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Hemogenic endothelial (HE) cells are specialized endothelial cells to give rise to hematopoietic stem/progenitor cells during hematopoietic development. The underlying mechanisms that regulate endothelial-to-hematopoietic transition (EHT) of human HE cells are not fully understand. Here, we identified platelet endothelial aggregation receptor-1 (PEAR1) as a novel regulator of early hematopoietic development in human pluripotent stem cells (hPSCs). We found that the expression of PEAP1 was elevated during hematopoietic development. A subpopulation of PEAR1

Gallium hydrides stabilised by primary and secondary amines are scarce due to their propensity to eliminate dihydrogen. Consequently, their reactivity has received limited attention. The synthesis of two novel gallium hydride complexes HGa(THF)[ON(H)O] and H2 Ga[μ2 -ON(H)O]Ga[ON(H)O] ([ON(H)O]2- =N,N-bis(3,5-di-tert-butyl-2-phenoxy)amine) is described and their reactivity towards aldehydes and ketones is explored. These reactions afford alkoxide-bridged dimers through 1,2-hydrogallation reactions. The gallium hydrides can be regenerated through Ga-O/B-H metathesis from the reaction of such dimers with pinacol borane (HBpin) or 9-borabicyclo[3.3.1]nonane (9-BBN). These observations allowed us to target the catalytic reduction of carbonyl substrates (aldehydes, ketones and carbon dioxide) with low catalyst loadings at room temperature.

Chlorhexidine skin antisepsis is frequently recommended for most surgical procedures; however, it is unclear if these recommendations should apply to surgery involving traumatic contaminated wounds where povidone-iodine has previously been preferred. We aimed to compare the effect of aqueous 10% povidone-iodine versus aqueous 4% chlorhexidine gluconate on the risk of surgical site infection in patients who required surgery for an open fracture.We conducted a multiple-period, cluster-randomised, crossover trial (Aqueous-PREP) at 14 hospitals in Canada, Spain, and the USA. Eligible patients were adults aged 18 years or older with an open extremity fracture treated with a surgical fixation implant. For inclusion, the open fracture required formal surgical debridement within 72 h of the injury. Participating sites were randomly assigned (1:1) to use either aqueous 10% povidone-iodine or aqueous 4% chlorhexidine gluconate immediately before surgical incision; sites then alternated between the study interventions every 2 months. Participants, health-care providers, and study personnel were aware of the treatment assignment due to the colour of the solutions. The outcome adjudicators and data analysts were masked to treatment allocation. The primary outcome was surgical site infection, guided by the 2017 US Centers for Disease Control and Prevention National Healthcare Safety Network reporting criteria, which included superficial incisional infection within 30 days or deep incisional or organ space infection within 90 days of surgery. The primary analyses followed the intention-to-treat principle and included all participants in the groups to which they were randomly assigned. This study is registered with ClinicalTrials.gov, NCT03385304.Between April 8, 2018, and June 8, 2021, 3619 patients were assessed for eligibility and 1683 were enrolled and randomly assigned to povidone-iodine (n=847) or chlorhexidine gluconate (n=836). The trial's adjudication committee determined that 45 participants were ineligible, leaving 1638 participants in the primary analysis, with 828 in the povidone-iodine group and 810 in the chlorhexidine gluconate group (mean age 44·9 years [SD 18·0]; 629 [38%] were female and 1009 [62%] were male). Among 1571 participants in whom the primary outcome was known, a surgical site infection occurred in 59 (7%) of 787 participants in the povidone-iodine group and 58 (7%) of 784 in the chlorhexidine gluconate group (odds ratio 1·11, 95% CI 0·74 to 1·65; p=0·61; risk difference 0·6%, 95% CI -1·4 to 3·4).For patients who require surgical fixation of an open fracture, either aqueous 10% povidone-iodine or aqueous 4% chlorhexidine gluconate can be selected for skin antisepsis on the basis of solution availability, patient contraindications, or product cost. These findings might also have implications for antisepsis of other traumatic wounds.US Department of Defense, Canadian Institutes of Health Research, McMaster University Surgical Associates, PSI Foundation.

