A major use of the 1000 Genomes Project (1000 GP) data is genotype imputation in genome-wide association studies (GWAS). Here we develop a method to estimate haplotypes from low-coverage sequencing data that can take advantage of single-nucleotide polymorphism (SNP) microarray genotypes on the same samples. First the SNP array data are phased to build a backbone (or 'scaffold') of haplotypes across each chromosome. We then phase the sequence data 'onto' this haplotype scaffold. This approach can take advantage of relatedness between sequenced and non-sequenced samples to improve accuracy. We use this method to create a new 1000 GP haplotype reference set for use by the human genetic community. Using a set of validation genotypes at SNP and bi-allelic indels we show that these haplotypes have lower genotype discordance and improved imputation performance into downstream GWAS samples, especially at low-frequency variants.

© 2018, Tsinghua University Press and Springer-Verlag GmbH Germany. Self-assembling peptides (SAPs) are synthetic bioinspired biomaterials that can be feasibly multi-functionalized for cell transplantation and/or drug delivery therapies. Despite their superior biocompatibility and ease of scaling-up for production, they are unfortunately hampered by weak mechanical properties due to transient non-covalent interactions among and within the self-assembled peptide chains, thus limiting their potential applications as fillers, hemostat solutions, and fragile scaffolds for soft tissues. Here, we have developed and characterized a cross-linking strategy that increases both the stiffness and the tailorability of SAP hydrogels, enabling the preparation of transparent flexible threads, discs, channels, and hemispherical constructs. Empirical and computational results, in close agreement with each other, confirmed that the cross-linking reaction does not affect the previously self-assembled secondary structures. In vitro tests also provided a first hint of satisfactory biocompatibility by favoring viability and differentiation of human neural stem cells. This work could bring self-assembling peptide technology to many applications that have been precluded so far, especially in regenerative medicine.

Introduction

To provide a systematic review and meta-analysis of randomized controlled trials (RCT) comparing semi-rigid ureteroscopic lithotripsy (URS) with laparoscopic ureterolithotomy (LU) for the treatment of the large proximal ureteral stone.

Materials and methods

A systematic literature review was performed in June 2015 using the PubMed, Scopus, and Web of Science databases to identify relevant studies. Article selection proceeded according to the search strategy based on Preferred Reporting Items for Systematic Reviews and Meta-analysis criteria.

Results

Six RCT including 646 patients were analyzed, 325 URS cases (50.3%) and 321 LU cases (49.7%). URS provided a significantly shorter operative time (weighted mean difference [WMD] = -31.26 min; 95%CI -46.88 to -15.64; <0.0001) and length of hospital stay (WMD = -1.48 days; 95%CI -2.78 to -0.18; p=0.03) than LU. There were no significant differences in terms of overall complications (OR = 0.78; 95%CI 0.21-2.92; p=0.71) and major complications - Clavien ≥3 - (OR = 1.79; 95%CI 0.59-5.42; p=0.30). LU led to a significantly higher initial stone-free rate (OR = 8.65; 95%CI 4.18-17.91; p<0.00001) and final stone-free rate (OR = 6.41; 95%CI 2.24-18.32; p=0.0005) than URS. There was a significantly higher need for auxiliary procedures in URS cases (OR = 6.58; 95%CI 3.42-12.68; p<0.00001).

Conclusions

Outcomes with LU for larger proximal ureteral calculi are favorable compared to semi-rigid URS and should be considered as a first-line alternative if flexible ureteroscopy is not available. Utilization of flexible ureteroscopy in conjunction with semi-rigid ureteroscopy may impact these outcomes, and deserves further systematic evaluation.

