Functional Validation of Different Alternative Splicing Variants of the Chrysanthemum lavandulifolium ClNUM1 Gene in Tobacco.

The Asteraceae are widely distributed throughout the world, with diverse functions and large genomes. Many of these genes remain undiscovered and unstudied. In this study, we discovered a new gene ClNUM1 in Chrysanthemum lavandulifolium and studied its function. In this study, bioinformatics, RT-qPCR, paraffin sectioning, and tobacco transgenics were utilized to bioinformatically analyze and functionally study the three variable splice variants of the unknown gene ClNUM1 cloned from C. lavandulifolium. The results showed that ClNUM1.1 and ClNUM1.2 had selective 3' splicing and selective 5' splicing, and ClNUM1.3 had selective 5' splicing. When the corresponding transgenic tobacco plants were subjected to abiotic stress treatment, in the tobacco seedlings, the ClNUM1.1 gene and the ClNUM1.2 gene enhanced salt and low-temperature tolerance and the ClNUM1.3 gene enhanced low-temperature tolerance; in mature tobacco plants, the ClNUM1.1 gene was able to enhance salt and low-temperature tolerance, and the ClNUM1.2 and ClNUM1.3 genes were able to enhance low-temperature tolerance. In summary, there are differences in the functions of the different splice variants and the different seedling stages of transgenic tobacco, but all of them enhanced the resistance of tobacco to a certain extent. The analysis and functional characterization of the ClNUM1 gene provided new potential genes and research directions for abiotic resistance breeding in Chrysanthemum.

Deficiency of Pdk1 drives heart failure by impairing taurine homeostasis through Slc6a6.

3-Phosphoinositide-dependent protein kinase-1 (Pdk1) as a serine/threonine protein kinase plays a critical role in multiple signaling pathways. Analysis of the gene expression omnibus database showed that Pdk1 was significantly downregulated in patients with heart diseases. Gene set enrichment analysis of the proteomics dataset identified apoptotic- and metabolism-related signaling pathways directly targeted by Pdk1. Previously, our research indicated that Pdk1 deletion-induced metabolic changes might be involved in the pathogenesis of heart failure; however, the underlying mechanism remains elusive. Here, we demonstrated that deficiency of Pdk1 resulted in apoptosis, oxidative damage, and disturbed metabolism, both in vivo and in vitro. Furthermore, profiling of metabonomics by 1 H-NMR demonstrated that taurine was the major differential metabolite in the heart of Pdk1-knockout mice. Taurine treatment significantly reduced the reactive oxygen species production and apoptosis, improved cardiac function, and prolonged the survival time in Pdk1 deficient mice. Proteomic screening identified solute carrier family 6 member 6 (Slc6a6) as the downstream that altered taurine levels in Pdk1-expression cells. Consistently, cellular apoptosis and oxidative damage were rescued by Slc6a6 in abnormal Pdk1 expression cells. These findings collectively suggest that Pdk1 deficiency induces heart failure via disturbances in taurine homeostasis, triggered by Slc6a6.

High expression of NOTCH2 in gastric adenocarcinoma: A novel early diagnostic target.

BACKGROUND AND AIM: NOTCH2 is overexpressed in gastric cancer (GC), and its enhanced activity is significantly correlated with worse tumor characteristics. We aim to analyze the clinicopathologic correlation between NOTCH2 and the molecular typing of GC by immunohistochemistry and by transcriptional sequencing. METHODS: In this immunohistochemical study, we detected NOTCH2, EBER, P53, HER2, MLH1, MSH2, PMS2, and MSH6 and evaluated the association of NOTCH2 with clinical and histopathological features in a large single-institutional series of gastric adenocarcinomas (n = 488). The correlation was also investigated between immunohistochemical results and survival outcomes. RESULTS: High NOTCH2 expression (2+/3+) was found in 139/488 (27.5%) samples analyzed. NOTCH2 expression was correlated with early stage T1 (P < 0.0001), GC in the fundus (P = 0.0364), and positive P53 status (P = 0.0019). We did not find an association between NOTCH2 and HER2, microsatellite instability, EBER, and overall survival. Through RNA sequencing, it was revealed that NOTCH2 plays an important biological function in the pathogenesis and development of GC. CONCLUSIONS: Our findings suggested that NOTCH2 may be a potential diagnostic target for GC due to the fact that its high expression is closely associated with the early stages of cancer.

mEosEM withstands osmium staining and Epon embedding for super-resolution CLEM.