To extend the frontier of genome editing and enable editing of repetitive elements of mammalian genomes, we made use of a set of dead-Cas9 base editor (dBE) variants that allow editing at tens of thousands of loci per cell by overcoming the cell death associated with DNA double-strand breaks and single-strand breaks. We used a set of gRNAs targeting repetitive elements—ranging in target copy number from about 32 to 161 000 per cell. dBEs enabled survival after large-scale base editing, allowing targeted mutations at up to ∼13 200 and ∼12 200 loci in 293T and human induced pluripotent stem cells (hiPSCs), respectively, three orders of magnitude greater than previously recorded. These dBEs can overcome current on-target mutation and toxicity barriers that prevent cell survival after large-scale genome engineering.

Overall, dermatologic surgery performed in the outpatient setting is very low risk to patients and safer than similar procedures performed under general anesthesia, and is also more cost-effective. There are several approaches to mitigating the risk of complications while optimizing patient outcomes. Strict oversight of the dermatology clinic helps to ensure team members all adhere to standards of care. Vial safety, strict hand hygiene, limiting the use of topical antibiotics, generally continuing all blood thinners perioperatively, and prebiopsy photographs are all examples of approaches to help maximize patient safety.

Established in 2014, the American Academy of Orthopaedic Surgeons (AAOS) Resident Assembly (RA) has served as a mode of "bidirectional communication" between AAOS and a combined resident body. Training and education initiatives relevant to the current issues facing residency training can be passed up to and directly addressed by the leadership of AAOS, whereas AAOS recruitment and membership initiatives can be disseminated to the full resident body through the RA. Since its inception in 2014, the RA has grown markedly, with representation from most MD and DO residency programs in the United States and Canada. It also has included an increasing number of medical students from Orthopaedic Surgery Interest Groups to directly take part in RA activities. For the past half decade, the RA has served as a partner for the AAOS in addition to a valuable recruitment tool to engage the broadest diversity of potential orthopaedic leaders at their earliest stages of training. This work is a review of the development of the RA over its first half decade, as well as a discussion of its future goals in line with AAOS priorities.

We have exploited the repetitive nature of transposable elements of the human genome to generate synthetic circuits. Transposable elements such as LINE-1 and Alu have successfully replicated in mammalian genomes throughout evolution to reach a copy number ranging from thousands to more than a million. Targeting these repetitive elements with programmable DNA nucleases such as CRISPR-Cas9 rapidly induce extremely high levels of cell death. We use this genotoxic feature to build synthetic biocontainment circuits: CRISPR defense system (CRISPR-DS) capable of preventing CRISPR genome editing, and we introduce the proof-of-concept of CRISPR Safety-Switch, an inducible, stringent and non-leaky kill-switch capable of clearing out cell lines resistant to DNA breaks.

BACKGROUND Recent data have shown an increased risk for rejection, fibrosis progression, and death in liver transplantation (LT) recipients with preformed or de novo HLA donor-specific alloantibodies (DSA). However, the role of non-HLA autoantibodies and the interaction between HLA DSA and non-HLA autoantibodies remains uncharacterized. METHODS We analyzed 1269 primary LT recipients from 1 of 2000 to 4 of 2009 with known HLA DSA status for angiotensin II type-1 receptor and endothelin-1 type A receptor autoantibodies pre-LT, and year 1 post-LT. RESULTS Preformed non-HLA autoantibodies alone did not impact outcomes. In multivariable modeling, the combination of preformed non-HLA autoantibodies and HLA-DSA were associated with an increased risk for death (hazard ratio [HR], 1.66; P = 0.02) especially if the HLA DSA was of the IgG3 subclass (HR, 2.28; P = 0.01). A single de novo non-HLA autoantibody was associated with an increased risk for T cell-mediated rejection or antibody-mediated rejection (68% vs 41%, P = 0.01) and fibrosis progression (HR, 1.84; P = 0.02). Biopsies with de novo non-HLA autoantibodies revealed a new sinusoidal C4d staining pattern when compared with HLA DSA (71% vs 3%; P < 0.001). Liver sinusoidal endothelial cell activation and stellate cell activation was increased in patients with non-HLA autoantibodies in the location of C4d positivity. CONCLUSIONS A non-HLA autoantibody combined with a preformed HLA DSA is associated with an increased mortality risk. Isolated de novo anti-angiotensin II type-1 receptor and anti-endothelin-1 type A receptor autoantibodies are associated with an increased risk of rejection and fibrosis progression. The novel location of C4d staining in proximity to liver sinusoidal endothelial cell capillarization and stellate cell activation demonstrates allograft injury in proximity to non-HLA autoantibody binding.