Canonical Wnt signaling plays multiple roles critical to normal craniofacial development while its dysregulation is known to be involved in structural birth defects of the face. However, when and how Wnt signaling influences phenotypic variation, including those associated with disease, remains unclear. One potential mechanism is via Wnt signaling's role in the patterning of an early facial signaling center, the frontonasal ectodermal zone (FEZ), and its subsequent regulation of early facial morphogenesis. For example, Wnt signaling may directly alter the shape and/or magnitude of expression of the sonic hedgehog (SHH) domain in the FEZ. To test this idea, we used a replication-competent avian sarcoma retrovirus (RCAS) encoding Wnt3a to modulate its expression in the facial mesenchyme. We then quantified and compared ontogenetic changes in treated to untreated embryos in the three-dimensional (3D) shape of both the SHH expression domain of the FEZ, and the morphology of the facial primordia and brain using iodine-contrast microcomputed tomography imaging and 3D geometric morphometrics (3DGM). We found that increased Wnt3a expression in early stages of head development produces correlated variation in shape between both structural and signaling levels of analysis. In addition, altered Wnt3a activation disrupted the integration between the forebrain and other neural tube derivatives. These results show that activation of Wnt signaling influences facial shape through its impact on the forebrain and SHH expression in the FEZ, and highlights the close relationship between morphogenesis of the forebrain and midface.

Lipofectamine reagents are widely accepted as "gold-standard" for the safe delivery of exogenous DNA or RNA into cells. Despite this, a satisfactory mechanism-based explanation of their superior efficacy has remained mostly elusive thus far. Here we apply a straightforward combination of live cell imaging, single-particle tracking microscopy, and quantitative transfection-efficiency assays on live cells to unveil the intracellular trafficking mechanism of Lipofectamine/DNA complexes. We find that Lipofectamine, contrary to alternative formulations, is able to efficiently avoid active intracellular transport along microtubules, and the subsequent entrapment and degradation of the payload within acidic/digestive lysosomal compartments. This result is achieved by random Brownian motion of Lipofectamine-containing vesicles within the cytoplasm. We demonstrate here that Brownian diffusion is an efficient route for Lipofectamine/DNA complexes to avoid metabolic degradation, thus leading to optimal transfection. By contrast, active transport along microtubules results in DNA degradation and subsequent poor transfection. Intracellular trafficking, endosomal escape and lysosomal degradation appear therefore as highly interdependent phenomena, in such a way that they should be viewed as a single barrier on the route for efficient transfection. As a matter of fact, they should be evaluated in their entirety for the development of optimized non-viral gene delivery vectors.

INTRODUCTION: The ureteral access sheath (UAS) is a medical device that enables repeated entrance into the ureter and collecting system during retrograde intrarenal surgery (RIRS). Its impact on stone-free rates, ureteral injuries, operative time, and postoperative complications remains controversial. Therefore, we performed a systematic review and meta-analysis comparing RIRS with versus without UAS for urolithiasis management. PURPOSE: To compare outcomes from retrograde intrarenal surgery (RIRS) for stone extraction with or without ureteral access sheath (UAS); evaluating stone-free rate (SFR), ureteral injuries, operative time, and postoperative complications. MATERIALS AND METHODS: We systematically searched PubMed, Embase, and Cochrane Library in June 2024 for randomized controlled trials (RCTs) evaluating the efficacy and safety outcomes of UAS use in RIRS for urolithiasis treatment. Articles published between 2014 and 2024 were included. Pooled risk ratios (RRs) and mean differences (MDs) were calculated for binary and continuous outcomes, respectively. RESULTS: Five RCTs comprising 466 procedures were included. Of these, 246 (52.7%) utilized UAS. The follow-up ranged from 1 week to 1 month. UAS reduced the incidence of postoperative fever (RR 0.49; 95% confidence interval [CI] 0.29-0.84; p=0.009), and postoperative infection (RR 0.50; 95% CI 0.30-0.83; p=0.008). There were no significant differences between groups in terms of SFR (RR 1.05; 95% CI 0.99-1.11; p=0.10), ureteral injuries (RR 1.29; 95% CI 0.95-1.75; p=0.11), operative time (MD 3.56 minutes; 95% CI -4.15 to 11.27 minutes; p=0.36), or length of stay (MD 0.32 days; 95% CI -0.42 to 1.07 days; p=0.40). CONCLUSION: UAS leads to a lower rate of post-operative fever and infection. However, UAS did not significantly reduce or increase the SFR or the rate of ureteral injuries during RIRS for patients with urolithiasis. The use of UAS should be considered to decrease the risk of infectious complications, particularly in those who may be at higher risk for such complications.

Multicomponent lipoplexes have recently emerged as especially promising transfection candidates, as they are from 10 to 100 times more efficient than binary complexes usually employed for gene delivery purposes. Previously, we investigated a number of chemical-physical properties of DNA-lipid complexes that were proposed to affect transfection efficiency (TE) of lipoplexes, such as nanoscale structure, size, surface potential, DNA-protection ability and DNA release from complexes upon interaction with cellular lipids. Although some minor differences between multicomponent and binary lipoplexes were found, they did not correlate clearly with efficiency. Instead, here we show that a marked difference between the cell internalization mechanism of binary and multicomponent lipoplexes does exist. Multicomponent lipoplexes significantly transfect cells at 4 °C, when endocytosis does not take place suggesting that they can enter cells via a temperature-independent mechanism. Confocal fluorescence microscopy experiments showed the existence of a correlation between endosomal escape and TE. Multicomponent lipoplexes exhibited a distinctive ability of endosomal escape and release DNA into the nucleus, whereas, poorly efficient binary lipoplexes exhibited minor, if any, endosomal rupture ability and remained confined in perinuclear late endosomes. Stopped-flow mixing measurements showed that the fusion rates of multicomponent cationic liposomes with anionic vesicles, used as model systems of cell membranes, were definitely shorter than those of binary liposomes. As either lipoplex uptake and endosomal escape involve fusion between lipoplex and cellular membranes, we suggest that a mechanism of lipoplex-cellular membrane interaction, driven by lipid mixing between cationic and anionic cellular lipids, does explain the TE boost of multicomponent lipoplexes.

The utility of using a protammine/DNA complex coated with a lipid envelope made of cationic 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) for transfecting CHO (Chinese hamster ovary cells), HEK293 (human embryonic kidney cells), NIH 3T3 (mouse embryonal cells), and A17 (murine cancer cells) cells was examined. The widely used DOTAP/DNA lipoplex was employed as a reference. In all the tested cell lines lipid/protamine/DNA (LPD) nanoparticles were more efficient in transfecting cells than lipoplexes even though the lipid composition of the lipid envelope was the same in both devices. Physical-chemical properties were found to control the ability of nanocarriers to release DNA upon interaction with cellular membranes. LPD complexes easily release their DNA payload, while lipoplexes remain largely intact and accumulate at the cell nucleus. Collectively, these data explain why LPD nanoparticles often exhibit superior performances compared to lipoplexes in trasfecting cells and represent a promising class of nanocarriers for gene delivery.

INTRODUCTION:: To demonstrate the entire surgeon's point of view of a prone split-leg (PSL) endoscopic guided percutaneous nephrolithotomy (ePCNL) recorded with a GoPro® camera for standardization of the essential technical steps towards a successful procedure (1). MATERIALS AND METHODS:: A 40y.o female patient presented with right flank pain for three years. She had previously been submitted to shock wave lithotripsy without success. Non-contrast computed tomography (NCCT) revealed a 2cm stone in the renal pelvis with 1400HU and stone-to-skin distance of 11cm (Guy's Stone Score 1). PCNL approach was chosen for providing higher chances of stone-free after a single procedure. Informed consent was obtained. The PSL ePCNL was uneventful with a single access in a mid-pole. The surgeon had a Full HD GoPro Hero 4® camera mounted on his head, controlled by the surgical staff with a remote control. All essential surgical steps were recorded. RESULTS:: Operative time was 90 minutes. Hemoglobin drop was 0.7g/dL. The post-operative NCCT scan was stone-free. The patient was discharged 24h after surgery. Kidney stent was left with a string and removed after 5days. The camera worked properly and didn't cause any kind of discomfort to the surgeon. The quality of the recorded movie was excellent. CONCLUSION:: By recording the surgeon's perspective of an endoscopic urological procedure, we were able to provide a comprehensive understanding of the surgical technique by assembling the endoscopic, fluoroscopic, and operative field views. The GoPro® camera proved to be an interesting tool to document surgical procedures without compromising outcomes and has great potential for educational purposes.