A highly efficient transgene knock-in technology in clinically relevant cell types.

Inefficient knock-in of transgene cargos limits the potential of cell-based medicines. In this study, we used a CRISPR nuclease that targets a site within an exon of an essential gene and designed a cargo template so that correct knock-in would retain essential gene function while also integrating the transgene(s) of interest. Cells with non-productive insertions and deletions would undergo negative selection. This technology, called SLEEK (SeLection by Essential-gene Exon Knock-in), achieved knock-in efficiencies of more than 90% in clinically relevant cell types without impacting long-term viability or expansion. SLEEK knock-in rates in T cells are more efficient than state-of-the-art TRAC knock-in with AAV6 and surpass more than 90% efficiency even with non-viral DNA cargos. As a clinical application, natural killer cells generated from induced pluripotent stem cells containing SLEEK knock-in of CD16 and mbIL-15 show substantially improved tumor killing and persistence in vivo.

Increased Rates of Late Periprosthetic Fractures in Larger Hydroxyapatite-Coated Cementless Stems: Are Collared Stems a Better Alternative for Total Hip Arthroplasty?

BACKGROUND: Cementless hip stems are widely used for total hip arthroplasty (THA) and have demonstrated excellent survivorship. This study aimed to investigate the effects of stem size and calcar collars on rates of revision due to periprosthetic fracture. METHODS: All primary THA procedures recorded by the Australian Orthopaedic Association National Joint Replacement Registry (AOANJRR) from September 1999 to December 2021 for a diagnosis of osteoarthritis using a single cementless hip stem with modern bearings were included. The primary outcome measure was revision due to periprosthetic fracture. Stems were divided into 2 groups for comparison, large (size 14 to 20) and small-medium (6 to 13). A subanalysis was performed for collared stems. A total of 59,518 primary THA procedures were included. RESULTS: The cumulative percent revision for periprosthetic fracture was significantly higher for large stems compared to small-medium stems (hazard ratio [HR] = 1.57 [95% confidence interval {CI} 1.18, 2.09] P = .002). Furthermore, collared stems had significantly lower revision rates due to late periprosthetic fracture compared to collarless variants (2 week + HR = 4.55 [95% CI 3.23, 6.42], P < .001). Large collarless stems were found to have greater revision rates due to fracture compared to small-medium collarless stems (HR = 1.55 [95% CI 1.13, 2.12] P = .006), but no difference was found between collared groups (HR = 1.37 [95% CI 0.68, 2.78] P = .382). CONCLUSION: Large cementless hip stems have a higher rate of revision due to periprosthetic fracture compared to small-medium stems. Using a collared stem reduces the rate of periprosthetic fracture.

Cross-National Generalizability of WISC-V and CHC Broad Ability Constructs across France, Spain, and the US.

The Cattell-Horn-Carroll (CHC) model is based on psychometric cognitive ability research and is the most empirically supported model of cognitive ability constructs. This study is one in a series of cross-national comparisons investigating the equivalence and generalizability of psychological constructs which align with the CHC model. Previous research exploring the cross-cultural generalizability of cognitive ability measures concluded that the factor analytic models of cognitive abilities generalize across cultures and are compatible with well-established CHC constructs. The equivalence of the psychological constructs, as measured by the Wechsler Intelligence Scale for Children-Fifth Edition (WISC-V), has been established across English-speaking samples. However, few studies have explored the equivalence of psychological constructs across non-English speaking, nationally representative samples. This study explored the equivalence of the WISC-V five-factor model across standardization samples from France, Spain, and the US. The five-factor scoring model demonstrated excellent fit across the three samples independently. Factorial invariance was investigated and the results demonstrated strict factorial invariance across France, Spain, and the US. The results provide further support for the generalizability of CHC constructs across Western cultural populations that speak different languages and support the continued use and development of the CHC model as a common nomenclature and blueprint for cognitive ability researchers and test developers. Suggestions for future research on the CHC model of intelligence are discussed.

Seizure reduction in TSC2-mutant mouse model by an mTOR catalytic inhibitor.