There is increasing demand for single-stranded DNA (ssDNA) of lengths >200 nucleotides (nt) in synthetic biology, biological imaging and bionanotechnology. Existing methods to produce high-purity long ssDNA face limitations in scalability, complexity of protocol steps and/or yield. We present a rapid, high-yielding and user-friendly method for in vitro production of high-purity ssDNA with lengths up to at least seven kilobases. Polymerase chain reaction (PCR) with a forward primer bearing a methanol-responsive polymer generates a tagged amplicon that enables selective precipitation of the modified strand under denaturing conditions. We demonstrate that ssDNA is recoverable in ∼40–50 min (time after PCR) with >70% yield with respect to the input PCR amplicon, or up to 70 pmol per 100 μl PCR reaction. We demonstrate that the recovered ssDNA can be used for CRISPR/Cas9 homology directed repair in human cells, DNA-origami folding and fluorescent in-situ hybridization.

Browsers are widely used on personal computers, laptops and mobile devices. In this chapter, we seek to determine and compare which forensic artifacts can be recovered from Google Chrome, Mozilla Firefox, their respective private modes, and TOR. Our analysis was primarily conducted using FTK in order to replicate the process and abilities of a digital forensics lab with limited resources. After identical data generation across all browsers and modes of browsing in a controlled virtual environment, forensic images were captured then analyzed. This research not only extends the current field of digital forensics for which artifacts can be found in which locations, but also confirms various claims in regards to the privacy of private browsing modes. As expected, all data was recovered from regular browsing modes, very minimal data from private browsing, and almost no artifacts from TOR.

To extend the frontier of genome editing and enable the radical redesign of mammalian genomes, we developed a set of dead-Cas9 base editor (dBE) variants that allow editing at tens of thousands of loci per cell by overcoming the cell death associated with DNA double-strand breaks (DSBs) and single-strand breaks (SSBs). We used a set of gRNAs targeting repetitive elements – ranging in target copy number from about 31 to 124,000 per cell. dBEs enabled survival after large-scale base editing, allowing targeted mutations at up to ~13,200 and ~2610 loci in 293T and human induced pluripotent stem cells (hiPSCs), respectively, three orders of magnitude greater than previously recorded. These dBEs can overcome current on-target mutation and toxicity barriers that prevent cell survival after large-scale genome engineering.One Sentence SummaryBase editing with reduced DNA nicking allows for the simultaneous editing of >10,000 loci in human cells.

We present a rapid, scalable, user-friendly method for in vitro production of high-purity single-stranded DNA (ssDNA) ranging from 89–3315 nucleotides in length. PCR with a forward primer bearing a methanol-responsive polymer generates a tagged amplicon that enables selective precipitation of the modified strand under denaturing conditions. We demonstrate that the recovered ssDNA can be used for CRISPR/Cas9 homology-directed repair in human cells, DNA-origami folding, and fluorescent in situ hybridization.

International audience; Multiplex genome editing is the simultaneous introduction of multiple distinct modifications to a given genome. Though in its infancy, maturation of this field will facilitate powerful new biomedical research approaches and will enable a host of far-reaching biological engineering applications, including new therapeutic modalities and industrial applications, as well as “genome writing” and de-extinction efforts. In this Perspective, we focus on multiplex editing of large eukaryotic genomes. We describe the current state of multiplexed genome editing, the current limits of our ability to multiplex edits, and provide perspective on the many applications that fully realized multiplex editing technologies would enable in higher eukaryotic genomes. We offer a broad look at future directions, covering emergent CRISPR-based technologies, advances in intracellular delivery, and new DNA assembly approaches that may enable future genome editing on a massively multiplexed scale.

