Publications by authors named "Christopher J Wilson"

74 Publications

Noise Evaluation of Arthroplasty Theaters: Results From the NEAT Study.

J Arthroplasty 2020 Nov 21. Epub 2020 Nov 21.

Orthopaedic Department, Dorset County Hospital, Dorchester, UK.

Aims: The aim of this study was to define the levels of noise exposure for the surgeon, assistant, scrub nurse, and anesthetist during total hip and knee arthroplasty surgery. In addition, we sought to determine whether the noise exposure during these procedures reaches or exceeds the action values set out by the U.K. Noise at Work Regulations (2005).

Materials And Methods: Individual noise exposure during arthroplasty hip and knee surgery was recorded using a personal noise Dosemeter System model 22 (DM22) (Pulsar instruments, Filey, U.K.). Recordings were taken in real-time during five separate theater sessions. Each theater session included two arthroplasty procedures and lasted approximately 4 hrs. Personal noise exposure was expressed in terms of peak sound pressure and an average noise exposure over an 8-hour time-period to reflect the noise experienced by the ear over a working day.

Results: In all three sessions involving total hip replacement surgery, the peak sound pressure, for the operating surgeon exceeded the exposure action values set out by the U.K. Noise at Work Regulations. Theater sessions involving total knee replacement surgery did not exceed any exposure action values for LCPeak or LEPd.

Conclusion: Arthroplasty surgery is a working environment with significant noise exposure. We recommend any surgeon or theater member who is concerned about the noise generated in their theater to have noise levels formally assessed using appropriately positioned recording devices.
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http://dx.doi.org/10.1016/j.arth.2020.11.026DOI Listing
November 2020

CT Validation of Intraoperative Implant Position and Knee Alignment as Determined by the MAKO Total Knee Arthroplasty System.

J Knee Surg 2020 Mar 4. Epub 2020 Mar 4.

Department of Orthopaedics, Flinders Medical Centre, Adelaide, South Australia, Australia.

Robotic-assisted technology in total knee arthroplasty (TKA) aims to increase implantation accuracy, with real-time data being used to estimate intraoperative component alignment. Postoperatively, Perth computed tomography (CT) protocol is a valid measurement technique in determining both femoral and tibial component alignments. The aim of this study was to evaluate the accuracy of intraoperative component alignment by robotic-assisted TKA through CT validation. A total of 33 patients underwent TKA using the MAKO robotic-assisted TKA system. Intraoperative measurements of both femoral and tibial component placements, as well as limb alignment as determined by the MAKO software were recorded. Independent postoperative Perth CT protocol was obtained ( = 29) and compared with intraoperative values. Mean absolute difference between intraoperative and postoperative measurements for the femoral component were 1.17 degrees (1.10) in the coronal plane, 1.79 degrees (1.12) in the sagittal plane, and 1.90 degrees (1.88) in the transverse plane. Mean absolute difference between intraoperative and postoperative measurements for the tibial component were 1.03 degrees (0.76) in the coronal plane and 1.78 degrees (1.20) in the sagittal plane. Mean absolute difference of limb alignment was 1.29 degrees (1.25), with 93.10% of measurements ≤3 degrees of postoperative CT measurements. Overall, intraoperatively measured component alignment as estimated by the MAKO robotic-assisted TKA system is comparable to CT-based measurements.
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http://dx.doi.org/10.1055/s-0040-1701447DOI Listing
March 2020

Discovery of a Brain-Penetrant ATP-Competitive Inhibitor of the Mechanistic Target of Rapamycin (mTOR) for CNS Disorders.

J Med Chem 2020 02 31;63(3):1068-1083. Epub 2020 Jan 31.

Chemical Biology and Therapeutics , Novartis Institutes for BioMedical Research , 181 Massachusetts Ave , Cambridge , Massachusetts 02139 , United States.

Recent clinical evaluation of everolimus for seizure reduction in patients with tuberous sclerosis complex (TSC), a disease with overactivated mechanistic target of rapamycin (mTOR) signaling, has demonstrated the therapeutic value of mTOR inhibitors for central nervous system (CNS) indications. Given that everolimus is an incomplete inhibitor of the mTOR function, we sought to develop a new mTOR inhibitor that has improved properties and is suitable for CNS disorders. Starting from an in-house purine-based compound, optimization of the physicochemical properties of a thiazolopyrimidine series led to the discovery of the small molecule , a potent and selective brain-penetrant ATP-competitive mTOR inhibitor. In neuronal cell-based models of mTOR hyperactivity, corrected the mTOR pathway activity and the resulting neuronal overgrowth phenotype. The new mTOR inhibitor showed good brain exposure and significantly improved the survival rate of mice with neuronal-specific ablation of the Tsc1 gene. These results demonstrate the potential utility of this tool compound to test therapeutic hypotheses that depend on mTOR hyperactivity in the CNS.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01398DOI Listing
February 2020

Structural analysis of lecithin:cholesterol acyltransferase bound to high density lipoprotein particles.

Commun Biol 2020 01 15;3(1):28. Epub 2020 Jan 15.

Departments of Biological Sciences and of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN, 47907, USA.

