The 5'UTR of HCoV-OC43 adopts a topologically constrained structure to intrinsically repress translation.

The emergence of SARS-CoV-2, which is responsible for the COVID-19 pandemic, has highlighted the need for rapid characterization of viral mechanisms associated with cellular pathogenesis. Viral UTRs represent conserved genomic elements that contribute to such mechanisms. Structural details of most CoV UTRs are not available, however. Experimental approaches are needed to allow for the facile generation of high-quality viral RNA tertiary structural models, which can facilitate comparative mechanistic efforts. By integrating experimental and computational techniques, we herein report the efficient characterization of conserved RNA structures within the 5'UTR of the HCoV-OC43 genome, a lab-tractable model coronavirus. We provide evidence that the 5'UTR folds into a structure with well-defined stem-loops (SLs) as determined by chemical probing and direct detection of hydrogen bonds by NMR. We combine experimental base-pair restraints with global structural information from SAXS to generate a 3D model that reveals that SL1-4 adopts a topologically constrained structure wherein SLs 3 and 4 coaxially stack. Coaxial stacking is mediated by short linker nucleotides and allows SLs 1 to 2 to sample different cojoint orientations by pivoting about the SL3,4 helical axis. To evaluate the functional relevance of the SL3,4 coaxial helix, we engineered luciferase reporter constructs harboring the HCoV-OC43 5'UTR with mutations designed to abrogate coaxial stacking. Our results reveal that the SL3,4 helix intrinsically represses translation efficiency since the destabilizing mutations correlate with increased luciferase expression relative to wildtype without affecting reporter mRNA levels, thus highlighting how the 5'UTR structure contributes to the viral mechanism.

The RNA-binding protein AUF1 facilitates Akt phosphorylation at the membrane.

Mammalian target of rapamycin (mTOR), which is part of mTOR complex 1 (mTORC1) and mTORC2, controls cellular metabolism in response to levels of nutrients and other growth signals. A hallmark of mTORC2 activation is the phosphorylation of Akt, which becomes upregulated in cancer. How mTORC2 modulates Akt phosphorylation remains poorly understood. Here, we found that the RNA-binding protein, AUF1 (ARE/poly(U)-binding/degradation factor 1), modulates mTORC2/Akt signaling. We determined that AUF1 is required for phosphorylation of Akt at Thr308, Thr450, and Ser473 and that AUF1 also mediates phosphorylation of the mTORC2-modulated metabolic enzyme glutamine fructose-6-phosphate amidotransferase 1 at Ser243. In addition, AUF1 immunoprecipitation followed by quantitative RT-PCR revealed that the mRNAs of Akt, glutamine fructose-6-phosphate amidotransferase 1, and the mTORC2 component SIN1 associate with AUF1. Furthermore, expression of the p40 and p45, but not the p37 or p42, isoforms of AUF1 specifically mediate Akt phosphorylation. In the absence of AUF1, subcellular fractionation indicated that Akt fails to localize to the membrane. However, ectopic expression of a membrane-targeted allele of Akt is sufficient to allow Akt-Ser473 phosphorylation despite AUF1 depletion. Finally, conditions that enhance mTORC2 signaling, such as acute glutamine withdrawal, augment AUF1 phosphorylation, whereas mTOR inhibition abolishes AUF1 phosphorylation. Our findings unravel a role for AUF1 in promoting membrane localization of Akt to facilitate its phosphorylation on this cellular compartment. Targeting AUF1 could have therapeutic benefit for cancers with upregulated mTORC2/Akt signaling.

Hepatectomy After Conversion Therapy Using Tyrosine Kinase Inhibitors Plus Anti-PD-1 Antibody Therapy for Patients with Unresectable Hepatocellular Carcinoma.