A proposed chronic antibody-mediated rejection (AMR) score has recently predicted 50%10-year death-censored allograft loss in patients with donor-specific alloantibodies (DSA) mean florescence intensity (MFI) greater than 10 000 and requires confirmation in patients with lower MFI (1000-10 000).All patients who underwent liver transplantation from January 2000 to April 2009, had DSA (MFI ≥1000) in serum 10 to 14 months postliver transplantation, and had a protocolized liver biopsy were evaluated (n = 230). The previously proposed chronic AMR (cAMR) score was used to risk-stratify putative chronic AMR in DSA+ patients with MFI from 1000 to 10 000.The MFI distribution of DSA+ recipients were as follows: 66% had MFI 1000 to 4999, 14% had MFI 5000 to 10 000, and 20% had MFI greater than 10 000. The cAMR score distribution on 1-year protocol liver biopsy found that 41% had a score less than 13; 27% a score of 13 to 27.5, and 32% a score greater than 27.5. MFI correlated with 1-year cAMR category (13, 46% vs 21% and27.5, 29% vs 42% when MFI was 1000-10 000 vs MFI10 000; P = 0.047). In patients with a cAMR score less than 13, 10-year death-censored allograft survival was 96% to 100% regardless of MFI (P = NS). The risk of allograft loss increased in patients with a cAMR score greater than 13 (P = 0.004) in DSA+ patients with MFI 1000 to 10 000. DSA MFI greater than 10 000 versus MFI 1000 to 10 000 at 1 year was also more likely to persist at 5 years (95% vs 68%; P0.0001).Validation of the previously proposed cAMR score in a separate cohort predicts death-censored long-term allograft failure in DSA+ patients regardless of MFI, and higher MFI at 1 year predicts DSA persistence at 5 years.

Human pluripotent stem cells (PSCs) hold great potential for regenerative medicine and currently are being used as a research tool for basic discovery and disease modeling. To evaluate the role of a single genetic variant, a system of genome editing is needed to precisely mutate any desired DNA sequence in isolation and measure its effect on phenotype when compared to the isogenic parental PSC from which it was derived. This protocol describes the general targeting schemes used by researchers to edit PSCs to knock out, knock-in, or precisely alter a single nucleotide, covering conditions for electroporation, clonal isolation, and screening of edited PSCs for the targeted mutation. These recent advances simplify the procedure for genome editing, allowing individual researchers to induce nearly any desired mutation to further study its function or to reverse a disease-causing variant for future applications in regenerative medicine.

Since the year 2000 the Waikato Institute of Technology has been reviewing the list of “Notable and Historic” trees of New Zealand first published by the Forest Service in 1974. Since that time 29.7% of those trees have been lost – 53% of those losses due to development, 29% to natural causes such as storms and disease, and a further 18% that could not be located. Despite this tree loss New Zealand has an impressive list of trees of international significance, many of which are regarded as the largest of their species in the world.

Grain growth of nanocrystalline aluminum ( nc Al) in trimodal Al metal-matrix-composites (MMCs) during hot forging was investigated. The nc Al phase formed through cryomilling of inert gas-atomized powders in liquid nitrogen has an average grain size down to 21 nm, exhibits excellent thermal stability. However, substantial grain growth of nc Al up to 63 nm was observed when the Al MMCs were thermo-mechanically processed even at relatively low temperatures. Grain growth of the cryomilled nc Al phase in trimodal Al MMCs after hot forging was documented with respect to temperature ranging from 175 °C to 287 °C, true strain ranging from 0.4 to 1.35 and strain rate ranging from 0.1 to 0.5 s −1 . Hollow cone dark field imaging technique was employed to provide statistically confident measurements of nc Al grain size that ranged from 21 to 63 nm. An increase in forging temperature and an increase in true strain were correlated with an increase in grain size of nc Al. Results were correlated to devise a phenomenological grain growth model for forging that takes strain, strain rate and temperature into consideration. Activation energy for the grain growth during thermo-mechanical hot-forging was determined to be 35 kJ/mol, approximately a quarter of activation energy for bulk diffusion of Al and a half of activation energy for static recrystallization.

