigated subconjunctival fibrosis to extend filtering blebs survival after GFS in vivo, where miR-146a decreased expression levels of NF-KB-SMAD4-related genes, such as fibronectin, collagen Iα, NF-KB, IL-1ß, TNF-α, SMAD4, and α-smooth muscle actin(α-SMA). Additionally, SMAD4 is a key target gene in the process of miR-146a inhibiting fibrosis. CONCLUSIONS: MiR-146a effectively reduced TGF-ß1-induced fibrosis in rat Tenon's capsule fibroblasts in vitro and in vivo, potentially through the NF-KB-SMAD4 signaling pathway. MiR-146a shows promise as a novel therapeutic target for preventing fibrosis and improving the success rate of GFS.

Fly-scan high-throughput coded ptychographic microscopy via active micro-vibration and rolling-shutter distortion correction.

Recent advancements in ptychography have demonstrated the potential of coded ptychography (CP) for high-resolution optical imaging in a lensless configuration. However, CP suffers imaging throughput limitations due to scanning inefficiencies. To address this, we propose what we believe is a novel 'fly-scan' scanning strategy utilizing two eccentric rotating mass (ERM) vibration motors for high-throughput coded ptychographic microscopy. The intrinsic continuity of the 'fly-scan' technique effectively eliminates the scanning overhead typically encountered during data acquisition. Additionally, its randomized scanning trajectory considerably reduces periodic artifacts in image reconstruction. We also developed what we believe to be a novel rolling-shutter distortion correction algorithm to fix the rolling-shutter effects. We built up a low-cost, DIY-made prototype platform and validated our approach with various samples including a resolution target, a quantitative phase target, a thick potato sample and biospecimens. The reported platform may offer a cost-effective and turnkey solution for high-throughput bio-imaging.

In vitro assessment of the bacterial stress response and resistance evolution during multidrug-resistant bacterial invasion of the Xenopus tropicalis intestinal tract under typical stresses.

The intestinal microbiome might be both a sink and source of resistance genes (RGs). To investigate the impact of environmental stress on the disturbance of exogenous multidrug-resistant bacteria (mARB) within the indigenous microbiome and proliferation of RGs, an intestinal conjugative system was established to simulate the invasion of mARB into the intestinal microbiota in vitro. Oxytetracycline (OTC) and heavy metals (Zn, Cu, Pb), commonly encountered in aquaculture, were selected as typical stresses for investigation. Adenosine 5'-triphosphate (ATP), hydroxyl radical (OH·-) and extracellular polymeric substance (EPS) were measured to investigate their influence on the acceptance of RGs by intestinal bacteria. The results showed that the transfer and diffusion of RGs under typical combined stressors were greater than those under a single stressor. Combined effect of OTC and heavy metals (Zn, Cu) significantly increased the activity and extracellular EPS content of bacteria in the intestinal conjugative system, increasing intI3 and RG abundance. OTC induced a notable inhibitory response in Citrobacter and exerted the proportion of Citrobacter and Carnobacterium in microbiota. The introduction of stressors stimulates the proliferation and dissemination of RGs within the intestinal environment. These results enhance our comprehension of the typical stresses effect on the RGs dispersal in the intestine.

Precisely Constructing Renal-Clearable and LAP-Activatable Ratiometric Molecular Probes for Early Diagnosis of Acute and Chronic Kidney Injury Via Optimizing Asymmetric DPP Dyes.

Fluorescence analysis is an increasingly important contributor to the early diagnosis of kidney diseases. To achieve precise visualization of the kidneys and early diagnosis of related diseases, an asymmetric pyrrolopyrrolidone (DPP) dye platform with C-aromatic substituents and N-lipophilic/hydrophilic modification was constructed. Based on these, we developed the renal-clearable, water-soluble, and kidney injury biomarker leucine aminopeptidase (LAP) activated ratiometric fluorescent probe DPP-S-L. In the mouse model of cisplatin-induced acute kidney injury and during the development of type 2 diabetes to diabetic kidney disease, we visualized for the first time the upregulation of LAP in the kidney and urine by dual-channel ratiometric fluorescence signal and diagnosed the kidney injury earlier and more sensitively than blood/urine enzyme detection and tissue analysis. This study showcases an excellent asymmetric DPP dye platform and renal-clearable ratiometric fluorescent probe design strategy that is extended to determination and visualization of other biomarkers for early disease diagnosis.

