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Warm-ups are an essential element of exercise and sport performance. Although research has demonstrated benefits associated with active warm-ups, research investigating the effects of a passive warm-up on subsequent performance is limited. The purpose of this study is to compare the effects of an active, post-activation potentiation (PAP) warm-up to a passive warm-up on vertical jump performance. Study participants were randomly assigned to one of two groups. Each group performed both the active and passive warm-ups, in reverse order, to control for order effect. Participants in this study consisted of 20 college students (men and women) between the ages of 18 and 35 years who were well-trained and anaerobically fit. A repeated measures ANOVA revealed a statistically significant main effect difference in vertical jump measures (F=16.148, p

T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells1–3. Here we developed a clinical-grade approach based on CRISPR–Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRβ). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen–HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial (NCT03970382). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.

Intronic G4C2 hexanucleotide repeat expansions (HRE) of C9orf72 are the most common cause of familial variants of frontotemporal dementia/amyotrophic lateral sclerosis (FTD/ALS). G4C2 HREs in C9orf72 undergo non-canonical repeat-associated translation, producing dipeptide repeat (DPR) proteins, with various deleterious impacts on cellular homeostasis. While five different DPRs are produced, poly(glycine-arginine) (GR) is amongst the most toxic and is the only DPR to accumulate in the associated clinically relevant anatomical locations of the brain. Previous work has demonstrated the profound effects of a poly(GR) model of C9orf72 FTD/ALS, including motor impairment, memory deficits, neurodegeneration, and neuroinflammation. Neuroinflammation is hypothesized to be a driving factor in the disease course; microglia activation is present prior to symptom onset and persists throughout the disease. Here, using an established mouse model of C9orf72 FTD/ALS we investigate the contributions of the nod-like receptor pyrin-containing 3 (NLRP3) inflammasome in the pathogenesis of FTD/ALS. We find that inflammasome-mediated neuroinflammation is increased with microglial activation, cleavage of caspase-1, production of IL-1β and upregulation of Cxcl10 in the brain of C9orf72 FTD/ALS mice. Excitingly, we find that genetic ablation of Nlrp3 significantly improved survival, protected behavioral deficits and prevented neurodegeneration suggesting a novel mechanism involving HRE-mediated induction of innate immunity. The findings provide experimental evidence of the integral role of HRE in inflammasome-mediated innate immunity in the C9orf72 variant of FTD/ALS pathogenesis and suggest the NLRP3 inflammasome as a therapeutic target.

Tin-117 m (Sn-117m) is used to treat dogs with osteoarthritic joints by radiosynoviorthesis. The decay process for Sn-117m is internal conversion wherein IC electrons and auger electrons provide the therapeutic effect. Additionally, the most prominent gamma emission is 158.6 keV. The effective dose rate received by a person interacting at close distances with a treated dog is needed to determine the person's total dose and thus regulatory compliance. Simple measurement of the dose rate at a given distance does not provide an accurate measurement of the effective dose to a person due to the non-uniform nature of the radiation field at close distances. MNCP models of the interactions of five ages of humans at three distances were created to determine the effective dose rates using the methodology from NRC Regulatory Guide 8.40. Ratios of the effective dose rate to the person to the measured dose rate at 1 m from the same source were calculated.

Increasing use of modified live virus (MLV) vaccines presents challenges to interpret positive results of porcine reproductive and respiratory syndrome virus (PRRSV) screening PCR that can detect both wild-type and vaccine strains. Instead, vaccine-specific PCR provides a convenient tool to detect vaccine-like virus from a sample. Here we report the development and validation of a real-time RT-PCR specific for PRRSGard® , a newly available commercial PRRSV-2 MLV vaccine. Analytical specificity, sensitivity and diagnostic performance of PRRSGard PCR were evaluated and compared to a commercial PRRSV screening PCR (reference PCR). PRRSGard and reference PCRs did not cross-react with any of the 27 non-PRRSV swine pathogens. PRRSGard PCR did not cross-react with other PRRSV-2 vaccine viruses and 31 laboratory and field PRRSV-2 isolates representing various genetic lineages of PRRSV-2. PRRSGard and reference PCRs consistently detected up to 10-6 and 10-5 dilutions of PRRSGard vaccine virus, respectively. Based on testing serial dilutions of in vitro transcribed RNA, the 95% limit of detection of PRRSGard PCR was 16 genomic copies/reaction with CT cut-off value of 36 and 7 genomic copies/reaction with CT cut-off value of 37. Diagnostic performance of PRRSGard PCR was evaluated using 846 clinical samples (684 serum and 162 oral fluid samples). Compared to the reference screening PCR, diagnostic sensitivity, specificity and agreement of PRRSGard PCR were 95.34%, 98.85% and 97.52% with cut-off CT value of 36 and 98.14%, 96.56% and 97.16% with cut-off CT value of 37. In addition, PRRSGard PCR was able to detect PRRSGard vaccine virus in a sample even with the co-presence of another PRRSV strain. In summary, in contrast to a reference screening PCR that detects both vaccine and field PRRSV strains, PRRSGard PCR provides a convenient tool to specifically detect PRRSGard vaccine-like virus and to inform PRRSV vaccination protocols.