Lecithin:cholesterol acyltransferase (LCAT) catalyzes a critical step of reverse cholesterol transport by esterifying cholesterol in high density lipoprotein (HDL) particles. LCAT is activated by apolipoprotein A-I (ApoA-I), which forms a double belt around HDL, however the manner in which LCAT engages its lipidic substrates and ApoA-I in HDL is poorly understood. Here, we used negative stain electron microscopy, crosslinking, and hydrogen-deuterium exchange studies to refine the molecular details of the LCAT-HDL complex. Our data are consistent with LCAT preferentially binding to the edge of discoidal HDL near the boundary between helix 5 and 6 of ApoA-I in a manner that creates a path from the lipid bilayer to the active site of LCAT. Our results provide not only an explanation why LCAT activity diminishes as HDL particles mature, but also direct support for the anti-parallel double belt model of HDL, with LCAT binding preferentially to the helix 4/6 region.
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http://dx.doi.org/10.1038/s42003-019-0749-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6962161PMC
January 2020

Ventricular Fibrillation Cardiac Arrest in Young Female from Diffuse Left Anterior Descending Coronary Vasospasm.

Clin Pract Cases Emerg Med 2019 Nov 14;3(4):395-397. Epub 2019 Oct 14.

Geisinger Commonwealth School of Medicine, Geisinger Wyoming Valley, Department of Emergency Medicine, Danville, Pennsylvania.

This is a case of the most severe and potentially fatal complication of coronary artery vasospasm. We report a case of a 40-year-old female presenting to the emergency department (ED) via emergency medical services with chest pain. The patient experienced a ventricular fibrillation cardiac arrest while in the ED. Post-defibrillation electrocardiogram showed changes suggestive of an ST-elevation myocardial infarction (STEMI). Cardiac catheterization showed severe left anterior descending spasm with no evidence of disease. Coronary vasospasm is a consideration in the differential causes of ventricular fibrillation and STEMI seen in the ED.
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http://dx.doi.org/10.5811/cpcem.2019.9.43762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6861043PMC
November 2019

The Role of Perspective Taking on Attention: A Review of the Special Issue on the Reflexive Attentional Shift Phenomenon.

Vision (Basel) 2019 Oct 9;3(4). Epub 2019 Oct 9.

Department of Psychology Politics and Sociology, Sheffield Hallam University, Sheffield S10 2BP, UK.

Attention is a process that alters how cognitive resources are allocated, and it allows individuals to efficiently process information at the attended location. The presence of visual or auditory cues in the environment can direct the focus of attention toward certain stimuli even if the cued stimuli are not the individual's primary target. Samson et al. demonstrated that seeing another person in the scene (i.e., a person-like cue) caused a delay in responding to target stimuli not visible to that person: "alter-centric intrusion." This phenomenon, they argue, is dependent upon the fact that the cue used resembled a person as opposed to a more generic directional indicator. The characteristics of the cue are the core of the debate of this special issue. Some maintain that the perceptual-directional characteristics of the cue are sufficient to generate the bias while others argue that the cuing is stronger when the cue has social characteristics (relates to what another individual can perceive). The research contained in this issue confirms that human attention is biased by the presence of a directional cue. We discuss and compare the different studies. The pattern that emerges seems to suggest that the social relevance of the cue is necessary in some contexts but not in others, depending on the cognitive demand of the experimental task. One possibility is that the social mechanisms are involved in perspective taking when the task is cognitively demanding, while they may not play a role in automatic attention allocation.
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http://dx.doi.org/10.3390/vision3040052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6969940PMC
October 2019

Accuracy of Bone Resection in MAKO Total Knee Robotic-Assisted Surgery.

J Knee Surg 2019 Nov 6. Epub 2019 Nov 6.

Department of Orthopaedics, Flinders Medical Centre, Adelaide, South Australia, Australia.

Accurate component positioning and planning is vital to prevent malalignment of total knee arthroplasty (TKA) as malalignment is associated with an increased rate of polyethylene wear and revision arthroplasty. The MAKO total knee robotic arm-assisted surgery (Stryker, Kalamazoo, MI) uses a preoperative computed tomography scan of the patient's knee and three-dimensional planning to size and orientate implants prior to bone resection. The aim of this study was to determine the accuracy of the MAKO Total Knee system in achieving the preoperative plan for bone resection and final limb coronal alignment. A series of 45 consecutive cases was performed using the MAKO Total Knee system and Triathlon Total Knee implant (Stryker) between April 2018 and May 2019. The difference between what was planned and what was achieved for bone resection and coronal limb alignment was calculated. A total of 37 patients had their data captured using the MAKO system software. Mean difference from the plan for distal femoral cuts was 0.38mm (0.32) deep/proud, anterior femoral cuts 0.44mm (0.27) deep/proud and tibial cuts 0.37mm (0.30) deep/proud. In total, 99 out of 105 (94.29%) of bone resections were within 1mm of the plan. Mean absolute difference in final limb coronal alignment was 0.78° (0.78), with 78.13% being ≤1.00° of the plan, and 100% being ≤3.00° of the plan. The accuracy in achieving preoperatively planned bone resection and final limb coronal alignment using the MAKO Total Knee system is high. Future research is planned to look at whether this is associated with decreased rates of polyethylene wear and revision arthroplasty.
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http://dx.doi.org/10.1055/s-0039-1700570DOI Listing
November 2019

Encapsulation of hydrophobic components in dendrimersomes and decoration of their surface with proteins and nucleic acids.

Proc Natl Acad Sci U S A 2019 07 15;116(31):15378-15385. Epub 2019 Jul 15.