Lack of access to health care coupled with a severe shortage of physicians is on the forefront of much debate nationally and internationally. Rural areas often suffer the most. Innovations in medical education, such as the creation of rural immersion rotations, are attempting to solve this health crisis. The purpose of this study was to investigate and analyze the narrative writings of 36 fourth-year medical students and primary care residents that participated in a rural immersion rotation in the Mississippi Delta. A grounded theory data analysis led to the emergence of four central themes: Positive refl ections of the experience, community health, limited access to health care, and alteration of prior perceptions. The fi ndings of this research provide rich data from the perspective of the medical student and resident. This study concluded that the use of narrative inquiry was benefi cial within the rural immersion experience, and the experience positively infl uenced and impacted medical students and residents.

This paper describes a methodology based on hollow-cone dark-field (HCDF) transmission electron microscopy (TEM) to study dislocation structures in both nano- and micro-crystalline grains. Although the conventional approach based on a two-beam condition has been commonly used to acquire weak-beam dark-field (WBDF) TEM images for dislocation structure characterization, it is very challenging to employ this technique to study nanocrystalline materials, especially when the grains are less than 100 nm in diameter. Compared to the conventional two-beam approach, the method described in this paper is more conducive for obtaining high-quality WBDF-TEM images. Furthermore, the method is suitable for studying samples with both nanocrystalline and coarse-grains. A trimodal Al metal-matrix-composite (MMC) consisting of B4C particles, a nanocrystalline Al (NC-Al) phase, and a coarse-grained Al (CG-Al) phase has been reported to exhibit an extremely high strength and tailorable ductility. The dislocations in both NC-Al and CG-Al phases of the trimodal Al MMCs at different fabrication stages were examined using the HCDF method described. The influence of the dislocation density in both NC-Al and CG-Al phases on the strength and ductility of the composite is also discussed.

Human induced pluripotent stem cells (iPSCs) and genome editing provide a precise way to generate gene-corrected cells for disease modeling and cell therapies. Human iPSCs generated from sickle cell disease (SCD) patients have a homozygous missense point mutation in the HBB gene encoding adult β-globin proteins, and are used as a model system to improve strategies of human gene therapy. We demonstrate that the CRISPR/Cas9 system designer nuclease is much more efficient in stimulating gene targeting of the endogenous HBB locus near the SCD point mutation in human iPSCs than zinc finger nucleases and TALENs. Using a specific guide RNA and Cas9, we readily corrected one allele of the SCD HBB gene in human iPSCs by homologous recombination with a donor DNA template containing the wild-type HBB DNA and a selection cassette that was subsequently removed to avoid possible interference of HBB transcription and translation. We chose targeted iPSC clones that have one corrected and one disrupted SCD allele for erythroid differentiation assays, using an improved xeno-free and feeder-free culture condition we recently established. Erythrocytes from either the corrected or its parental (uncorrected) iPSC line were generated with similar efficiencies. Currently ∼6%–10% of these differentiated erythrocytes indeed lacked nuclei, characteristic of further matured erythrocytes called reticulocytes. We also detected the 16-kDa β-globin protein expressed from the corrected HBB allele in the erythrocytes differentiated from genome-edited iPSCs. Our results represent a significant step toward the clinical applications of genome editing using patient-derived iPSCs to generate disease-free cells for cell and gene therapies. Stem Cells 2015;33:1470–1479

Efficient and precise genome editing is crucial for realizing the full research and therapeutic potential of human induced pluripotent stem cells (iPSCs). Engineered nucleases including CRISPR/Cas9 and transcription activator like effector nucleases (TALENs) provide powerful tools for enhancing gene-targeting efficiency. In this study, we investigated the relative efficiencies of CRISPR/Cas9 and TALENs in human iPSC lines for inducing both homologous donor-based precise genome editing and nonhomologous end joining (NHEJ)-mediated gene disruption. Significantly higher frequencies of NHEJ-mediated insertions/deletions were detected at several endogenous loci using CRISPR/Cas9 than using TALENs, especially at nonexpressed targets in iPSCs. In contrast, comparable efficiencies of inducing homologous donor-based genome editing were observed at disease-associated loci in iPSCs. In addition, we investigated the specificity of guide RNAs used in the CRISPR/Cas9 system in targeting disease-associated point mutations in patient-specific iPSCs. Using myeloproliferative neoplasm patient-derived iPSCs that carry an acquired JAK2-V617F point mutation and α1-antitrypsin (AAT) deficiency patient-derived iPSCs that carry an inherited Z-AAT point mutation, we demonstrate that Cas9 can specifically target either the mutant or the wild-type allele with little disruption at the other allele differing by a single nucleotide. Overall, our results demonstrate the advantages of the CRISPR/Cas9 system in allele-specific genome targeting and in NHEJ-mediated gene disruption.