Background: Adoptive T-cell therapies would benefit from accurate computational prediction of T cell receptor (TCR) specificity to its target antigen. Despite the diversity of complementarity determining region (CDR) among T cells, many sequence features of CDRs are conserved. Conserved CDR sequences allow for shared specificity, that is, recognition of the same antigen, potentially increasing the pool of candidate TCRs available for treating patients. Utilizing sequence similarity, enrichment of V-genes, CDR lengths, and evidence of clonal expansion, several computational algorithms have been developed to predict the specificity of TCRs. Using neoantigen-specific TCRs (neoTCRs) in our phase 1 clinical trial (NCT039703820), we investigated the shared specificity of TCRs in the context of personalized autologous T cell therapy for cancer patients. Methods: Leveraging a high throughput TCR discovery and validation platform, neoantigen-specific T cells were isolated from patient peripheral blood mononuclear cells (PBMCs) and their neoTCR sequences were identified. RNA-seq was performed on tumor biopsies and TCR sequences derived from tumor-infiltrating lymphocytes (TILs) were extracted using MiXCR software. TCR specificity algorithms TCRdist3, GLIPH2, and TCRmatch were then used to identify specificity groups within and between neoTCR sequences derived from patient PBMCs, TCR sequences derived from TILs, and publicly available TCR sequences of known specificities. Results: NeoTCRs identified during our phase 1 clinical trial and their known neoantigens provided an experimentally validated benchmark for testing accuracy of TCR specificity prediction. TCR specificity algorithms accurately clustered neoTCR β chains into groups of shared antigen specificity. TCR chain sequences detected from TILs with high similarity to TCR sequences of neoTCRs from PMBCs were found, suggesting shared antigen specificity between circulating T cells and those found in the tumor. In addition, TCR specificity algorithms identified a public TCR sequence known to recognize an HPV-derived epitope presented by HLA-A*02:01 with high similarity to a TCR sequence identified in a tumor biopsy of an HPV16+ HLA-A*02:01+ patient with head and neck cancer. Conclusion: Neoantigens and their associated neoTCRs identified by our TCR discovery and validation platform can be used as a benchmark for TCR specificity prediction algorithms. TCR specificity algorithms provide insight into TCRs with predicted shared specificity in the blood and within the tumor. Accurate identification of TIL TCRs with shared specificity to neoTCRs could inform on tumor trafficking and aid in therapeutic product selection. Similarly, accurate specificity matching of TIL TCRs to public TCRs with known targets could aid in identification of efficacious targets within the tumor. Citation Format: Tyler Borrman, Eric Stawiski, Zheng Pan, Chad Smith, Susan Foy, Stefanie J. Mandl. TCR specificity prediction of circulating T cells and TILs in personalized adoptive neoTCR T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3125.