BACKGROUND: Combined treatment with tyrosine kinase inhibitors (TKI) plus anti-PD-1 antibodies showed high anti-tumor efficacy and made conversion resection possible for patients with unresectable hepatocellular carcinoma (HCC). However, long-term survival has not been reported. METHODS: A cohort of consecutive patients who received combined TKI/anti-PD-1 antibodies as first-line treatment for initially unresectable HCC at the authors' hospital between August 2018 and September 2020 was eligible for this study. Patients who were responding to systemic therapy and met the criteria for hepatectomy underwent liver resection with curative intention. The study also investigated the association of clinical factors with successful conversion resection and postoperative recurrence. RESULTS: The study enrolled 101 patients including 24 patients (23.8 %) who underwent R0 resection a median of 3.9 months (interquartile range: 2.5-5.9 months) after initiation of systemic therapy. Patients with an Eastern cooperative oncology group performance status of 0, fewer intrahepatic tumors, or a radiographic response to systemic therapy were more likely to be able to receive curative resection. After a median follow-up period of 21.5 months, hepatectomy was independently associated with a favorable overall survival (hazard ratio [HR], 0.050; 95 % confidence interval [CI], 0.007-0.365; P = 0.003). For the 24 patients who underwent surgery, the 12-month recurrence-free survival and overall survival rates were respectively 75% and 95.8%. Achieving a pathologic complete response (n = 10) to systemic therapy was associated with a favorable recurrence-free survival after resection, with a trend toward significance (HR, 0.345; 95% CI, 0.067-1.785; P = 0.187). CONCLUSIONS: Selected patients with initially unresectable HCC can undergo hepatectomy after systemic therapy with combined TKI/anti-PD-1 antibodies. In this study, conversion resection was associated with a favorable prognosis.

Ratiometric Fluorescent Sensor Based on Tb(III) Functionalized Metal-Organic Framework for Formic Acid.

Formic acid is a common chemical raw material, the effective detection of which is of importance to food safety and environmental quality. In this work, the lanthanide functionalized dual-emission metal-organic framework (TH25) was prepared as a ratiometric fluorescent sensor for formic acid. This ratiometric sensor has a good detection performance with high selectivity, sensitivity, and reproducibility. Together with a low limit of detection of 2.1 ppm, these characters promise the ability to sense at low levels as well as a practical detection ability. This work provides ideas for the design and synthesis of effective chemical sensors for organic acids.

Functional analyses of mammalian virus 5'UTR-derived, small RNAs that regulate virus translation.

Enterovirus A71 (EV-A711) RNA contains an internal ribosomal entry site (IRES) to direct cap-independent translation. IRES-dependent translation requires the host's translation initiation factors and IRES-associated trans-acting factors (ITAFs). We previously showed that hnRNP A1, the mRNA stability factor HuR, and the RISC subunit Argonaute 2 (Ago2) are ITAFs that associate with stem loop II (SL-II) of the IRES and promote IRES-dependent translation. By contrast, the mRNA decay factor AUF1 is a negative-acting ITAF that also binds SL-II. Moreover, the small RNA-processing enzyme Dicer produces at least four virus-derived, small RNAs (vsRNAs 1-4) from the EV-A71 5'UTR in infected cells. One of these, vsRNA1, derived from SL-II, inhibits IRES activity via an unknown mechanism. In vitro RNA-binding assays revealed that vsRNA1 can alter association of Ago2, HuR, and AUF1 with SL-II. This presents a possible mechanism by which vsRNA1 could control association of ITAFs with the IRES and modulate viral translation. Here, we describe methods for functional analyses of vsRNA1-mediated regulation of IRES activity. These methods should be applicable to other virus-derived, small RNAs as well.

Impact of sampling and storage stress on the recovery of airborne SARS-CoV-2 virus surrogate captured by filtration.