Summary Culture-expanded human mesenchymal stem cells (MSCs) are increasingly used in clinics, yet full characterization of the genomic compositions of these cells is lacking. We present a whole-genome investigation on the genetic dynamics of cultured MSCs under ex vivo establishment (passage 1 [p1]) and serial expansion (p8 and p13). We detected no significant changes in copy-number alterations (CNAs) and low levels of single-nucleotide changes (SNCs) until p8. Strikingly, a significant number (677) of SNCs were found in p13 MSCs. Using a sensitive Droplet Digital PCR assay, we tested the nonsynonymous SNCs detected by whole-genome sequencing and found that they were preexisting low-frequency mutations in uncultured mononuclear cells (∼0.01%) and early-passage MSCs (0.1%–1% at p1 and p8) but reached 17%–36% in p13. Our data demonstrate that human MSCs maintain a stable genomic composition in the early stages of ex vivo culture but are subject to clonal growth upon extended expansion., Graphical Abstract, Highlights • Whole-genome investigation reveals variances of cultured MSCs along serial passages • Human MSCs maintain a stable genomic composition in early stages of ex vivo culture • Human MSCs are subject to clonal growth upon extended expansion • The “new” SNCs present in the expanded MSCs preexist in the early cell population, Wang, Cheng, and colleagues present a whole-genome analysis on genetic dynamics of cultured MSCs under ex vivo establishment (passage 1) and serial expansion (passages 8 and 13). The data demonstrate that human MSCs maintain a stable genomic composition in the early stages of ex vivo culture but are subject to clonal growth upon extended expansion.

Our ability to precisely and efficiently edit mammalian and plant genomes has been significantly improved in recent years, partially due to increasing use of designer nucleases that recognize a pre-determined DNA sequence, make a specific DNA double-strand break, and stimulate gene targeting. A pair of zinc finger nucleases (ZFNs) or transcription activator–like effector nucleases (TALENs) that recognize two adjacent unique DNA sequences dimerize through the fused FokI nuclease domain and cut in the middle of target DNA sequences. We report here that increasing the length of recognition DNA sequences by TALENs or ZFNs does not necessarily translate to a higher efficiency or specificity. We also discover that one subunit of ZFNs and one subunit of TALENs can form a pair of hybrid nucleases with expanded specificity at two diverse targets, and stimulate gene targeting in multiple cell types including human induced pluripotent stem (iPS) cells with improved efficiency.

Pluripotent stem cells (PSCs), defined by their capacity for self-renewal and differentiation into all cell types, are an integral tool for basic biological research and disease modeling. However, full use of PSCs for research and regenerative medicine requires the ability to precisely edit their DNA to correct disease-causing mutations and for functional analysis of genetic variations. Recent advances in DNA editing of human stem cells (including PSCs) have benefited from the use of designer nucleases capable of making double-strand breaks (DSBs) at specific sequences that stimulate endogenous DNA repair. The clustered, regularly interspaced short palindromic repeats (CRISPR)–Cas9 system has become the preferred designer nuclease for genome editing in human PSCs and other cell types. Here we describe the principles for designing a single guide RNA to uniquely target a gene of interest and describe strategies for disrupting, inserting, or replacing a specific DNA sequence in human PSCs. The improvements in efficiency and ease provided by these techniques allow individuals to precisely engineer PSCs in a way previously limited to large institutes and core facilities.