Background: NeoTCR-P1 is a personalized autologous T cell therapy for treatment of patients with solid tumors. Neoantigen-specific T cell receptors (neoTCRs) were isolated from the patients’ circulating CD8 T cells followed by non-viral precision genome engineering into autologous apheresis-derived T cells for infusion back into the patient. We present comprehensive immune monitoring data for 2 patients treated in a first-in-human Phase 1 clinical trial (NCT03970382). Each patient was infused with their own personalized cell therapy product consisting of 3 unique neoTCRs targeting neoantigens identified as truncal in screening biopsies. Methods: We identified infused neoTCR specific T cells in the blood by using patient-specific peptide HLA reagents with single cell RNA seq and in the tumor using RNA scope probes or bulk transcriptome analysis. Results: Patient 0503, a patient with MSS colorectal cancer, was treated with 3 unique neoTCRs (1.3x109 neoTCR+ cells) which targeted 2 distinct neoantigens presented by HLA-B*39:01 or HLA-C*12:02. Analysis of longitudinal biopsies confirmed that targeted mutations were truncal, with both neoantigens present pre- and post-dosing. NeoTCR+ T cells were identified in the tumor by RNA seq and imaging, with neoTCR+ T cells also expressing Granzyme B. Furthermore, single cell RNA seq of neoTCR+ T cells from the pre-infusion product as compared neoTCR T cells isolated from the patients’ blood 2 months post-infusion demonstrated evidence of antigen engagement in 1 out of 3 neoTCR T cell products, with a shift from a predominantly Tcm/Tem-like phenotype pre-infusion to a Teff-like state post-infusion. This neoTCR T cell product also increased slightly in the blood from 1.1 to 3.4% between month 1 and 2, while the other 2 T cell products remained predominantly in the Tcm-like phenotype and remained stable in the blood between month 1 and 2. Patient 0612, a patient with melanoma, was treated with 3 unique neoTCRs (4.0x109 neoTCR+ cells) which targeted 3 distinct neoantigens presented by HLA-B*08:01 or HLA-C*07:02. Longitudinal biopsies support the assessment that 2/3 mutations were truncal and 1 targeted mutation was sub-clonal based on their presence in each biopsy. Retrospective analysis showed HLA loss of heterozygosity at HLA-C07:02 in all 3 biopsies, affecting 2/3 infused TCR products. The remaining TCR showed evidence of a shift from a predominantly Tcm-like phenotype pre-infusion to more differentiated Teff/Tcm-em phenotypes post-infusion compared to the 2 TCRs targeting mutations expressed by the lost HLA allele. Conclusion: These case studies illustrate for the first time tracking of 3 unique personalized T cell products within a single patient. Shifts in the T cell phenotype of the neoTCR T cell products post-infusion for some, but not all, of the products suggest early signs of T cell engagement with the target. Citation Format: Susan Foy, Cliff Wang, Benjamin Yuen, Sara Said, Boi Quach, Jonathan Hoover, Saparya Nayak, Theresa Hunter, Zheng Pan, Chad Smith, Tyler Borrman, Eric Stawiski, Alphonsus H. C. Ng, Yue Lu, James Heath, Stefanie Mandl. Monitoring of neoantigens and adoptively transferred personalized neoTCR T cell populations in the tumor and PBMCs of patients with solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 901.

Intronic G4C2hexanucleotide repeat expansions (HRE) ofC9orf72are the most common cause of familial variants of frontotemporal dementia/amyotrophic lateral sclerosis (FTD/ALS). G4C2HREs inC9orf72undergo non-canonical repeat-associated translation, producing dipeptide repeat (DPR) proteins, with various deleterious impacts on cellular homeostasis. While five different DPRs are produced, poly(glycine-arginine) (GR) is amongst the most toxic and is the only DPR to accumulate in the associated clinically relevant anatomical locations of the brain. Previous work has demonstrated the profound effects of a poly(GR) model ofC9orf72FTD/ALS, including motor impairment, memory deficits, neurodegeneration, and neuroinflammation. Neuroinflammation is hypothesized to be a driving factor in the disease course; microglia activation is present prior to symptom onset and persists throughout the disease. Here, using an established mouse model ofC9orf72FTD/ALS we investigate the contributions of the nod-like receptor pyrin-containing 3 (NLRP3) inflammasome in the pathogenesis of FTD/ALS. We find that inflammasome-mediated neuroinflammation is increased with microglial activation, cleavage of caspase-1, production of IL-1β and upregulation ofCxcl10in the brain ofC9orf72FTD/ALS mice. Excitingly, we find that genetic ablation ofNlrp3significantly improved survival, protected behavioral deficits and prevented neurodegeneration suggesting a novel mechanism involving HRE-mediated induction of innate immunity. The findings provide experimental evidence of the integral role of HRE in inflammasome-mediated innate immunity in theC9orf72variant of FTD/ALS pathogenesis and suggest the NLRP3 inflammasome as a therapeutic target.

