Phosphorylcholine-based copolymer as synthetic vector for gene delivery

Gene therapy has a great potential for the treatment of a wide range of diseases. However, the development of a safe and efficient delivery vector is the major obstacle for gene therapy. Recently synthesized 2 - (dimethylamino) ethyl methacrylate 2-(methacryloxloxyethyl phosphorylcholine) (DMA-MPC) diblock copolymer was investigated in this work as a novel non-viral vector for gene delivery. It has been previously demonstrated that the cationic DMA block can condense DNA efficiently. The zwitterionic PC head groups are found naturally in the outer leaflet of biomembranes and are extremely biocompatible. It is thus proposed here that the MPC can act as a new steric stabilizer to the system. Different compositions of DMA-MPC diblock copolymers were evaluated. The MPC block with minimum length 30 monomeric units can successfully provide steric stabilization to the system, and reduce nonspecific cellular interaction by providing a steric barrier to the DNA complexes. However, long MPC chain can hinder the interaction between cationic DMA and DNA, leading to the formation of loosely condensed complexes which were more susceptible to enzymatic degradation. Therefore the composition of the copolymer must be carefully adjusted so that the DNA condensing and steric stabilization effect are well balanced. In order to investigate the cellular uptake mechanism DMA homopolymerDNA complexes, the effect of different endocytosis inhibitors was examined. Microtubules and actin filaments were involved in the uptake of DNA complexes, suggesting that the complexes were internalised by endocytosis. Both the clathrin- and caveolae- mediated pathway were responsible for the uptake of DNA complexes, and the former appeared to be the main route of entry. Finally, folic acid ligand was incorporated into the DMA-MPC copolymer in order to improve the specific targeting. Initial data showed that there was selective uptake of the folate conjugated system in folate receptor expressing cells possibly via receptor mediated endocytosis. However, parameters such as the optimum length of MPC component, number of ligands per DNA complex and the composition of the system need to be further investigated in order to maximize the specificity and transfection efficiency

Aberrant Transferrin and Ferritin Upregulation Elicits Iron Accumulation and Oxidative Inflammaging Causing Ferroptosis and Undermines Estradiol Biosynthesis in Aging Rat Ovaries by Upregulating NF-Κb-Activated Inducible Nitric Oxide Synthase: First Demonstration of an Intricate Mechanism

We report herein a novel mechanism, unraveled by proteomics and validated by in vitro and in vivo studies, of the aberrant aging-associated upregulation of ovarian transferrin and ferritin in rat ovaries. The ovarian mass and serum estradiol titer plummeted while the ovarian labile ferrous iron and total iron levels escalated with age in rats. Oxidative stress markers, such as nitrite/nitrate, 3-nitrotyrosine, and 4-hydroxy-2-nonenal, accumulated in the aging ovaries due to an aberrant upregulation of the ovarian transferrin, ferritin light/heavy chains, and iron regulatory protein 2(IRP2)-mediated transferrin receptor 1 (TfR1). Ferritin inhibited estradiol biosynthesis in ovarian granulosa cells in vitro via the upregulation of a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and p65/p50-induced oxidative and inflammatory factor inducible nitric oxide synthase (iNOS). An in vivo study demonstrated how the age-associated activation of NF-κB induced the upregulation of iNOS and the tumor necrosis factor α (TNFα). The downregulation of the keap1-mediated nuclear factor erythroid 2-related factor 2 (Nrf2), that induced a decrease in glutathione peroxidase 4 (GPX4), was observed. The aberrant transferrin and ferritin upregulation triggered an iron accumulation via the upregulation of an IRP2-induced TfR1. This culminates in NF-κB-iNOS-mediated ovarian oxi-inflamm-aging and serum estradiol decrement in naturally aging rats. The iron accumulation and the effect on ferroptosis-related proteins including the GPX4, TfR1, Nrf2, Keap1, and ferritin heavy chain, as in testicular ferroptosis, indicated the triggering of ferroptosis. In young rats, an intraovarian injection of an adenovirus, which expressed iron regulatory proteins, upregulated the ovarian NF-κB/iNOS and downregulated the GPX4. These novel findings have contributed to a prompt translational research on the ovarian aging-associated iron metabolism and aging-associated ovarian diseases

Phosphorylcholine-based copolymer as synthetic vector for gene delivery

Gene therapy has a great potential for the treatment of a wide range of diseases. However, the development of a safe and efficient delivery vector is the major obstacle for gene therapy. Recently synthesized 2 - (dimethylamino) ethyl methacrylate 2-(methacryloxloxyethyl phosphorylcholine) (DMA-MPC) diblock copolymer was investigated in this work as a novel non-viral vector for gene delivery. It has been previously demonstrated that the cationic DMA block can condense DNA efficiently. The zwitterionic PC head groups are found naturally in the outer leaflet of biomembranes and are extremely biocompatible. It is thus proposed here that the MPC can act as a new steric stabilizer to the system. Different compositions of DMA-MPC diblock copolymers were evaluated. The MPC block with minimum length 30 monomeric units can successfully provide steric stabilization to the system, and reduce nonspecific cellular interaction by providing a steric barrier to the DNA complexes. However, long MPC chain can hinder the interaction between cationic DMA and DNA, leading to the formation of loosely condensed complexes which were more susceptible to enzymatic degradation. Therefore the composition of the copolymer must be carefully adjusted so that the DNA condensing and steric stabilization effect are well balanced. In order to investigate the cellular uptake mechanism DMA homopolymerDNA complexes, the effect of different endocytosis inhibitors was examined. Microtubules and actin filaments were involved in the uptake of DNA complexes, suggesting that the complexes were internalised by endocytosis. Both the clathrin- and caveolae- mediated pathway were responsible for the uptake of DNA complexes, and the former appeared to be the main route of entry. Finally, folic acid ligand was incorporated into the DMA-MPC copolymer in order to improve the specific targeting. Initial data showed that there was selective uptake of the folate conjugated system in folate receptor expressing cells possibly via receptor mediated endocytosis. However, parameters such as the optimum length of MPC component, number of ligands per DNA complex and the composition of the system need to be further investigated in order to maximize the specificity and transfection efficiency.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Exome-wide association analysis reveals novel coding sequence variants associated with lipid traits in Chinese

