CRISPR-Cas9 screening identifies INTS3 as an anti-apoptotic RNA-binding protein and therapeutic target for colorectal cancer.

Growing evidences indicate that RNA-binding proteins (RBPs) play critical roles in regulating the RNA splicing, polyadenylation, stability, localization, translation, and turnover. Abnormal expression of RBPs can promote tumorigenesis. Here, we performed a CRISPR screen using an RBP pooled CRISPR knockout library and identified 27 potential RBPs with role in supporting colorectal cancer (CRC) survival. We found that the deletion/depletion of INTS3 triggered apoptosis in CRC. The in vitro experiments and RNA sequencing revealed that INTS3 destabilized pro-apoptotic gene transcripts and contributed to the survival of CRC cells. INTS3 loss delayed CRC cells growth in vivo. Furthermore, delivery of DOTAP/cholesterol-mshINTS3 nanoparticles inhibited CRC tumor growth. Collectively, our work highlights the role of INTS3 in supporting CRC survival and provides several novel therapeutic targets for treatment.

OPEN STOMATA 1 phosphorylates CYCLIC NUCLEOTIDE-GATED CHANNELs to trigger Ca2+ signaling for abscisic acid-induced stomatal closure in Arabidopsis.

Multiple cyclic nucleotide-gated channels (CNGCs) are abscisic acid (ABA)-activated Ca2+ channels in Arabidopsis (Arabidopsis thaliana) guard cells. In particular, CNGC5, CNGC6, CNGC9, and CNGC12 are essential for ABA-specific cytosolic Ca2+ signaling and stomatal movements. However, the mechanisms underlying ABA-mediated regulation of CNGCs and Ca2+ signaling are still unknown. In this study, we identified the Ca2+-independent protein kinase OPEN STOMATA 1 (OST1) as a CNGC activator in Arabidopsis. OST1-targeted phosphorylation sites were identified in CNGC5, CNGC6, CNGC9, and CNGC12. These CNGCs were strongly inhibited by Ser-to-Ala mutations and fully activated by Ser-to-Asp mutations at the OST1-targeted sites. The overexpression of individual inactive CNGCs (iCNGCs) under the UBIQUITIN10 promoter in wild-type Arabidopsis conferred a strong dominant-negative-like ABA-insensitive stomatal closure phenotype. In contrast, expressing active CNGCs (aCNGCs) under their respective native promoters in the cngc5-1 cngc6-2 cngc9-1 cngc12-1 quadruple mutant fully restored ABA-activated cytosolic Ca2+ oscillations and Ca2+ currents in guard cells, and rescued the ABA-insensitive stomatal movement mutant phenotypes. Thus, we uncovered that ABA elicits cytosolic Ca2+ signaling via an OST1-CNGC module, in which OST1 functions as a convergence point of the Ca2+-dependent and -independent pathways in Arabidopsis guard cells.

Seismic Performance and Engineering Application Investigation of a New Alternative Retainer.

Focusing on the dilemma that the traditional lateral shear keys are ineffectual in limiting the displacement and repair of small-to-medium spanning highway bridges, this paper briefly describes the necessity of considering fiber-reinforced polymer concrete with the shear keys design, and studies the seismic performance of an alternative retainer that focuses on three functions of "limiting displacement", "energy consumption", and "alternative link". In order to study the anti-seismic effectiveness under the seismic loads, four alternative retainer specimens with different sizes were designed. The quasi-static tests were carried out on four specimens, respectively. The seismic damage mode of the quasi-static alternative retainer was investigated. We examined the influence of the designed parameter of the alternative retainer on the anti-seismic effectiveness of the alternative retainer. Taking a two-span simply supported girder bridge, for example, the comparison between the seismic response of the bridge with retainers and without is analyzed based on a consideration of the sliding plate rubber bearings and the test results of the new retainers. The results show that the failure mode of the new alternative retainers is a two-stage process involving the alternative links: firstly shear failure and then the overall retainer damages, which is convenient to retrofit and reinforce post-earthquake. The thickness of the web of the alternative link, as a sensitive factor, influences the bearing capacity of the new retainers, yield displacement, ultimate displacement, ductility coefficient and overall energy consumption. The height of the alternative link will merely influence the ultimate bearing capacity, and transverse replacement of the main girder with the new alternative retainers is greatly reduced compared to without retainers, and the seismic response increase in the pier is gentle.

