Diagnostic tools for the detection of protein-misfolding diseases  are limited. Gold nanoparticles (AuNPs) facilitate sensitive diagnostic techniques via visual color change for the identification of a variety of targets. In parallel, recently developed quaking-induced conversion (QuIC) assays leverage protein-amplification and fluorescent signaling for the accurate detection of misfolded proteins. Here, we combine AuNP and QuIC technologies for the visual detection of amplified misfolded prion proteins from tissues of wild white-tailed deer infected with chronic wasting disease (CWD), a prion disease of cervids. Our newly developed assay, MN-QuIC, enables both naked-eye and light-absorbance measurements for detection of misfolded prions. MN-QuIC leverages basic laboratory equipment that is cost-effective and portable, thus facilitating real-time prion diagnostics across a variety of settings. In addition to laboratory-based tests, we deployed to a rural field-station in southeastern Minnesota and tested for CWD on site. We successfully demonstrated that MN-QuIC is functional in a non-traditional laboratory setting by performing a blinded analysis in the field and correctly identifying all CWD positive and CWD not-detected deer at the field site in 24 h, thus documenting the portability of the assay. White-tailed deer tissues used to validate MN-QuIC included medial retropharyngeal lymph nodes, parotid lymph nodes, and palatine tonsils. Importantly, all of the white-tailed deer (n = 63) were independently tested using ELISA, IHC, and/or RT-QuIC technologies and results secured with MN-QuIC were 95.7% and 100% consistent with these tests for positive and non-detected animals, respectively. We hypothesize that electrostatic forces help govern the AuNP/prion interactions and conclude that MN-QuIC has great potential for sensitive, field-deployable diagnostics for CWD, with future potential diagnostic applications for a variety of proteopathies. Given well-documented diagnostic limitations surrounding proteopathies in both animals and humans, , it is imperative to develop improved diagnostic assays11. With respect to prion diseases (a proteopathy subclass caused by infectious proteins), tests that could be deployed in a variety of settings  would greatly aid the detection of infectious prions thus limiting their spread. It is within this framework that we approach the development of diagnostic tools for CWD of cervids, a model neurodegenerative disorder with urgent needs for portable diagnostic assays that would facilitate rapid detection, thus preventing CWD prions from entering human and animal food-chains.A common feature of many neurodegenerative diseases is the presence of misfolded proteins that accumulate within the central nervous system, ultimately contributing to advanced neurodegeneration and death. Misfolded protein diseases (proteopathies) impact a wide variety of mammals, including Creutzfeldt-Jakob disease (CJD), Alzheimer’s disease, and Parkinson's disease in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, pituitary pars intermedia dysfunction (PPID) in horses and chronic wasting disease (CWD) in cervids12. CWD impacts cervids across North America, Scandinavia, and South Korea14 . The disease continues to spread to new cervid populations, and there are increasing health concerns for both humans and animals exposed to various CWD prion strains15. All mammals have native functioning cellular prion protein (PrP) distributed throughout various tissues and playing essential roles in a variety of physiological functions, especially those of the central nervous system16. Like other protein misfolding neurodegenerative disorders, native prions in cervids adopt conformations of misfolded prions (PrPCWD) .Similar to CJD in humans, which progresses rapidly and is always fatal, and BSE in cattle, CWD is a prion disease or Transmissible Spongiform Encephalopathy (TSE) that is 100% fatal to infected animals and has no treatments or prevention methods11. Commonly utilized TSE diagnostic assays rely heavily on antibody-based enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) technologies that are expensive, time-consuming, and require substantial training and expertise to operate6. In addition, a major limitation of ELISA and IHC assays is that the antibodies routinely used cannot differentiate between native PrP and misfolded TSE-associated prion proteins (PrPTSE) thus requiring protein digestion to enrich for PrPTSE, a methodology that may impact diagnostic sensitivity through the destruction of particular TSE-affiliated PrP strains20. Collectively, these antibody-based assays are limited in the identification of early-stage TSE infections, and they are primarily used on tissues collected post-mortem.Current diagnostic methods for the detection of protein-misfolding diseases, including CWD and other TSEs, are limited21, end-point quaking-induced conversion (EP-QuIC)23 and real-time quaking-induced conversion (RT-QuIC)27. Of these prion amplification methods, both EP-QuIC and RT-QuIC  measurements. Despite the advantages of EP-QuIC and RT-QuIC, there still remain major limitations, including the need for expensive and large laboratory equipment and complex strategies for visualizing and analyzing results, thus limiting access to diagnostic tests. In short, more effective TSE diagnostic methods that leverage small and portable equipment with easily interpretable results are needed to rapidly detect various TSEs in widespread surveillance and prevent additional spread and introduction of TSE prions into human and animal food chains. This is especially true for CWD, as the disease continues to expand across both farmed and wild cervid populations.The detection of prion seeding activity was recently enhanced by various assays involving the amplification of protein misfolding in vitro, including protein misfolding cyclic amplification (PMCA)30, drinking water safety31, and food safety32. Due to localized surface plasmon resonances (density fluctuation of conduction electrons)33, AuNPs have optical absorption peaks that are sensitive to the environment at the AuNP’s surface37. These unique optical properties make plasmonic nanoparticles useful in color-based detection assays39. Previous studies have indicated affinities of prion proteins with a variety of bare and functionalized metals, including gold42. One limitation of AuNPs is that they are susceptible to nonspecific binding43. Real biological samples are not homogeneous and have many different proteins, ions, and other organic molecules associated with them that can induce AuNPs to spontaneously aggregate making effective AuNP diagnostics challenging. To overcome this, we sought to demonstrate the diagnostic utility of gold nanoparticles for detecting misfolded PrPCWD within QuIC amplified products of CWD positive and negative white-tailed deer (WTD) tissues in homogeneous recombinant hamster prion protein substrates.In parallel to diagnostic advancements based on prion and protein amplification methods, gold nanoparticles (AuNPs) have been increasingly used for medical applications including disease diagnosticsCWD using both visual and spectroscopic methods  that can detect the presence or absence of misfolded PrPods Fig. . This me45. The presence of fibril formation was examined in all reaction mixtures by adding and quantifying ThT post hoc , two sets of reaction mixtures seeded with and without spontaneously misfolded rPrP prion fibrils were processed following modified RT-QuIC protocols without ThThoc Fig. a. ThT flhoc Fig. a. We hyphoc Fig. a.Figure 39. DLS experiments were performed on three AuNP solutions , and average effective particle size was determined for each sample , we then investigated the potential of MN-QuIC for CWD diagnostics using PrPCWD positive and negative WTD lymphoid tissues. We used homogenates of independently confirmed CWD positive and negative WTD medial retropharyngeal lymph nodes (RPLN) (Table 27.Understanding that misfolded prions can induce rPrP misfolding and subsequent amplificationN) Table . IndepenN) Table a27.Figur23. To test this, homogenates from the same set of samples tested with RT-QuIC  to determine the presence of CJD prion seeding activityTo quantify our observations and measure statistical differences, the absorbance spectrum of the AuNPs was measured from 400 to 800 nm using a 96-well plate reader. In the resulting absorbance spectrum, AuNP solutions combined with QuIC-amplified CWD positive samples had absorbance peaks near 516 nm Fig. c, simila48. Because the theoretical isoelectric point (pI) of our rPrP is around pH 8.949, rPrP is positively charged in the pH 7.4 AuNP buffer whereas citrate capped AuNPs are negatively charged even at pHs well below our buffer50. Thus at pH 7.4, there exists an electrostatic attractive force between AuNPs and native rPrP that contributes to their interactions (AuNP aggregation and the color change). The charge on the protein changes when the pH of the environment is altered and thus the interaction between AuNP and rPrP is disrupted. When the pH of a solution is raised closer to the IP of the rPrP, the charge of the protein will become closer to neutral, decreasing the force of attraction between AuNP and rPrP. We showed that as the pH of the AuNP solution was raised