Blood lipids are important risk factors for coronary artery disease (CAD). Here we perform an exome-wide association study by genotyping 12,685 Chinese, using a custom Illumina HumanExome BeadChip, to identify additional loci influencing lipid levels. Single-variant association analysis on 65,671 single nucleotide polymorphisms reveals 19 loci associated with lipids at exome-wide significance (P<2.69 × 10−7), including three Asian-specific coding variants in known genes (CETP p.Asp459Gly, PCSK9 p.Arg93Cys and LDLR p.Arg257Trp). Furthermore, missense variants at two novel loci—PNPLA3 p.Ile148Met and PKD1L3 p.Thr429Ser—also influence levels of triglycerides and low-density lipoprotein cholesterol, respectively. Another novel gene, TEAD2, is found to be associated with high-density lipoprotein cholesterol through gene-based association analysis. Most of these newly identified coding variants show suggestive association (P<0.05) with CAD. These findings demonstrate that exome-wide genotyping on samples of non-European ancestry can identify additional population-specific possible causal variants, shedding light on novel lipid biology and CAD

The surgical safety checklist and patient outcomes after surgery: a prospective observational cohort study, systematic review and meta-analysis

© 2017 British Journal of Anaesthesia Background: The surgical safety checklist is widely used to improve the quality of perioperative care. However, clinicians continue to debate the clinical effectiveness of this tool. Methods: Prospective analysis of data from the International Surgical Outcomes Study (ISOS), an international observational study of elective in-patient surgery, accompanied by a systematic review and meta-analysis of published literature. The exposure was surgical safety checklist use. The primary outcome was in-hospital mortality and the secondary outcome was postoperative complications. In the ISOS cohort, a multivariable multi-level generalized linear model was used to test associations. To further contextualise these findings, we included the results from the ISOS cohort in a meta-analysis. Results are reported as odds ratios (OR) with 95% confidence intervals. Results: We included 44 814 patients from 497 hospitals in 27 countries in the ISOS analysis. There were 40 245 (89.8%) patients exposed to the checklist, whilst 7508 (16.8%) sustained ≥1 postoperative complications and 207 (0.5%) died before hospital discharge. Checklist exposure was associated with reduced mortality [odds ratio (OR) 0.49 (0.32–0.77); P\u3c0.01], but no difference in complication rates [OR 1.02 (0.88–1.19); P=0.75]. In a systematic review, we screened 3732 records and identified 11 eligible studies of 453 292 patients including the ISOS cohort. Checklist exposure was associated with both reduced postoperative mortality [OR 0.75 (0.62–0.92); P\u3c0.01; I2=87%] and reduced complication rates [OR 0.73 (0.61–0.88); P\u3c0.01; I2=89%). Conclusions: Patients exposed to a surgical safety checklist experience better postoperative outcomes, but this could simply reflect wider quality of care in hospitals where checklist use is routine

Prospective observational cohort study on grading the severity of postoperative complications in global surgery research

Background The Clavien–Dindo classification is perhaps the most widely used approach for reporting postoperative complications in clinical trials. This system classifies complication severity by the treatment provided. However, it is unclear whether the Clavien–Dindo system can be used internationally in studies across differing healthcare systems in high- (HICs) and low- and middle-income countries (LMICs). Methods This was a secondary analysis of the International Surgical Outcomes Study (ISOS), a prospective observational cohort study of elective surgery in adults. Data collection occurred over a 7-day period. Severity of complications was graded using Clavien–Dindo and the simpler ISOS grading (mild, moderate or severe, based on guided investigator judgement). Severity grading was compared using the intraclass correlation coefficient (ICC). Data are presented as frequencies and ICC values (with 95 per cent c.i.). The analysis was stratified by income status of the country, comparing HICs with LMICs. Results A total of 44 814 patients were recruited from 474 hospitals in 27 countries (19 HICs and 8 LMICs). Some 7508 patients (16·8 per cent) experienced at least one postoperative complication, equivalent to 11 664 complications in total. Using the ISOS classification, 5504 of 11 664 complications (47·2 per cent) were graded as mild, 4244 (36·4 per cent) as moderate and 1916 (16·4 per cent) as severe. Using Clavien–Dindo, 6781 of 11 664 complications (58·1 per cent) were graded as I or II, 1740 (14·9 per cent) as III, 2408 (20·6 per cent) as IV and 735 (6·3 per cent) as V. Agreement between classification systems was poor overall (ICC 0·41, 95 per cent c.i. 0·20 to 0·55), and in LMICs (ICC 0·23, 0·05 to 0·38) and HICs (ICC 0·46, 0·25 to 0·59). Conclusion Caution is recommended when using a treatment approach to grade complications in global surgery studies, as this may introduce bias unintentionally

Critical care admission following elective surgery was not associated with survival benefit: prospective analysis of data from 27 countries

This was an investigator initiated study funded by Nestle Health Sciences through an unrestricted research grant, and by a National Institute for Health Research (UK) Professorship held by RP. The study was sponsored by Queen Mary University of London