Super-resolution correlative light and electron microscopy (SR-CLEM) is a powerful approach for imaging specific molecules at the nanoscale in the context of the cellular ultrastructure. Epon epoxy resin embedding offers advantages for SR-CLEM, including ultrastructural preservation and high quality sectioning. However, Epon embedding eliminates fluorescence from most fluorescent proteins. We describe a photocontrollable fluorescent protein, mEosEM, that can survive Epon embedding after osmium tetroxide (OsO4) treatment for improved SR-CLEM.

Application of Dexmedetomidine in Surgical Anesthesia for Gastric Cancer and Its Effects on IL-1ß, IL-6, TNF-α and CRP.

This study was performed to analyze the application of dexmedetomidine (Dex) in anesthesia for gastric cancer surgery and its effect on serum inflammatory factors in patients. In this regard, a total of 78 patients with gastric cancer who were hospitalized in our hospital from January 2020 to September 2023 and received general intravenous anesthesia were randomly divided into two groups (n=39 in each group). The conventional group was given the same volume of 0.9% sodium chloride solution 10min before induction of anesthesia, and the Dex group was given Dex1µg/kg intravenous pump 10min before induction of anesthesia. The hemodynamics, serum levels of IL-1ß, IL-6, TNF-α, CRP, propofol, remifentanil, and the total incidence of adverse reactions were compared between the two groups at different periods. The results showed that the mean arterial pressure (MAP), heart rate (HR), serum IL-1ß, IL-6, TNF-α and CRP in the Dex group were compared with those in the routine group (P>0.05). MAP and HR in T1, T2 and T3Dex groups were lower than those in the conventional group (P<0.05). The serum levels of IL-1ß, IL-6, TNF-α and CRP in T4 and T5 of the Dex group were lower than those of the routine group (P<0.05). The dosage of propofol and remifentanil in the Dex group was lower than those in the conventional group (P<0.05). The total incidence of adverse reactions in the Dex group (5.13%) was compared with that in the conventional group (10.26%), P>0.05. It was concluded that Dex can effectively maintain the stability of hemodynamics during gastric cancer surgery, reduce the dosage of propofol and other anesthetic drugs, reduce inflammation, and has a certain safety without obvious adverse reactions.

Risk assessment of combined exposure to lead, cadmium, and total mercury among the elderly in Shanghai, China.

Lead (Pb), cadmium (Cd) and total mercury (THg) are toxic heavy metals (THMs) that are widely present in the environment and can cause substantial health problems. However, previous risk assessment studies have rarely focused on the elderly population and have usually targeted a single heavy metal, which might underestimate the long-term accumulative and synergistic effects of THMs in humans. Based on the food frequency questionnaire and inductively coupled plasma mass spectrometry, this study assessed external and internal exposures to Pb, Cd and THg in 1747 elderly people in Shanghai. Probabilistic risk assessment with the relative potential factor (RPF) model was used to assess the neurotoxicity and nephrotoxicity risks of combined THMs exposures. The mean external exposures of Pb, Cd and THg in Shanghai elderly were 46.8, 27.2 and 4.9 µg/day, respectively. Plant-based foods are the main source of Pb and THg exposure, while Cd is mainly from animal-based foods. The mean concentrations of Pb, Cd and THg were 23.3, 1.1 and 2.3 µg/L in the whole blood, and 6.2, 1.0 and 2.0 µg/L in the morning urine, respectively. Combined exposure to THMs leading to 10.0 % and 7.1 % of Shanghai elderly at risk of neurotoxicity and nephrotoxicity. The results of this study have important implications for understanding the profiles of Pb, Cd and THg exposure in the elderly living in Shanghai and provide data support for risk assessment and control of nephrotoxicity and neurotoxicity from combined THMs exposure in the elderly.