Although the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) technique has dramatically lowered the cost and increased the speed of generating genetically engineered mice, success depends on using guide RNAs and donor DNAs which direct efficient knock-out (KO) or knock-in (KI). By Sanger sequencing DNA from blastocysts previously injected with the same CRISPR components intended to produce the engineered mice, one can test the effectiveness of different guide RNAs and donor DNAs. We describe in detail here a simple, rapid (three days), inexpensive protocol, for amplifying DNA from blastocysts to determine the results of CRISPR point mutation KIs. Using it, we show that (1) the rate of KI seen in blastocysts is similar to that seen in mice for a given guide RNA/donor DNA pair, (2) a donor complementary to the variable portion of a guide integrated in a more all-or-none fashion, (3) donor DNAs can be used simultaneously to integrate two different mutations into the same locus, and (4) by placing silent mutations about every 6 to 10 bp between the Cas9 cut site and the desired mutation(s), the desired mutation(s) can be incorporated into genomic DNA over 30 bp away from the cut at the same high efficiency as close to the cut.

Channel modeling often provides a basis for the design and deployment of wireless technology. Engineers design systems to operate under certain expected channel conditions. Channel models are typically based on the statistics of a collection of many measurements performed by channel sounders in nominally similar radio-propagation environments. In 2016, researchers at the US Department of Commerce National Institute of Standards and Technology (NIST) and the National Telecommunications and Information Administration Institute for Telecommunication Sciences (ITS) began a collaboration to conduct a series of channel sounder verifications to identify sources of uncertainty due to systematic and random effects in a channel sounder hardware. This report describes conducted-channel measurements designed to focus on errors within the channel sounding hardware as a base-line test of the channel sounder’s performance. Repeat measurements and an analysis of the random components of uncertainty were performed. Sample results are reported and analyzed. The work concludes with guidance and best-practice procedures with the intent of allowing users to perform similar verifications of their channel sounders.

Previous evidence has suggested that dietary supplementation with a bioactive dietary polyphenol preparation (BDPP) rescues impairment of hippocampus-dependent memory in a mouse model of sleep deprivation (SD). In the current study, we extend our previous evidence and demonstrate that a mechanism by which dietary BDPP protects against SD-mediated cognitive impairment is via mechanisms that involve phosphorylation of the mammalian target of rapamycin complex 1 and its direct downstream targets, including the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) and the ribosomal protein S6 kinase β-1 (p70S6K). In additional mechanistic studies in vitro, we identified the brain bioavailable phenolic metabolites derived from the metabolism of dietary BDPP that are responsible for the attenuation of SD-mediated memory impairments. On the basis of high-throughput bioavailability studies of brain bioavailable metabolites after dietary BDPP treatment, we found that select polyphenol metabolites [ e.g., cyanidin-3'- O-glucoside and 3-(3'-hydroxyphenyl) propionic acid] were able to rescue mTOR and p70S6K phosphorylation in primary cortico-hippocampal neuronal cultures, as well as rescue 4E-BP1 phosphorylation in response to treatment with 4EGI-1, a specific inhibitor of eIF4E-eIF4G interaction. Our findings reveal a previously unknown role for dietary polyphenols in the rescue of SD-mediated memory impairments via mechanisms involving the promotion of protein translation.-Frolinger, T., Smith, C., Cobo, C. F., Sims, S., Brathwaite, J., de Boer, S., Huang, J., Pasinetti, G. M. Dietary polyphenols promote resilience against sleep deprivation-induced cognitive impairment by activating protein translation.

