Functional mechanism and clinical implications of miR-141 in human cancers.

Cancer is caused by the abnormal proliferation of local tissue cells under the control of many oncogenic factors. MicroRNAs (miRNAs) are a class of evolutionarily conserved, approximately 22-nucleotide noncoding small RNAs that influence transcriptional regulationby binding to the 3'-untranslated region of target messenger RNA. As a member of the miRNA family, miR-141 acts as a suppressor or an oncomiR in various cancers and regulates cancer cell proliferation, apoptosis, invasion, and metastasis through a variety of signaling pathways, such as phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) and constitutive activation of nuclear factor-κB (NF-κB). Target gene validation and pathway analysis have provided mechanistic insight into the role of this miRNA in different tissues. This review also outlines novel findings that suggest miR-141 may be useful as a noninvasive biomarker and as a therapeutic target in several cancers.

[Pollution Characterization, Source Identification, and Health Risks of Atmospheric Particle-Bound Heavy Metals in PM2.5 in Zhengzhou City: Based on High-resolution Data].

In order to study the pollution characteristics and sources of heavy metals in urban atmospheric PM2.5, 21 elements in atmospheric PM2.5 in Zhengzhou City were detected using an online metal analyzer during July and October 2017 and January and April 2018, and the changes in heavy metal concentrations were analyzed. Heavy metals were traced by enrichment factors, principal component analysis, and potential source function. The US EPA risk assessment model was used to assess their health risks. The results showed that:the concentrations of K, Zn, Mn, Pb, Cu, As, Cr, and Se increased with the increase in pollution level. The results of enrichment factors and principal component analysis showed that the main sources of heavy metals were crust, mixed combustion, industry, and motor vehicles. The characteristic radar charts showed that the pollution dominated by crustal sources mainly occurred in spring and winter, whereas the pollution dominated by mixed combustion sources mainly occurred in winter. Pb, As, and Ni were greatly affected by the transport of a fen nutrient-laden plain, Beijing-Tianjin-Hebei, and southern Henan, whereas Cd was greatly affected by the northwest region of the sampling site. As presented a significant carcinogenic risk in both adults and children, whereas Pb and Sb presented a significant non-carcinogenic risk in children.

Efficacy of Clopidogrel and Clinical Outcome When Clopidogrel Is Coadministered With Atorvastatin and Lansoprazole: A Prospective, Randomized, Controlled Trial.

This prospective, randomized, nonblind, controlled trial evaluated the effects of clopidogrel on platelet function upon coadministration with atorvastatin and lansoprazole. One hundred four adult patients with non-ST-segment elevated acute coronary syndrome (NSTE-ACS) who underwent percutaneous coronary intervention (PCI) with drug-eluting stent implantation were included. All patients were treated with standard dual antiplatelet therapy (DAPT) plus rosuvastatin 10  mg daily after the operation. On the sixth day after PCI, patients were randomly divided into 4 groups, Group A: DAPT + atorvastatin 20  mg daily (a change from rosuvastatin to atorvastatin) + lansoprazole 30  mg daily, Group B: DAPT + atorvastatin 20  mg daily (a change from rosuvastatin to atorvastatin), Group C: DAPT + lansoprazole 30  mg daily (continuing to take rosuvastatin), Group D is the control group. Additional drugs were used according to the situation of patients. Platelet function and concentrations of platelet activation markers (granular membrane protein 140 (P-selectin), thromboxane B2 (TXB2), and human soluble cluster of differentiation 40 ligand (sCD40L)) were assessed before randomization and at 15- and 30-day follow-up visits. All patients were maintained on treatment for 6 months and observed for bleeding and ischemic events. A total of 104 patients were enrolled, 27 patients in group A, 26 patients in Group B/C, 25 patients in Group D separately, and all the patients were analyzed. There were no differences in platelet function and the levels of platelet activation markers (P-selectin, TXB2, and sCD40L) among or within the 4 groups at the 3 time points of interest (P > 0.05). In the subsequent 6 months, no significant bleeding events occurred, and 12 patients experienced ischemic events, these results were also not significantly different among the groups (P > 0.05). In patients diagnosed with NSTE-ACS who have had drug-eluting stent implantation, simultaneously administering clopidogrel, atorvastatin, and lansoprazole did not decrease the antiplatelet efficacy of clopidogrel or increase adverse event frequency over 6 months.