closer to the IP of native rPrP, the absorbance peak of the AuNP-rPrP solution decreased from 530 nm  and plate reader (RT-QuIC), respectively. Within 24 h both MN-QuIC and RT-QuIC detected seeding for replicate(s) from 10–2 to 10–6 (Table CWD seeding activity at a 10–1 dilution (eight replicates with no amyloid formation), perhaps due to a higher concentration of inhibitors . However, four of eight replicates for the 10–1 dilution were positive using MN-QuIC.Table 14 there is an urgent need to develop widely accessible diagnostic tools for CWD. Historically, AuNP colorimetric assays have been limited because of spontaneous AuNP aggregation issues when used with biological samples43. Here, we combined QuIC amplification of CWD prions with the simplicity of gold nanoparticles to effectively eliminate spontaneous AuNP aggregation challenges and to enable the visualization of positive vs. negative CWD lymph node and palatine tonsil WTD samples. We hypothesized that the conformational differences between the native rPrP substrate and misfolded prion fibrils would influence the aggregation of AuNPs in solution.Given the continued spread of CWD among cervid populations throughout North America, Northern Europe, and South KoreaBy injecting post-QuIC-amplified protein solutions into AuNPs, we were able to clearly distinguish (via visible color change) between CWD positive and CWD negative medial retropharyngeal lymph node and palatine tonsil tissues. AuNP solutions for these positive and negative samples appeared red and purple, respectively. We further confirmed that the color change was a result of the aggregation of AuNPs by conducting DLS and TEM experiments that compared the effective particle sizes in the presence of native and misfolded rPrP. We observed that the AuNP aggregation was governed by electrostatic interactions by altering the pH of the solution. Finally, we demonstrated proof-of-concept experiments for the practical utility of MN-QuIC by successfully identifying CWD-infected WTD tissues at a DNR field station in southeastern MN.27. It is important to note that where tissue types differ, we are not only comparing diagnostic test performance, but also the probability that a certain tissue type will contain detectable levels of PrPCWD as compared to RPLN. Yet, tissue distribution of PrPCWD can vary based on route of infection, stage of infection, and prion strain56. We also recognize that under conditions of natural infection, the assumption that ELISA and IHC are a "gold standard" for diagnostic test comparison may be inappropriate given that these tests are imperfect57. Knowing these limitations, estimates of diagnostic test performance warrant further evaluation using methods that can overcome the constraints related to reliance on a “gold standard” comparison60.We estimated the sensitivity and specificity of MN-QuIC for CWD detection  in comparison to diagnosis by ELISA/IHC using RPLNs, the standard of practice for CWD diagnosis in WTD Table 27. It isCWD seeding activity down to 10–6 homogenate solutions  and a low rate of amyloid formation on RT-QuIC, likely indicating relatively low levels of PrPCWD within the tissue. Using MN-QuIC, sample 926  was found to be negative on RPLN  or different subsamples from the same tissue61.Of all samples tested, there were only two CWD positive RPLNs (sample process IDs 923 and 926) where MN-QuIC was not consistent with ELISA or RT-QuIC results Table . For theLN Table , howeverLN Table . Tissue 6, results from MN-QuIC can be visualized with the naked eye or quantified using simply light-absorbance readings. Because MN-QuIC is a protein amplification method, it can be adapted to examine any tissue or biological sample that existing and future RT-QuIC protocols use, thus giving MN-QuIC wide versatility. Studies have shown that amplification-based assays, such as RT-QuIC, are more sensitive than conventional ELISA62. Given that MN-QuIC has similar sensitivity to RT-QuIC . When including reagent and production costs for recombinant hamster protein, the approximate MN-QuIC cost per sample is nearly the same as RT-QuIC . At the time of publication, the cost of equipment for MN-QuIC is just 20% (~ $6000 USD) of that which is needed for RT-QuIC  and slightly over 50% of that used in low-throughput ELISA . By eliminating the need for time consuming preparation and/or the relatively expensive visualization strategies used in EP-QuIC, RT-QuIC, ELISA, IHC and/or PMCA, MN-QuIC provides new opportunities for widely accessible and sensitive CWD diagnostics.The primary laboratory equipment for MN-QuIC consists of a tissue homogenizer, temperature-controlled shaker, and if desired (but not necessary), a spectrometer for light absorbance readings in addition to visual observations. Compared to EP-QuIC/RT-QuIC and PMCA, which leverage ThT fluorescence and antibody-based Western blotting, respectively67. Our work demonstrates that AuNPs can open promising avenues for the identification of misfolded prions. Because prion proteins have strong interactions with simply functionalized metallic surfaces, besides AuNPs, we envision a broad range of metallic nanoparticles with various materials and shapes to be useful in detection. Additionally, substrate-based nanostructures exhibiting optical resonances could be useful in detecting conformational changes via other sensing modalities such as surface-enhanced infrared absorption spectroscopy69 to further improve the speed and accuracy of prion detection. Examining other areas of the electromagnetic spectrum, such as tera-Hertz sensing, could also lead to improved detection70.AuNPs have been used in a variety of advanced sensing applications15, any field-based diagnostic assay that avoids producing false-negative is preferred.To demonstrate field deployment capabilities of the MN-QuIC assay, we collected medial retropharyngeal lymph nodes, parotid lymph nodes, and palatine tonsil samples from 13 WTD at a rural DNR field station. Using both pooled and individual tissues of these 13 individuals, MN-QuIC was 100% successful in identifying three CWD positive and 10 CWD not-detected animals. The successful field-based classification of these animals provides clear proof-of-concept demonstration of MN-QuIC's utility as a portable and sensitive field test. We note that our MN-QuIC analysis has a low false-negative rate, and has yet to produce a statistically significant false positive result. These observations are critically important when considering MN-QuIC as a field-based diagnostic tool for CWD. Moreover, any positive result can be independently validated using downstream RT-QuIC, ELISA, and/or IHC testing. Given growing concerns of CWD prion strain variation and risks to human and animal health71. Future studies should be performed using MN-QuIC on large sample sets to better characterize MN-QuIC's sensitivity and specificity for specific tissues. Additional work will focus on leveraging MN-QuIC for CWD diagnostics using a variety of antemortem biological samples . RT-QuIC amplification assays using samples acquired from living deer have recently been reported72 and these assays could be readily combined with MN-QuIC to provide field-deployable antemortem tests of both wild and farmed cervids. Moreover, MN-QuIC may have potential food-safety test applications given the recent documentation of RT-QuIC-based detection of CWD prions in WTD muscles used for human and animal consumption26.RPLN and palatine tonsils collected from WTD were the basis for the laboratory-based analyses conducted herein because these tissues are ideal for early and accurate identification of CWD infection, with tonsils additionally having antemortem applications74 could also play a role. Various studies have shown that both native and misfolded PrP bind various metal ions and bulk metals including gold42. Our research reveals AuNPs stabilized with a simple citrate capping readily interact with the truncated rPrP substrate that is used as the primary substrate for a growing variety of QuIC assays. Future structural analyses focused on native rPrP and prion fibrils75 could provide further insight into how native rPrP, but not misfolded rPrP fibrils, influence AuNP aggregation.The mechanisms underlying interactions between AuNPs and various prion proteins are underexplored, given the results reported herein a detailed characterization of AuNP–prion interactions is highly desirable. Based on the available knowledge in the field, we hypothesized that as the pH approaches the prion’s isoelectric point, the electrostatic force of attraction between the negatively charged citrate capped AuNP and the protein would decrease. Our analyses revealed that as the pH neared the theoretical isoelectric point of rPrP, the wavelength of the peak absorbance of the AuNPs spiked with native (CWD negative) protein decreased. We also found that pH alterations had little effect on AuNP solutions without protein, indicating that the effect of pH on native rPrP-spiked AuNP solutions was not caused by intrinsic AuNP aggregation in response to the changing pH. QuIC-amplified CWD positive solutions did not change with varying pH because of fibril formation effects on free-floating prion concentration. Therefore, the difference in interactions between CWD negative and positive solutions is likely governed