A Study on the Dual Thermo- and pH-Responsive Behaviors of Well-Defined Star-like Block Copolymers Synthesize by Combining of RAFT Polymerization and Thiol-Ene Click Reaction.

A series of stimuli-responsive star-like block copolymers are synthesized via the combination of reversible addition, fragmentation chain transfer (RAFT) polymerization, and photo-initiated thiol-ene (PITE) click reaction. The controllable block ratio and block sequence, narrow distribution of molecular weight, and customized arm numbers of the star-shaped copolymers reveal the feasibility and benefits of combination of RAFT polymerization and PITE click reaction for synthesis of well-defined star-like (co)polymers. A clear insight into the relationship among the arm number, block sequence, and block ratio of the star-like block copolymers and their stimuli-responsive aggregation behavior was achieved via dynamic light scattering and UV-vis spectroscopy study. Notably, the star-like poly(acrylic acid)-b-poly(2-(dimethylamino) ethyl methacrylate) (star-PAA-b-PDMAEMA) shows higher lower critical solution temperature (LCST) compared to star-PDMAEMA-b-PAA with the same arm number and block ratio due to the inner charged PAA segments at pH > IEP. In addition, for star-like PAA-b-PDMAEMA, higher PAA content enhances the hydrophilicity of the polymer in basic solution and leads to the LCST increase, except for star-PAA1-b-PDMAEMA4 at pH = 9.0 (≈IEP). For star-PDMAEMA-b-PAA, the PAA content shows minimal effect on their LCSTs, except for the polymer in solution with pH = 9.5, which is far from their IEP. The star-like block copolymers with well-defined structure and tunable composition, especially the facile-controlled block sequence, bring us a challenging opportunity to control the stimuli-responsive properties of star-like block copolymers.

MiR-128-3p accelerates cardiovascular calcification and insulin resistance through ISL1-dependent Wnt pathway in type 2 diabetes mellitus rats.

Vascular calcification is highly prevalent in patients with type 2 diabetes mellitus (T2DM), one of the most common chronic diseases with high morbidity and mortality. In recent years, microRNAs have been widely reported as potential biomarkers for the diagnosis and treatment of T2DM. We hypothesized that miR-128-3p is associated with cardiovascular calcification and insulin resistance (IR) in rats with T2DM by targeting ISL1 via the Wnt pathway. Microarray analysis was adopted to identify differentially expressed genes related to T2DM. T2DM models were induced in rats. Blood samples from normal and T2DM rats were used to detect islet ß-cell function, islet sensitivity, and calcium content. Next, islet tissues were obtained to identify the expression of miR-128-3p, ISL1, and the Wnt signaling pathway- and apoptosis-related genes. Finally, apoptosis of islet ß-cells was determined by flow cytometry. Through microarray analysis of GSE27382 and GSE23343, ISL1 was found to be downregulated in T2DM. In blood samples from T2DM rats, basic biochemical indicators, IR, and calcium content were increased, and islet sensitivity and islet ß-cell function were decreased. Furthermore, upregulation of miR-128-3p and ISL1 gene silencing promoted the expression of Wnt-1, ß-catenin, GSK-3ß, and Bax and the phosphorylation of ß-catenin and GSK-3ß, inhibited c-fos, PDX-1, and Bcl-2 expression, and enhanced cell apoptosis. The key findings of our study demonstrate that miR-128-3p aggravates cardiovascular calcification and IR in T2DM rats by downregulating ISL1 through the activation of the Wnt pathway. Thus, miR-128-3p may serve as a potential target for the treatment of T2DM.

Correlation between overexpression of connective tissue growth factor, tumor progression, and clinical prognosis in endometrial cancer patients.