Oxytetracycline and heavy metals promote the migration of resistance genes in the intestinal microbiome by plasmid transfer.

Horizontal gene transfer (HGT) has been considered the most important pathway to introduce antibiotic resistance genes (ARGs), which seriously threatens human health and biological security. The presence of ARGs in the aquatic environment and their effect on the intestinal micro-ecosystem of aquatic animals can occur easily. To investigate the HGT potential and rule of exogenous ARGs in the intestinal flora, a visual conjugative model was developed, including the donor of dual-fluorescent bacterium and the recipient of Xenopus tropicalis intestinal microbiome. Some common pollutants of oxytetracycline (OTC) and three heavy metals (Zn, Cu and Pb) were selected as the stressor. The multi-techniques of flow cytometry (FCM), scanning electron microscopy (SEM), atomic force microscopy (AFM), single-cell Raman spectroscopy with sorting (SCRSS) and indicator analysis were used in this study. The results showed that ARG transfer could occur more easily under stressors. Moreover, the conjugation efficiency mainly depended on the viability of the intestinal bacteria. The mechanisms of OTC and heavy metal stressing conjugation included the upregulation of ompC, traJ, traG and the downregulation of korA gene. Moreover, the enzymatic activities of SOD, CAT, GSH-PX increased and the bacterial surface appearance also changed. The predominant recipient was identified as Citrobacter freundi by SCRSS, in which the abundance and quantity of ARG after conjugation were higher than those before. Therefore, since the diversity of potential recipients in the intestine are very high, the migration of invasive ARGs in the microbiome should be given more attention to prevent its potential risks to public health.

Matrix Metalloproteinases in Glaucoma: An Updated Overview.

Matrix metalloproteinases (MMPs) are important regulators of the extracellular matrix (ECM) and are involved in many stages of cellular growth and development. An imbalance of MMP expression is also the basis of many diseases, including eye diseases, such as diabetic retinopathy (DR), glaucoma, dry eye, corneal ulcer, keratoconus. This paper describes the role of MMPs in the glaucoma and their role in the glaucomatous trabecular meshwork (TM), aqueous outflow channel, retina, and optic nerve (ON). This review also summarizes several treatments for glaucoma that target MMPs imbalance and suggests that MMPs may represent a viable therapeutic target for glaucoma.

On methodology and application of smoothed particle hydrodynamics in fluid, solid and biomechanics.

Smoothed particle hydrodynamics (SPH), as one of the earliest meshfree methods, has broad prospects in modeling a wide range of problems in engineering and science, including extremely large deformation problems such as explosion and high velocity impact. This paper aims to provide a comprehensive overview on the recent advances of SPH method in the fields of fluid, solid, and biomechanics. First, the theory of SPH is described, and improved algorithms of SPH with high accuracy are summarized, such as the finite particle method (FPM). Techniques used in SPH method for simulating fluid, solid and biomechanics problems are discussed. The δ-SPH method and Godunov SPH (GSPH) based on the Riemann model are described for handling instability issues in fluid dynamics. Next, the interface contact algorithm for fluid-structure interaction is also discussed. The common algorithms for improving the tensile instability and the framework of total Lagrangian SPH are examined for challenging tasks in solid mechanics. In terms of biomechanics, the governing equations and the coupling forces based on SPH method are exemplified. Then, various typical engineering applications and recent advances are elaborated. The application of fluid mainly depicts the interaction between fluid and rigid body as well as elastomer, while some complicated fluid-structure interaction ocean engineering problems are also presented. In the aspect of solid dynamics, galaxy, geotechnical mechanics, explosion and impact, and additive manufacturing are summarized. Furthermore, the recent advancements of SPH method in biomechanics, such as hemodynamically and gut health, are discussed in general. In addition, to overcome the limitations of computational efficiency and computational scale, the multiscale adaptive resolution, the parallel algorithm and the automated mesh generation are addressed. The development of SPH software in China and abroad is also summarized. Finally, the challenging task of SPH method in the future is summarized. In future research work, the establishment of multi-scale coupled SPH model and deep learning technology in solid and biodynamics will be the focus of expanding the engineering applications of SPH methods.