by electrostatic forces and rPrP concentration effects. However, other factors such as hydrophobic interactions76. It has been proposed that advances in CWD diagnostics will yield technologies that are useful for a broad range of neurodegenerative diseases6. RT-QuIC protocols have already been developed for a number of sample types allowing for antemortem tests72. These and future amplification methods could readily be combined with MN-QuIC. Additionally, QuIC amplification protocols have been developed for a variety of other protein misfolding diseases including scrapie in sheep77, BSE in cattle78, and Alzheimer’s79, Parkinson’s80, and CJD in humans23. Thus, we posit that the combination of AuNP technology with protein amplification assays has great potential for the development of versatile neurodegenerative disease diagnostic platforms. By eliminating the need for expensive/complicated visualization schemes, our hybrid assay technology has the potential to greatly increase access to neurodegenerative disease diagnostics. It is our vision that in the future, variations of this AuNP-based protein amplification/detection assay could be deployed in medical clinics around the world to aid in neurodegenerative diagnosis and early application of therapeutics.MN-QuIC holds great promise not only for the visual detection of CWD-positive samples but also for the detection of other protein misfolding diseases. The need for inexpensive, sensitive, widely deployable diagnostics for neurodegenerative diseases is only growing as neurodegenerative diseases are predicted to greatly increase in the next decades27; Tables 27.60 WTD tissues  were selected for laboratory-based RT-QuIC and MN-QuIC analyses Tables  and S5. 27 The substrate is derived from a truncated form (amino acids 90–231) of the Syrian hamster PRNP gene cloned into the pET41-a( +) expression vector and was expressed in Rosetta (DE3) E. coli. The original clone was provided by the National Institutes of Health Rocky Mountain Laboratory. Many studies have utilized haPrP for RT-QuIC45. Recombinant hamster PrP was used in this study due to its seemingly universal proclivity to misfold in the presence of infectious prions for a large variety of species.Recombinant hamster PrP (HaPrP90-231) production and purification followed the methods in Schwabenlander et al.27 . 1ul of N-2 Supplement ) was added to 99ul dilution of SDS (for MN-QuIC reactions N-2 was excluded from SDS). 2 μL of the diluent were added to each well containing 98 uL of master mix. Plates for RT-QuIC were amplified on a FLUOstar® Omega plate reader . Fluorescent readings were taken at ~ 45 min increments.For QuIC analysis (both MN-QuIC and RT-QuIC), a master mix was made to the following specifications: 1X PBS, 1 mM Ethylenediaminetetraacetic acid (EDTA), 170 mM NaCl, 10 μM thioflavin T (ThT), and 0.1 mg/mL rPrP. In instances where the end reaction would be analyzed using AuNPs, ThT could be excluded. The 10% tissue homogenates (prepared as described above) were further diluted 100-fold in 0.1% Sodium Dodecyl Sulfate (SDS) using methods from Schwabenlander et al.22 and Vendramelli et al.23, although with slight modifications. Plates were prepared identically to those amplified on the plate reader (see protocol above). Instead of shaking on a plate reader, reactions were performed on a ThermoMixer® C equipped with SmartBlock plate and Thermotop  at 48 °C for 24 h at 600 RPM (60 s shake and 60 s rest). We selected a 24 h run time based on independent RT-QuIC results for RPLNs and palatine tonsils from CWD positive WTD reported in Schwabenlander et al.27, including those examined herein, showing significant seeding activity within 9 to 24 h  to produce QuIC-based prion amplifications as previously reported by Cheng et al.4 h Fig. . The resSpontaneous misfolding of recombinant prion protein was generated similarly standard RT-QuIC but with unfiltered recombinant proteins and reagents. For these reactions, no infectious seed was necessary. The spontaneously misfolded material was used to seed reactions for the dynamic light scattering experiment, described below.81 and Zhang et al.39 AuNPs were buffer exchanged using 530ul of stock solution that was centrifuged in 1.6 mL tubes at 13,800 g for 10 min. 490ul of supernatant was removed and the undisturbed pellet was resuspended with 320ul of a low concentration phosphate buffer  made of 10 mM Na2HPO4(Anhydrous), 2.7 mM KCl, 1.8 mM KH2PO4). After the quaking/incubation steps, protein solutions were diluted to 50% in MN-QuIC buffer (pH 7.2), consisting of 1X PBS with the addition of final concentrations of 1 mM EDTA, 170 mM NaCl, 1.266 mM sodium phosphate. Forty microliters of the protein diluted 50% in MN-QuIC buffer were then added to the 360ul AuNP solution with ample mixing  with stock concentrations ranging from 2.45 nM to 2.7 nM. AuNP protocols were modified from Špringer et al.For AuNP visualization experiments performed to determine higher throughput capacity Fig. , S4, prolow. Additionally, a blank with no protein was produced by adding 40ul of MN-QuIC buffer to 360ul of AuNPs in PBSlow. For native rPrP samples, color change was observed within 1 min of rPrP addition. No color change was observed in spontaneously misfolded rPrP samples at any time length. Dynamic light scattering measurements of all samples were taken after 5 min of protein addition using a Microtract NanoFlex Dynamic Light Scattering Particle Analyzer , and measurement times were 60 s. Five measurements were taken for each sample and then averaged.Spontaneously misfolded rPrP samples (described above) were produced from solutions of rPrP with no seed added. In addition to these samples, a 96-well RT-QuIC reaction was performed with half the wells consisting of native rPrP seeded with spontaneously misfolded rPrP, and half consisting of native rPrP with no seed. The 96-well plate was then amplified using QuIC protocols described above. Post-amplification, seeded samples were confirmed to have fibrillation while the non-seeded samples were confirmed to not have fibrillation based on ThT binding (described above). Seeded and non-seeded samples were diluted to 50% in MN-QuIC buffer, and 40ul of these solutions were added to 360ul of AuNPs in PBSTEM measurements were performed using a Tecnai G2 F30 Transmission Electron Microscope. Images were recorded on a Gatan K2 Summit direct electron detector. 20ul of post-amplified positive and negative rPrP were added to separate volumes of 200ul of AuNPs prepared as described above. Each protein solution was added to separate uncharged TEM grids. Stain was not used because it was unnecessary to view the AuNPs.low. Protein solutions were added as previously described. It can be noted that the peak absorbance for the tris buffer solution below pH 7.4 is still not as high as the peak shifts in pH 7.4 PBSlow. This is likely due to the differences in tris and PBSlow buffers.In order to test the effects of pH on the interaction of rPrP with AuNP, five different 10 mM tris-buffer solutions with pHs ranging from 7.2 to 9.0 were created. Tris was used to give buffering for the desired pH range. AuNPs were buffer exchanged as described above except tris-buffer was used instead of PBSTo examine effects of rPrP substrate concentration Fig. , six dif82. Wilson confidence intervals are appropriate for small sample sizes when values of p are close to 0 or 183. Note for sensitivity calculations, Animal 832 was considered positive because of it’s MN-QuIC tonsil result (Table www.graphpad.com) was used for conducting statistical analysis. Three technical replicates were used to demonstrate the potential application of AuNP on spontaneously misfolded rPrP. For initial trials on RPLN tissues from eight (four positive and four negative) animals, four and three technical replicates were used for RT-QuIC and AuNPs, respectively. For plate-based protocols, we tested palatine tonsils from ten positive and ten negative animals using four and eight replicates for RT-QuIC and AuNPs, respectively. We also tested 40 medial retropharyngeal lymph nodes . White-tailed deer were euthanized for annual culling efforts to control the spread of CWD in Minnesota following Minnesota Department of Natural Resources state regulations and euthanasia guidelines established by the Animal Care and Use Committee of the American Society of Mammalogists84. All methods and all experimental procedures carried out during the course of this research followed University of Minnesota guidelines and regulations as approved by the Institutional Biosafety Committee under protocol #1912-37662H. This study was also carried out in compliance with the ARRIVE guidelines (https://arriveguidelines.org).No white-tailed deer were euthanized specifically for the research conducted herein and all tissues were secured from dead animals or loaned for our analyses. For these reasons, the research activities conducted herein are exempt from review by the University of Minnesota Institutional Animal Care and Use Committee (as specified White-tailed deer were euthanized by the state of Minnesota for routine annual culling efforts to control the spread of CWD and were not sampled specifically for the current study. Tissue samples were provided by the Minnesota Department of Natural Resources.Supplementary Information.