Derivation of pluripotent stem cells (iPSCs) induced from somatic cell types and the subsequent genetic modifications of disease-specific or patient-specific iPSCs are crucial steps in their applications for disease modeling as well as future cell and gene therapies. Conventional procedures of these processes require co-culture with primary mouse embryonic fibroblasts (MEFs) to support self-renewal and clonal growth of human iPSCs as well as embryonic stem cells (ESCs). However, the variability of MEF quality affects the efficiencies of all these steps. Furthermore, animal sourced feeders may hinder the clinical applications of human stem cells. In order to overcome these hurdles, we established immortalized human feeder cell lines by stably expressing human telomerase reverse transcriptase, Wnt3a, and drug resistance genes in adult mesenchymal stem cells. Here, we show that these immortalized human feeders support efficient derivation of virus-free, integration-free human iPSCs and long-term expansion of human iPSCs and ESCs. Moreover, the drug-resistance feature of these feeders also supports nonviral gene transfer and expression at a high efficiency, mediated by piggyBac DNA transposition. Importantly, these human feeders exhibit superior ability over MEFs in supporting homologous recombination-mediated gene targeting in human iPSCs, allowing us to efficiently target a transgene into the AAVS1 safe harbor locus in recently derived integration-free iPSCs. Our results have great implications in disease modeling and translational applications of human iPSCs, as these engineered human cell lines provide a more efficient tool for genetic modifications and a safer alternative for supporting self-renewal of human iPSCs and ESCs.

This article was written to describe a collaborative effort between the University of Mississippi Medical Center and the Delta Health Alliance to increase primary care providers in the 18 counties designated as the Mississippi Delta. Journals compiled by trainees were analyzed to reveal issues significant to practicing in a rural area from the perspective of medical students and residents who participated in the program. Patient noncompliance, provider sensitivity to cost, continuity of care, and quality of the doctor-patient relationship were all identified as significant issues by the trainees.