Environmental air sampling of the SARS-CoV-2 virus in occupational and community settings is pertinent to reduce and monitor the spread of the COVID pandemic. However, there is a general lack of standardized procedures for airborne virus sampling and limited knowledge of how sampling and storage stress impact the recovery of captured airborne viruses. Since filtration is one of the commonly used methods to capture airborne viruses, this study analyzed the effect of sampling and storage stress on SARS-CoV-2 surrogate virus (human coronavirus OC43, or HCoV-OC43) captured by filters. HCoV-OC43, a simulant of the SARS-CoV-2, was aerosolized and captured by PTFE-laminated filters. The impact of sampling stress was evaluated by comparing the RNA yields recovered when sampled at 3 L/min and 10 L/min and for 10 min and 60 min; in one set of experiments, additional stress was added by passing clean air through filters with the virus for 1, 5, and 15 hr. The impact of storage stress was designed to examine RNA recovery from filters at room temperature (25 °C) and refrigerated conditions (4 °C) for up to 1 week of storage. To our knowledge, this is the first report on using HCoV-OC43 aerosol in air sampling experiments, and the mode diameter of the virus aerosolized from the growth medium was 40-60 nm as determined by SMPS + CPC system (TSI Inc.) and MiniWRAS (Grimm Inc.) measurements. No significant difference was found in virus recovery between the two sampling flow rates and different sampling times (p > 0.05). However, storage at room temperature (25 °C) yielded ∼2x less RNA than immediate processing and storage at refrigerated conditions (4 °C). Therefore, it is recommended to store filter samples with viruses at 4 °C up to 1 week if the immediate analysis is not feasible. Although the laminated PTFE filter used in this work purposefully does not include a non-PTFE backing, the general recommendations for handling and storing filter samples with viral particles are likely to apply to other filter types.

[Effects of sperm DNA fragmentation index on semen parameters and pregnancy outcomes in intrauterine insemination].

Objective: To analyze the correlation of the sperm DNA fragmentation index (DFI) level with semen parameters and pregnancy outcomes of artificial insemination of the husband (AIH) in the cycle of intrauterine insemination (IUI). METHODS: We collected the clinical data on 777 cases of IUI, including female clinical indicators, male semen parameters, sperm DFI and pregnancy outcomes. According to the DFI level, we divided the patients into three groups: DFI < 15%, 15% ≤ DFI < 30% and DFI ≥ 30%. RESULTS: The sperm DFI level was significantly elevated with the increased age of the males (P = 0.002) and closely related to the total number of motile sperm (P = 0.002) and total sperm motility (P = 0.000) before treatment, as well as to sperm concentration (P = 0.000), total sperm motility (P = 0.001) and total number of progressively motile sperm (P = 0.000) after density gradient centrifugation. The rate of clinical pregnancy was decreased in the DFI ≥ 30% group. There were no statistically significant differences between sperm DFI and the rates of clinical pregnancy and abortion. CONCLUSIONS: Male age significantly affects the sperm DFI level. Sperm DFI is closely related to sperm motility and total number of progressively motile sperm, but not to the rates of clinical pregnancy and abortion in patients undergoing IUI. IUI can be used as an effective method of assisted reproduction for male infertility./.

MOF-derived Ni3S4 Encapsulated in 3D Conductive Network for High-Performance Supercapacitor.

Transition-metal sulfide is a good kind of material for supercapacitors because of the large capacity. Nevertheless, the low electroconductivity, slow reaction kinetics, and limited active centers lead to poor electrochemical properties such as long-term cycling stability. In the present work, nano nickel metal-organic framework (Ni-MOF) was constructed by using the nitrogen-rich functional group ligand 2,4,6-tris(3,5-dicarboxylphenylamino)-1,3,5-triazin and compounded with carbon nanotubes (CNTs) to prepare Ni-MOF/CNTs composite, which was used as a precursor to prepare the MOFs-derived NC/Ni-Ni3S4/CNTs composite with the Ni3S4 uniformly distributed in the three-dimensional (3D) conductive network. The rich nitrogen doping and 3D conductive network constructed by CNTs improved the conductivity, prompted the rapid entry of electrolyte, and improved the reaction kinetics of NC/Ni-Ni3S4/CNTs, thus obtained excellent specific capacitance, coulomb efficiency, and cyclic stability. The specific capacitance of NC/Ni-Ni3S4/CNTs is 1489.9 F/g at 1 A/g, which remains 800 F/g at 10 A/g, showing good rate performance.