Simultaneous determination of five estrogens and four androgens in water samples by online solid-phase extraction coupled with high-performance liquid chromatography-tandem mass spectrometry.

A novel method for simultaneous determination of five estrogens and four androgens by online solid-phase extraction (SPE) coupled with high-performance liquid chromatography-tandem mass spectrometry (HPLC-ESI-MS/MS) in water samples was developed. An aliquot of 50 mL water sample after filtration was injected directly into autosampler and the analytes were preconcentrated on a NG1 online SPE column. After cleanup step the analytes were eluted in back flush mode and then separated on a liquid chromatography column. The experimental parameters, such as sample loading flow rate, cleanup condition and elution time, were optimized in detail. Estrogens and androgens were detected in negative and positive mode, respectively. High ionization efficiency of all the analytes was achieved by adding of 1‰ ammonia in the mobile phase. The recoveries ranged from 31.8% to 119.0% and the inter-day RSDs ranged from 2.7% to 19.6%. The limits of detections (LODs) were between 0.1 and 2.5 ng/L. The proposed method was successfully applied to the analysis of three types of water samples, including river water, influent and effluent water from a wastewater treatment plant (WWTP). The recoveries of androgens were not that good and a further study is being planned to improve the sensitivity for them. The proposed method is simple, sensitive and suitable for simultaneous analysis and monitoring of estrogens and androgens in water samples.

[Expression profiles of IL-10, TNF-a, and SOCS3 in placenta of pregnant women with intrahepatic cholestasis].

OBJECTIVE: To detect the expression profiles of suppressor of cytokine signaling 3 (SOCS3), interleukin (IL)-10, and tumor necrosis factor-alpha (TNF-a) in the placenta of women with intrahepatic cholestasis of pregnancy (ICP) and determine the clinical significance of the differential expressions. METHODS: Placentas were collected from 37 ICP gravidas who delivered through cesarean section at the First Teaching Hospital of Xingjiang Medical University from October 2010 to May 2011 and from 35 healthy pregnant women (controls). SOCS3, TNF-a, and IL-10 protein levels were detected by immunoblotting and the Envision immunohistochemical method. RESULTS: TNF-a and IL-10 expression was detected in placentas of both groups, and was present mainly in the cytoplasm of trophoeytes. IL-10 expression was obviously lower in the ICP placentas than in the control placentas; meanwhile, TNF-a expression was obviously higher than in the control placentas (Z=-2.63, P less than 0.01). SOCS3 protein was significantly more abundant in the control placentas than in the ICP placentas. Furthermore, SOCS3 and IL-10 placental expressions were positively correlated (r=0.494, P less than 0.01), but there was a negative correlation between SOCS3 and TNF-a placental expressions (r=-0.472, P less than 0.01). CONCLUSION: In ICP, an increase of the type 1 cytokine, TNF-a, is associated with decreases of the type 2 cytokine, IL-10, and of SOCS3, which may reduce the secretion of IL-10. Furthermore, SOCS3 may contribute to ICP pathogenesis by modulating the Th1/Th2 cytokine balance.

PEG-modulated column chromatography for purification of recombinant adeno-associated virus serotype 9.

Column chromatography has been described for purification of recombinant adeno-associated viral vectors (rAAV) serotypes 1, 2, 5, 6 and 8. Some of these purification processes have been used in manufacturing pre-clinical grade and clinical grade rAAV vectors. Recently, recombinant AAV9 has been reported to be highly efficient in transducing cardiac muscle in animal models. Systemic or cardiac gene delivery and other applications may require large quantities of rAAV9 vectors, thus a scalable method supporting large scale purification of rAAV9 is needed for clinical development. However, column chromatography-based purification has not been reported to date for rAAV9. This study reports a polyethylene glycol (PEG) modulated chromatography process for purification of AAV9 vectors. Inclusion of PEG in chromatography buffers modulated rAAV9 elution profiles in a manner that resulted in significantly improved resin binding capacity, vector purity and yield. PEG-modulated methods were developed and optimized for hydroxyapatite and ion exchange chromatography, and shown to result in vectors of high purity and functional activity.