What contributes to the success of improvement programs designed to reduce time to surgery (TTS) for adult patients with hip fractures?In this systematic review of 69 studies, 49 programs were associated with significant decreases in TTS, and 20 programs were not. Common themes among successful improvement strategies  were cataloged according to Expert Recommendations for Implementing Change, and contextual factors contributing to failed experimental results were evaluated.These findings suggest that many of the assessed improvement programs show promise for reducing hip fracture TTS across different clinical settings in the near future. This systematic review catalogs improvement programs designed to decrease time to surgery for hip fractures, identifies their results, and categorizes program strategies according to the Expert Recommendations for Implementing Change. Longer time to surgery (TTS) for hip fractures has been associated with higher rates of postoperative complications and mortality. Given that more than 300 000 adults are hospitalized for hip fractures in the United States each year, various improvement programs have been implemented to reduce TTS with variable results, attributed to contextual patient- and system-level factors.To catalog TTS improvement programs, identify their results, and categorize program strategies according to Expert Recommendations for Implementing Change (ERIC), highlighting components of successful improvement programs within their associated contexts and seeking to guide health care systems in implementing programs designed to reduce TTS.A systematic review was conducted per the Preferred Reporting Items for Systematic Reviews and Meta-analyses guideline. Three databases  were searched for studies published between 2000 and 2021 that reported on improvement programs for hip fracture TTS. Observational studies in high-income country settings, including patients with surgical, low-impact, nonpathological hip fractures aged 50 years or older, were considered for review. Improvement programs were assessed for their association with decreased TTS, and ERIC strategies were matched to improvement program components.Preliminary literature searches yielded 1683 articles, of which 69 articles were included for final analysis. Among the 69 improvement programs, 49 were associated with significantly decreased TTS, and 20 programs did not report significant decreases in TTS. Among 49 successful improvement programs, the 5 most common ERIC strategies were (1) assess for readiness and identify barriers and facilitators, (2) develop a formal implementation blueprint, (3) identify and prepare champions, (4) promote network weaving, and (5) develop resource-sharing agreements.In this systematic review, certain components  are common among improvement programs that were associated with reducing TTS and may inform the approach of hospital systems developing similar programs. Other strategies had mixed results, suggesting local contextual factors  may affect their success. To contextualize the success of a given improvement program across different clinical settings, subsequent investigation must elucidate the association between interventional success and facility-level factors influencing TTS, such as hospital census and type, teaching status, annual surgical volume, and other factors. In response, hospitals have implemented programs dedicated to modifying these factors, particularly at the facility-level scale, with the goal of optimizing TTS and thereby improving patient outcomes.Among older patients with hip fracture, mortality in the first year following fracture surgery ranges from 15% to 36%, approximately 4 times higher than that of younger patients with hip fractures. The risk for surgical complications and mortality among patients with hip fracture is dependent on multiple factors, including scope and severity of injury, course of treatment, and patient characteristics.23 or acute coronary syndrome24) by mapping improvement program strategies. A Cochrane review25 found that such tailored improvement can address implementation barriers and improve professional practice and health care outcomes more effectively than untailored strategies. This systematic approach to classifying improvement strategies has not yet been applied to the management of hip fractures. The aims of our study were to (1) catalog improvement programs aimed at reducing TTS and (2) use the ERIC taxonomy to characterize the improvement strategies most commonly used in promising programs. This approach highlights successful improvement program components to provide strategic recommendations for future TTS improvement implementation.Across other sectors of medicine, the Expert Recommendations for Implementing Change (ERIC) framework has been used to address barriers to achieving a desired health services outcomes  methods and was registered with Prospero (CRD42021267021). Two authors (P.T. and J.L.L) conducted literature searches from peer-reviewed journals in 3 medical databases  to identify improvement programs for which TTS was an experimental outcome. Search strategies included terms such as hip fracture, time to treatment, surgery, and intervention  conducted several stages of text review, consisting of abstract screening, full-text review, and data extraction prior to final article selection. Two researchers independently reviewed eligible texts, according to the predetermined selection criteria, and voted to include or exclude a given text. In the event of a voting conflict, a third reviewer independently resolved disagreements.For each eligible study, data on study design, years of data collection, sample size, improvement program components, results of analysis and variable-outcome associations were extracted onto an Excel spreadsheet and cross-checked for accuracy.27 was used to assess the methodological quality of both randomized and nonrandomized studies. This checklist comprises 27 questions across 5 domains: (1) reporting; (2) external validity; (3) internal validity, bias; (4) internal validity, confounding (selection bias); and (5) power. It assigns points to each study accordingly. Studies earning 20 or more points are considered having good individual methodological quality, while studies assigned 14 points or fewer are considered having poor methodological quality and high risk of bias.28The 1998 Downs and Black Quality Assessment Checklist29 in 2012, maps implementation strategies across studies to mitigate contextual barriers to implementation.30 This method, used across other sectors of medicine, creates a standardized and potentially generalizable approach to improvement efforts.25 For example, Ebben et al23 used ERIC strategies to match improvement program components across studies and optimize emergency department response to time-sensitive conditions such as asthma, pulmonary embolism, triage, acute gastroenteritis, and foot and ankle issues. Similarly, Doucette et al24 used ERIC strategies to effectively match improvement program components and improve outcomes for acute coronary syndrome and myocardial injury.The ERIC project, introduced by Powell et al31 The improvement programs identified for our review were partitioned into program components and were independently classified by 2 coauthors (B.F.S. and P.T.) according to concise definitions obtained in literature for each of the 73 ERIC strategies.32 Any discrepancies between researchers’ ERIC classifications were discussed and resolved. Descriptive statistics were used to calculate the rate of specific ERIC strategies used among improvement programs that were associated with reduced TTS.To develop the taxonomy of strategies, extracted data were classified according to 73 ERIC strategies, organized by hierarchical clustering into 9 domain groupings.103 Among the final 69 improvement programs, 49 programs significantly decreased TTS103 and 20 programs102 did not. Delayed TTS cutoffs were most often defined dichotomously as more than 24 hours, more than 36 hours, more than 48 hours, or more than 72 hours, although some studies collected continuous data.A total of 1683 studies were eligible for abstract screening based on literature searches. 190 studies underwent full-text review, and 69 studies were selected for final analysis .33,34,3527 Each study collected and used patient medical records. TTS was also calculated according to hospital medical records and/or databases.Among the 69 total studies scored, no study earned a checklist score of lower than 20 points, indicating a relatively high-quality body of studies and low risk of bias.Among the 49 improvement programs associated with significant decreases in TTS, each program had a mean (SD) of 9 (4) strategies defined under ERIC. The 20 improvement programs that did not significantly decrease TTS had a mean (SD) of 8 (2) strategies, from which the 5 improvement programs that were associated with increases in TTS had a mean (SD) of 7 (2) strategies. Successful improvement programs most often had program components corresponding to 3 ERIC domains: (1) use of evaluative and iterative strategies ; (2) development of stakeholder interrelationships ; and (3) support of clinicians  . Specifi104 Novel improvement programs have demonstrated potential in reducing in-hospital and 30-day mortality rates as well as decreasing the length of hospital stay.36 Recent studies have further explored the results of improvement programs designed to reduce TTS for hip fracture patients. However, poor consistency in implementation methods has impeded efforts for generalizable strategies for improvement efforts to address hip fracture TTS.31 In this systematic review, we cataloged and identified program results using the ERIC classification system. These results highlighted commonalities across studies and provided the foundation for guiding the implementation of future TTS optimization programs in health systems. While designing TTS improvement programs, health care systems may utilize the findings of this review to reference the individual programs we reviewed that bear similarities to the needs of their clinical settings. Additionally, common ERIC strategies and domains of successful programs described in this review can provide nuanced insight into the strategy and design of future TTS improvement programs.As a result of the strong body of evidence supporting the association of TTS with morbidity and mortality, various organizations  have recommended surgical treatment within 48 hours of presentation for patients sustaining hip fractures.The results of our review indicated that, of the studies included, successful TTS improvement programs had a mean (SD) of 9 (4) strategies defined under ERIC. These studies suggest that a comprehensive strategic approach, using multiple, diverse implementation strategies across different domains, can be beneficial in reducing TTS. Given the studies that did not significantly improve or failed to improve TTS used a mean (SD) of 8 (2) and 7 (2) strategies, respectively, it appears that more implementation strategies were associated with improved TTS. The most prevalent ERIC domains among successful improvement programs were the use of evaluative and iterative strategies (100% rate), development of stakeholder interrelationships (71.4% rate), and support of clinicians (67.3% rate). This pattern suggests the need for increased efficient preoperative communication and partnerships between medical and surgical teams to reduce TTS. Furthermore, the 100% rate of the ERIC strategies assessing for readiness and identify barriers and facilitators and developing a formal implementation blueprint among the 49 successful improvement programs indicates that preemptive strategic planning appears to be positively associated with the success of a given improvement strategy.31  assess the readiness of a given study site to implement an improvement program; (2) assess barriers that could impede implementation efforts; and (3) assess strengths for implementation42 confounding comorbidities63)  may affect the success of improvement programs and should be a consideration of future TTS improvement initiatives.ERIC strategies common to successful programs were also found among the 20 studies that did not report significant decreases in TTS . Howeverities63) . These sIn this review, studies did not commonly include information on facility-level factors when describing the environment of hospital and health care settings. For hospital and health system settings that are already advanced prior to implementation of a given improvement program, smaller margins of program success will be reflected in study results regardless of a given program’s true potential. Therefore, the environment of a health system provides essential context for the performance of a given improvement program. This lack of comprehensive facility-level factor data impeded our ability to provide context for the success of a given improvement program based upon its environment.Another consideration for our review is the inclusion of studies that only focused on TTS as a main outcome. Several systemwide improvement programs have instead focused on mortality and length of hospital stay as primary outcomes, without also considering changes in TTS. It may be beneficial to consider the replication of these successful programs, with a focus on TTS improvement.This study has limitations. The ability to gauge the potential of improvement programs was limited by inconsistencies in defining delayed TTS cutoffs between studies. For example, the relative success of an improvement program that reduced TTS to less than 48 hours may depend on the delayed TTS cutoff decided for that study. Similarly, an improvement program aiming to increase surgical intervention within the first 24 hours of patient admission may report smaller margins of program success than an improvement program aiming promote surgical intervention within the first 3 days of patient admission.It is important to note the heterogeneity of the study age cutoff as a limitation to this review. While geriatric age is typically defined as those 65 years and older, this study included all reports with a patient population aged 50 years and older. This allowed for a more robust analysis of systematic improvements while adding a level of heterogeneity to the study population. Future studies may wish to investigate improvement programs of geriatric populations specifically to identify targeted systematic improvements for this population.Furthermore, our review was limited by a lack of data regarding program failure, as only 5 included studies described increased TTS results. No failed improvement program provided reasoning for why a program should not be pursued for further implementation efforts in the clinical setting. Thus, the available data informing future improvement program strategies may be limited by publication bias. This publication bias may further occlude research teams from providing reasoning as to why a given improvement program fails and, instead, encourage researchers to focus on cases for future program promise.Moreover, several studies included TTS data during the improvement program’s implementation as well as after implementation, while other studies merged these data into an overall postimplementation category, limiting an assessment of rates of reduced TTS. Additionally, this review focused on timely surgical management of femoral neck, intertrochanteric, and subtrochanteric fractures only. This was done to minimize confounding considerations for surgical management and to simplify comparisons of improvement program data. Nevertheless, future studies should aim to replicate improvement program results across other hip fracture types to confirm TTS program generalizability. Also, while this systematic review included studies with levels of evidence III and IV, ie controlled trials without randomization and cohort studies, it excluded case reports and case studies to reduce confounding sample sizes; varying sample sizes among our included studies may skew the success rates presented for any given improvement program. Additionally, while each improvement program was independently read and cataloged according to ERIC strategies by 2 authors, and any conflicts were independently reviewed and resolved by a third author, study classifications may be subject to, albeit minimal, manual human error.In this review of improvement programs for TTS for hip fracture, we summarized the results of novel programs. Generally, the 69 improvement programs possessed the capability to decrease TTS. Particular promise for replicable success was demonstrated by the 49 improvement programs that were associated with significant TTS decreases. Among these successful programs, general themes were ascertained regarding common ERIC strategies and domains. Future study designs may incorporate these common implementation strategies from previously successful programs to inform and structure novel improvement programs. Finally, this review suggests a need for understanding the facility-level and patient-level factors of potential implementation settings to provide necessary context associated with the success of a given improvement program. By highlighting promising strategies and bodies of work, this review may therefore inform the direction of future TTS improvement programs to promote the development of both generalizable and context-specific strategies for improvement programs in the future.


