Dioctadecyldimethylammonium:monoolein nanocarriers for efficient in vitro gene silencing

This study describes a novel liposomal formulation for siRNA delivery, based on the mixture of the neutral lipid monoolein (MO) and cationic lipids of the dioctadecyldimethylammonium (DODA) family. The cationic lipids dioctadecyldimethylammonium bromide (DODAB) and chloride (DODAC) were compared in order to identify which one will most efficiently induce gene silencing. MO has a fluidizing effect on DODAC and DODAB liposomes, although it was more homogeneously distributed in DODAC bilayers. All MO-based liposomal formulations were able to efficiently encapsulate siRNA. Stable lipoplexes of small size (100-160 nm) with a positive surface charge (>+45 mV) were formed. A more uniform MO incorporation in DODAC:MO may explain an increase of the fusogenic potential of these liposomes. The siRNA-lipoplexes were readily internalized by human nonsmall cell lung carcinoma (H1299) cells, in an energy dependent process. DODAB:MO nanocarriers showed a higher internalization efficiency in comparison to DODAC:MO lipoplexes, and were also more efficient in promoting gene silencing. MO had a similar gene silencing ability as the commonly used helper lipid 1,2-dioleyl-3-phosphatidylethanolamine (DOPE), but with much lower cytotoxicity. Taking in consideration all the results presented, DODAB:MO liposomes are the most promising tested formulation for systemic siRNA delivery.This work was supported by FEDER through POFC - COMPETE and by national funds from FCT through the projects PEst-C/BIA/UI4050/2011 (CBM.A), PEst-C/FIS/UI0607/2011 (CFUM), and PTDC/QUI/69795/2006, while Ana Oliveira holds scholarship SFRH/BD/68588/2010. Eloi Feitosa thanks FAPESP (2011/03566-0) and CNPq (303030/2012-7), and Renata D. Adati thanks FAPESP for scholarship (2011/07414-0). K. Raemdonck is a postdoctoral fellow of the Research Foundation - Flanders (FWO-Vlaanderen). We acknowledge NanoDelivery-I&D em Bionanotecnologia, Lda. for access to their equipment

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Analysis of a Standardized Technique for Laparoscopic Cuff Closure following 1924 Total Laparoscopic Hysterectomies

Objective. To review the vaginal cuff complications from a large series of total laparoscopic hysterectomies in which the laparoscopic culdotomy closure was highly standardized. Methods. Retrospective cohort study (Canadian Task Force Classification II-3) of consecutive total and radical laparoscopic hysterectomy patients with all culdotomy closures performed laparoscopically was conducted using three guidelines: placement of all sutures 5 mm deep from the vaginal edge with a 5 mm interval, incorporation of the uterosacral ligaments with the pubocervical fascia at each angle, and, whenever possible, suturing the bladder peritoneum over the vaginal cuff edge utilizing two suture types of comparable tensile strength. Four outcomes are reviewed: dehiscence, bleeding, infection, and adhesions. Results. Of 1924 patients undergoing total laparoscopic hysterectomy, 44 patients (2.29%) experienced a vaginal cuff complication, with 19 (0.99%) requiring reoperation. Five patients (0.26%) had dehiscence after sexual penetration on days 30–83, with 3 requiring reoperation. Thirteen patients (0.68%) developed bleeding, with 9 (0.47%) requiring reoperation. Twenty-three (1.20%) patients developed infections, with 4 (0.21%) requiring reoperation. Three patients (0.16%) developed obstructive small bowel adhesions to the cuff requiring laparoscopic lysis. Conclusion. A running 5 mm deep × 5 mm apart culdotomy closure that incorporates the uterosacral ligaments with the pubocervical fascia, with reperitonealization when possible, appears to be associated with few postoperative vaginal cuff complications

Concordance between tropism measured phenotypically and inferred genotypically using the Geno2pheno_(coreceptor) and webPSSM algorithms.

<p>(<b>A</b>) Concordance for subtype B (black bars) and non-B subtype (grey bars) strains with Geno2Pheno (G2P) at different FPR cutoffs and webPSSM. (<b>B</b>) Concordance with Geno2pheno_(coreceptor) with a FPR set at 10% (black bars) and webPSSM (grey bars) for different HIV-1 subtypes. The webPSSM X4/R5 matrix was used for all subtypes, except for subtype C, for which the subtype C SI/NSI matrix was used.</p

Characteristics of phenotypic assays developed for determination of HIV-1 coreceptor usage.

<p>Abbreviations: ESTA: Enhanced Sensitivity Trofile Assay; Env: Envelope; eGFP: enhanced Green Fluorescent protein; X4: CXCX4-using strains; R5:CCR5-using strains.</p

Study design/RVA design.

<p>Viral RNA was extracted from patient plasma RT-PCR amplified. Env amplicons spanning the Env ectodomain were further amplified through an inner PCR. Five independent PCRs were pooled to minimize PCR-selection. Recombinant viruses were produced by co-transfecting HEK293T cells with Afe I-linearized, luciferase-tagged, Env-deleted, viral backbone and patient-derived PCR amplicon. Normalized amounts of recombinant viruses were used to infect U87.CD4.CCR5 or U87.CD4.CXCR4 indicator cells. Infection was monitored by quantifying luminescence in the cell lysates. Depending on the outcome of the infection, viruses were classified as either CCR5 tropic, CXCR4 tropic or dual/mixed. The same patient-derived PCR amplicon used for viral production was sequenced and tropism inferred by Geno2Pheno_[coreceptor] and webPSSM algorithms. The phenotypic and genotypic results were compared. Abbreviations: Env EC: Env ectodomain; gp41-TM-CT: gp41 Transmembrane+cytoplasmic tail.</p

Detection of minority CXCR4 and CCR5 using variants within mixed viral populations.

<p>Mixtures containing known proportions of pNLAD8 and pNL4-3 (100∶0, i.e. pure NLAD8, 99∶1, 97.5∶2.5, 95∶5, 90∶10, 80∶20, 50∶50, 20∶80, 10∶90, 5∶95, 2.5∶97.5, 1∶99, 0∶100, i.e. pure NL4-3) were PCR-amplified and used to generate recombinant viruses. U87.CD4.CCR5 and U87.CD4.CXCR4 indicator cells were infected with serial 2-fold dilutions (x-axis) of mixtures (z-axis) to determine the threshold for detection of minority variants. Infection was quantified 48 hours after infection by measuring luciferase activity in cell lysates (y-axis). Black bars report infection of U87.CD4.CXCR4 cells and grey bars report infection of U87.CD4.CCR5 cells. Panels A and B report the same data, oriented to focus on NL4-3 minority variants (A) or on NLAD8 minority variants (B).</p