OBJECTIVE: Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder caused by autosomal-dominant pathogenic variants in either the TSC1 or TSC2 gene, and it is characterized by hamartomas in multiple organs, such as skin, kidney, lung, and brain. These changes can result in epilepsy, learning disabilities, and behavioral complications, among others. The mechanistic link between TSC and the mechanistic target of the rapamycin (mTOR) pathway is well established, thus mTOR inhibitors can potentially be used to treat the clinical manifestations of the disorder, including epilepsy. METHODS: In this study, we tested the efficacy of a novel mTOR catalytic inhibitor (here named Tool Compound 1 or TC1) previously reported to be more brain-penetrant compared with other mTOR inhibitors. Using a well-characterized hypomorphic Tsc2 mouse model, which displays a translationally relevant seizure phenotype, we tested the efficacy of TC1. RESULTS: Our results show that chronic treatment with this novel mTOR catalytic inhibitor (TC1), which affects both the mTORC1 and mTORC2 signaling complexes, reduces seizure burden, and extends the survival of Tsc2 hypomorphic mice, restoring species typical weight gain over development. INTERPRETATION: Novel mTOR catalytic inhibitor TC1 exhibits a promising therapeutic option in the treatment of TSC.

Identification of Brain-Penetrant ATP-Competitive mTOR Inhibitors for CNS Syndromes.

The allosteric inhibitor of the mechanistic target of rapamycin (mTOR) everolimus reduces seizures in tuberous sclerosis complex (TSC) patients through partial inhibition of mTOR functions. Due to its limited brain permeability, we sought to develop a catalytic mTOR inhibitor optimized for central nervous system (CNS) indications. We recently reported an mTOR inhibitor (1) that is able to block mTOR functions in the mouse brain and extend the survival of mice with neuronal-specific ablation of the Tsc1 gene. However, 1 showed the risk of genotoxicity in vitro. Through structure-activity relationship (SAR) optimization, we identified compounds 9 and 11 without genotoxicity risk. In neuronal cell-based models of mTOR hyperactivity, both corrected aberrant mTOR activity and significantly improved the survival rate of mice in the Tsc1 gene knockout model. Unfortunately, 9 and 11 showed limited oral exposures in higher species and dose-limiting toxicities in cynomolgus macaque, respectively. However, they remain optimal tools to explore mTOR hyperactivity in CNS disease models.

The Impact of a Dedicated Multidisciplinary Team Approach for Prosthetic Joint Infections of the Lower Limb.

Introduction: Prosthetic joint infections (PJI) of the hip and knee have significant morbidity and mortality, and present with varying local, host and microbiological factors. Given the broad presentation and complexity of PJI's, we developed a dedicated multidisciplinary team (MDT) to manage this complex patient cohort, and report on our early outcomes. Methods: This was a retrospective observational study of all patients diagnosed with a prosthetic joint infection of the hip or knee (n = 71) at our institution during a 4.5-year period. Patients treated after development of the MDT (post-MDT) (n = 44), were compared to a control group prior establishment of the MDT (pre-MDT) (n = 27). Results: 85.2% of individuals in the pre-MDT, and 85.7% of individuals in the post-MDT group were considered cured at a minimum 2 years post-operatively according to the Delphi-based definition. The total number of admissions to hospital (2.44 vs. 1.84) and total number of antibiotics used (3.37 vs. 2.75) decreased in the post-MDT group; however, differences were not considered statistically significant. Discussion: Implementation of a dedicated MDT in the management of individuals with PJI's of the lower limb at our hospital has allowed early and effective collaboration between healthcare personnel, with early promising results. Given the broad nature of PJI, future studies are ongoing to determine modifiable risk factors to reduce the incidence and improve outcomes of individuals with PJI's where systems can then be implemented into already established MDTs to achieve the best clinical outcome for our patients. Supplementary Information: The online version contains supplementary material available at 10.1007/s43465-023-00842-5.

Measurement invariance of the Wechsler Intelligence Scale for children, fifth edition, in Australian and New Zealand and U.S. standardization samples.

Measurement invariance underlies construct validity generalization in psychology and must be demonstrated prior to any cross-population comparison of means and validity correlations. The purpose of this study was to evaluate the measurement invariance of the Wechsler Intelligence Scale for Children-Fifth Edition (WISC-V) across Australia and New Zealand (A&NZ) versus the U.S. normative samples. The WISC-V is the most widely used assessment of intelligence in children. Participants were census matched, nationally representative samples from A&NZ (n = 528) and the United States (n = 2,200) who completed the WISC-V standardization version. Baseline model estimation was conducted to ensure the same model showed acceptable fit in both samples separately. Measurement invariance was then examined across A&NZ and United States. The five-factor scoring model described in the test manual showed excellent fit in both samples. Results showed that the WISC-V demonstrated strict metric measurement invariance across the A&NZ and U.S. samples. Further, the results were consistent with the Cattell-Horn-Carroll (CHC) framework of cognitive abilities, indicating the generalizability of cognitive abilities across cultures. Small but significant differences in visual spatial latent means were found across females, highlighting the importance of local normative data. These findings suggest that the WISC-V scores can be meaningfully compared across A&NZ and United States and that the constructs, which align with CHC theory, and associated construct validity research, generalize across countries. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

The effect of polyethylene thickness on revision rates of contemporary cruciate retaining knee replacements. A registry analysis.

BACKGROUND: An attribute that may influence knee replacement survivorship is tibial polyethylene (PE) insert thickness. Previous studies have suggested thin polyethylene made from ultra-high molecular weight polyethylene (UHMWPE) leads to higher rates of revision surgery. This study aimed to determine if modern polyethylene thickness is associated with altered survivorship of primary total knee arthroplasty (TKA) procedures. METHODS: A retrospective analysis of data from Australian Orthopaedic Association National Joint Replacement Registry (AOANJRR) was done on well performing total knee arthroplasty prostheses used in Australia from 1999-2018. Six of the best performing minimally stabilized prostheses were examined and categorized into three PE thickness subgroups: A (≤10 mm), B (11-14 mm) and C (≥15 mm). There were 185,539 TKA procedures, of which 64.3% (n = 119,382) were ≤ 10 mm, 33.5% (n = 62,173) 11-14 mm, and 2.2% (n = 3984) ≥ 15 mm. Differences in revision rates were analysed for all causes, including loosening, wear, and instability. RESULTS: At 14 years, respective cumulative point revision (CPR) was A: 4.8, B: 4.2 and C: 6.0. The thickest polyethylene group (≥15 mm) had a higher rate of revision for any reason compared to both 11-14 mm and ≤ 10 mm groups. When non-XLPE was analysed the ≤ 10 mm group had higher rates of revision compared to the 11-14 mm group, but this difference was not seen with XLPE. CONCLUSION: Higher rates of revision were seen overall in the thicker PE group (≥15 mm). This group also had higher rates of revision for loosening, instability, and infection. The use of a thicker insert may be a sign of surgical complexity, but is associated with increased revision.

Home Information and Communication Technology Use and Student Academic Performance: Encouraging Results for Uncertain Times.

This study set out to examine the associations of certain information communication technology (ICT) factors in the home environment with academic performance. We employed existing data sets collated by Pearson Clinical Assessment in 2016 which included the WIAT-III A&NZ (Wechsler Individual Achievement Test - Australian and New Zealand Standardised, Third Edition) completed by 714 students aged between 4 and 18 years old, and the home environment questionnaire (HEQ) completed by the parents of those children. Sequential multiple regression models were used to analyze the complex interactions between home ICT factors and measures of student reading, writing, mathematical, and oral ability. The findings of this study indicate that after accounting for the known powerful predictors of household income and parental education: (a) a student's access to an ICT rich home environment, (b) their aptitude in using home ICT, and (c) their recreational use of home ICT, are largely unrelated to academic performance. We observed some small positive correlations between academic performance and child ICT affinity, but also comparably sized negative associations with use of social media and educational TV viewing. Encouragingly, we propose that these findings suggest that increasing levels of ICT use and access in the home are unlikely to be detrimental to academic progress. These results provide important information for parents and educators given the impact of the Coronavirus global pandemic and the near world-wide adoption of ICT for home-schooling.

CALITAS: A CRISPR-Cas-aware ALigner for In silico off-TArget Search.

We describe CALITAS, a CRISPR-Cas-aware aligner and integrated off-target search algorithm. CALITAS uses a modified and CRISPR-tuned version of the Needleman-Wunsch algorithm. It supports an unlimited number of mismatches and gaps and allows protospacer adjacent motif (PAM) mismatches or PAMless searches. CALITAS also includes an exhaustive search routine to scan genomes and genome variants provided with a standard Variant Call Format file. By default, CALITAS returns a single best alignment for a given off-target site, which is a significant improvement compared to other off-target algorithms, and it enables off-targets to be referenced directly using alignment coordinates. We validate and compare CALITAS using a selected set of target sites, as well as experimentally derived specificity data sets. In summary, CALITAS is a new tool for precise and relevant alignments and identification of candidate off-target sites across a genome. We believe it is the state of the art for CRISPR-Cas specificity assessments.

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

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

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

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

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

AIMS: The aim of this study was to define the levels of noise exposure for the surgeon, assistant, scrub nurse, and anesthetist during total hip and knee arthroplasty surgery. In addition, we sought to determine whether the noise exposure during these procedures reaches or exceeds the action values set out by the U.K. Noise at Work Regulations (2005). MATERIALS AND METHODS: Individual noise exposure during arthroplasty hip and knee surgery was recorded using a personal noise Dosemeter System model 22 (DM22) (Pulsar instruments, Filey, U.K.). Recordings were taken in real-time during five separate theater sessions. Each theater session included two arthroplasty procedures and lasted approximately 4 hrs. Personal noise exposure was expressed in terms of peak sound pressure and an average noise exposure over an 8-hour time-period to reflect the noise experienced by the ear over a working day. RESULTS: In all three sessions involving total hip replacement surgery, the peak sound pressure, for the operating surgeon exceeded the exposure action values set out by the U.K. Noise at Work Regulations. Theater sessions involving total knee replacement surgery did not exceed any exposure action values for LCPeak or LEPd. CONCLUSION: Arthroplasty surgery is a working environment with significant noise exposure. We recommend any surgeon or theater member who is concerned about the noise generated in their theater to have noise levels formally assessed using appropriately positioned recording devices.

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

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

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

Recent clinical evaluation of everolimus for seizure reduction in patients with tuberous sclerosis complex (TSC), a disease with overactivated mechanistic target of rapamycin (mTOR) signaling, has demonstrated the therapeutic value of mTOR inhibitors for central nervous system (CNS) indications. Given that everolimus is an incomplete inhibitor of the mTOR function, we sought to develop a new mTOR inhibitor that has improved properties and is suitable for CNS disorders. Starting from an in-house purine-based compound, optimization of the physicochemical properties of a thiazolopyrimidine series led to the discovery of the small molecule 7, a potent and selective brain-penetrant ATP-competitive mTOR inhibitor. In neuronal cell-based models of mTOR hyperactivity, 7 corrected the mTOR pathway activity and the resulting neuronal overgrowth phenotype. The new mTOR inhibitor 7 showed good brain exposure and significantly improved the survival rate of mice with neuronal-specific ablation of the Tsc1 gene. These results demonstrate the potential utility of this tool compound to test therapeutic hypotheses that depend on mTOR hyperactivity in the CNS.

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

This is a case of the most severe and potentially fatal complication of coronary artery vasospasm. We report a case of a 40-year-old female presenting to the emergency department (ED) via emergency medical services with chest pain. The patient experienced a ventricular fibrillation cardiac arrest while in the ED. Post-defibrillation electrocardiogram showed changes suggestive of an ST-elevation myocardial infarction (STEMI). Cardiac catheterization showed severe left anterior descending spasm with no evidence of disease. Coronary vasospasm is a consideration in the differential causes of ventricular fibrillation and STEMI seen in the ED.

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

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

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

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

Identification of Guide-Intrinsic Determinants of Cas9 Specificity.

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

Encoding biological recognition in a bicomponent cell-membrane mimic.

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