QKI gene drive the conversion of embryonic myoblast cells into mature muscle fiber and tissue. These genes activate other genetic factors and signaling pathways that are all implicated in the differentiation of muscle progenitor cells and the formation of mature, differentiated muscle tissue. Although future investigations will be necessary to elucidate the entire molecular mechanism involved, we believe that the discoveries reported herein will facilitate the genetic engineering of novel goat lines with enhanced musculature and, by extension, superior meat yield and quality, breeder profitability, and consumer satisfaction.Meat goats are highly valued as excellent sources of lean dietary muscle-protein. However, the molecular mechanisms by which muscle cells and tissues are generated and the genes and signaling pathways regulating this process in meat goats are poorly understood. The present study applied currently conventional molecular-genetics methods and determined that the major isoform of the QKI genes encode RNA-binding proteins regulating cell proliferation, differentiation, and apoptosis. The Goat QKI has six isoforms, but their roles in myogenesis are unclear. In this study, the six isoforms of the QKI gene were overexpressed in goat myoblast. Immunofluorescence, qPCR and Western blot were used to evaluate the effect of QKI on the differentiation of goat myoblast. An RNA-Seq was performed on the cells with the gain of the function from the major isoforms to screen differentially expressed genes (DEGs). The results show that six isoforms had different degrees of deletion in exons 6 and 7, and caused the appearance of different types of encoded amino acids. The expression levels of the QKI-1 and QKI-5 groups were upregulated in the biceps femoris and latissimus dorsi muscle tissues compared with those of the QKI-4, QKI-7, QKI-3 and QKI-6 groups. After 6 d of myoblast differentiation, QKI-5 and the myogenic differentiators MyoG, MyoD, and MyHC were upregulated. Compared to the negative control group, QKI promoted myotube differentiation and the myoblasts overexpressing QKI-5 formed large, abundant myotubes. In summary, we identified that the overexpression of the QKI gene promotes goat-myoblast differentiation and that QKI-5 is the major isoform, with a key role. The RNA-Seq screened 76 upregulated and 123 downregulated DEGs between the negative control and the QKI-5-overexpressing goat myoblasts after d 6 of differentiation. The GO and KEGG analyses associated the downregulated DEGs with muscle-related biological functions. Only the pathways related to muscle growth and development were enriched. This study provides a theoretical basis for further exploring the regulatory mechanism of QKI in skeletal-muscle development in goats.The  Capra hircus) and it is a research focus in genetic breeding. Lingqiu gray-backed goats are a typical local breed in Shanxi Province, China. They have several excellent production traits, such as strong adaptability and good meat quality. Meat yield and quality are closely related to skeletal-muscle growth and development. Hence, muscle growth and development are regarded as the bases of meat-production performance in livestock and poultry. Elucidating the mechanisms regulating goat-muscle-tissue development will allow the identification of their molecular-level effects on meat quality and production performance and lay the foundation for future genetic meat-goat breeding. Skeletal-muscle growth and development are highly coordinated and includes myoblast proliferation, differentiation, migration, and fusion [Meat-production performance is an economically important trait in meat goats , is a member of the STAR  family. The deletion of the upstream regulatory region of QKI results in tissue-specific QKI downregulation and leads to neuromyelin-development disorders. Mice with this genetic defect present with a trembling phenotype [QKI gene increases during myogenesis and regulate alternative splicing by binding the ACUAA motifs that are enriched in regions downstream of muscle-specific exons. When QKI binds upstream of the alternative splicing region, it promotes inclusion [QKI proteins are localized on mouse and human chromosomes 17 and 6, respectively; they encode the isoforms QKI-5, QKI-6 and QKI-7, which share the same K homology (KH) structural domain [QKI protein isoforms [QKI is a global splicing regulator during C2C12 muscle-cell differentiation [QKI is directly or indirectly involved in skeletal-muscle growth and development.The RBPs affect RNA structure and interactions by binding RNA. They participate in RNA transcription, processing, degradation and nuclear export. Thus, they regulate biological RNA function ,7,8. Thehenotype . The expnclusion . When itnclusion . The QKIl domain ,16. In mntiation . It is entiation . It alsontiation , early mntiation  and circntiation . Hence, QKI regulates goat skeletal-muscle-myoblast differentiation has never been reported. Although it is known that goat QKI has six isoforms, it is unclear which of these is upregulated in goats muscle. The aim of the present study, then, was to identify the major QKI isoforms by investigating their expression trends during goats-myoblast differentiation and to determine their regulatory effects on myogenesis. In addition, in order to further explore the molecular mechanism of QKI in goat-myoblast differentiation, RNA-seq was performed to screen for differentially expressed genes (DEGs). The discoveries reported herein could provide theoretical and practical bases for the regulation of skeletal-muscle growth and development in meat goats.To the best of our knowledge, the mechanism by which the RBP v/v) fetal bovine serum  and 1% (w/v) penicillin/streptomycin . When the myoblasts reached 90% confluence, the growth medium was replaced with differentiation medium consisting of DMEM, 2% (v/v) horse serum , and 1% (w/v) penicillin/streptomycin. The medium was changed every 2 d.All animal experiments were performed according to the animal procedures established by the Ministry of Agriculture of China and were approved by the Institutional Animal Care and Use Committee of Shanxi Agricultural University. Biceps femoris (BF) and longissimus dorsi (LD) muscle tissue was collected from Lingqiu gray-backed goats ≤ 1 mo old. One part of each tissue was isolated and cultured while the other was used for total RNA extraction. Myoblasts were isolated and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% .Total RNA was extracted from the goat BF tissues with TRIzol reagent. The RNA concentration was measured by nucleic-acid-protein assay . The extracted RNA was reverse-transcribed to cDNA with a PrimeScripthttps://www.ncbi.nlm.nih.gov, accessed on 1 April 2022) revealed six predicted goat QKI isoforms, namely, QKI-1, QKI-3, QKI-4, QKI-5, QKI-6 and QKI-7. A sequence alignment showed that the sequences of QKI-1 and QKI-5, of QKI-3 and QKI-6 and of QKI-4 and QKI-7 differed by 24 bases in the center of the coding region. All other sequences were identical. The qPCR primer pairs QKI-15, QKI-36 and QKI-47 were designed with Primer Premier v. 6.0   for full gene synthesis to obtain overexpression vectors for the six QKI isoforms. The vector was pHBLV-CMVIE-ZsGreen-T2A-puro and the 5′ and 3′ digestion sites were EcoRI and XbaI, respectively.A search of the NCBI database  . The seqTM 3000 transfection reagent (Thermo Fisher Scientific). The existing medium was replaced with fresh complete medium after 6 h of transfection. Cell cultures were collected after 48 h and 72 h transfection, passed through a 0.22-micrometer filter and centrifuged at 72,000× g and 4 °C for 2 h. The supernatant was discarded and the pellet was resuspended in 500 μL fresh culture medium to obtain the viral fluid. Successful infection of the goat myoblast by the viral fluid was observed under a fluorescent microscope  and appeared as green fluorescence. When the cells reached 90% confluence, they were subjected to cell-differentiation medium containing 2% (v/v) horse serum. That time point was recorded as day 0 of differentiation. The cell-differentiation medium was changed every 2 d. Total RNA and protein were collected from the cells at days 0 and 6 of differentiation. Cell morphology and myotube appearance were observed under a microscope.The myoblasts were cultured in six-well plates  before transfection. The goat myoblasts were used for cell transfection when they reached ~60% confluence. Before 24 h of transfection, 293T cells were inoculated into 10 cm cell-culture dishes  and co-transfected with recombinant plasmids and the packaging plasmids, pMD2G and psPAX2, via Lipofectaminev/v) Triton X-100 for 10 min, blocked with 5% (v/v) bovine serum albumin (BSA) and incubated with primary antibodies  at 4 °C overnight. The cells were then washed thrice with PBS, stained with fluorophore-conjugated secondary antibodies (CoraLite594-conjugated goat anti-rabbit immunoglobulin G (IgG) (H + L) 1:400) at room temperature for 1 h and incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min. Images of the cells were captured with a DMi8 microscope . The number of fusion cells per unit area of differentiated myotubes was analyzed. The fusion indices (% of nuclei in the myotubes) were determined with ImageJ 2.3.0 .After 6 d of differentiation, the cells were washed twice with phosphate-buffered saline (PBS), fixed in cold methanol for 10 min, permeabilized with 0.1%  and transferred to nitrocellulose membranes with a Trans-Blot turbo transfer system (Bio-Rad Laboratories). The membranes were blocked with 5% (w/v) nonfat dry milk in PBS and immunoblotted with the primary antibodies anti-QKI-5  and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH)  at 4 °C overnight. The membranes were then washed thrice in Tris-buffered saline plus Tween 20 (TBST) and incubated with secondary anti-IgG antibody  at room temperature for 1 h. Staining was performed in an Odyssey infrared imaging system .Protein was extracted from the myoblast with radioimmunoprecipitation assay (RIPA) lysis buffer on ice for 30 min. The protein fractions were collected by centrifugation at 10,000× QKI-5-overexpressing and negative control groups were collected at 6 d of differentiation. Total RNA was extracted with TRIzol reagent  following the manufacturer’s protocol. The