Systemic elimination of de novo capsid protein synthesis from replication-competent AAV contamination in the liver.

The capsid protein synthesis in targeted tissues resulting from residual contaminating replication-competent adeno-associated virus particles (rcAAV) remains a concern for hazardous immune responses that shut down the factor IX expression in the hemophilia B clinical trial. To systematically reduce/eliminate the effects of potential contaminating rcAAV particles, we designed a novel adeno-associated virus (AAV) helper (pH22mir) with a microRNA binding cassette containing multiple copies of liver-specific (hsa-mir-122) and hematopoietic-specific (has-mir-142-3p) sequences to specifically control cap gene expression. In 293 cells, the rep and cap gene from pH22mir functioned similarly to that of conventional helper pH22. The vector yields and compositions from pH22mir and pH22 were indistinguishable. The performance of vector produced in this new system was comparable to that of similar vectors produced by conventional methods. In the human hepatic cell line, the capsid expression was reduced significantly from cap-mir cassette driven by a cytomegalovirus promoter. In the liver, 99.9% of capsid expression could be suppressed and no cap expression could be detected by western blot. In summary, we demonstrated a new concept in reducing de novo capsid synthesis in the targeted tissue. This strategy may not only help AAV vectors in controlling undesirable capsid gene expression, but can also be adopted for lentiviral or adenoviral vector production.

Undetectable transcription of cap in a clinical AAV vector: implications for preformed capsid in immune responses.

In a gene therapy clinical trial for hemophilia B, adeno-associated virus 2 (AAV2) capsid-specific CD8(+) T cells were previously implicated in the elimination of vector-transduced hepatocytes, resulting in loss of human factor IX (hFIX) transgene expression. To test the hypothesis that expression of AAV2 cap DNA impurities in the AAV2-hFIX vector was the source of epitopes presented on transduced cells, transcription of cap was assessed by quantitative reverse transcription-PCR (Q-RT-PCR) following transduction of target cells with the vector used in the clinical trial. Transcriptional profiling was also performed for residual Amp(R), and adenovirus E2A and E4. Although trace amounts of DNA impurities were present in the clinical vector, transcription of these sequences was not detected after transduction of human hepatocytes, nor in mice administered a dose 26-fold above the highest dose administered in the clinical study. Two methods used to minimize encapsidated DNA impurities in the clinical vector were: (i) a vector (cis) production plasmid with a backbone exceeding the packaging limit of AAV; and (ii) a vector purification step that achieved separation of the vector from vector-related impurities (e.g., empty capsids). In conclusion, residual cap expression was undetectable following transduction with AAV2-hFIX clinical vectors. Preformed capsid protein is implicated as the source of epitopes recognized by CD8(+) T cells that eliminated vector-transduced cells in the clinical study.

Reversal of blindness in animal models of leber congenital amaurosis using optimized AAV2-mediated gene transfer.

We evaluated the safety and efficacy of an optimized adeno-associated virus (AAV; AAV2.RPE65) in animal models of the RPE65 form of Leber congenital amaurosis (LCA). Protein expression was optimized by addition of a modified Kozak sequence at the translational start site of hRPE65. Modifications in AAV production and delivery included use of a long stuffer sequence to prevent reverse packaging from the AAV inverted-terminal repeats, and co-injection with a surfactant. The latter allows consistent and predictable delivery of a given dose of vector. We observed improved electroretinograms (ERGs) and visual acuity in Rpe65 mutant mice. This has not been reported previously using AAV2 vectors. Subretinal delivery of 8.25 x 10(10) vector genomes in affected dogs was well tolerated both locally and systemically, and treated animals showed improved visual behavior and pupillary responses, and reduced nystagmus within 2 weeks of injection. ERG responses confirmed the reversal of visual deficit. Immunohistochemistry confirmed transduction of retinal pigment epithelium cells and there was minimal toxicity to the retina as judged by histopathologic analysis. The data demonstrate that AAV2.RPE65 delivers the RPE65 transgene efficiently and quickly to the appropriate target cells in vivo in animal models. This vector holds great promise for treatment of LCA due to RPE65 mutations.