Chalcone flavokawain A attenuates TGF-ß1-induced fibrotic pathology via inhibition of ROS/Smad3 signaling pathways and induction of Nrf2/ARE-mediated antioxidant genes in vascular smooth muscle cells.

TGF-ß1 plays a crucial role in the pathogenesis of vascular fibrotic diseases. Chalcones are reportedly cancer chemo-preventive food components that are rich in fruits and vegetables. In this study, flavokawain A (FKA, 2-30 µM), a naturally occurring chalcone in kava extracts, was evaluated for its anti-fibrotic and antioxidant properties in TGF-ß1-stimulated vascular smooth muscle (A7r5) cells, as well as its underlying molecular mechanism of action. Immunofluorescence data showed down-regulated F-actin expression with FKA treatment in TGF-ß1-stimulated A7r5 cells. Western blotting demonstrated that FKA treatment suppressed the expression of α-SMA and fibronectin proteins under TGF-ß1 stimulation. Findings from wound-healing and invasion experiments showed that FKA inhibits TGF-ß1-mediated migration and invasion. Western blotting demonstrated that treatment with FKA down-regulated MMP-9 and MMP-2 and up-regulated TIMP-1 expression. Further evidence showed that FKA decreased TGF-ß1-mediated phosphorylation and the transcriptional activity of Smad3. TGF-ß1-induced excessive ROS production was remarkably reversed by FKA treatment in A7r5 cells, and inhibition by FKA or N-acetylcysteine (NAC) substantially diminished TGF-ß1-induced p-Smad3 activation and wound-healing migration. Interestingly, FKA-mediated antioxidant properties were associated with increased nuclear translocation of Nrf2 and elevated antioxidant response element (ARE) luciferase activity. Activation of Nrf2/ARE signaling was accompanied by the induction of HO-1, NQO-1 and γ-GCLC genes in FKA-treated A7r5 cells. Notably, silencing of Nrf2 (siRNA transfection) significantly diminished the FKA-mediated antioxidant effects, indicating that FKA may inhibit TGF-ß1-induced fibrosis through suppressing ROS generation in A7r5 cells. Our results suggested that anti-fibrotic and antioxidant activities of the chalcone flavokawain A may contribute to the development of food-based chemo-preventive drugs for fibrotic diseases.

Anticancer activities of chalcone flavokawain B from Alpinia pricei Hayata in human lung adenocarcinoma (A549) cells via induction of reactive oxygen species-mediated apoptotic and autophagic cell death.

Chalcones found in fruits and vegetables have promising cancer chemopreventive properties. This study attempts to identify the anticancer efficacies of chalcone flavokawain B (FKB) in the rhizomes of Alpinia pricei Hayata by examining key molecular events in non-small-cell lung cancer (A549) cells. Our results indicated that in human A549 cells, FKB (0-15 µg/ml) decreases cell viability and colony formation, dysregulates the Bax:B-cell lymphoma 2 ratio and increases apoptotic DNA fragmentation. Mitochondrial (caspase-9/-3 and poly ADP ribose polymerase [PARP]) signaling was found to be involved in FKB-induced apoptosis. In addition, FKB-induced reactive oxygen species (ROS) generation, and N-acetylcysteine attenuated FKB-induced apoptotic cell death. Moreover, FKB triggered autophagy, as evidenced by the improved acidic vesicular organelle formation, lipidated light chain 3 (microtubule-related light chain 3) accumulation, and ATG7 expression and the decreased mammalian target of rapamycin phosphorylation. Furthermore, FKB suppressed ROS-mediated ATG4B expression. Inhibiting autophagy using 3-methyladenine/chloroquine diminished FKB-induced cell death, indicating that autophagy is triggered as a death mechanism by FKB. In summary, FKB has a crucial role in the execution and propagation of ROS-mediated apoptotic and autophagic cell death of lung adenocarcinoma cells.

