Colorimetric and fluorescent probes for cysteine detection: Applications in food safety and cellular imaging.

In this study, three BODIPY-based fluorescent probes were designed and synthesized. The ultraviolet-visible spectra, fluorescence spectra, smartphone color recognition application and bioimaging were utilized to evaluate the capacity of the probes. By comparing key parameters, BDP-SIN had optimal performances including fastest response (10 min), highest signal-to-noise ratio (815 times) and lowest limit of detection (LOD = 49 nM). The recovery rate ranged from 92.04 % to 103.25 %. Meanwhile, BDP-SIN was triumphantly employed for determination of Cys in different daily food samples. Moreover, the test strips and microporous filter membrane loaded with BDP-SIN were developed for the portable real-time visualization and quantitative detection of Cys in food samples, which the contents ranged from 0.27 µM to 0.49 µM. Besides, BDP-SIN could image Cys in the living cells and mice. The novelty of this work was that developed an effective tool for researching the roles of Cys in food industry and living organisms.

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.

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.

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.

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.

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.

LncRNA-OIS1 regulates DPP4 activation to modulate senescence induced by RAS.

Oncogene-induced senescence (OIS), provoked in response to oncogenic activation, is considered an important tumor suppressor mechanism. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nt without a protein-coding capacity. Functional studies showed that deregulated lncRNA expression promote tumorigenesis and metastasis and that lncRNAs may exhibit tumor-suppressive and oncogenic function. Here, we first identified lncRNAs that were differentially expressed between senescent and non-senescent human fibroblast cells. Using RNA interference, we performed a loss-function screen targeting the differentially expressed lncRNAs, and identified lncRNA-OIS1 (lncRNA#32, AC008063.3 or ENSG00000233397) as a lncRNA required for OIS. Knockdown of lncRNA-OIS1 triggered bypass of senescence, higher proliferation rate, lower abundance of the cell-cycle inhibitor CDKN1A and high expression of cell-cycle-associated genes. Subcellular inspection of lncRNA-OIS1 indicated nuclear and cytosolic localization in both normal culture conditions as well as following oncogene induction. Interestingly, silencing lncRNA-OIS1 diminished the senescent-associated induction of a nearby gene (Dipeptidyl Peptidase 4, DPP4) with established role in tumor suppression. Intriguingly, similar to lncRNA-OIS1, silencing DPP4 caused senescence bypass, and ectopic expression of DPP4 in lncRNA-OIS1 knockdown cells restored the senescent phenotype. Thus, our data indicate that lncRNA-OIS1 links oncogenic induction and senescence with the activation of the tumor suppressor DPP4.

Transcription Impacts the Efficiency of mRNA Translation via Co-transcriptional N6-adenosine Methylation.

Transcription and translation are two main pillars of gene expression. Due to the different timings, spots of action, and mechanisms of regulation, these processes are mainly regarded as distinct and generally uncoupled, despite serving a common purpose. Here, we sought for a possible connection between transcription and translation. Employing an unbiased screen of multiple human promoters, we identified a positive effect of TATA box on translation and a general coupling between mRNA expression and translational efficiency. Using a CRISPR-Cas9-mediated approach, genome-wide analyses, and in vitro experiments, we show that the rate of transcription regulates the efficiency of translation. Furthermore, we demonstrate that m6A modification of mRNAs is co-transcriptional and depends upon the dynamics of the transcribing RNAPII. Suboptimal transcription rates lead to elevated m6A content, which may result in reduced translation. This study uncovers a general and widespread link between transcription and translation that is governed by epigenetic modification of mRNAs.

Functional genetic screens for enhancer elements in the human genome using CRISPR-Cas9.

Systematic identification of noncoding regulatory elements has, to date, mainly relied on large-scale reporter assays that do not reproduce endogenous conditions. We present two distinct CRISPR-Cas9 genetic screens to identify and characterize functional enhancers in their native context. Our strategy is to target Cas9 to transcription factor binding sites in enhancer regions. We identified several functional enhancer elements and characterized the role of two of them in mediating p53 (TP53) and ERα (ESR1) gene regulation. Moreover, we show that a genomic CRISPR-Cas9 tiling screen can precisely map functional domains within enhancer elements. Our approach expands the utility of CRISPR-Cas9 to elucidate the functions of the noncoding genome.

Adenosine triphosphate-binding cassette transporter genes up-regulation in untreated hepatocellular carcinoma is mediated by cellular microRNAs.

UNLABELLED: Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are drug efflux pumps responsible for the multidrug resistance phenotype causing hepatocellular carcinoma (HCC) treatment failure. Here we studied the expression of 15 ABC transporters relevant for multidrug resistance in 19 paired HCC patient samples (16 untreated, 3 treated by chemotherapeutics). Twelve ABC transporters showed up-regulation in HCC compared with adjacent healthy liver. These include ABCA2, ABCB1, ABCB6, ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, ABCC10, ABCC11, ABCC12, and ABCE1. The expression profile and function of some of these transporters have not been associated with HCC thus far. Because cellular microRNAs (miRNAs) are involved in posttranscriptional gene silencing, we hypothesized that regulation of ABC expression in HCC might be mediated by miRNAs. To study this, miRNAs were profiled and dysregulation of 90 miRNAs was shown in HCC compared with healthy liver, including up-regulation of 11 and down-regulation of 79. miRNA target sites in ABC genes were bioinformatically predicted and experimentally verified in vitro using luciferase reporter assays. In total, 13 cellular miRNAs were confirmed that target ABCA1, ABCC1, ABCC5, ABCC10, and ABCE1 genes and mediate changes in gene expression. Correlation analysis between ABC and miRNA expression in individual patients revealed an inverse relationship, providing an indication for miRNA regulation of ABC genes in HCC. CONCLUSION: Up-regulation of ABC transporters in HCC occurs prior to chemotherapeutic treatment and is associated with miRNA down-regulation. Up-regulation of five ABC genes appears to be mediated by 13 cellular miRNAs in HCC patient samples. miRNA-based gene therapy may be a novel and promising way to affect the ABC profile and overcome clinical multidrug resistance.