Insertion of Hemin into Metal-Organic Frameworks: Mimicking Natural Peroxidase Microenvironment for the Rapid Ultrasensitive Detection of Uranium.

Constructing enzyme-like active sites in mimic enzyme systems is critical for achieving catalytic performances comparable to natural enzymes and can shed light on the natural development of enzymes. In this study, we described a specific hemin-based mimetic enzyme, which was facilely synthesized by the assembly of zeolitic imidazolate framework-l (ZIF-l) and hemin. The obtained hemin-based mimetic enzyme (denoted as ZIF-l-hemin) displayed enhanced peroxidase activity compared to free hemin in solution. Such excellent activity originated from the ZIF-l framework mimicking the active site cavity microenvironment of horseradish peroxidase in terms of axially coordinated histidine and distal histidine. Additionally, the constructed peroxidase mimetic was extremely resistant to a variety of severe circumstances that would normally denature natural enzymes. These characteristics made ZIF-l-hemin a potential platform for the colorimetric sensor of uranium (UO22+) with wide linear ranges (0.25-40 µM) and low limits of detection (0.079 µM). Moreover, the detection mechanism demonstrated that the coordination of uranyl ion with imidazole of ZIF-l-hemin reduced the catalytic efficiency of ZIF-l-hemin. The current work not only proposed a novel approach for fabricating artificial peroxidase but also offered facile colorimetric methods for selective radionuclide detection.

Full-length transcriptome reconstruction reveals genetic differences in hybrids of Oryza sativa and Oryza punctata with different ploidy and genome compositions.

BACKGROUND: Allopolyploid breeding is an efficient technique for improving the low seed setting rate of autotetraploids in plant breeding and one of the most promising breeding methods. However, there have been few comprehensive studies of the posttranscriptional mechanism in allopolyploids. RESULTS: By crossing cultivated rice (Oryza sativa, genome AA) with wild rice (Oryza punctata, genome BB), we created hybrid rice lines with different ploidy and genome compositions [diploid hybrid F01 (AB), allotetraploid hybrid F02 (AABB) and F03 (AAAB)]. The genetic differences of the hybrids and the mechanism of allopolyploid breeding dominance were revealed through morphological and cytological observations and single molecule real-time sequencing techniques. The tissues and organs of allotetraploid hybrid F02 exhibited "gigantism" and the highest levels of fertility. The numbers of non-redundant transcripts, gene loci and new isoforms in the polyploid rice lines were higher and the isoform lengths greater than those of the diploid line. Moreover, alternative splicing (AS) events occurred twice as often in the polyploid rice lines than the diploid line. During these events, intron retention dominated. Furthermore, a large number of new genes and isoforms specific to the lines of different ploidy were discovered. CONCLUSIONS: The results indicated that alternative polyadenylation (APA) and AS events contributed to the complexity and superiority of polyploids in the activity of translation regulators, nucleic acid binding transcription factor activities and the regulation of molecular function. Therefore, these APA and AS events in allopolyploid rice were found to play a role in regulation. Our study provides new germplasm for polyploid rice breeding and reveals complex regulatory mechanisms that may be related to heterosis and fertility.

Room temperature fabrication of magnetic covalent organic frameworks for analyzing sulfonamide residues in animal-derived foods.