Establishment of a minimally invasive distal traumatic optic neuropathy model in mice to investigate cascade reactions of retinal glial cells.

Traumatic optic neuropathy (TON) is a complication of craniocerebral, orbital and facial injuries, leading to irreversible vision loss. At present, there is no reliable, widely used animal model, although it has been confirmed that TON can cause the loss of retinal ganglion cells (RGC). However, the cascade reaction of retinal glial cells underlying TON is unclear. Therefore, the establishment of an animal model to explore the pathological mechanism of TON would be of great interest to the scientific community. In this study, we propose a novel mouse model utilizing a 3D stereotaxic apparatus combined with a 27G needle to evaluate damage to the optic nerve by micro-CT, anatomy, SD-OCT and F-VEP. Immunofluorescence, western blotting, qPCR experiments were conducted to investigate the loss of RGCs and activation or inactivation of microglia, astrocytes and Müller glial cells in the retina from the first week to the fourth week after modeling. The results showed that this minimally invasive method caused damage to the distal optic nerve and loss of RGC after optic nerve injury. Microglia cells were found to be activated from the first week to the third week; however, they were inactivated at the fourth week; astrocytes were activated at the second week of injury, while Müller glial cells were gradually inactivated following injury. In conclusion, this method can be used as a novel animal model of distal TON, that results in a series of cascade reactions of retinal glial cells, which will provide a basis for future studies aimed at exploring the mechanism of TON and the search for effective treatment methods.

Notoginsenoside R1 Promotes Proliferation and Osteogenic Differentiation of hPDLSCs via Wnt/ß-Catenin Signaling Pathway.

Purpose: To investigate the roles of Notoginsenoside R1 (NG-R1) on the proliferation and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and explore its possible mechanism. Methods: hPDLSCs were isolated and, then characterized by flow cytometry. Cell-counting kit-8 (CCK-8) and colony assays were used to validate the effect of different NG-R1 concentrations on hPDLSCs proliferation and the optimal concentration was determined. Quantitative detection of alkaline phosphatase (ALP) activity at optimal concentration and the mineralization of the cells was investigated by Alizarin Red S staining. qRT-PCR and Western blot were utilized to examine the factors expression levels of ALP, Runx Family Transcription Factor 2 (RUNX2), Collagen I (Col-1) and catenin beta 1 (CTNNB1; ß-catenin). In addition, the tankyrase inhibitor XAV-939 was used to explore NG-R1's role in canonical Wnt signaling. Results: hPDLSCs were positive for surface antigens CD90 while negative for CD34 and CD45, which indicated that we have successfully isolated the hPDLSCs. Furthermore, a concentration of 20µmol NG-R1 dramatically enhanced hPDLSCs proliferation, ALP activity, and mineral deposition. ALP, RUNX2, COL-1, and ß-catenin expression were all rised in comparison to control group. After XAV-939 was added to disrupt the canonical Wnt signaling, the impact of NG-R1 appeared to be reversed. Conclusion: These findings suggest that NG-R1 can stimulate osteogenic differentiation of hPDLSCs, which is probably attributable to canonical Wnt signaling activation.

Parental psychological control and adolescent social problems: The mediating effect of emotion regulation.

This study aimed to investigate relationships among parental psychological control, adolescent emotion regulation, and social problems in China. In total, 1,145 adolescents aged 12-15 years participated in the study, which used the Parental Psychological Control Scale, Adolescent Problem Behavior Scale, and Emotion Regulation Scale. The results indicated the following: (1) Compared with only-child teens, adolescents in multi-child families had significant social problems; (2) parental psychological control significantly predicted adolescents' social problems; (3) there was a partially mediating effect of adolescents' emotion regulation between parental psychological control and adolescents' social problems.

Effect of Puerarin on Osteogenic Differentiation in vitro and on New Bone Formation in vivo.