PACT Pharma has developed an ultra-sensitive approach to validate predicted neoantigens and the cognate T cell receptors (neoTCRs) from tumor specific somatic mutations by capturing neoantigen-specific T cells from peripheral blood. This process is used in clinical trials (NCT03970382) of personalized neoTCR-T therapy for persons with solid cancers. Using the state of the art prediction pipeline and screening process, more than one hundred twenty neoantigens were predicted from 5 type of solid cancers that were validated by characterizing cognate T cells and their close to 200 unique TCRs captured from the blood of the same individual. These validated neoepitopes of 8 to 11 amino acids represent broad HLA class I coverage with >30 alleles to date. Our analysis revealed that mutations can occur in all positions within the epitopes. Epitope immunogenicity is potentially affected by different mechanisms including mutation position, agretopicity, as well as HLA interacting positions and/or by interactions between mutated residues and its cognate TCRs. It was observed that these validated neoepitopes comprise broad ranges of predicted HLA binding affinities, stability, and neoantigen expression levels. The analysis presented here offers insights to enable machine learning to advance rules for epitope selection and prioritization that may be important for immunological approaches to address a broad range of diseases, including cancer. Citation Format: Zheng Pan, Olivier Dalmas, Songming Peng, Kyle Jacoby, Barbara Sennino, Yan Ma, Chad Smith, Amin Momin, Allison Xu, Katharine Heeringa, Jonathan Johnston, Duo An, Boi Quach, William Lu, Diana Nguyen, Andrew Conroy, Bhamini Purandare, Eva Huang, Eric Stawiski, Alex Franzusoff, Stefanie Mandl. Tumor neoantigen profiling with validated patient-specific TCR characterization to improve neoepitope prediction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2177.

Purpose The development and implementation of a residency project advisory board (RPAB) to manage multiple pharmacy residents’ yearlong projects across several residency programs are described. Summary Preceptor and resident feedback during our annual residency program review and strategic planning sessions suggested the implementation of a more-coordinated approach to the identification, selection, and oversight of all components of the residency project process. A panel of 7 department leaders actively engaged in residency training and performance improvement was formed to evaluate the residency project process and provide recommendations for change. These 7 individuals would eventually constitute the RPAB. The primary objective of the RPAB at Aurora Health Care is to provide oversight and a structured framework for the selection and execution of multiple residents’ yearlong projects across all residency programs within our organization. Key roles of the RPAB include developing expectations, coordinating residency project ideas, and providing oversight and feedback. The development and implementation of the RPAB resulted in a significant overhaul of our entire yearlong resident project process. Trends toward success were realized after the first year of implementation, including consistent expectations, increased clarity and engagement in resident project ideas, and more projects meeting anticipated endpoints. Conclusion The development and implementation of an RPAB have provided a framework to optimize the organization, progression, and outcomes of multiple pharmacy resident yearlong projects in all residency programs across our pharmacy enterprise.

Israeli entrepreneur and inventor Dov Moran envisioned the creation of a mobile device that was a small, stand-alone, fully functional mobile phone that could be slipped into a variety of enclosures, or “jackets,” that would provide added functionality and better reflect the personalities of its users. As the development of the Modu phone began to take shape, Moran and his team decided that to ensure the success of the new phone's much anticipated launch, Modu would develop and market the accessory jackets itself. The question now was which of the eight jackets to develop and what factors should be considered in making that decision. The case is about how to estimate optimal product-line extensions after accounting for experience curve and cannibalization effects of products that share similar features, cost, and price. This will require quantitative analysis that estimates the effect of the experience curve and cannibalization on cost, revenues, and ultimately, profit. The issue is how to optimize profits by choosing an ideal set of products. Understand the importance of quantitative analysis in launching product-line extensions while taking into account demand and cost side interactions Combine both qualitative and quantitative data in choosing a targeted segment Reflect on the strategic and financial considerations in choosing a segment for a new technology product Evaluate the implications of experience curve and cannibalization when introducing product-line extensions and their impact on the decision under consideration

Background CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as “two-donor floxing” method). Results We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. Conclusion We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.

Optimizing physical activity (PA) in children is paramount to attenuate the incidence of chronic disease and to improve social and cognitive health. Limited research exists examining the observed PA patterns during school leisure times in children from the U.S. The purpose of this study was to examine the observed PA patterns of children during three school leisure times: before school, during lunch, and after school. The SOPLAY instrument was used to observe PA during the three leisure times across six weeks at four elementary schools in the U.S. Observer PA counts were stratified by sex, PA intensity (sedentary, walking, and very active), and leisure time. Multi-level models were employed to examine the effect of leisure time and PA intensity on observer PA counts, adjusting for day and school-level clustering. Lunch displayed the greatest number of counts for sedentary, walking, and very active PA intensities (p < 0.001). In boys, a greater number of observer counts in the very active PA intensity were recorded compared to the sedentary intensity before school (β = 10.34, p = 0.04) and during lunch (β = 15.57, p < 0.001). Girls did not display any differences among the PA intensities within before school and lunch leisure times (p > 0.05). After school displayed the fewest counts for walking and very active PA in both sexes (p < 0.05). An emphasis should be placed on increasing walking and very active PA intensities before school and during lunch in girls and after school in both sexes. Keywords: after school, before school, lunch, SOPLAY, systematic observation

CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the method’s success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA–2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (

Background: Extensive evidence-based literature supports the use of intracameral (IC) moxifloxacin for the prevention of postoperative endophthalmitis after cataract surgery. The Aurora Pharmacy Packaging Center (APPC) has developed a process for centrally preparing IC moxifloxacin. Purpose: The aim of this study was to evaluate the centralized preparation of IC moxifloxacin production for quality assurance and to quantify a potential reduction in costs. Methods: The APPC’s compounding procedure of IC moxifloxacin was evaluated using United States Pharmacopeia (USP) Convention 797 standard and compared with practices described in evidence-based literature. Patients who received IC moxifloxacin intraoperatively from one of 3 ophthalmologists during cataract surgery performed between February 15, 2016, and August 15, 2016, were identified using electronic health records. Cost savings were calculated by reviewing costs associated with drug supplies used by the APPC. Results: The APPC process for the centralized preparation of IC moxifloxacin was deemed compliant with USP 797’s sterile compounding standards. USP 797 validation criteria included proper sterile technique, equipment, room sterility and pressure, beyond use dating, and storage. Implementation of the centralized production of moxifloxacin reduced the direct product cost per surgery from $140 to $20 (a cost savings of $120 per surgery). There were 459 cataract surgeries analyzed during the study period, resulting in a savings of $55 080 over 6 months. Conclusion: The APPC’s centralized compounding procedure was found to be compliant with pharmacy compounding standards and to yield significant cost savings.

The purpose of this study was to document timing and habitat of alosines (Hickory Shad Alosa mediocris, American Shad A. sapidissima, Alewife A. pseudoharengus, and Blueback Herring A. aestivalis) and Striped Bass Morone saxatilis in the lower Tar River, a North Carolina coastal watershed partially restricted by an unused industrial dam. Alosines and Striped Bass are usually separated in time or space during spawning activity but occasionally overlap in some systems. Results show that both eggs and larvae of Hickory Shad and Striped Bass predominated catches, followed by American Shad. Alewife and Blueback Herring had negligible catches of eggs and larvae, but a separate study conducted 48 river kilometers downstream of the study area collected larvae of both species. Eggs and larvae of target species overlapped spatially and were most abundant in downstream tributaries, which seasonally reached hypoxic to anoxic conditions. The origins of spawning are unknown, but the collection of eggs suggests ...

Previous studies performed in cell lines have shown that the heat shock protein, DNAJB6, protects against the proteotoxic effects of mutant huntingtin (mut-Htt) via direct interaction with mut-Htt. However, these studies were performed primarily using in vitro models and cell lines. We report that when expressed in primary neurons, DNAJB6 induces cell death. Neurotoxicity is observed with both the DNAJB6a isoform, which is strictly nuclear, and the DNAJB6b isoform, which is predominantly cytoplasmic, suggesting that neurotoxicity is mediated in the nucleus. However, when co-expressed in primary neurons with mut-Htt, DNAJB6 protects against mut-Htt neurotoxicity. This suggests that the contrasting effect of DNAJB6 on neuronal viability depends on the presence or absence of proteotoxic stress. Neurotoxicity of DNAJB6 cannot be prevented by inhibition of glycogen synthase kinase 3 beta (GSK3β) or c-Jun N-terminal kinase (JNK) but is prevented by pharmacological inhibition of cyclin-dependent kinases (CDKs). Expression of dominant-negative forms of CDK2 or CDK4, or of p21(CIP1), the physiological inhibitor of CDKs, also inhibits DNAJB6 neurotoxicity. DNAJB6 neurotoxicity can also be inhibited by histone deacetylase-4 (HDAC4), which interacts with DNAJB6 and which has previously been described to inhibit cell cycle progression. These results conclude that neurotoxicity resulting from elevated DNAJB6 is cell cycle dependent.