Roy & Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323;

Reconstructing the functions of living cells using nonnatural components is one of the great challenges of natural sciences. Compartmentalization, encapsulation, and surface decoration of globular assemblies, known as vesicles, represent key early steps in the reconstitution of synthetic cells. Here we report that vesicles self-assembled from amphiphilic Janus dendrimers, called dendrimersomes, encapsulate high concentrations of hydrophobic components and do so more efficiently than commercially available stealth liposomes assembled from phospholipid components. Multilayer onion-like dendrimersomes demonstrate a particularly high capacity for loading low-molecular weight compounds and even folded proteins. Coassembly of amphiphilic Janus dendrimers with metal-chelating ligands conjugated to amphiphilic Janus dendrimers generates dendrimersomes that selectively display folded proteins on their periphery in an oriented manner. A modular strategy for tethering nucleic acids to the surface of dendrimersomes is also demonstrated. These findings augment the functional capabilities of dendrimersomes to serve as versatile biological membrane mimics.
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http://dx.doi.org/10.1073/pnas.1904868116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681758PMC
July 2019

Identification of Guide-Intrinsic Determinants of Cas9 Specificity.

CRISPR J 2019 06;2:172-185

Editas Medicine, Cambridge, Massaschusetts.

Considerable effort has been devoted to developing a comprehensive understanding of CRISPR nuclease specificity. predictions and multiple genome-wide cellular and biochemical approaches have revealed a basic understanding of the Cas9 specificity profile. However, none of these approaches has delivered a model that allows accurate prediction of a CRISPR nuclease's ability to cleave a site based entirely on the sequence of the guide RNA (gRNA) and the target. We describe a library-based biochemical assay that directly reports the cleavage efficiency of a particular Cas9-guide complex by measuring both uncleaved and cleaved target molecules over a wide range of mismatched library members. We applied our assay using libraries of targets to evaluate the specificity of Cas9 under a variety of experimental conditions. Surprisingly, our data show an unexpectedly high variation in the random gRNA:target DNA mismatch tolerance when cleaving with different gRNAs, indicating guide-intrinsic mismatch permissiveness and challenging the assumption of universal specificity models. We use data generated by our assay to create the first off-target, guide-specific cleavage models. The barcoded libraries of targets approach is rapid, highly modular, and capable of generating protein- and guide-specific models, as well as illuminating the biophysics of Cas9 binding versus cutting. These models may be useful in identifying potential off-targets, and the gRNA-intrinsic nature of mismatch tolerance argues for coupling these specificity models with orthogonal methods for a more complete assessment of gRNA specificity.
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http://dx.doi.org/10.1089/crispr.2019.0009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6694761PMC
June 2019

3D Printing of Thermoresponsive Polyisocyanide (PIC) Hydrogels as Bioink and Fugitive Material for Tissue Engineering.

Polymers (Basel) 2018 May 21;10(5). Epub 2018 May 21.

Faculty of Materials Science and Engineering, Warsaw University of Technology, 141 Woloska str., 02-507 Warsaw, Poland.

Despite the rapid and great developments in the field of 3D hydrogel printing, a major ongoing challenge is represented by the development of new processable materials that can be effectively used for bioink formulation. In this work, we present an approach to 3D deposit, a new class of fully-synthetic, biocompatible PolyIsoCyanide (PIC) hydrogels that exhibit a reverse gelation temperature close to physiological conditions (37 °C). Being fully-synthetic, PIC hydrogels are particularly attractive for tissue engineering, as their properties-such as hydrogel stiffness, polymer solubility, and gelation kinetics-can be precisely tailored according to process requirements. Here, for the first time, we demonstrate the feasibility of both 3D printing PIC hydrogels and of creating dual PIC-Gelatin MethAcrylate (GelMA) hydrogel systems. Furthermore, we propose the use of PIC as fugitive hydrogel to template structures within GelMA hydrogels. The presented approach represents a robust and valid alternative to other commercial thermosensitive systems-such as those based on Pluronic F127-for the fabrication of 3D hydrogels through additive manufacturing technologies to be used as advanced platforms in tissue engineering.
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http://dx.doi.org/10.3390/polym10050555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6415434PMC
May 2018

Encoding biological recognition in a bicomponent cell-membrane mimic.

Proc Natl Acad Sci U S A 2019 03 28;116(12):5376-5382. Epub 2019 Feb 28.

Department of Chemistry, Roy & Diana Vagelos Laboratories, University of Pennsylvania, Philadelphia, PA 19104-6323;

Self-assembling dendrimers have facilitated the discovery of periodic and quasiperiodic arrays of supramolecular architectures and the diverse functions derived from them. Examples are liquid quasicrystals and their approximants plus helical columns and spheres, including some that disregard chirality. The same periodic and quasiperiodic arrays were subsequently found in block copolymers, surfactants, lipids, glycolipids, and other complex molecules. Here we report the discovery of lamellar and hexagonal periodic arrays on the surface of vesicles generated from sequence-defined bicomponent monodisperse oligomers containing lipid and glycolipid mimics. These vesicles, known as glycodendrimersomes, act as cell-membrane mimics with hierarchical morphologies resembling bicomponent rafts. These nanosegregated morphologies diminish sugar-sugar interactions enabling stronger binding to sugar-binding proteins than densely packed arrangements of sugars. Importantly, this provides a mechanism to encode the reactivity of sugars via their interaction with sugar-binding proteins. The observed sugar phase-separated hierarchical arrays with lamellar and hexagonal morphologies that encode biological recognition are among the most complex architectures yet discovered in soft matter. The enhanced reactivity of the sugar displays likely has applications in material science and nanomedicine, with potential to evolve into related technologies.
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http://dx.doi.org/10.1073/pnas.1821924116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6431222PMC
March 2019

Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10.