RNA integrity was evaluated with the RNA Nano 6000 assay kit and a Bioanalyzer 2100 system . Total RNA from each sample was used in Illumina sequencing at Novogene Bioinformatics Technology Co., Ltd. . The mRNA was purified from the total RNA using poly-T oligo-attached magnetic beads. To select cDNA fragments 370–420 bp in length, the library fragments were purified in an AMPure XP system . A PCR was then performed using Phusion high-fidelity DNA polymerase, universal PCR primers and index (X) primer. The PCR products were purified in the AMPure XP system and library quality was assessed in the Agilent Bioanalyzer 2100 system.Goat myoblasts from the The index-coded samples were clustered in a cBot cluster-generation system with a TruSeq PE cluster kit v3-cBot-HS  according to the manufacturer’s instructions. The library preparations were then sequenced on an Illumina NovaSeq platform (Illumina) and 150-base-pair paired-end reads were generated.https://github.com/mskcc-cwl/fastp_0.19.7, accessed on 21 October 2022). Clean reads were obtained by removing reads containing adapter or poly-N and low-quality reads. The Q20, the Q30 and the GC content of the clean data were calculated. The reference genome index was built with HiSat2 v. 2.0.5 . Paired-end clean reads were aligned to the goat reference genome. The reference genome and the gene-model-annotation files were directly downloaded from the genome website .Raw reads in fastq format were processed in fastp v. 0.19.7  was used to count the reads mapped to each gene. The fragments per kilobase of transcript per million mapped reads (FPKM) was calculated for each gene based on the length of the gene and the read counts mapped to it. Analysis of differentially expressed genes was performed using the DESeq2 v. 1.20.0 package in R . The p-values were subjected to the Benjamini and Hochberg correction to control for the false-discovery rate (FDR). Genes with adjusted p-value ≤ 0.05 were deemed differentially expressed.FeatureCounts v. 1.5.0-p3  enrichment analysis of the DEGs was performed using the clusterProfiler package in R and the gene-length bias was corrected. The GO terms with corrected QKI isoforms obtained from NCBI  were used to translate the base sequence in the coding region of each isoform into an amino-acid sequence. We then compared the structures of the different splice variants, as shown in QKI isoforms. At the exon-6-translation starting position, QKI-1, QKI-3, and QKI-4 lost eight amino acids, but their amino-acid domains were unchanged. Starting from the exon-7-amino-acid sequence, the amino-acid structure of each isoform underwent certain changes relative to QKI-1 and QKI-5. The QKI-3 and QKI-6 were missing 16 amino acids, while the QKI-4 and QKI-7 were missing 22 amino acids. These changes caused different types of amino acid to be encoded.We compared the coding sequence (CDS) nucleotides of the goat rom NCBI A. We fouQKI was upregulated in the BF muscle compared with the LD muscle. The expression was significantly increased in the QKI-1, QKI-5 group relative to the QKI-4, QKI-7 and QKI-3, QKI-6 groups (QKI isoforms in the BF myoblasts. The expression level of the QKI-15 group was significantly higher than those of the other isoforms during the myoblast differentiation. The highest expression level was determined for d 6 of the differentiation (The qPCR showed that the 6 groups A. The trntiation B.QKI isoforms were overexpressed during the myoblast differentiation for 6 days. The myotubes were subjected to immunofluorescence to compare the relative myoblast differentiation. The myoblasts transfected with QKI-5 overexpression formed larger and more numerous myotubes than those transfected with the negative control (NC)  C. The fuisoforms D. Hence,pression E. The QKo the NC F.The transcriptome sequencing of all the samples showed that there were ≥5.82 G of clean reads after the original data filtering, sequencing-error-rate examination, and GC content distribution. The Q30 scores for the clean bases were all >92.29% in all six samples . The cleACTA1, CAV3, TNNC1, TNNC2, TNNI1, TNNT1 and TNNT3 were highly significantly differentially expressed in relation to muscle growth and development. The distribution of the DEGS in the two treatment groups is displayed in a volcano plot | > 1 and padj < 0.05) disclosed 199 DEGs between the OE and NC groups. Of these, 76 were upregulated and 123 were downregulated. ano plot A. The DEThe DEGs were annotated according to the molecular-function, cellular-component, and biological-process GO categories. The 10 most significantly enriched pathways in each function are shown in p < 0.05) enriched among all the DEGs, including hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), cardiac-muscle contraction, focal adhesion, and MAPK signaling  between the experimental and control groups. The use of RNA high-throughput sequencing (RNA-Seq) is indispensable in the analysis of differential gene expression and alternative mRNA splicing at the transcriptome level. Here, we applied second-generation high-throughput sequencing on the NC goat myoblasts and those overexpressing QKI-5. We found that the Q30 was >92% and the GC base content was in the range of 51.32–52.33%. The data quality is considered satisfactory when Q30 > 80% and the GC base content is in the range of 50–60%. To determine which genes and sequenced fragments are transcribed, quality-controlled clean reads must be compared against a reference genome. Here, >92% of the clean reads were successfully mapped to the goat genome. Hence, the data were adequate for the subsequent analysis.To clarify the molecular mechanism by which QKI may be related to muscle-tissue development. There were far more downregulated than upregulated DEGs enriched in biological function, including muscle contraction and skeletal-muscle development. Hence, QKI may participate upstream of the muscle-development signaling pathway. The genes downstream of QKI might be critical for muscle growth and development. The proteins TNNC1, TNNC2, TNNI1 and TNNT3 are all troponins (Tn). Furthermore, TnC, TnI and TnT comprise Tn, which collaborates with tropomyosin to regulate muscle contraction. The Tn complexes to form a calcium-sensitive molecular switch on striated muscle filaments. It regulates striated muscle contraction in response to changes in intracellular calcium concentration [TNNT3 is vital for growing muscle and C2C12 expression and the alternative spliceosomes that gradually increase with myotube differentiation. Therefore, TNNT3 performs important biological functions in muscle growth and development [TNNC2 plays a critical role in skeletal muscle contraction. The TNNC2 gene is expressed during the myoblast differentiation and skeletal muscle development [TNNI1 genes are switched on during skeletal-muscle myogenesis and are expressed during the early periods of development [QKI gene in myoblasts, which suggests that the QKI gene can inhibit the expression of genes related to muscle development. Future research should elucidate the mechanism of the DEGs associated with muscle growth and development.In the present study, we attempted to identify the key genes and signaling pathways related to muscle growth and development. A gene ontology (GO) analysis showed that most of the DEGs regulated muscle function in general and muscle contraction in particular. Thus, ntration  and affentration ,32. In melopment ; TNNC2 pelopment ; TNNI1 gelopment . These rQKI was associated with dilated cardiomyopathy (DCM) and cardiac fibrosis [CAV-3, is of great importance in skeletal-muscle development and regulation and controls the focal-adhesion signaling pathway. Furthermore, CAV-3 is a muscle-specific protein that maintains muscle health and repairs damage [CAV-3 knockout mice presented with a loss of membrane cellars in their muscle fibers and pathological changes in their skeletal muscle cells. Therefore, CAV-3 is vital to muscle-tissue development.The KEGG analysis helped to identify the 20 most significantly enriched pathways related to the DEGs, including hypertrophic cardiomyopathy, dilated cardiomyopathy and cardiac-muscle contraction. These processes are associated with muscle development. Furthermore, fibrosis . Hence, fibrosis . We idenfibrosis . Focal afibrosis . The DEGs damage . The CAVNFATC2 and IGF1 were significantly enriched in the MAPK signaling pathway. The NFATC2 isoforms are important for skeletal-muscle growth [IGF1, encodes a single-chain polypeptide hormone. This protein affects muscle development by regulating the synthesis of skeletal-muscle proteins. It plays vital roles in growth and metabolism [IGF1 isoforms generated by alternative splicing affect muscle-cell proliferation and differentiation [The MAPK signaling pathway is ubiquitous and evolutionarily conserved and controls cell proliferation, motility, oxidative stress, survival and apoptosis ,42. The e growth . A membetabolism . Variousntiation . TherefoQKI isoforms, QKI-5 had the highest expression level and the strongest ability to promote goat-myoblast differentiation. Hence, it was identified as the major isoform in this process. The overexpression of QKI-5 and its subsequent induction of differentiation confirmed that QKI promotes goat-myoblast differentiation. The RNA-Seq identified 199 DEGs and the GO- and KEGG-enrichment analyses showed that QKI regulated multiple downstream genes and signaling pathways associated with muscle growth and development. However, the underlying molecular mechanisms by which QKI transcription regulates muscle development in goats are currently not clear and require further investigation.In conclusion, we demonstrated that out of six different goat