A magnetic solid phase extraction method based on magnetic covalent organic frameworks (TpBD@Fe3 O4 ; 2,4,6-triformylphloroglucinol (Tp) and benzidine (BD)) combined with high performance liquid chromatography has been developed to detect the sulfonamides including sulfadiazine, sulfamerazine, sulfamethazine, and sulfamethoxazole in milk and meat. TpBD@Fe3 O4 were synthesized at room temperature under mild reaction conditions with a simple and rapid operation. The TpBD@Fe3 O4 exhibited higher extraction efficiency because of the π-π and electrostatic interactions between the benzene ring structure of the TpBD and the sulfonamide molecules. The extraction conditions including the dosage of adsorbents, the type and dosage of eluent, the elution time, and the pH of the sample solution were fully optimized. The detection results showed good linearity over a wide range (50-5 × 104 ng/mL) and low detection limits (3.39-5.77 ng/mL) for the sulfonamide targets. The practicability of this magnetic solidphase extraction-high-performance liquid chromatography method was further evaluated by analyzing milk and meat samples, with recoveries of the targets of 71.6-110.8% in milk and 71.9-109.7% in pork. The successful detection of sulfonamides residues has demonstrated the TpBD@Fe3 O4 excellent practical potential for analyzing pharmaceutical residues in animal-derived foods.

Heterogeneous distribution of PD-L1 expression in the IASLC/ATS/ERS classification of lung adenocarcinoma.

BACKGROUND: PD-L1 expression in tumor cells can predict the efficacy of PD-1/PD-L1 inhibitors and prognosis in patients. However, the correlation between the PD-L1 expression and the novel lung adenocarcinoma classification are obscure. METHODS: 94 lung adenocarcinoma cases were reviewed in the Third Affiliated Hospital of Soochow University from January to December 2019. PD-L1 (DAKO 22C3) was used to test the PD-L1 expression in lung cancer tissue. RESULT: TPS was used to interpret the PD-L1 expression. The negative, low positive and high positive of PD-L1 were 52 cases (55.30%), 29 cases (30.90%) and 13 cases (13.80%). The subtype ratio of acinar, lepidic and solid in adenocarcinoma were correlation with the PD-L1 TPS (r = - 0.37, P < 0.001; r = - 0.22, P = 0.013; r = 0.68, P < 0.001). The results of χ2 test showed the PD-L1 expression had the significant difference with gender (P = 0.027), age (P = 0.018), smoking history (P = 0.021), lymph node metastasis (P = 0.001), TNM stage (P = 0.002), acinar structure (P = 0.017) and solid structure (P < 0.001). Multi-factor linear regression results suggested that solid structure, TNM stage and smoking history were associated with PD-L1 expression (P < 0.05). The solid structure showed more capability to PD-L1 expression (ß = 0.398). CONCLUSION: PD-L1 expression was heterogeneity in lung adenocarcinoma. The solid structure, TNM stage and smoking history were correlation to up-regulation of PD-L1 expression, and solid structure was the most importance factor.

Integration of FGF21 Signaling and Metabolomics in High-Fat Diet-Induced Obesity.

Sex differences in obesity have been well established, but the metabolic mechanism underlying these differences remains unclear. In the present study, we determined the expression levels of endogenous fibroblast growth factor 21 (FGF21) and its related receptors in male and female mice that were fed a high-fat diet (HFD) for 12 weeks. We also analyzed the metabolic changes in serum and livers using a nuclear magnetic resonance-based metabolomics approach. Reverse transcription polymerase chain reaction and western blotting results revealed that the levels of FGFR1, FGFR2, and co-factor ß-klotho were upregulated in female mice to alleviate FGF21 resistance induced by HFD. The metabolomics results demonstrated that the serum and liver metabolic patterns of HFD-fed male mice were significantly separated from those of the female HFD-fed group and the normal diet group. Furthermore, low-density lipoprotein/very low density lipoprotein and betaine levels were associated with the resistance of exogenous HFD in female mice. These findings imply that sex-based differences in metabolism and susceptibility to obesity might be mediated by the FGF21 signaling pathway.

The Protective Effect of Basic Fibroblast Growth Factor in Intestine of db/db Mice: A 1H NMR-Based Metabolomics Investigation.