Nat Med 2019 02 21;25(2):229-233. Epub 2019 Jan 21.

Editas Medicine, Cambridge, MA, USA.

Leber congenital amaurosis type 10 is a severe retinal dystrophy caused by mutations in the CEP290 gene. We developed EDIT-101, a candidate genome-editing therapeutic, to remove the aberrant splice donor created by the IVS26 mutation in the CEP290 gene and restore normal CEP290 expression. Key to this therapeutic, we identified a pair of Staphylococcus aureus Cas9 guide RNAs that were highly active and specific to the human CEP290 target sequence. In vitro experiments in human cells and retinal explants demonstrated the molecular mechanism of action and nuclease specificity. Subretinal delivery of EDIT-101 in humanized CEP290 mice showed rapid and sustained CEP290 gene editing. A comparable surrogate non-human primate (NHP) vector also achieved productive editing of the NHP CEP290 gene at levels that met the target therapeutic threshold, and demonstrated the ability of CRISPR/Cas9 to edit somatic primate cells in vivo. These results support further development of EDIT-101 for LCA10 and additional CRISPR-based medicines for other inherited retinal disorders.
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http://dx.doi.org/10.1038/s41591-018-0327-9DOI Listing
February 2019

Screening Libraries of Amphiphilic Janus Dendrimers Based on Natural Phenolic Acids to Discover Monodisperse Unilamellar Dendrimersomes.

Biomacromolecules 2019 02 7;20(2):712-727. Epub 2018 Nov 7.

Roy & Diana Vagelos Laboratories, Department of Chemistry , University of Pennsylvania , Philadelphia , Pennsylvania 19104-6323 , United States.

Natural, including plant, and synthetic phenolic acids are employed as building blocks for the synthesis of constitutional isomeric libraries of self-assembling dendrons and dendrimers that are the simplest examples of programmed synthetic macromolecules. Amphiphilic Janus dendrimers are synthesized from a diversity of building blocks including natural phenolic acids. They self-assemble in water or buffer into vesicular dendrimersomes employed as biological membrane mimics, hybrid and synthetic cells. These dendrimersomes are predominantly uni- or multilamellar vesicles with size and polydispersity that is predicted by their primary structure. However, in numerous cases, unilamellar dendrimersomes completely free of multilamellar assemblies are desirable. Here, we report the synthesis and structural analysis of a library containing 13 amphiphilic Janus dendrimers containing linear and branched alkyl chains on their hydrophobic part. They were prepared by an optimized iterative modular synthesis starting from natural phenolic acids. Monodisperse dendrimersomes were prepared by injection and giant polydisperse by hydration. Both were structurally characterized to select the molecular design principles that provide unilamellar dendrimersomes in higher yields and shorter reaction times than under previously used reaction conditions. These dendrimersomes are expected to provide important tools for synthetic cell biology, encapsulation, and delivery.
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http://dx.doi.org/10.1021/acs.biomac.8b01405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6571140PMC
February 2019

Clinical Evidence for Use of a Noninvasive Biosensor for Tear Glucose as an Alternative to Painful Finger-Prick for Diabetes Management Utilizing a Biopolymer Coating.

Biomacromolecules 2018 11 25;19(11):4504-4511. Epub 2018 Oct 25.

NovioSense B.V., NovioTech Campus, Transistorweg 5 , Nijmegen 6534 AT , The Netherlands.

Diabetes is a metabolic condition that is exponentially increasing worldwide. Current monitoring methods for diabetes are invasive, painful, and expensive. Herein, we present the first multipatient clinical trial that demonstrates clearly that tear fluid may be a valuable marker for systemic glucose measurements. The NovioSense Glucose Sensor, worn under the lower eye lid (inferior conjunctival fornix), is reported to continuously measure glucose levels in the basal tear fluid with good correlation to blood glucose values, showing clear clinical feasibility in both animals and humans. Furthermore, the polysaccharide coated device previously reported by our laboratory when worn, does not induce pain or irritation. In a phase II clinical trial, six patients with type 1 Diabetes Mellitus were enrolled and the capability of the device to measure glucose in the tear fluid was evaluated. The NovioSense Glucose Sensor gives a stable signal and the results correlate well to blood glucose values obtained from finger-prick measurements determined by consensus error grid analysis.
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http://dx.doi.org/10.1021/acs.biomac.8b01429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6234487PMC
November 2018

Publisher Correction: Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells.

Nat Commun 2018 08 28;9(1):3542. Epub 2018 Aug 28.

Editas Medicine, 11 Hurley St., Cambridge, MA 02141, USA.

The original HTML version of this Article incorrectly listed an affiliation of Josh Tycko as 'Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA', instead of the correct 'Present address: Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA'. It also incorrectly listed an affiliation of this author as 'Present address: Arrakis Therapeutics, 35 Gatehouse Dr., Waltham, MA, 02451, USA'.The original HTML version incorrectly listed an affiliation of Luis A. Barrera as 'Present address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, 06511, USA', instead of the correct 'Present address: Arrakis Therapeutics, 35 Gatehouse Dr., Waltham, MA 02451, USA'.Finally, the original HTML version incorrectly omitted an affiliation of Nicholas C. Huston: 'Present address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511, USA'.This has been corrected in the HTML version of the Article. The PDF version was correct from the time of publication.
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http://dx.doi.org/10.1038/s41467-018-06029-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6113259PMC
August 2018

Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells.

Nat Commun 2018 07 27;9(1):2962. Epub 2018 Jul 27.

Editas Medicine, 11 Hurley St., Cambridge, MA, 02141, USA.