Separation of adeno-associated virus type 2 empty particles from genome containing vectors by anion-exchange column chromatography.

Adeno-associated virus (AAV) empty capsids typically co-purify with genome containing AAV2 vectors purified by column chromatography. This study describes a method to remove empty capsids from genome containing vector particles by anion exchange chromatography. The separation is based on the slightly less anionic character of empty particles compared to vectors. Detailed methods to achieve AAV2 vector purification and particle separation using cation exchange resin POROS 50HS followed by anion exchange resin Q-Sepharose(xl) are described. Chromatographic separation of AAV2 particles was achieved using gradients based on sodium acetate and ammonium acetate, and was optimal at pH 8.5. Efficient removal of particle surface nucleic acid impurities was found to be important to achieve good particle separation. In a large scale experiment performed using partially purified vector containing a mixture of 1.56 x 10(14)vg and 2.52 x 10(15) empty capsids as a starting material, the optimized anion exchange chromatography method resulted in a vector peak of 1.15 x 10(14)vg containing 0.25 x 10(14) empty capsids, corresponding to 74% vector yield and 86-fold reduction in empty capsids in the vector product.

Identification of factors that contribute to recombinant AAV2 particle aggregation and methods to prevent its occurrence during vector purification and formulation.

Aggregation of recombinant AAV2 results in reduced yield during purification and may have deleterious effects on vector transduction efficiency, biodistribution and immunogenicity following in vivo administration. Studies to elucidate the mechanism of vector aggregation and methods to prevent its occurrence are reported. In excipient screening studies, the sugars sorbitol, sucrose, mannitol, trehalose, or glycerol at concentrations of up to 5% (w/v), or surfactants Tween 80 or Pluronic F68, did not prevent aggregation. Aggregation was prevented by the use of various salts at concentrations corresponding to solution ionic strengths of >200 mM. AAV2 vectors purified by double cesium chloride gradient centrifugation, cation-exchange chromatography, or combined chromatography and gradient centrifugation each demonstrated a similar requirement for ionic strength to prevent aggregation. AAV2 vectors concentrated to 6.7 x 10(13) vector genome (vg)/mL in neutral-buffered isotonic saline resulted in 59+/-6.0% recovery of nonaggregated material compared to 96+/-4.4% recovery in an isotonic formulation with elevated ionic strength. The latter showed no aggregation following storage or after 10 freeze-thaw cycles at -20 degrees C. AAV2 vectors stored for an extended period in an elevated ionic strength formulation retained a high infectivity titer (13 vg/infectious unit) and transduction efficiency. Nuclease digestion of purified AAV2 vectors reduced aggregation, implicating trace amounts of vector surface nucleic acids in interparticle binding.

Recombinant adeno-associated virus: formulation challenges and strategies for a gene therapy vector.

Recombinant adeno-associated virus (AAV)-based vectors capable of expressing therapeutic gene products in vivo have shown significant promise for human gene therapy. One challenge facing the field is the development of vector formulations to achieve optimal vector safety, stability and efficacy. Formulation challenges for AAV vectors can be divided into those relating to maintaining vector activity during purification and storage, and those relating to efficient target tissue transduction in vivo. AAV vectors are potentially susceptible to loss of activity through aggregation, proteolysis and oxidation, as well as through non-specific binding to product contact materials used for vector purification and storage. These deleterious changes need to be thoroughly characterized, and the conditions and excipients to prevent them need to be identified. For in vivo administration, major vector formulation challenges include optimization of efficiency and specificity of target tissue transduction, and the ability to overcome host immune responses.