OBJECTIVE: To investigate the correlation between overexpression of connective tissue growth factor (CTGF), tumor progression, and clinical prognosis in patients with endometrial cancer. METHODS: Tumor samples were obtained from 198 patients with endometrial cancer who underwent hysterectomy, and 50 samples were collected from normal endometrial tissue. Immunohistochemical staining was performed on all samples. Scoring was carried out by two independent pathologists experienced in evaluating immunohistochemical staining. RESULTS: Normal endometrial specimens exhibited little or no CTGF immunoreactivity. CTGF was localized mainly in the cytoplasm of the tumor cells. Of the endometrial cancer specimens examined, 95 (48%) of 198 patients were negative for CTGF, whereas 103 (52%) of 198 patients were positive for CTGF. No significant correlation was noted between the level of CTGF and patient age (P=0.81), blood pressure (P=0.76), blood glucose (P=0.51) or vascular/lymphatic invasion (P=0.15). However, positive CTGF expression showed a strong association with the level of CA125 (P=0.02), histologic grade (P=0.004), depth of myometrial invasion (P=0.028), and FIGO stage (P=0.025). Independent predictive value for overall survival was shown for positive CTGF expression (P<0.001), lymph node status (P<0.001) as well as lymphovascular invasion (P=0.02). CONCLUSION: CTGF is an independent prognostic factor of endometrial cancer. CTGF expression may play a critical role in progression of endometrial cancer and is significantly associated with poor prognosis.

MiR-195-5p targeting FGF2 inhibits malignant biological behaviors of endometrial carcinoma HEC-1B cells / 中国肿瘤生物治疗杂志

@# Objective: To explore the molecular mechanism of miR-195-5p targeting FGF2 to inhibit the proliferation, apoptosis, invasion and migration of endometrial cancer HEC-1B cells. Methods: After culture and transfection, HEC-1B cells were divided into 4 groups: HEC-1B group, miR-195-5p mimic group, pLV-FGF2 group and miR-195-5p+FGF2 group. The expressions of miR-195-5p and mRNA levels of FGF2 were detected by qRT-PCR. The targeted relationship of miR-195-5p and FGF2 was verified by luciferase assay. The protein expression of FGF2 was examined by Western blotting; Proliferation of HEC-1B cells was measured by CCK-8; Apoptosis was tested by flow cytometry; HEC-1B cell invasion was detected by transwell, and migration was measured by scratch assay. Results: Compared with HEC-1B group, the expression of miR-195-5p in miR-195-5p mimic group was elevated while FGF2 mRNA level was declined (all P<0.01). Luciferase assay indicated that FGF2 was a target of miR-195-5p. Compared with HEC-1B group, the protein level of FGF2 in miR-195-5p mimic group was decreased, and the protein levels of FGF2 in pLV-FGF2 group were enhanced (P<0.01). The protein levels of FGF2 in miR-195-5p+FGF2 group were lower than that in pLV-FGF2 group (all P<0.01). The proliferation in miR-195-5p mimic group was lower than HEC-1B group (P<0.01), while the proliferation in pLV-FGF2 group was higher than that in HEC-1B group (all P<0.01). Compared with HEC-1B group, apoptosis in miR-195-5p mimic group was increased, and apoptosis in pLV-FGF2 group was decreased (P<0.01); moreover, apoptosis in miR-195-5p+FGF2 group was higher than that in pLV-FGF2 group (P<0.01). Compared with HEC-1B group, the number of invasive cells per field and the rate of wound healing in miR195-5p mimic group were decreased, while those in pLV-FGF2 group was enhanced (P<0.01); moreover, the number of invasive cells per field and the rate of wound healing in miR-195-5p+FGF2 group was lower than those in pLV-FGF2 group (all P<0.01). Conclusion: miR-195-5p inhibits proliferation, invasion and migration and promotes apoptosis of endometrial cancer HEC-1B cells by targeting FGF2, and could be used as a treatment target of endometrial cancer.

Adaptive Neural Tracking Control for Switched High-Order Stochastic Nonlinear Systems.

This paper deals with adaptive neural tracking control design for a class of switched high-order stochastic nonlinear systems with unknown uncertainties and arbitrary deterministic switching. The considered issues are: 1) completely unknown uncertainties; 2) stochastic disturbances; and 3) high-order nonstrict-feedback system structure. The considered mathematical models can represent many practical systems in the actual engineering. By adopting the approximation ability of neural networks, common stochastic Lyapunov function method together with adding an improved power integrator technique, an adaptive state feedback controller with multiple adaptive laws is systematically designed for the systems. Subsequently, a controller with only two adaptive laws is proposed to solve the problem of over parameterization. Under the designed controllers, all the signals in the closed-loop system are bounded-input bounded-output stable in probability, and the system output can almost surely track the target trajectory within a specified bounded error. Finally, simulation results are presented to show the effectiveness of the proposed approaches.