Therapeutic genome editing with Staphylococcus aureus Cas9 (SaCas9) requires a rigorous understanding of its potential off-target activity in the human genome. Here we report a high-throughput screening approach to measure SaCas9 genome editing variation in human cells across a large repertoire of 88,692 single guide RNAs (sgRNAs) paired with matched or mismatched target sites in a synthetic cassette. We incorporate randomized barcodes that enable whitelisting of correctly synthesized molecules for further downstream analysis, in order to circumvent the limitation of oligonucleotide synthesis errors. We find SaCas9 sgRNAs with 21-mer or 22-mer spacer sequences are generally more active, although high efficiency 20-mer spacers are markedly less tolerant of mismatches. Using this dataset, we developed an SaCas9 specificity model that performs robustly in ranking off-target sites. The barcoded pairwise library screen enabled high-fidelity recovery of guide-target relationships, providing a scalable framework for the investigation of CRISPR enzyme properties and general nucleic acid interactions.
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http://dx.doi.org/10.1038/s41467-018-05391-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6063963PMC
July 2018

Effects of Disease-Causing Mutations on the Conformation of Human Apolipoprotein A-I in Model Lipoproteins.

Biochemistry 2018 07 13;57(30):4583-4596. Epub 2018 Jul 13.

Department of Chemistry and Chemical Biology , Northeastern University , 360 Huntington Avenue , Boston , Massachusetts 02115 , United States.

Plasma high-density lipoproteins (HDLs) are protein-lipid nanoparticles that transport lipids and protect against atherosclerosis. Human apolipoprotein A-I (apoA-I) is the principal HDL protein whose mutations can cause either aberrant lipid metabolism or amyloid disease. Hydrogen-deuterium exchange (HDX) mass spectrometry (MS) was used to study the apoA-I conformation in model discoidal lipoproteins similar in size to large plasma HDL. We examined how point mutations associated with hereditary amyloidosis (F71Y and L170P) or atherosclerosis (L159R) influence the local apoA-I conformation in model lipoproteins. Unlike other apoA-I forms, the large particles showed minimal conformational heterogeneity, suggesting a fully extended protein conformation. Mutation-induced structural perturbations in lipid-bound protein were attenuated compared to the free protein and indicated close coupling between the two belt-forming apoA-I molecules. These perturbations propagated to distant lipoprotein sites, either increasing or decreasing their protection. This HDX MS study of large model HDL, compared with previous studies of smaller particles, ascertained that apoA-I's central region helps accommodate the protein conformation to lipoproteins of various sizes. This study also reveals that the effects of mutations on lipoprotein conformational dynamics are much weaker than those in a lipid-free protein. Interestingly, the mutation-induced perturbations propagate to distant sites nearly 10 nm away and alter their protection in ways that cannot be predicted from the lipoprotein structure and stability. We propose that long-range mutational effects are mediated by both protein and lipid and can influence lipoprotein functionality.
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http://dx.doi.org/10.1021/acs.biochem.8b00538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6067968PMC
July 2018

UDiTaS™, a genome editing detection method for indels and genome rearrangements.

BMC Genomics 2018 03 21;19(1):212. Epub 2018 Mar 21.

Editas Medicine, 11 Hurley Street, Cambridge, MA, 02141, USA.

Background: Understanding the diversity of repair outcomes after introducing a genomic cut is essential for realizing the therapeutic potential of genomic editing technologies. Targeted PCR amplification combined with Next Generation Sequencing (NGS) or enzymatic digestion, while broadly used in the genome editing field, has critical limitations for detecting and quantifying structural variants such as large deletions (greater than approximately 100 base pairs), inversions, and translocations.

Results: To overcome these limitations, we have developed a Uni-Directional Targeted Sequencing methodology, UDiTaS, that is quantitative, removes biases associated with variable-length PCR amplification, and can measure structural changes in addition to small insertion and deletion events (indels), all in a single reaction. We have applied UDiTaS to a variety of samples, including those treated with a clinically relevant pair of S. aureus Cas9 single guide RNAs (sgRNAs) targeting CEP290, and a pair of S. pyogenes Cas9 sgRNAs at T-cell relevant loci. In both cases, we have simultaneously measured small and large edits, including inversions and translocations, exemplifying UDiTaS as a valuable tool for the analysis of genome editing outcomes.

Conclusions: UDiTaS is a robust and streamlined sequencing method useful for measuring small indels as well as structural rearrangements, like translocations, in a single reaction. UDiTaS is especially useful for pre-clinical and clinical application of gene editing to measure on- and off-target editing, large and small.
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http://dx.doi.org/10.1186/s12864-018-4561-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5861650PMC
March 2018

Polyisocyanopeptide hydrogels: A novel thermo-responsive hydrogel supporting pre-vascularization and the development of organotypic structures.

Acta Biomater 2018 04 15;70:129-139. Epub 2018 Feb 15.