Human iPSCs provide renewable cell sources for human biology and disease research and the potential for developing gene and cell therapy. Realization of this potential will rely in part on our ability to precisely edit or engineer the human genome in an efficient way. Recent developments in designer endonuclease technologies such as zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9 endonuclease have provided ways to significantly improve genome editing efficiency in human iPSCs. These endonucleases make a double-stranded break (DSB) at a predetermined DNA sequence and trigger natural DNA repair processes such as nonhomologous end joining (NHEJ) or homologous recombination (HR) with a donor DNA template. Among these existing approaches, RNA-guided CRISPR/Cas9 is the most user-friendly and versatile system, and it has been applied in both animal models and cell lines (Cong et al., 2013; Hsu et al., 2014; Mali et al., 2013). The most commonly used system consists of a single polypeptide endonuclease Cas9 complexed with a single guide RNA (gRNA) that provides complementarity to 20-nucleotide target DNA sequence. However, the specificity and efficiency of this approach in human iPSCs have not been studied in detail (Cong et al., 2013; Ding et al., 2013; Mali et al., 2013; Yang et al., 2013). Some analyses using cancer cell lines reported higher-than-expected levels of off-target mutagenesis by Cas9-gRNAs (Fu et al., 2013; Hsu et al., 2013), raising concerns about the practical applicability of this approach in therapeutic contexts. Some recent studies, including one on human adult stem cells, showed a minimal level of off-target effects by CRISPR/Cas9 (Schwank et al., 2013). However, these existing analyses of off-target effects and mutational load in gene-corrected stem cells have been restricted to checking predicted off target sites and are therefore limited in scope. To assess the value of this type of gene editing approach for therapeutic applications, it is critical to rigorously examine whether it is possible to generate gene-edited cell lines with minimal mutational load. To this end, we have conducted whole-genome sequencing of four iPSC clones successfully targeted at the AAVS1 locus, a “safe harbor” in the human genome that is used for stable transgene expression in a variety of contexts. To generate the lines, we used an integration-free human iPSC line, BC1, whose genomic integrity has been characterized in detail by next-generation sequencing (Cheng et al., 2012) and targeted a GFP expression cassette into the AAVS1 site with either a previously reported Cas9-gRNA combination or a pair of improved heterodimeric TALENs (Mali et al., 2013; Yan et al., 2013) (Table S1 and Supplemental Experimental Procedures available online). Twenty days after transfection of the donor plasmid and either the TALENs or Cas9-gRNA into BC1, we harvested four clones with confirmed targeted integration (hCas9-C4, hCas9-C16, TALEN-C3, and TALEN-C6; Table S1 and Supplemental Experimental Procedures) and the parental BC1 iPSCs for whole-genome sequencing. The sequencing reads, ranging from 83 Gbps to 100 Gbps from each targeted clone, were first aligned to the human hg19 reference genome to enable identification of single-nucleotide variants (SNVs) and small indels (Table S1). Our analysis identified ≥4.2 million SNVs and ≥500,000 indels in each genome (Table S1) in comparison to the hg19 reference genome, suggesting that it is a rigorous data set that covers the genome in sufficient depth to detect sequence variants. The “germ-line” variants (present in BC1 parental iPSCs and different from hg19) were readily detectable in each targeted cell line (80%%–88%), indicating that the sensitivity of variant detection in our analysis is high (Table S1). The variations from each targeted clone were then compared to the BC1 parental iPSCs to enable the generation of a list of potential variations arising during the gene editing process, which we then confirmed using genomic PCR and Sanger sequencing. We confirmed 62 out of 69 SNVs tested for an overall confirmation rate of 90%, and based on that we estimate that the total SNVs in the four iPSC clones range between 217 and 281 and that the total indels range between 7 and 12 (Table S1). Overall the genomic variation levels in TALEN- and Cas9-targeted groups were comparable. One important consideration is how many of these detected SNVs and indels were the results of off-target mutagenesis by the engineered endonucleases. To address this question, we generated a list of 3,665 (Cas9) and 238 (TALEN) putative off-target positions by using the EMBOSS fuzznuc software package. Each candidate SNV and indel was compared to this list and none of them are within a potential off-target region (Table S1), consistent with previous analyses looking at predicted off-target sites. Our analysis also shows that each SNV and indel is unique and that none of them occurred in more than one cell line. The absence of recurring mutations and the fact that none of the mutations resides in any putative off-target site by bioinformatic prediction strongly suggest that these mutations were randomly accumulated during regular cell expansion and are not direct results of off-target activities by Cas9 or TALENs. Our results from whole-genome sequencing analysis of Cas9- and TALEN-targeted human iPSC clones demonstrate that these engineered endonucleases provide efficient genome-editing tools with high specificity. It remains to be clarified whether the higher off-target rates observed in cancer cell lines are due to the overexpression of gRNAs and Cas9 protein and/or due to exacerbated and faulty DNA repair in these cell types. The higher specificity observed in human iPSCs, combined with the rapid development of next-generation sequencing technology, makes it possible to characterize and isolate high quality genome-edited stem cell clones with minimal mutational load. The guiding principle established with human iPSCs will likely be applicable to other types of stem cells and come with improvements in gene transfer and targeting efficiencies. Our current study of gene targeting in human iPSCs will help to establish better models for human biology and disease research and to provide proof-of-principle for future gene therapy.

Methodologies have been developed for modeling both gas dynamics and heat transfer inside the carbon fiber rope (CFR) for applications in the space shuttle reusable solid rocket motor joints. Specifically, the CFR is modeled using an equivalent rectangular duct with a cross-section area, friction factor and heat transfer coefficient such that this duct has the same amount of mass flow rate, pressure drop, and heat transfer rate as the CFR. An equation for the friction factor is derived based on the Darcy-Forschheimer law and the heat transfer coefficient is obtained from pipe flow correlations. The pressure, temperature and velocity of the gas inside the CFR are calculated using the one-dimensional Navier-Stokes equations. Various subscale tests, both cold flow and hot flow, have been carried out to validate and refine this CFR model. In particular, the following three types of testing were used: (1) cold flow in a RSRM nozzle-to-case joint geometry, (2) cold flow in a RSRM nozzle joint No. 2 geometry, and (3) hot flow in a RSRM nozzle joint environment simulator. The predicted pressure and temperature history are compared with experimental measurements. The effects of various input parameters for the model are discussed in detail.