Diabetic enteropathy (DE) is a diabetic complication and affects the quality of life for which there are limited therapies. In this study, db/db mice were administered with a basic fibroblast growth factor (bFGF) to explore its therapeutic effect on the intestine. 1H NMR-based metabolomics was applied to investigate the metabolic pattern. H&E and PAS staining were used to observe the morphological phenotypes related to intestinal barrier function. Tight junction proteins such as Zo-1 and Occluding were successively tested by immunofluorescence and real-time PCR. We found that bFGF treatment significantly restored intestinal barrier function. In addition, the administration of bFGF decreased the levels of inflammatory cytokines in the cecum. Metabolomic results show that bFGF remodeled metabolic phenotypes of the colon, cecum, and small intestine in db/db mice, including energy metabolism, short chain fatty acid metabolism, amino acid metabolism, and choline metabolism. Hence, this study indicates that the bFGF has a protective effect in diabetic bowel disease by restoring intestinal barrier function, reducing inflammatory infiltration, and remodeling metabolic function.

Knockdown of Long Non-coding RNA LINC00200 Inhibits Gastric Cancer Progression by Regulating miR-143-3p/SERPINE1 Axis.

BACKGROUND: An increasing number of studies have found that long non-coding RNAs (lncRNAs) play an important role in carcinogenesis and tumor progression, whereas their molecular mechanisms of function remain largely unknown. AIMS: This study was aimed to explore the biological function and underlying mechanism of a new lncRNA LINC00200 in gastric cancer (GC). METHODS: qRT-PCR analysis was conducted to examine the LINC00200 expression level in both GC tissues and cell lines. Functional assays were carried out to detect the effect of LINC00200 on GC cell proliferation, invasion and migration. The interaction between LINC00200 and miR-143-3p was confirmed by luciferase reporter assays. Rescue assays were performed to confirm the influence of LINC00200-miR-143-3p-SERPINE1 axis on GC development. RESULTS: LINC00200 was found to be upregulated in GC tissues and cell lines. Moreover, knockdown of LINC00200 suppressed GC cell proliferation, invasion and migration in vitro and inhibited tumorigenesis in mouse xenografts. Finally, mechanism research indicated that LINC00200 functioned as a ceRNA to sponge for miR-143-3p, thus leading to the disinhibition of its target gene SERPINE1. CONCLUSIONS: LINC00200 is significantly overexpressed in GC and accelerates GC progression through regulating miR-143-3p/SERPINE1 axis. Our results may provide a potential diagnostic biomarker and therapeutic target for the management of GC patients.

Fast Super-Resolution Imaging Technique and Immediate Early Nanostructure Capturing by a Photoconvertible Fluorescent Protein.

Low temporal resolution and limited photocontrollable fluorescent protein probes have restricted the widespread application of single-molecule localization microscopy (SMLM). In the current study, we developed a new photoconvertible fluorescent protein (PCFP), pcStar, and quick single molecule-guided Bayesian localization microscopy (Quick-SIMBA). The combination of pcStar and Quick-SIMBA achieved the highest temporal resolution (0.1-0.25 s) with large field-of-view (76 × 9.4 µm2 -76 × 31.4 µm2) among the SMLM methods, which enabled the dynamic movements of the endoplasmic reticulum dense tubular matrix to be resolved. Moreover, pcStar extended the application of SMLM to imaging the immediate early nanostructures in Drosophila embryos and revealed a specific "parallel three-pillar" structure in the neuronal-glial cell junction, helping to elucidate glial cell "locking" and support of neurons during Drosophila embryogenesis.

NLRC3 negatively regulates CD4+ T cells and impacts protective immunity during Mycobacterium tuberculosis infection.

NLRC3, a member of the NLR family, has been reported as a negative regulator of inflammatory signaling pathways in innate immune cells. However, the direct role of NLRC3 in modulation of CD4+ T-cell responses in infectious diseases has not been studied. In the present study, we showed that NLRC3 plays an intrinsic role by suppressing the CD4+ T cell phenotype in lung and spleen, including differentiation, activation, and proliferation. NLRC3 deficiency in CD4+ T cells enhanced the protective immune response against Mycobacterium tuberculosis infection. Finally, we demonstrated that NLRC3 deficiency promoted the activation, proliferation, and cytokine production of CD4+ T cells via negatively regulating the NF-κB and MEK-ERK signaling pathways. This study reveals a critical role of NLRC3 as a direct regulator of the adaptive immune response and its protective effects on immunity during M. tuberculosis infection. Our findings also suggested that NLRC3 serves as a potential target for therapeutic intervention against tuberculosis.

Controllable and high-performance immobilized enzyme reactor: DNA-directed immobilization of multienzyme in polyamidoamine dendrimer-functionalized capillaries.

In recent years, CE-integrated immobilized enzyme reactors (IMERs) for single-enzyme immobilization have attracted considerable attention. However, there has been little research on multienzyme immobilization in CE. Here, we introduce a method for fabricating a CE-integrated IMER, using DNA-directed immobilization to fix glucose oxidase and horseradish peroxidase in the capillary, which had been functionalized with polyamidoamine dendrimer (PAMAM). Owing to the reversibility of DNA hybridization, the reactor is capable of dynamic immobilization. Moreover, by introducing the PAMAM, the loading capacity of the IMER is greatly enhanced, and the PAMAM can spontaneously form complexes with DNA and then contribute to the efficiency and stability of the reactor. After 25 days storage, the prepared IMER ultimately retained approximately 70% of its initial activity. We also used the IMER to detect glucose, and the favorable linearity was obtained over the concentration range of 0.78-12.5 mM, with an LOD of 0.39 mM, demonstrating that the CE-integrated IMER can be applied to actual samples. We believe that this strategy can be extended to other multienzyme immobilization systems, and CE-integrated IMERs are potentially useful in a wide range of biochemical research applications.

Prognostic impact of D2-plus lymphadenectomy and optimal extent of lymphadenectomy in advanced gastric antral carcinoma: Propensity score matching analysis.

OBJECTIVE: To investigate the prognostic impact of D2-plus lymphadenectomy including the posterior (No. 8p, No. 12b/p, No. 13, and No. 14v), and para-aortic (No. 16a2, and No. 16b1) lymph nodes (LNs) in subtotal gastrectomy for advanced gastric antral carcinoma. METHODS: A total of 203 patients with advanced gastric cancer (GC) located in the antrum, who underwent R0 gastrectomy with D2 or D2-plus lymphadenectomy between January 2003 and December 2011 were enrolled. Propensity score matching was used to reduce the strength of the confounding factors to accurately evaluate prognoses. The therapeutic value index (TVI) was calculate to evaluate the survival benefit of dissecting each LN station. RESULTS: Of 102 patients with D2-plus lymphadenectomy, 21 (20.59%) were pathologically identified as having LN metastases beyond the extent of D2 lymphadenectomy. After matching, the overall survival (OS) was significantly better in the D2-plus than the D2 group (P=0.030). In the multivariate survival analysis, D2-plus lymphadenectomy (hazard ratio, 0.516; P=0.006) was confirmed to significantly improve the survival rate. In the logistic regression analysis, pN stage [odds ratio (OR), 2.533; 95% confidence interval (95% CI), 1.368-4.691; P=0.003] and extent of LNs metastasis (OR, 5.965; 95% CI, 1.335-26.650; P=0.019) were identified as independent risk factors for LN metastases beyond the extent of D2 lymphadenectomy. The TVI of patient with metastasis to LNs station was 7.1 (No. 8p), 5.7 (No. 12p), 5.1 (No. 13), and 7.1 (both No. 16a2 and No. 16b1), respectively. CONCLUSIONS: D2-plus lymphadenectomy may improve the prognoses of some patients with advanced GC located in the antrum, especially for No. 8p, No. 12b, No. 13, and No. 16.

CRNDE promotes cell tongue squamous cell carcinoma cell growth and invasion through suppressing miR-384.