Tissue Biology Research Unit, Department of Surgery, University Children's Hospital Zurich, August Forel Str. 7, CH-8008 Zurich, Switzerland. Electronic address:

Molecular and mechanical interactions with the 3D extracellular matrix are essential for cell functions such as survival, proliferation, migration, and differentiation. Thermo-responsive biomimetic polyisocyanopeptide (PIC) hydrogels are promising new candidates for 3D cell, tissue, and organ cultures. This is a synthetic, thermo-responsive and stress-stiffening material synthesized via polymerization of the corresponding monomers using a nickel perchlorate as a catalyst. It can be tailored to meet various demands of cells by modulating its stiffness and through the decoration of the polymer with short GRGDS peptides using copper free click chemistry. These peptides make the hydrogels biocompatible by mimicking the binding sites of certain integrins. This study focuses on the optimization of the PIC polymer properties for efficient cell, tissue and organ development. Screening for the optimal stiffness of the hydrogel and the ideal concentration of the GRGDS ligand conjugated with the polymer, enabled cell proliferation, migration and differentiation of various primary cell types of human origin. We demonstrate that fibroblasts, endothelial cells, adipose-derived stem cells and melanoma cells, do survive, thrive and differentiate in optimized PIC hydrogels. Importantly, these hydrogels support the spontaneous formation of complex structures like blood capillaries in vitro. Additionally, we utilized the thermo-responsive properties of the hydrogels for a rapid and gentle recovery of viable cells. Finally, we show that organotypic structures of human origin grown in PIC hydrogels can be successfully transplanted subcutaneously onto immune-compromised rats, on which they survive and integrate into the surrounding tissue.

Statement Of Significance: Molecular and mechanical interactions with the surrounding environment are essential for cell functions. Although 2D culture systems greatly contributed to our understanding of complex biological phenomena, they cannot substitute for crucial interaction that take place in 3D. 3D culture systems aim to overcome limitations of the 2D cultures and answer new questions about cell functions. Thermo-responsive biomimetic polyisocyanopeptide (PIC) hydrogels are promising new candidates for 3D cell, tissue, and organ cultures. They are synthetic and can be tailor to meet certain experimental demands. Additionally, they are characterized by strain-stiffening, a feature crucial for cell behaviour, but rare in hydrogels. Their thermos-responsive properties enable quick recovery of the cells by a simple procedure of lowering the temperature.
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http://dx.doi.org/10.1016/j.actbio.2018.01.042DOI Listing
April 2018

Exploring functional pairing between surface glycoconjugates and human galectins using programmable glycodendrimersomes.

Proc Natl Acad Sci U S A 2018 03 30;115(11):E2509-E2518. Epub 2018 Jan 30.

Roy & Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323;

Precise translation of glycan-encoded information into cellular activity depends critically on highly specific functional pairing between glycans and their human lectin counter receptors. Sulfoglycolipids, such as sulfatides, are important glycolipid components of the biological membranes found in the nervous and immune systems. The optimal molecular and spatial design aspects of sulfated and nonsulfated glycans with high specificity for lectin-mediated bridging are unknown. To elucidate how different molecular and spatial aspects combine to ensure the high specificity of lectin-mediated bridging, a bottom-up toolbox is devised. To this end, negatively surface-charged glycodendrimersomes (GDSs), of different nanoscale dimensions, containing sulfo-lactose groups are self-assembled in buffer from a synthetic sulfatide mimic: Janus glycodendrimer (JGD) containing a 3'--sulfo-lactose headgroup. Also prepared for comparative analysis are GDSs with nonsulfated lactose, a common epitope of human membranes. These self-assembled GDSs are employed in aggregation assays with 15 galectins, comprising disease-related human galectins, and other natural and engineered variants from four families, having homodimeric, heterodimeric, and chimera architectures. There are pronounced differences in aggregation capacity between human homodimeric and heterodimeric galectins, and also with respect to their responsiveness to the charge of carbohydrate-derived ligand. Assays reveal strong differential impact of ligand surface charge and density, as well as lectin concentration and structure, on the extent of surface cross-linking. These findings demonstrate how synthetic JGD-headgroup tailoring teamed with protein engineering and network assays can help explain how molecular matchmaking operates in the cellular context of glycan and lectin complexity.
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http://dx.doi.org/10.1073/pnas.1720055115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5856548PMC
March 2018

Knee instability as the primary cause of failure following Total Knee Arthroplasty (TKA): A systematic review on the patient, surgical and implant characteristics of revised TKA patients.

Knee 2017 Dec 29;24(6):1271-1281. Epub 2017 Sep 29.

College of Medicine and Public Health, Flinders University, Adelaide, South Australia, Australia; The International Musculoskeletal Research Institute Inc., 13 Laffers Road, Belair, South Australia 5052, Australia. Electronic address:

Background: The aim of this review was to systematically assess the current evidence available regarding knee instability after TKA to identify time to failure between primary and revision TKA. In addition, we considered the patient, surgical and implant characteristics of primary TKA patients revised for knee instability, and investigated methods used for knee instability diagnosis.

Methods: A systematic search of six databases and the unpublished literature was performed. Studies referring to instability in post-operative primary TKA patients, reporting on revision TKA due to instability, and published or available between 2005 to 30-Mar-2015 were eligible for inclusion. Quantitative data for continuous variables were pooled in statistical meta-analyses.

Results: A total of 1841 unique studies were identified, 42 of which met the selection criteria and a total of 22 studies included in the review. Time to failure between primary and revision TKA was 44.7months (95% CI [33.8, 55.7]), and the weighted mean age at time of revision surgery was 67.6years (95% CI [65.38, 69.75]). A gender distribution was identified, with approximately 16.4% more females revised for instability, however this was unable to be corrected for the baseline population. The majority of studies used a combination of radiographic and clinical testing to diagnose knee instability.

Conclusion: Research on knee instability following primary TKA reported early failure and subsequent revision knee surgery. The need for revision due to instability was frequently reported in a younger patient cohort and most commonly in female TKA patients. Early revision at a younger age highlights the severe implications of an unstable knee.
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http://dx.doi.org/10.1016/j.knee.2017.08.060DOI Listing
December 2017

Attentional interference is modulated by salience not sentience.