Antrodia camphorata inhibits epithelial-to-mesenchymal transition by targeting multiple pathways in triple-negative breast cancers.

Antrodia camphorata (AC) exhibits potential for engendering cell-cycle arrest as well as prompting apoptosis and metastasis inhibition in triple-negative breast cancer (TNBC) cells. We performed the current study to explore the anti-epithelial-to-mesenchymal transition (EMT) properties of fermented AC broth in TNBC cells. Our results illustrated that noncytotoxic concentrations of AC (20-60 µg/ml) reversed the morphological changes (fibroblastic-to-epithelial phenotype) as well as the EMT by upregulating the observed E-cadherin expression. Furthermore, we discovered treatment with AC substantially inhibit the Twist expression in human TNBC (MDA-MB-231) cells as well as in those that were transfected with Twist. In addition, we determined AC to decrease the observed Wnt/ß-catenin nuclear translocation through a pathway determined to be dependent on GSK3ß. Notably, AC treatment consistently inhibited the EMT by downregulating mesenchymal marker proteins like N-cadherin, vimentin, Snail, ZEB-1, and fibronectin; at that same time upregulating epithelial marker proteins like occludin and ZO-1. Bioluminescence imaging that was executed in vivo demonstrated AC substantially suppressed breast cancer metastasis to the lungs. Notably, we found that western blot analysis confirmed that AC decreased lung metastasis as demonstrated by upregulation of E-cadherin expression in biopsied lung tissue. Together with our results support the anti-EMT activity of AC, indicating AC as having the potential for acting as an anticancer agent for the treatment of human TNBC treatment.

Intake of Molecular Hydrogen in Drinking Water Increases Membrane Transporters, p-Glycoprotein, and Multidrug Resistance-Associated Protein 2 without Affecting Xenobiotic-Metabolizing Enzymes in Rat Liver.

Molecular hydrogen (H2) has been shown to have antioxidant and anti-inflammatory activities that may reduce the development and progression of many diseases. In this study, hydrogen-rich water (HRW) was obtained by reacting hybrid magnesium-carbon hydrogen storage materials with water. Then, the effects of intake of HRW on the activities of xenobiotic-metabolizing enzymes, membrane transporters, and oxidative stress in rats were investigated. Rats were given HRW ad libitum for four weeks. The results showed that intake of HRW had no significant effect on the activities of various cytochrome P450 (CYP) enzymes (CYP1A1, 1A2, 2B, 2C, 2D, 2E1, 3A, and 4A), glutathione-S-transferase, and Uridine 5'-diphospho (UDP)-glucuronosyltransferase. Except for a mild lower plasma glucose concentration, intake of HRW had no effect on other plasma biochemical parameters in rats. p-Glycoprotein and multidrug resistance-associated protein (Mrp) 2 protein expressions in liver were elevated after intake of HRW. However, HRW had no significant effects on glutathione, glutathione peroxidase, or lipid peroxidation in liver. The results from this study suggest that consumption of HRW may not affect xenobiotic metabolism or oxidative stress in liver. However, intake of HRW may increase the efflux of xenobiotics or toxic substances from the liver into bile by enhancing p-glycoprotein and Mrp2 protein expressions.

Additive Promotion of Viral Internal Ribosome Entry Site-Mediated Translation by Far Upstream Element-Binding Protein 1 and an Enterovirus 71-Induced Cleavage Product.

The 5' untranslated region (5' UTR) of the enterovirus 71 (EV71) RNA genome contains an internal ribosome entry site (IRES) that is indispensable for viral protein translation. Due to the limited coding capacity of their RNA genomes, EV71 and other picornaviruses typically recruit host factors, known as IRES trans-acting factors (ITAFs), to mediate IRES-dependent translation. Here, we show that EV71 viral proteinase 2A is capable of cleaving far upstream element-binding protein 1 (FBP1), a positive ITAF that directly binds to the EV71 5' UTR linker region to promote viral IRES-driven translation. The cleavage occurs at the Gly-371 residue of FBP1 during the EV71 infection process, and this generates a functional cleavage product, FBP11-371. Interestingly, the cleavage product acts to promote viral IRES activity. Footprinting analysis and gel mobility shift assay results showed that FBP11-371 similarly binds to the EV71 5' UTR linker region, but at a different site from full-length FBP1; moreover, FBP1 and FBP11-371 were found to act additively to promote IRES-mediated translation and virus yield. Our findings expand the current understanding of virus-host interactions with regard to viral recruitment and modulation of ITAFs, and provide new insights into translational control during viral infection.

Overexpression of MTERF4 promotes the amyloidogenic processing of APP by inhibiting ADAM10.

Alzheimer's disease (AD) is characterized by the deposition of ß-amyloid (Aß) peptide in the brain, which is produced by the proteolysis of ß-amyloid precursor protein (APP). Recently, the mitochondrial transcription factor 4 (MTERF4), a member of the MTERF family, was implicated in regulating mitochondrial DNA transcription and directly in controlling mitochondrial ribosomal translation. The present study identified a novel role for MTERF4 in shifting APP processing toward the amyloidogenic pathway. The levels of MTERF4 protein were significantly increased in the hippocampus of APP/PS1 mice. In addition, the overexpression of MTERF4 induced a significant increase in the levels of APP protein and secreted Aß42 in HEK293-APPswe cells compared with control cells. Further, MTERF4 overexpression shifted APP processing from α-to ß-cleavage, as indicated by decreased C83 levels and elevated C99 levels. Finally, the MTERF4 overexpression suppressed a disintegrin and metalloproteinase 10 (ADAM10) expression via a transcriptional mechanism. Taken together, these results suggest that MTERF4 promotes the amyloidogenic processing of APP by inhibiting ADAM10 in HEK293-APPswe cells; therefore, MTERF4 may play an important role in the pathogenesis of AD.

Antrodia camphorata attenuates cigarette smoke-induced ROS production, DNA damage, apoptosis, and inflammation in vascular smooth muscle cells, and atherosclerosis in ApoE-deficient mice.

Cigarette smoke exposure activates several cellular mechanisms predisposing to atherosclerosis, including oxidative stress, dyslipidemia, and vascular inflammation. Antrodia camphorata, a renowned medicinal mushroom in Taiwan, has been investigated for its antioxidant, anti-inflammatory, and antiatherosclerotic properties in cigarette smoke extracts (CSE)-treated vascular smooth muscle cells (SMCs), and ApoE-deficient mice. Fermented culture broth of Antrodia camphorata (AC, 200-800 µg/mL) possesses effective antioxidant activity against CSE-induced ROS production. Treatment of SMCs (A7r5) with AC (30-120 µg/mL) remarkably ameliorated CSE-induced morphological aberrations and cell death. Suppressed ROS levels by AC corroborate with substantial inhibition of CSE-induced DNA damage in AC-treated A7r5 cells. We found CSE-induced apoptosis through increased Bax/Bcl-2 ratio, was substantially inhibited by AC in A7r5 cells. Notably, upregulated SOD and catalase expressions in AC-treated A7r5 cells perhaps contributed to eradicate the CSE-induced ROS generation, and prevents DNA damage and apoptosis. Besides, AC suppressed AP-1 activity by inhibiting the c-Fos/c-Jun expressions, and NF-κB activation through inhibition of I-κBα degradation against CSE-stimulation. This anti-inflammatory property of AC was accompanied by suppressed CSE-induced VEGF, PDGF, and EGR-1 overexpressions in A7r5 cells. Furthermore, AC protects lung fibroblast (MRC-5) cells from CSE-induced cell death. In vivo data showed that AC oral administration (0.6 mg/d/8-wk) prevents CSE-accelerated atherosclerosis in ApoE-deficient mice. This antiatherosclerotic property was associated with increased serum total antioxidant status, and decreased total cholesterol and triacylglycerol levels. Thus, Antrodia camphorata may be useful for prevention of CSE-induced oxidative stress and diseases. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 2070-2084, 2017.

A cytoplasmic RNA virus generates functional viral small RNAs and regulates viral IRES activity in mammalian cells.

The roles of virus-derived small RNAs (vsRNAs) have been studied in plants and insects. However, the generation and function of small RNAs from cytoplasmic RNA viruses in mammalian cells remain unexplored. This study describes four vsRNAs that were detected in enterovirus 71-infected cells using next-generation sequencing and northern blots. Viral infection produced substantial levels (>10(5) copy numbers per cell) of vsRNA1, one of the four vsRNAs. We also demonstrated that Dicer is involved in vsRNA1 generation in infected cells. vsRNA1 overexpression inhibited viral translation and internal ribosomal entry site (IRES) activity in infected cells. Conversely, blocking vsRNA1 enhanced viral yield and viral protein synthesis. We also present evidence that vsRNA1 targets stem-loop II of the viral 5' untranslated region and inhibits the activity of the IRES through this sequence-specific targeting. Our study demonstrates the ability of a cytoplasmic RNA virus to generate functional vsRNA in mammalian cells. In addition, we also demonstrate a potential novel mechanism for a positive-stranded RNA virus to regulate viral translation: generating a vsRNA that targets the IRES.

Combinatorial mRNA binding by AUF1 and Argonaute 2 controls decay of selected target mRNAs.

The RNA-binding protein AUF1 binds AU-rich elements in 3'-untranslated regions to regulate mRNA degradation and/or translation. Many of these mRNAs are predicted microRNA targets as well. An emerging theme in post-transcriptional control of gene expression is that RNA-binding proteins and microRNAs co-regulate mRNAs. Recent experiments and bioinformatic analyses suggest this type of co-regulation may be widespread across the transcriptome. Here, we identified mRNA targets of AUF1 from a complex pool of cellular mRNAs and examined a subset of these mRNAs to explore the links between RNA binding and mRNA degradation for both AUF1 and Argonaute 2 (AGO2), which is an essential effector of microRNA-induced gene silencing. Depending on the specific mRNA examined, AUF1 and AGO2 binding is proportional/cooperative, reciprocal/competitive or independent. For most mRNAs in which AUF1 affects their decay rates, mRNA degradation requires AGO2. Thus, AUF1 and AGO2 present mRNA-specific allosteric binding relationships for co-regulation of mRNA degradation.

[Pharmacokinetics and MR imaging of SPIO-shRNA dual functional molecular probe in vivo].

In this study, we investigated the pharmacokinetics parameters of SPIO-shRNA dual functional molecular probe and observed the main organ distribution by MRI in vivo. Eighteen New Zealand white rabbits were randomly divided into three groups and injected intravenously with different doses of SPIO-shRNA molecular probe, respectively. The blood samples were collected to analyze the pharmacokinetic parameters by measuring the iron content at 30 minutes before and after the injection. Twenty-four Kun Ming (KM) mice were randomly divided into 4 groups: the control group was injected intravenously with physiological saline 200 µL per mouse via the tail vein, the other 3 groups were injected intravenously with different doses of SPIO-shRNA molecular probe. MRI observation was performed in 24 hours, and the liver, spleen, kidney, brain and muscle were collected for iron quantification with Prussian blue staining to determine distribution of the SPIO-shRNA molecular probe in the main organ in vivo. Our results suggest that the molecular probe blood half-life is more than 3 hours. The data of MRI suggest the probe was distributed in liver and spleen, and the MRI signal was reduced with the increase in probe's doses (P < 0.05). The results of Prussian blue staining confirmed the results of MRI. Most of the probe could escape the phagocytosis of mononuclear phagocyte system. Our data provide the pharmacokinetic and distribution of SPIO-shRNA molecular probe in organs. Meanwhile, it suggests the choice of the time and dose of probe for MR imaging of tumor in vivo.