Golf is a global game enjoyed yearly by tens of millions. Effective ways to improve golf performance, especially putting, the club used the most, is of value to all golfers. Hence, the purpose of the present prospective study was to further the knowledge of the impact of approach-avoidance achievement goals on golf putting performance. Participants (N = 175) from university run golf courses participated in a two-round qualifying putting tournament with 81 participants making the cut to the championship round. Achievement goals, at the dispositional level, from Elliot and McGregor’s (2001) 2 × 2 framework (i.e., mastery-approach, masteryavoidance, performance-approach, and performance avoidance) were collected 5 weeks before the tournament, and then, at the contextual level, minutes before each round. Results indicated that those who made the cut line had significantly greater endorsement of the mastery-approach goal and lower endorsement of the performance-avoidance goal compared with those who missed the...

Background This study seeks to quantify variation in healthcare utilization and per capita costs using system-defined geographic regions based on enrollee residence within the Military Health System (MHS). Methods Data for fiscal years 2007 – 2010 were obtained from the Military Health System under a data sharing agreement with the Defense Health Agency (DHA). DHA manages all aspects of the Department of Defense Military Health System, including TRICARE. Adjusted rates were calculated for per capita costs and for two procedures with high interest to the MHS- back surgery and Cesarean sections for TRICARE Prime and Plus enrollees. Coefficients of variation (CoV) and interquartile ranges (IQR) were calculated and analyzed using residence catchment area as the geographic unit. Catchment areas anchored by a Military Treatment Facility (MTF) were compared to catchment areas not anchored by a MTF. Results Variation, as measured by CoV, was 0.37 for back surgery and 0.13 for C-sections in FY 2010- comparable to rates documented in other healthcare systems. The 2010 CoV (and average cost) for per capita costs was 0.26 ($3,479.51). Procedure rates were generally lower and CoVs higher in regions anchored by a MTF compared with regions not anchored by a MTF, based on both system-wide comparisons and comparisons of neighboring areas. Conclusions In spite of its centrally managed system and relatively healthy beneficiaries with very robust health benefits, the MHS is not immune to unexplained variation in utilization and cost of healthcare.

An essential aspect of genetically-engineered mice (GEM) is the ability to produce live animals after the appropriate injection procedure. Animals are produced by implantation of manipulated embryos into pseudopregnant females for gestation, parturition, and growth to the weaning stage. This study was carried out to test whether the anesthesia used during surgery could affect the number of pups produced. Anesthetics commonly used for implant surgery include tribromoethanol (Avertin) delivered by intraperitoneal (IP) injection, IP-injected ketamine:xylazine or ketamine:medetomidine mix, and inhaled isoflurane. To determine if the anesthesia used might affect the number of animals produced, each anesthetic agent was tested in implant surgeries and the numbers of pups produced using both wild-type and GEM embryos were assessed. Parallel studies were conducted in institutions in the EU and in the USA. Based on a direct comparison of pregnancy status, number of pups born, and number of pups weaned for each agent, we found no statistical differences among the three anesthetics. We conclude that all three anesthetic agents tested are equally useful for implantation surgery.

The selection of an appropriate and effective pedagogy has been a central theme in management education for decades. There currently exists a wide range of pedagogical options designed to match course content with the most appropriate technique(s) for effective learning outcomes. Most recently, a variety of experiential learning methods have been employed to provide students with real-life experiences and applications in the overall class design. Live case analysis is typically identified as one of a series of options within the domain of experiential learning methods. This paper examines the live case approach as a tool for achieving desired outcomes in management education. Perspectives are offered from multiple stakeholder groups that highlight both the challenges and prospects in the use of this method of teaching. Results demonstrate the usefulness of the live case approach for achieving assessment objectives and measuring important program outcomes.

The production of chimeric mice is a complex process, requiring the careful coordination of tissue culture cell growth, production of a large number (30-75) of competent blastocysts and the availability of appropriately timed pseudo pregnant female mice. Failure at any of these steps can impinge upon the rapid production of chimeras. One potential improvement for the efficient generation of chimeric mice would be the utilization of cryopreserved embryos suitable for injection. C57Bl/6 morulae were frozen using a standard 2-step protocol with ethylene glycol as the cryopreservation agent. We determined that cryopreserved morulae could thaw, culture to blastocyst stage in KSOM media and survive injection at rates equivalent to control embryos. Cryopreserved morulae were also equivalent to controls at all later stages in the process of production of chimeric mice, including birth rate, percentage chimerism of resulting animals and ability to produce germline progeny. Hence, cryopreservation of morulae for blastocyst injection is a suitable option to enhance the efficiency of chimeric mouse generation.