Online or hybrid exercise programs incorporating interactive methods  could enhance RNL and quality of life of children with CP. Thus, well-designed online or hybrid exercise programs should be developed and provided for children with CP to enhance overall quality of life during the pandemic.Due to the ongoing COVID-19 pandemic, many online programs for social meetings, education, leisure, and physical activities have been developed and provided; however, children with cerebral palsy (CP) cannot enjoy online programs in the same way that those without disabilities can. The aim of this study was to investigate the differences in reintegration to normal living (RNL), social interaction, and quality of life among school-age children with CP after participation in a game-based online–offline hybrid group exercise program. The current study was conducted on 26 children with CP who participated in a hybrid exercise program. The RNL, social interaction, and quality of life were measured before and after the six-week program. The scores of RNL and quality of life were improved ( The ongoing COVID-19 pandemic has affected people’s lives and led to changes in the daily lives of the worldwide population. People with disabilities who have pre-existing barriers are more vulnerable to unexpected pandemics than people without disabilities. Specifically, children with disabilities and their caregivers have struggled more with changes and adapting to unanticipated pandemic situations than the general population has . Social-In addition, the lack of physical activity and regular rehabilitation treatment due to COVID-19 negatively affect physical development and the attenuation of physical strength, such as muscle loss and muscle atrophy among children with CP, which may cause other problems such as lethargy and social isolation , which hPhysical activity is also a significant predictor of satisfaction with the performance of the daily activities of people with disabilities. The Reintegration to Normal Living (RNL) Index has been used to assess personal satisfaction with social, physical, and psychological functions in the everyday activities of people with disabilities. The RNL is particularly important to understanding the daily life of people with disabilities since it shows how one can actively participate in some domains of human life, such as mobility, self-care, daily activity, and recreational activity . McVeighDespite the known benefits of participation in physical activities, the physical activity levels of people with or without disabilities have dropped due to the COVID-19 pandemic ,23. As aTherefore, the purpose of this study was to explore the effectiveness of the program and investigate the differences in RNL, social interaction levels, and quality of life among school-age children with CP after participation in a game-based online–offline hybrid group exercise program. The hypotheses were as follows.Hypothesis 1.There will be differences in RNL among children with CP before and after participation in a blended (online and offline) group exercise program.Hypothesis 2.There will be differences in social interaction levels among children with CP before and after participation in the blended group exercise program.Hypothesis 3.There will be differences in the quality of life of children with CP before and after participation in the blended group exercise program.The pre–post research design was employed to observe the association between a six- week game-based online–offline hybrid group exercise program for children with CP to explore the effects of the program on children’s RNL, social interaction levels, and quality of life.Participants were recruited from children with CP who were provided wheelchairs from a mobility-improvement project of a welfare foundation. Among them, only children whose parents or caregivers could support online–offline exercise participation were able to participate in this program due to its requirement of both online and offline participation. Thirty-three children voluntarily participated in a group exercise program for CP organized by a G fitness center for children with disabilities in Seoul, South Korea, but only 26 children participated in all six-week sessions and completed both pre- and post-program surveys. Participants were allowed to continue any rehabilitation treatments that they had undergone before participating the program.Throughout the program, there were unpredictable changing schedules due to the government’s COVID-19 policy, making it difficult for the children’s parents and caregivers to follow the changed schedules to support their child’s participation. Additionally, some parents experienced challenges in providing support for their child’s online participation due to a lack of technical knowledge, which also caused an increase in the dropout rate. Moreover, children with disabilities or chronic conditions, including children with CP, are often considered vulnerable during such pandemics by their parents or caregivers ,33; thusParticipants’ ages ranged from 6 to 15 years , with 57.7% identifying as male. The degree of brain lesion disability was reported by parents or caregivers based on the Korean disability rating system, which was assessed by the Modified Barthel Index . The paThe purpose of the study was explained to the parents and caregivers, and informed consent was obtained from them. Parents/caregivers completed questionnaires assessing children’s RNL, social interaction levels, and quality of life before starting the program. Parents/caregivers of children with CP who participated in the exercise program were asked to complete the self-administered questionnaires via email or Google online survey.The game-based online–offline hybrid group exercise program was conducted for 6 weeks every Sunday between September 2020 to October 2020. The program consisted of four online sessions  and two offline sessions (4th week and 5th week), which lasted for 50 min each. The online sessions were conducted using video conferencing platforms, and the offline sessions were held at a gym in Seoul, South Korea. Participants were divided into five classes (5 to 7 children) which eventually became five teams for competition prior to beginning the program. The exercise program focused on a different set of physical activities using metal cage-shaped exercise equipment allowing height and weight adjustments to fix the resistance bands according to the physical characteristics of individual users. The equipment was developed by a team of medical doctors with national funding to support the exercise participation of people with disabilities and was used for exercise programs by wheelchair users . Each grEach online session included (1) a warm-up exercise (stretching), (2) five movements of muscle-strengthening exercises focused on the upper body using resistance bands fixed on a metal cage-shaped exercise instrument, and (3) a cool-down exercise. Strengthening exercises included shoulder-press, pull-down, and trunk exercises  that werThe participants’ level of community reintegration was measured with the Reintegration to Normal Living Index (RNLI), which consists of 11 items in eight domains . The RNLAlthough RNLI includes an item indirectly asking the sociality of the respondents, we used four additional items to identify changes in participants’ ability to interact with various social contexts. The items measuring the social interaction levels of the participants were modified from the socialization part of the Children’s Assessment of Participation and Enjoyment (CAPE)  used in For assessing the quality of life, the work of Charlifue et al.  for peopThe pre- and post-survey questionnaire for this study consisted of a total of 18 questions  and general characteristic questions. Demographic information  was also asked in the pre-survey .W(26) = 0.982, p = 0.913) and quality of life (W(26) = 0.970, p = 0.620). However, the social interaction levels did not meet the criteria for the normality assumption; thus, the Wilcoxon signed-rank test was used to identify the differences between pre- and post-program scores. A paired t-test analysis comparing pre- and post-program scores was conducted to identify changes in the RNL and quality of life of children with CP before and after participating in the exercise program. We adopted a statistical significance level at p≤ 0.05. The differences between pre- and post-program tests were presented using means, standard deviations, t-values for the paired t-test, z-values for the Wilcoxon signed-rank test, and p-values.The data collected in this study were analyzed using IBM SPSS Statistics for Windows, version 25.0  Frequency analysis and technical statistics were conducted to identify demographic information, participation in exercise programs, RNL, social interaction levels, and quality of life of the participants. Before statistical analysis, the Shapiro–Wilk test was performed to assess the normality of continuous data. The results did not show evidence of non-normality of RNL (SD = 0.46), and that of the post-test was 3.40 (SD = 0.51), which were significantly different (t(25) = −2.210, p < 0.05). The average values were compared to see the differences in the detailed items of the RNLI before and after participation in exercise programs for children with CP. In detail, only one item, the ability to deal with life events, showed a significant positive difference before and after participating exercise program (t(25) = 2.379, p < 0.05). However, the scores of nine RNLI items improved after participating in the program, while those of two items remained same  and the post-test  were not significantly different, z = −1.051, p = 0.293 , and the mean of the post-test, 7.50 (SD = 1.39), were significantly different (t(25) = −3.765, p < 0.01; t(25) = −3434, p < 0.01), satisfaction with physical health (t(25) = −2.929, p ≤ 0.01), and satisfaction with psychological health, emotions and mood (t(25) = −3.353, p < 0.01).The paired t-test was conducted to determine the difference in the quality of life before and after participation in exercise programs for children with CP. The mean of the pre-test, 6.20  have been emerging; however, the research shows conflicting results of the satisfaction and effectiveness of the services ,52. ThusIn conclusion, this study focused on exploring the positive changes in participation related to daily living in the social context and quality of life through the hybrid exercise program rather than the enhancement of physical functions of children with CP, which seems small but meaningful; the results of the study could open doors for future research. More studies should be conducted to enhance the quality of online or digital-related exercise programs for children with disabilities and to explore the statistical effectiveness of the programs, which will eventually enable children with disabilities to enjoy the benefits of exercise.