Tongue squamous cell carcinoma (TSCC) is the most common type of oral cancer and is an aggressive head and neck malignancy. Increasing studies have demonstrated that long noncoding RNAs (lncRNAs) play important roles in diverse biological cell processes, such as cell development, fate decisions, cell differentiation, cell migration, and invasion. In our study, we showed that long noncoding RNA colorectal neoplasia differentially expressed (CRNDE) expression was upregulated in TSCC cell lines and tissues. Overexpression of CRNDE increased the TSCC cell proliferation, cell cycle, and cell invasion. Moreover, ectopic expression of CRNDE inhibited the miR-384 expression in the SCC1 cell and increased the Kirsten Ras (KRAS), cell division cycle 42, and insulin receptor substrate 1 expression, which were the direct target genes of miR-384. We demonstrated that the miR-384 expression was downregulated in the TSCC samples compared with the paired adjacent nontumor samples. The expression of CRNDE was negatively correlated with the expression of miR-384 in the TSCC samples. Overexpression of miR-384 suppressed TSCC cell proliferation, cell cycle, and invasion. Furthermore, we demonstrated that CRNDE promoted TSCC cell proliferation and invasion through inhibiting miR-384 expression. These results suggested that CRNDE acts as an oncogene in the development of TSCC, which partially occurs through inhibiting miR-384 expression.

Hsa_circ_0009361 acts as the sponge of miR-582 to suppress colorectal cancer progression by regulating APC2 expression.

Circular RNA (circRNA) plays an important role in the development of human malignant tumors. Recently, an increasing number of circRNAs have been identified and investigated in various tumors. However, the expression pattern and biological function of circRNAs in colorectal cancer (CRC) still remain largely unexplored. In the present study, hsa_circ_0009361 was significantly down-regulated in CRC tissues and cells. Low expression level of hsa_circ_0009361 promoted the proliferation, epithelial-mesenchymal transition (EMT), migration, and invasion of CRC cells. Hsa_circ_0009361 was identified as the sponge of miR-582 by fluorescence in situ hybridization (FISH), RNA immunoprecipitation (RIP), and luciferase reporter assays. Overexpression of hsa_circ_0009361 up-regulated the expression of adenomatous polyposis coli 2 (APC2) and inhibited the activity of the Wnt/ß-catenin pathway by competitively combining with miR-582. Exogenous miR-582 and APC2 interventions could reverse the multiple biological functions mediated by hsa_circ_0009361 in CRC cells. In vivo experiments also confirmed that hsa_circ_0009361 inhibited the growth and metastasis of CRC. Hsa_circ_0009361 acted as a tumor suppressive sponge of miR-582, which could up-regulate the expression of APC2, inhibit the Wnt/ß-catenin signaling, and suppress the growth and metastasis of CRC. Collectively, the hsa_circ_0009361/miR-582/APC2 network could be employed as a potential therapeutic target for CRC patients.

Highly photostable, reversibly photoswitchable fluorescent protein with high contrast ratio for live-cell superresolution microscopy.

Two long-standing problems for superresolution (SR) fluorescence microscopy are high illumination intensity and long acquisition time, which significantly hamper its application for live-cell imaging. Reversibly photoswitchable fluorescent proteins (RSFPs) have made it possible to dramatically lower the illumination intensities in saturated depletion-based SR techniques, such as saturated depletion nonlinear structured illumination microscopy (NL-SIM) and reversible saturable optical fluorescence transition microscopy. The characteristics of RSFPs most critical for SR live-cell imaging include, first, the integrated fluorescence signal across each switching cycle, which depends upon the absorption cross-section, effective quantum yield, and characteristic switching time from the fluorescent "on" to "off" state; second, the fluorescence contrast ratio of on/off states; and third, the photostability under excitation and depletion. Up to now, the RSFPs of the Dronpa and rsEGFP (reversibly switchable EGFP) families have been exploited for SR imaging. However, their limited number of switching cycles, relatively low fluorescence signal, and poor contrast ratio under physiological conditions ultimately restrict their utility in time-lapse live-cell imaging and their ability to reach the desired resolution at a reasonable signal-to-noise ratio. Here, we present a truly monomeric RSFP, Skylan-NS, whose properties are optimized for the recently developed patterned activation NL-SIM, which enables low-intensity (∼100 W/cm(2)) live-cell SR imaging at ∼60-nm resolution at subsecond acquisition times for tens of time points over broad field of view.