Acta Psychol (Amst) 2017 Jul 1;178:56-65. Epub 2017 Jun 1.

Department of Psychological Sciences, University of Liverpool, United Kingdom.

Spatial cueing of attention occurs when attention is oriented by the onset of a stimulus or by other information that creates a bias towards a particular location. The presence of a cue that orients attention can also interfere with participants' reporting of what they see. It has been suggested that this type of interference is stronger in the presence of socially-relevant cues, such as human faces or avatars, and is therefore indicative of a specialised role for perspective calculation within the social domain. However, there is also evidence that the effect is a domain-general form of processing that is elicited equally with non-social directional cues. The current paper comprises four experiments that systematically manipulated the social factors believed necessary to elicit the effect. The results show that interference persists when all social components are removed, and that visual processes are sufficient to explain this type of interference, thus supporting a domain-general perceptual interpretation of interference.
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http://dx.doi.org/10.1016/j.actpsy.2017.05.010DOI Listing
July 2017

Polymer Brush-Functionalized Chitosan Hydrogels as Antifouling Implant Coatings.

Biomacromolecules 2017 Jun 12;18(6):1983-1992. Epub 2017 May 12.

DWI-Leibniz Institute for Interactive Materials and Institute of Technical and Macromolecular Chemistry, RWTH Aachen University , Forckenbeckstraße 50, 52074 Aachen, Germany.

Implantable sensor devices require coatings that efficiently interface with the tissue environment to mediate biochemical analysis. In this regard, bioinspired polymer hydrogels offer an attractive and abundant source of coating materials. However, upon implantation these materials generally elicit inflammation and the foreign body reaction as a consequence of protein fouling on their surface and concomitant poor hemocompatibility. In this report we investigate a strategy to endow chitosan hydrogel coatings with antifouling properties by the grafting of polymer brushes in a "grafting-from" approach. Chitosan coatings were functionalized with polymer brushes of oligo(ethylene glycol) methyl ether methacrylate and 2-hydroxyethyl methacrylate using photoinduced single electron transfer living radical polymerization and the surfaces were thoroughly characterized by XPS, AFM, water contact angle goniometry, and in situ ellipsometry. The antifouling properties of these new bioinspired hydrogel-brush coatings were investigated by surface plasmon resonance. The influence of the modifications to the chitosan on hemocompatibility was assessed by contacting the surfaces with platelets and leukocytes. The coatings were hydrophilic and reached a thickness of up to 180 nm within 30 min of polymerization. The functionalization of the surface with polymer brushes significantly reduced the protein fouling and eliminated platelet activation and leukocyte adhesion. This methodology offers a facile route to functionalizing implantable sensor systems with antifouling coatings that improve hemocompatibility and pave the way for enhanced device integration in tissue.
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http://dx.doi.org/10.1021/acs.biomac.7b00516DOI Listing
June 2017

Structural stability and local dynamics in disease-causing mutants of human apolipoprotein a-I: what makes the protein amyloidogenic?

Amyloid 2017 Mar 31;24(sup1):11-12. Epub 2016 Dec 31.

a Department of Physiology & Biophysics , Boston University School of Medicine , Boston , MA , USA and.

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http://dx.doi.org/10.1080/13506129.2016.1269737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5557347PMC
March 2017

Jenkins-CI, an Open-Source Continuous Integration System, as a Scientific Data and Image-Processing Platform.

SLAS Discov 2017 03 13;22(3):238-249. Epub 2016 Dec 13.

3 Developmental and Molecular Pathways, NIBR, Postfach, Basel, Switzerland.

High-throughput screening generates large volumes of heterogeneous data that require a diverse set of computational tools for management, processing, and analysis. Building integrated, scalable, and robust computational workflows for such applications is challenging but highly valuable. Scientific data integration and pipelining facilitate standardized data processing, collaboration, and reuse of best practices. We describe how Jenkins-CI, an "off-the-shelf," open-source, continuous integration system, is used to build pipelines for processing images and associated data from high-content screening (HCS). Jenkins-CI provides numerous plugins for standard compute tasks, and its design allows the quick integration of external scientific applications. Using Jenkins-CI, we integrated CellProfiler, an open-source image-processing platform, with various HCS utilities and a high-performance Linux cluster. The platform is web-accessible, facilitates access and sharing of high-performance compute resources, and automates previously cumbersome data and image-processing tasks. Imaging pipelines developed using the desktop CellProfiler client can be managed and shared through a centralized Jenkins-CI repository. Pipelines and managed data are annotated to facilitate collaboration and reuse. Limitations with Jenkins-CI (primarily around the user interface) were addressed through the selection of helper plugins from the Jenkins-CI community.
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http://dx.doi.org/10.1177/1087057116679993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5322829PMC
March 2017

Blood Loss in Total Knee Arthroplasty.

J Knee Surg 2017 Jun 21;30(5):452-459. Epub 2016 Sep 21.

The International Musculoskeletal Research Institute Inc., Adelaide, South Australia, Australia.

Patients undergoing total knee arthroplasty (TKA) have expected blood loss during and after surgery. The morbidity associated with blood loss and the burden of blood transfusions in adult arthroplasty necessitates preoperative optimization as routine practice. Current literature remains inconclusive on which TKA surgical instrumentation techniques are effective in minimizing perioperative blood loss, and consequently lower transfusion rates. The primary objective of this retrospective review, of a prospective randomized cohort study, was to compare surgical and patient factors, and their influence on blood loss and transfusions rates, between one type of patient-specific instrumentation (PSI), navigated computer-assisted surgery (CAS), and conventional TKA surgical techniques.A cohort of 128 matched patients (38 PSI, 44 CAS, 46 conventional surgeries) were compared. Preoperative factors analyzed included; age, gender, body mass index, preoperative hemoglobin (Hb) (g/L), international normalized ratio, use of anticoagulants and comorbid bleeding diathesis. Maximal Hb drop and transfusion requirements were compared on day 1 to 3. Perioperative factors collected included: surgical time, tourniquet time, drain output, in situ drain time, order of tibia or femoral cut, and intraoperative loss from suction.The three groups did not differ on the preoperative patient demographics examined. The difference between preoperative Hb and the lowest postoperative Hb readings did not differ between study groups ( = 0.39).There are no statistically significant differences in blood loss when comparing PSI versus CAS versus conventional TKA. Although emerging evidence on PSI is encouraging, the PSI technique for TKA does not result in reduced blood loss. The study was registered with ClinicalTrials.gov: NCT01145157.
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http://dx.doi.org/10.1055/s-0036-1592147DOI Listing
June 2017

Atomic Layer Deposition of Ruthenium with TiN Interface for Sub-10 nm Advanced Interconnects beyond Copper.

ACS Appl Mater Interfaces 2016 Oct 21;8(39):26119-26125. Epub 2016 Sep 21.

imec, Kapeldreef 75, 3001 Leuven, Belgium.

Atomic layer deposition of ruthenium is studied as a barrierless metallization solution for future sub-10 nm interconnect technology nodes. We demonstrate the void-free filling in sub-10 nm wide single damascene lines using an ALD process in combination with 2.5 Å of ALD TiN interface and postdeposition annealing. At such small dimensions, the ruthenium effective resistance depends less on the scaling than that of Cu/barrier systems. Ruthenium effective resistance potentially crosses the Cu curve at 14 and 10 nm according to the semiempirical interconnect resistance model for advanced technology nodes. These extremely scaled ruthenium lines show excellent electromigration behavior. Time-dependent dielectric breakdown measurements reveal negligible ruthenium ion drift into low-κ dielectrics up to 200 °C, demonstrating that ruthenium can be used as a barrierless metallization in interconnects. These results indicate that ruthenium is highly promising as a replacement to Cu as the metallization solution for future technology nodes.
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http://dx.doi.org/10.1021/acsami.6b07181DOI Listing
October 2016

Association of MTOR Mutations With Developmental Brain Disorders, Including Megalencephaly, Focal Cortical Dysplasia, and Pigmentary Mosaicism.

JAMA Neurol 2016 07;73(7):836-845

Division of Genetic Medicine, Department of Pediatrics, University of Washington, Seattle, Washington, USA.

Importance: Focal cortical dysplasia (FCD), hemimegalencephaly, and megalencephaly constitute a spectrum of malformations of cortical development with shared neuropathologic features. These disorders are associated with significant childhood morbidity and mortality.

Objective: To identify the underlying molecular cause of FCD, hemimegalencephaly, and diffuse megalencephaly.

Design, Setting, And Participants: Patients with FCD, hemimegalencephaly, or megalencephaly (mean age, 11.7 years; range, 2-32 years) were recruited from Pediatric Hospital A. Meyer, the University of Hong Kong, and Seattle Children's Research Institute from June 2012 to June 2014. Whole-exome sequencing (WES) was performed on 8 children with FCD or hemimegalencephaly using standard-depth (50-60X) sequencing in peripheral samples (blood, saliva, or skin) from the affected child and their parents and deep (150-180X) sequencing in affected brain tissue. Targeted sequencing and WES were used to screen 93 children with molecularly unexplained diffuse or focal brain overgrowth. Histopathologic and functional assays of phosphatidylinositol 3-kinase-AKT (serine/threonine kinase)-mammalian target of rapamycin (mTOR) pathway activity in resected brain tissue and cultured neurons were performed to validate mutations.

Main Outcomes And Measures: Whole-exome sequencing and targeted sequencing identified variants associated with this spectrum of developmental brain disorders.

Results: Low-level mosaic mutations of MTOR were identified in brain tissue in 4 children with FCD type 2a with alternative allele fractions ranging from 0.012 to 0.086. Intermediate-level mosaic mutation of MTOR (p.Thr1977Ile) was also identified in 3 unrelated children with diffuse megalencephaly and pigmentary mosaicism in skin. Finally, a constitutional de novo mutation of MTOR (p.Glu1799Lys) was identified in 3 unrelated children with diffuse megalencephaly and intellectual disability. Molecular and functional analysis in 2 children with FCD2a from whom multiple affected brain tissue samples were available revealed a mutation gradient with an epicenter in the most epileptogenic area. When expressed in cultured neurons, all MTOR mutations identified here drive constitutive activation of mTOR complex 1 and enlarged neuronal size.

Conclusions And Relevance: In this study, mutations of MTOR were associated with a spectrum of brain overgrowth phenotypes extending from FCD type 2a to diffuse megalencephaly, distinguished by different mutations and levels of mosaicism. These mutations may be sufficient to cause cellular hypertrophy in cultured neurons and may provide a demonstration of the pattern of mosaicism in brain and substantiate the link between mosaic mutations of MTOR and pigmentary mosaicism in skin.
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http://dx.doi.org/10.1001/jamaneurol.2016.0363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4979321PMC
July 2016