Purpose: Puerarin (C21H20O10) is a phytoestrogen that possesses various pharmacological effect, and several researches have revealed the relationship between puerarin and bone metabolism. This study was aimed to evaluate the potential influence of puerarin on the proliferation and osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (BMSCs) as well as on new bone formation following rapid maxillary expansion (RME) model in rats. Methods: Rat BMSCs were adopted, and the cell proliferation was detected by cell-counting kit-8 (CCK-8) assay in vitro experiments. Alkaline phosphatase (ALP) activity and alizarin red staining were analyzed quantitatively to show extracellular matrix mineralization. The mRNA and protein expression levels were used to detect osteogenic differentiation of BMSCs. In vivo bone regeneration was analyzed in a rat RME model. Eighteen 6-week-old male Wistar rats were divided into 3 groups: group 1 without any treatment, group 2 received RME and saline solution (15mg/kg), group 3 received RME and puerarin solution (15mg/kg). After 2 weeks, micro-computed tomography (Micro-CT), hematoxylin and eosin (HE) staining, and Masson staining were used to detect the new bone formation and morphological changes. Besides, ALP and bone morphogenetic protein 2 (BMP2) expression levels in mid-palatal suture were evaluated by immunohistochemical staining. Results: The results showed that puerarin upregulates cell proliferation dose-dependently. ALP activity and mineralized matrix generation were clearly enhanced at certain specific concentrations (10-5 and 10-6 mol/L); the expression levels of the osteoblast-related genes and proteins were increased. The measurement of micro-CT imaging revealed that puerarin significantly promoted new bone formation. Concomitantly, the histological examinations showed that puerarin solution enhanced osteogenesis in mid-palatal suture. Conclusion: Those works indicated that puerarin regulates osteogenesis in vitro and exerts a beneficial impact on bone regeneration in vivo, revealing that puerarin treatment may become one of the potential keys for improving the stability and preventing relapse of RME.

Sinomenine Inhibits Orthodontic Tooth Movement and Root Resorption in Rats and Enhances Osteogenic Differentiation of PDLSCs.

Purpose: To investigate the effects of sinomenine on orthodontic tooth movement and root resorption in rats, as well as the effect of sinomenine on the osteogenesis of periodontal ligament stem cells (PDLSCs). Methods: Fifty-four male Wistar rats were randomly divided into 3 groups: control group, 20 mg/kg sinomenine group and 40 mg/kg sinomenine group. Fifty-gram orthodontic force was applied to all groups. Each group was injected intraperitoneally with corresponding concentration of sinomenine every day. After 14 days, all rats were sacrificed. Micro-computed tomography (micro-CT) scan was used to analyze tooth movement, root resorption and alveolar bone changes. The effect on periodontal tissue was analyzed by Masson, tartrate-resistant acid phosphatase (TRAP) and immunohistochemical staining. In vitro, PDLSCs were extracted and identified. The effect of sinomenine on proliferation was determined by cell-counting kit-8. The effect of sinomenine on osteogenesis was investigated by alkaline phosphatase (ALP) activity and alizarin red staining. qPCR and Western blotting were performed to explore the effects of sinomenine on the expression levels of ALP, runt-related transcription factor 2 (RUNX2), receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG). Results: The tooth movement and root resorption of sinomenine groups were reduced. Sinomenine decreased trabecular spacing on compression side and increased alveolar bone volume and trabecular thickness on tension side. TRAP-positive cells in sinomenine groups decreased significantly. The expressions of TNF-α and RANKL were decreased, while the expressions of OPG, RUNX2 and osteocalcin were up-regulated. In vitro, 0.1 M and 0.5 M sinomenine enhanced ALP activity, mineral deposition and the expression of ALP, RUNX2 and OPG, and reduced the expression of RANKL. Conclusion: Sinomenine could inhibit tooth movement, reduce root resorption, and exert a positive effect on bone formation in rats. Moreover, sinomenine promoted the osteogenesis of PDLSCs.

Building regulatory landscapes reveals that an enhancer can recruit cohesin to create contact domains, engage CTCF sites and activate distant genes.

Developmental gene expression is often controlled by distal regulatory DNA elements called enhancers. Distant enhancer action is restricted to structural chromosomal domains that are flanked by CTCF-associated boundaries and formed through cohesin chromatin loop extrusion. To better understand how enhancers, genes and CTCF boundaries together form structural domains and control expression, we used a bottom-up approach, building series of active regulatory landscapes in inactive chromatin. We demonstrate here that gene transcription levels and activity over time reduce with increased enhancer distance. The enhancer recruits cohesin to stimulate domain formation and engage flanking CTCF sites in loop formation. It requires cohesin exclusively for the activation of distant genes, not of proximal genes, with nearby CTCF boundaries supporting efficient long-range enhancer action. Our work supports a dual activity model for enhancers: its classic role of stimulating transcription initiation and elongation from target gene promoters and a role of recruiting cohesin for the creation of chromosomal domains, the engagement of CTCF sites in chromatin looping and the activation of distal target genes.

Noncanonical Wnt5a Signaling Suppresses Hippo/TAZ-Mediated Osteogenesis Partly Through the Canonical Wnt Pathway in SCAPs.

PURPOSE: Stem cells from the apical papilla (SCAPs) are promising seed cells for tissue regeneration medicine and possess the osteogenic differentiation potential. Wnt5a, a typical ligand of the noncanonical Wnt pathway, exhibits diverse roles in the regulation of osteogenesis. The transcriptional co-activator with PDZ-binding motif (TAZ, WWTR1) is a core regulator in the Hippo pathway and regulates stem behavior including osteogenic differentiation. This study aims to examine how Wnt5a regulates SCAPs osteogenesis and explore the precise mechanistic relationship between Wnt5a and TAZ. METHODS: SCAPs were isolated from developing apical papilla tissue of extracted human immature third molars in vitro. ALP staining, ALP activity and Alizarin red staining were used to evaluate osteogenic capacity. Osteogenic-related factors were assessed by qRT-PCR or Western blotting. Additionally, the receptor tyrosine kinase-like orphan receptor 2 (ROR2) was detected by immunocytofluorescence staining and silenced by small interfering RNA to verify the function of Wnt5a/ROR2 in TAZ-mediated osteogenesis. And we constructed TAZ-overexpression and ß-catenin-overexpression SCAPs generated by lentivirus to explore the precise mechanistic relationship between Wnt5a and TAZ. RESULTS: Wnt5a (100ng/mL) significantly suppressed ALP activity, mineralization nodules formation, expression of osteogenic-related factors. Meanwhile, it decreased the expression of TAZ mRNA and protein. TAZ overexpression promoted osteogenesis of SCAPs while Wnt5a could block TAZ-mediated osteogenesis. Furthermore, ROR2 siRNA (siROR2) was found to upregulate TAZ and canonical Wnt pathway signaling related molecules such as ß-catenin, GSK3ß and p-GSK3ß. The suppression of Wnt5a/ROR2 on osteogenesis was significantly reversed by ß-catenin overexpression through Wnt5a/ROR2/ß-catenin/TAZ pathway. CONCLUSION: Taken together, the present study demonstrates that Wnt5a suppresses TAZ-mediated osteogenesis of SCAPs and there may be a Wnt5a/ROR2/ß-catenin/TAZ pathway regulating osteogenesis of SCAPs. Moreover, Wnt5a could be a candidate for regulators in tissue regeneration.

Migration of antibiotic resistance genes and evolution of flora structure in the Xenopus tropicalis intestinal tract with combined exposure to roxithromycin and oxytetracycline.

The intestinal flora is one of the most important environments for antibiotic resistance development, owing to its diverse mix of bacteria. An excellent medicine model organism, Xenopus tropicalis, was selected to investigate the spread of antibiotic resistance genes (ARGs) in the intestinal bacterial community with single or combined exposure to roxithromycin (ROX) and oxytetracycline (OTC). Seventeen resistance genes (tetA, tetB, tetE, tetM, tetO, tetS, tetX, ermF, msrA, mefA, ereA, ereB, mphA, mphB, intI1, intI2, intI3) were detected in the intestines of Xenopus tropicalis living in three testing tanks (ROX tanks, OTC tanks, ROX + OTC tanks) and a blank tank for 20 days. The results showed that the relative abundance of total ARGs increased obviously in the tank with single stress but decreased in the tank with combined stress, and the genes encoding the macrolide antibiotic efflux pump (msrA), phosphatase (mphB) and integron (intI2, intI3) were the most sensitive. With the aid of AFM scanning, DNA was found to be scattered short chain in the blank, became extended or curled and then compacted with the stress from a single antibiotic, and was compacted and then fragmented with combined stress, which might be the reason for the variation of the abundance of ARGs with stress. The ratio of Firmicutes/Bacteroides related to diseases was increased by ROX and OTC. The very significant correlation between intI2 and intI3 with tetS (p ≤ 0.001) hinted at a high risk of ARG transmission in the intestines. Collectively, our results suggested that the relative abundance of intestinal ARGs could be changed depending on the intestinal microbiome and DNA structures upon exposure to antibiotics at environmental concentrations.

Tracking pesticide exposure to operating workers for risk assessment in seed coating with tebuconazole and carbofuran.

BACKGROUND: Coating seed with pesticides is an effective way to control plant pests, however, factory-based coating processes may carry a potential risk to operational workers of chemical exposure. To study the risk, carbofuran and tebuconazole were used to coat corn seed and their subsequent distribution on the bodies of workers was measured at manufacturers XFS and LS (Shanxi, China). Clothing was collected from workers during operations and analyzed using high-performance liquid chromatography. RESULTS: At XFS, dermal exposure to carbofuran was 4.83, 3.31 and 1.48 mg kg-1 , and exposure to tebuconazole was 6.88, 5.16 and 1.72 mg kg-1 for coating, packing and transport workers, respectively. At LS, dermal exposure to carbofuran was 2.32, 0.46 and 0.55 mg kg-1 , and exposure to tebuconazole was 1.69, 0.46 and 0.70 mg kg-1 , for coating, packing and transport workers, respectively. The level of pesticide exposure was significantly higher for seed-coating workers than for packing and transport workers. The main area of exposure was the hands for all workers and the lower limbs for packers; exposure was relatively uniform for pesticide handlers. Occupational risk was assessed based on margin of exposure (MOE). In seed-coating, the MOE was greater than 100 for tebuconazole, indicating no potential risk, but ranged from 0.25 to 2.88 for carbofuran, indicating the risk of a health impact. CONCLUSION: The level of exposure varied depending on type of operation undertaken and body parts of workers' body, but the risk of a health impact was highly associated with pesticide toxicity. This provides a guideline for workers in pesticide manufacturing to ensure safe operation of the seed-coating process. © 2021 Society of Chemical Industry.

A comprehensive enhancer screen identifies TRAM2 as a key and novel mediator of YAP oncogenesis.

BACKGROUND: Frequent activation of the co-transcriptional factor YAP is observed in a large number of solid tumors. Activated YAP associates with enhancer loci via TEAD4-DNA-binding protein and stimulates cancer aggressiveness. Although thousands of YAP/TEAD4 binding-sites are annotated, their functional importance is unknown. Here, we aim at further identification of enhancer elements that are required for YAP functions. RESULTS: We first apply genome-wide ChIP profiling of YAP to systematically identify enhancers that are bound by YAP/TEAD4. Next, we implement a genetic approach to uncover functions of YAP/TEAD4-associated enhancers, demonstrate its robustness, and use it to reveal a network of enhancers required for YAP-mediated proliferation. We focus on EnhancerTRAM2, as its target gene TRAM2 shows the strongest expression-correlation with YAP activity in nearly all tumor types. Interestingly, TRAM2 phenocopies the YAP-induced cell proliferation, migration, and invasion phenotypes and correlates with poor patient survival. Mechanistically, we identify FSTL-1 as a major direct client of TRAM2 that is involved in these phenotypes. Thus, TRAM2 is a key novel mediator of YAP-induced oncogenic proliferation and cellular invasiveness. CONCLUSIONS: YAP is a transcription co-factor that binds to thousands of enhancer loci and stimulates tumor aggressiveness. Using unbiased functional approaches, we dissect YAP enhancer network and characterize TRAM2 as a novel mediator of cellular proliferation, migration, and invasion. Our findings elucidate how YAP induces cancer aggressiveness and may assist diagnosis of cancer metastasis.

Transcription Dynamics Regulate Poly(A) Tails and Expression of the RNA Degradation Machinery to Balance mRNA Levels.

Gene expression is regulated by the rates of synthesis and degradation of mRNAs, but how these processes are coordinated is poorly understood. Here, we show that reduced transcription dynamics of specific genes leads to enhanced m6A deposition, preferential activity of the CCR4-Not complex, shortened poly(A) tails, and reduced stability of the respective mRNAs. These effects are also exerted by internal ribosome entry site (IRES) elements, which we found to be transcriptional pause sites. However, when transcription dynamics, and subsequently poly(A) tails, are globally altered, cells buffer mRNA levels by adjusting the expression of mRNA degradation machinery. Stress-provoked global impediment of transcription elongation leads to a dramatic inhibition of the mRNA degradation machinery and massive mRNA stabilization. Accordingly, globally enhanced transcription, such as following B cell activation or glucose stimulation, has the opposite effects. This study uncovers two molecular pathways that maintain balanced gene expression in mammalian cells by linking transcription to mRNA stability.

SLC1A3 contributes to L-asparaginase resistance in solid tumors.

L-asparaginase (ASNase) serves as an effective drug for adolescent acute lymphoblastic leukemia. However, many clinical trials indicated severe ASNase toxicity in patients with solid tumors, with resistant mechanisms not well understood. Here, we took a functional genetic approach and identified SLC1A3 as a novel contributor to ASNase resistance in cancer cells. In combination with ASNase, SLC1A3 inhibition caused cell cycle arrest or apoptosis, and myriads of metabolic vulnerabilities in tricarboxylic acid (TCA) cycle, urea cycle, nucleotides biosynthesis, energy production, redox homeostasis, and lipid biosynthesis. SLC1A3 is an aspartate and glutamate transporter, mainly expressed in brain tissues, but high expression levels were also observed in some tumor types. Here, we demonstrate that ASNase stimulates aspartate and glutamate consumptions, and their refilling through SLC1A3 promotes cancer cell proliferation. Lastly, in vivo experiments indicated that SLC1A3 expression promoted tumor development and metastasis while negating the suppressive effects of ASNase by fueling aspartate, glutamate, and glutamine metabolisms despite of asparagine shortage. Altogether, our findings identify a novel role for SLC1A3 in ASNase resistance and suggest that restrictive aspartate and glutamate uptake might improve ASNase efficacy with solid tumors.

Functional CRISPR screen identifies AP1-associated enhancer regulating FOXF1 to modulate oncogene-induced senescence.

BACKGROUND: Functional characterization of non-coding elements in the human genome is a major genomic challenge and the maturation of genome-editing technologies is revolutionizing our ability to achieve this task. Oncogene-induced senescence, a cellular state of irreversible proliferation arrest that is enforced following excessive oncogenic activity, is a major barrier against cancer transformation; therefore, bypassing oncogene-induced senescence is a critical step in tumorigenesis. Here, we aim at further identification of enhancer elements that are required for the establishment of this state. RESULTS: We first apply genome-wide profiling of enhancer-RNAs (eRNAs) to systematically identify enhancers that are activated upon oncogenic stress. DNA motif analysis of these enhancers indicates AP-1 as a major regulator of the transcriptional program induced by oncogene-induced senescence. We thus constructed a CRISPR-Cas9 sgRNA library designed to target senescence-induced enhancers that are putatively regulated by AP-1 and used it in a functional screen. We identify a critical enhancer that we name EnhAP1-OIS1 and validate that mutating the AP-1 binding site within this element results in oncogene-induced senescence bypass. Furthermore, we identify FOXF1 as the gene regulated by this enhancer and demonstrate that FOXF1 mediates EnhAP1-OIS1 effect on the senescence phenotype. CONCLUSIONS: Our study elucidates a novel cascade mediated by AP-1 and FOXF1 that regulates oncogene-induced senescence and further demonstrates the power of CRISPR-based functional genomic screens in deciphering the function of non-coding regulatory elements in the genome.