Patients with chronic myelogenous leukemia (CML) harboring the T315I mutation who progress to blast phase CML while on ponatinib may be successfully treated with asciminib monotherapy following induction therapy with cytotoxic chemotherapy. CML patients with the T315I mutation who progress to blast phase on ponatinib represent a challenging clinical scenario. Asciminib has shown efficacy in chronic phase CML patients with the T315I mutation. Asciminib should be considered for CML patients with the T315I mutation who progress to blast phase while on ponatinib. Imatinib was the first drug to specifically target and inhibit the BCR‐ABL fusion protein and was approved by the Food and Drug Administration for the treatment of CP CML in 2001. This targeted approach drastically altered the clinical course of CML, improving the overall survival from 4 to 5 years to that near age matched controls.Since the approval of imatinib, there are now 4 tyrosine kinase inhibitors approved by the US FDA for the first‐line treatment of CP CML: imatinib, nilotinib, dasatinib, and bosutinib. Similar to imatinib, these TKIs all target the ATP‐binding pocket of the ABL1 domain. Clinical trials with second generation TKIs have shown faster and deeper responses and decreased progression to BP CML, but have failed to show improvements in overall survival.Resistance to TKIs is thought to be due to point mutations in the ATP‐binding domain. In particular, the ABL1 T315I mutation, known as the “gatekeeper” mutation, confers resistance to all TKIs with the exception of ponatinib.Asciminib is a first‐in‐class STAMP  inhibitor. It is an allosteric inhibitor that binds to the myristoyl pocket of the BCR‐ABL1 protein to induce a conformational change and lock the BCR‐ABL protein in an inactive confirmation.These promising results prompted the ASCEMBL trial (NCT03106779), a phase 3, randomized, open‐label trial of 233 CP CML patients previously treated with at least 2 TKIs who were given asciminib 40 mg orally twice daily or bosutinib 500 mg orally daily.Since the introduction of imatinib and other small molecule TKIs, the rate of progression to blast phase has decreased from 20% to around 1%–1.5% per year.For patients who are found to have the T315I mutation upon progression to blast phase, ponatinib with or without cytotoxic chemotherapy is a viable option.2In July of 2018, a 62‐year‐old woman with no prior medical history presented to an outside hospital for gout where she was told her white blood cell count (WBC) was “very high” and her red blood cells (RBC) and platelets (plt) were “low.” She received a bone marrow biopsy, but never followed up afterwards. Six months later, she presented again to the outside hospital for shortness of breath and was found to have a pleural effusion. At that time, she had a WBC count of 354 K/cumm, plt count of 933 K/cumm, and was reportedly anemic. The differential and blast count at that time is unknown. She received several thoracenteses over the hospital course and was started on hydroxyurea. She was diagnosed with AP CML and was started on imatinib. Upon discharge at the outside institution, she had a WBC count of 144 k/cumm with 6% blasts, hemoglobin of 7.5 g/dl, and plt count of 1184 K/cumm.After taking imatinib for about 1 week, she presented to our institution with shortness of breath and back pain. Upon presentation, she was noted to have decreased breath sounds in the left lower lung field, splenomegaly, and bilateral lower extremity pitting edema. At that time, her WBC count had decreased to 79.4 K/cumm, hemoglobin of 6.6 g/dl, and plt count of 895 K/cumm. The differential showed 72% neutrophils, 4% bands, 6% lymphocytes, 2% monocytes, 3% eosinophils, 5% basophils, 4% myelocytes, and 4% blasts. Peripheral blood polymerase chain reaction (PCR) for BCR‐ABL1/ABL1 was 57.940% and detected the P210 BCR‐ABL1 fusion transcript. She was continued on imatinib 600 mg per day for AP CML. After 1 month, her WBC count normalized at 5.1 K/cumm, but she remained anemic (hemoglobin 7.8 g/dl) and with thrombocytosis (plt 846 K/cumm). After 6 months of imatinib therapy, her PCR BCR‐ABL1/ABL1 remained persistently elevated at 38.728%. ABL mutational testing was negative, and she was switched to dasatinib 100 mg daily.After 3 months of dasatinib, her BCR‐ABL1/ABL1 decreased to 12.567%; however she required several medications pauses and dose reductions due to episodes of grade 3‐4 neutropenia. Her BCR‐ABL1/ABL1 nadired at 10.701%, but after about 15 months was again elevated at 94.141%. ABL mutational testing done at that time showed a 944C➔T(T315I) mutation and she was switched to ponatinib.Ponatinib therapy was also complicated by cytopenias requiring several pauses and dose reductions. After about 3 months, her peripheral blood showed a WBC count of 54.8 K/cumm with 68% basophils. No blasts were seen. Bone marrow biopsy subsequently confirmed AP CML. It was thought that the progression to accelerated phase CML was likely the result of the numerous drug holidays due to cytopenias, and the patient was continued on ponatinib 45 mg daily.Her counts initially improved; however, 8 months after starting ponatinib, she was found to have a WBC of 35.9 K/cumm and 45% blasts in her peripheral blood confirming progression to BP CML. Bone marrow biopsy showed myeloid blasts harboring the Ph chromosome and the following karyotype: 46,XX,t7;17), t , which showed at least AP CML with 5%–10% blasts and focal marked marrow fibrosis Figure . Within At 6 weeks post induction, her BCR‐ABL1/ABL1 remained at 92.216% in the setting of persistent pancytopenia  without blasts. Given lack of molecular remission and the fact that she had progressed to blast crisis while on ponatinib, asciminib was requested and approved as part of the Managed Access Program (MAP) by Novartis. She was subsequently started on asciminib 200 mg oral twice daily, the approved dosing for patients with ABL T315I mutations in CP CML. Repeat bone marrow biopsy at 3 months post induction therapy (6 weeks after starting asciminib) showed complete response with no blasts and negative minimal residual disease by flow cytometry  in addition a mutation in BCOR on NGS. To our knowledge, the t has only been reported once in the literature and was in association with de novo or therapy‐related myelodysplastic syndromes or acute non‐lymphocytic leukemia.The response to TKI inhibition alone in BP is usually transient, demonstrating that while most cells continue to be sensitive to BCR‐ABL inhibition, there is a component of BCR‐ABL independent growth advantage. In our case, the patient did not receive TKI therapy during AML induction therapy due to the presence of the T315I mutation with prior progression on ponatinib and lack of FDA‐approved therapy. On induction therapy alone, she was not able to achieve a complete remission and her BCR‐ABL1/ABL1 remained above 90%. It was not until she started asciminib that she attained a complete response on bone marrow examination with MRD negativity. Unfortunately, due to logistical reasons, we were unable to also attain a BCR‐ABL1/ABL1 at this time. However, pathology did not note any chronic myeloid leukemia cells present.To the best of our knowledge, there are no published studies of BP CML patients treated with asciminib. Additionally, there are only limited studies of asciminib in CML patients with the T315I mutation. In the phase 1, CABL001X2101 (NCT02081378) previously mentioned, 28 CP and 5 AP CML patients with T315I mutation were included.Since then, a real‐world study of 31 heavily treated CP CML patients also treated with asciminib under the Novartis MAP program was published. In this study, 28 patients (90%) had received three or more prior TKIs including 11 (35%) who had received ponatinib. Of these, 22 patients had intolerance to and 9 patients had resistance to prior TKI.4This case report shows that asciminib, in combination with cytotoxic chemotherapy, may be an effective treatment for CML patients with the T315I mutation who progress to BP CML while on ponatinib. Consistent with prior studies, it also shows that hematologic toxicity, when present, seems to occur across multiple TKIs, in addition to, asciminib.S.T. drafted the manuscript. J.L. and X.Q. helped to draft the manuscript and proofread the paper. X.Q. provided the initial diagnosis and pathology images. S.T. and J.L. participated in the clinical treatment. All of the authors read and approved the final version of the manuscript.Sarah Tomassetti receives research funding from Novartis Pharmaceuticals. The other authors have no conflicts of interests to declare.Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy.