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Background: This study investigates the biomechanical efficacy of new 4-leg Sustained Dynamic Compression (SDC) NiTiNOL staples, hypothesized to offer superior stability and resilience to loading before fusion completion, compared with conventional hardware., Methods: Twenty sawbones left full foot models were divided into 4 treatment groups: (1) 4-leg Inline Staple, (2) 4-leg Inline Staple + 2-leg Staple, (3) 4-leg Inline Staple + Screw, and (4) Plate + Screw. An osteotomy was performed to simulate a Lapidus procedure, and the respective fixation methods were applied. Mechanical testing was conducted using a servo-hydraulic testing machine to evaluate constructs' load, contact force, contact area, and plantar gap., Results: The 4-leg Inline Staple + Screw group demonstrated significantly increased joint contact force, joint contact area, and decreased plantar gap compared with the Plate + Screw group, both before and after cyclic testing. All SDC-containing constructs exhibited post-cyclic joint contact areas that were 2.36 ×, 3.87 ×, and 5.49 × greater than the post-cyclic plate + screw group. Most notably, the 4-leg Inline Staple + Screw group maintained a plantar gap of less than 3 mm throughout the testing, unlike other groups., Conclusions: The 4-leg Inline SDC Staple, particularly when combined with a static screw, demonstrated biomechanical superiority over traditional plate and screw constructs in Lapidus procedures. These findings suggest a promising avenue for enhanced post-operative stability, which could translate into quicker patient recovery, improved fusion rates, and potentially lower non-union rates. Further clinical trials are warranted to validate these biomechanical advantages in patient outcomes., Level of Evidence: Therapeutic, Level V: Bench Testing ., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: J.J. and D.S. are paid employees of Enovis.

Relapse of acute myeloid leukemia (AML) is highly aggressive and often treatment refractory. We analyzed previously published AML relapse cohorts and found that 40% of relapses occur without changes in driver mutations, suggesting that non-genetic mechanisms drive relapse in a large proportion of cases. We therefore characterized epigenetic patterns of AML relapse using 26 matched diagnosis-relapse samples with ATAC-seq. This analysis identified a relapse-specific chromatin accessibility signature for mutationally stable AML, suggesting that AML undergoes epigenetic evolution at relapse independent of mutational changes. Analysis of leukemia stem cell (LSC) chromatin changes at relapse indicated that this leukemic compartment underwent significantly less epigenetic evolution than non-LSCs, while epigenetic changes in non-LSCs reflected overall evolution of the bulk leukemia. Finally, we used single-cell ATAC-seq paired with mitochondrial sequencing (mtscATAC) to map clones from diagnosis into relapse along with their epigenetic features. We found that distinct mitochondrially-defined clones exhibit more similar chromatin accessibility at relapse relative to diagnosis, demonstrating convergent epigenetic evolution in relapsed AML. These results demonstrate that epigenetic evolution is a feature of relapsed AML and that convergent epigenetic evolution can occur following treatment with induction chemotherapy., Competing Interests: KN, AA, TK, AE, MC No competing interests declared, CL consultant for Cartography Biosciences, AS founder of Immunai and Cartography Biosciences; receives research funding from Allogene Therapeutics and Merck Research Laboratories, RM on the Advisory Boards of Kodikaz Therapeutic Solutions, Orbital Therapeutics, and is an inventor on a number of patents related to CD47 cancer immunotherapy licensed to Gilead Sciences. Co-founder and equity holder of Pheast Therapeutics, MyeloGene, and Orbital Therapeutics, (© 2024, Nuno, Azizi et al.)

Poor CAR T persistence limits CAR T cell therapies for B cell malignancies and solid tumors 1,2 . The expression of memory-associated genes such as TCF7 (protein name TCF1) is linked to response and long-term persistence in patients 3-7 , thereby implicating memory programs in therapeutic efficacy. Here, we demonstrate that the pioneer transcription factor, FOXO1, is responsible for promoting memory programs and restraining exhaustion in human CAR T cells. Pharmacologic inhibition or gene editing of endogenous FOXO1 in human CAR T cells diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype, and impaired antitumor activity in vitro and in vivo . FOXO1 overexpression induced a gene expression program consistent with T cell memory and increased chromatin accessibility at FOXO1 binding motifs. FOXO1-overexpressing cells retained function, memory potential, and metabolic fitness during settings of chronic stimulation and exhibited enhanced persistence and antitumor activity in vivo . In contrast, TCF1 overexpression failed to enforce canonical memory programs or enhance CAR T cell potency. Importantly, endogenous FOXO1 activity correlated with CAR T and TIL responses in patients, underscoring its clinical relevance in cancer immunotherapy. Our results demonstrate that memory reprogramming through FOXO1 can enhance the persistence and potency of human CAR T cells and highlights the utility of pioneer factors, which bind condensed chromatin and induce local epigenetic remodeling, for optimizing therapeutic T cell states., Competing Interests: Competing interests: C.A.L. is a consultant to Cartography Biosciences. S.A.G. receives research funding from Novartis, Kite, Vertex, and Servier and consults for Novartis, Roche, GSK, Humanigen, CBMG, Eureka, Janssen/JNJ, and Jazz Pharmaceuticals and has advised for Novartis, Adaptimmune, TCR2, Cellctis, Juno, Vertex, Allogene, Jazz Pharmaceuticals, and Cabaletta. J.A.F. receives research funding from Tceleron (formerly Tmunity Therapeutics) and Danaher Corporation and consults for Retro Biosciences, and is a member of the Scientific Advisory Boards of Cartography Bio and Shennon Biotechnologies Inc. A.T.S. is a founder of Immunai and Cartography Biosciences and receives research funding from Allogene Therapeutics and Merck Research Laboratories. C.L.M. is a co-founder of and holds equity in Link Cell Therapies, is a co-founder of and holds equity in Cargo Therapeutics (formerly Syncopation Life Sciences), is a co-founder of and holds equity in Lyell Immunopharma, holds equity and consults for Mammoth and Ensoma, consults for Immatics, Nektar, and receives research funding from Tune Therapeutics. E.W.W. is a consultant for and holds equity in Lyell Immunopharma and consults for Umoja Immunopharma.

Competing Interests: The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Christopher Born, MD, reports personal fees from Stryker Trauma, Biointraface, BI Medical, and Illuminoss outside the submitted work. ICMJE forms for all authors are available online.

Although chimeric antigen receptor (CAR) T cells have altered the treatment landscape for B cell malignancies, the risk of on-target, off-tumour toxicity has hampered their development for solid tumours because most target antigens are shared with normal cells 1,2 . Researchers have attempted to apply Boolean-logic gating to CAR T cells to prevent toxicity 3-5 ; however, a truly safe and effective logic-gated CAR has remained elusive 6 . Here we describe an approach to CAR engineering in which we replace traditional CD3ζ domains with intracellular proximal T cell signalling molecules. We show that certain proximal signalling CARs, such as a ZAP-70 CAR, can activate T cells and eradicate tumours in vivo while bypassing upstream signalling proteins, including CD3ζ. The primary role of ZAP-70 is to phosphorylate LAT and SLP-76, which form a scaffold for signal propagation. We exploited the cooperative role of LAT and SLP-76 to engineer logic-gated intracellular network (LINK) CAR, a rapid and reversible Boolean-logic AND-gated CAR T cell platform that outperforms other systems in both efficacy and prevention of on-target, off-tumour toxicity. LINK CAR will expand the range of molecules that can be targeted with CAR T cells, and will enable these powerful therapeutic agents to be used for solid tumours and diverse diseases such as autoimmunity 7 and fibrosis 8 . In addition, this work shows that the internal signalling machinery of cells can be repurposed into surface receptors, which could open new avenues for cellular engineering., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

While cancers evolve during disease progression and in response to therapy, temporal dynamics remain difficult to study in humans due to the lack of consistent barcodes marking individual clones in vivo. We employ mitochondrial single-cell assay for transposase-accessible chromatin with sequencing to profile 163,279 cells from 9 patients with chronic lymphocytic leukemia (CLL) collected across disease course and utilize mitochondrial DNA (mtDNA) mutations as natural genetic markers of cancer clones. We observe stable propagation of mtDNA mutations over years in the absence of strong selective pressure, indicating clonal persistence, but dramatic changes following tight bottlenecks, including disease transformation and relapse posttherapy, paralleled by acquisition of copy-number variants and changes in chromatin accessibility and gene expression. Furthermore, we link CLL subclones to distinct chromatin states, providing insight into nongenetic sources of relapse. mtDNA mutations thus mirror disease history and provide naturally occurring genetic barcodes to enable patient-specific study of cancer subclonal dynamics., Significance: Single-cell multi-omic profiling of CLL reveals the utility of somatic mtDNA mutations as in vivo barcodes, which mark subclones that can evolve over time along with changes in accessible chromatin and gene expression profiles to capture dynamics of disease evolution. See related commentary by Hilton and Scott, p. 2965. This article is highlighted in the In This Issue feature, p. 2945., (©2021 American Association for Cancer Research.)

The germinal center (GC) response is critical for both effective adaptive immunity and establishing peripheral tolerance by limiting autoreactive B cells. Dysfunction in these processes can lead to defective immune responses to infection or contribute to autoimmune disease. To understand the gene regulatory principles underlying the GC response, we generated a single-cell transcriptomic and epigenomic atlas of the human tonsil, a widely studied and representative lymphoid tissue. We characterize diverse immune cell subsets and build a trajectory of dynamic gene expression and transcription factor activity during B cell activation, GC formation, and plasma cell differentiation. We subsequently leverage cell type–specific transcriptomic and epigenomic maps to interpret potential regulatory impact of genetic variants implicated in autoimmunity, revealing that many exhibit their greatest regulatory potential in GC-associated cellular populations. These included gene loci linked with known roles in GC biology ( IL21 , IL21R , IL4R , and BCL6 ) and transcription factors regulating B cell differentiation ( POU2AF1 and HHEX ). Together, these analyses provide a powerful new cell type–resolved resource for the interpretation of cellular and genetic causes underpinning autoimmune disease.

Objective: Decoding neural activity has been limited by the lack of tools available to record from large numbers of neurons across multiple cortical regions simultaneously with high temporal fidelity. To this end, we developed the Argo system to record cortical neural activity at high data rates., Approach: Here we demonstrate a massively parallel neural recording system based on platinum-iridium microwire electrode arrays bonded to a CMOS voltage amplifier array. The Argo system is the highest channel count in vivo neural recording system, supporting simultaneous recording from 65 536 channels, sampled at 32 kHz and 12-bit resolution. This system was designed for cortical recordings, compatible with both penetrating and surface microelectrodes., Main Results: We validated this system through initial bench testing to determine specific gain and noise characteristics of bonded microwires, followed by in-vivo experiments in both rat and sheep cortex. We recorded spiking activity from 791 neurons in rats and surface local field potential activity from over 30 000 channels in sheep., Significance: These are the largest channel count microwire-based recordings in both rat and sheep. While currently adapted for head-fixed recording, the microwire-CMOS architecture is well suited for clinical translation. Thus, this demonstration helps pave the way for a future high data rate intracortical implant.

Cell differentiation and function are regulated across multiple layers of gene regulation, including modulation of gene expression by changes in chromatin accessibility. However, differentiation is an asynchronous process precluding a temporal understanding of regulatory events leading to cell fate commitment. Here we developed simultaneous high-throughput ATAC and RNA expression with sequencing (SHARE-seq), a highly scalable approach for measurement of chromatin accessibility and gene expression in the same single cell, applicable to different tissues. Using 34,774 joint profiles from mouse skin, we develop a computational strategy to identify cis-regulatory interactions and define domains of regulatory chromatin (DORCs) that significantly overlap with super-enhancers. During lineage commitment, chromatin accessibility at DORCs precedes gene expression, suggesting that changes in chromatin accessibility may prime cells for lineage commitment. We computationally infer chromatin potential as a quantitative measure of chromatin lineage-priming and use it to predict cell fate outcomes. SHARE-seq is an extensible platform to study regulatory circuitry across diverse cells in tissues., Competing Interests: Declaration of Interests A.R. is a founder and equity holder of Celsius Therapeutics, is an equity holder in Immunitas Therapeutics, and until August 31, 2020, was an SAB member of Syros Pharmaceuticals, Neogene Therapeutics, Asimov, and ThermoFisher Scientific. From August 1, 2020, A.R. is an employee of Genentech. J.D.B. holds patents related to ATAC-seq and is an SAB member of Camp4 and seqWell. J.D.B., A.R., and S.M. submitted a provisional patent application based on this work., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Regulatory networks that maintain functional, differentiated cell states are often dysregulated in tumor development. Here, we use single-cell epigenomics to profile chromatin state transitions in a mouse model of lung adenocarcinoma (LUAD). We identify an epigenomic continuum representing loss of cellular identity and progression toward a metastatic state. We define co-accessible regulatory programs and infer key activating and repressive chromatin regulators of these cell states. Among these co-accessibility programs, we identify a pre-metastatic transition, characterized by activation of RUNX transcription factors, which mediates extracellular matrix remodeling to promote metastasis and is predictive of survival across human LUAD patients. Together, these results demonstrate the power of single-cell epigenomics to identify regulatory programs to uncover mechanisms and key biomarkers of tumor progression., Competing Interests: Declaration of Interests T.J. is a member of the Board of Directors of Amgen and Thermo Fisher Scientific. He is also a co-Founder of Dragonfly Therapeutics and T2 Biosystems. T.J. serves on the Scientific Advisory Board of Dragonfly Therapeutics, SQZ Biotech, and Skyhawk Therapeutics. None of these affiliations represent a conflict of interest with respect to the design or execution of this study or interpretation of data presented in this manuscript. T.J. laboratory currently also receives funding from the Johnson & Johnson Lung Cancer Initiative and The Lustgarten Foundation for Pancreatic Cancer Research, but this funding did not support the research described in this manuscript. J.D.B. holds patents related to ATAC-seq and single-cell ATAC-seq and serves on the Scientific Advisory Board of CAMP4 Therapeutics and seqWell. A.R. is a co-founder and equity holder of Celsius Therapeutics, an equity holder in Immunitas, and an SAB member of Thermo Fisher Scientific, Syros Pharmaceuticals, Asimov, and Neogene Therapeutics., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Recent innovations in single-cell Assay for Transposase Accessible Chromatin using sequencing (scATAC-seq) enable profiling of the epigenetic landscape of thousands of individual cells. scATAC-seq data analysis presents unique methodological challenges. scATAC-seq experiments sample DNA, which, due to low copy numbers (diploid in humans), lead to inherent data sparsity (1-10% of peaks detected per cell) compared to transcriptomic (scRNA-seq) data (10-45% of expressed genes detected per cell). Such challenges in data generation emphasize the need for informative features to assess cell heterogeneity at the chromatin level., Results: We present a benchmarking framework that is applied to 10 computational methods for scATAC-seq on 13 synthetic and real datasets from different assays, profiling cell types from diverse tissues and organisms. Methods for processing and featurizing scATAC-seq data were compared by their ability to discriminate cell types when combined with common unsupervised clustering approaches. We rank evaluated methods and discuss computational challenges associated with scATAC-seq analysis including inherently sparse data, determination of features, peak calling, the effects of sequencing coverage and noise, and clustering performance. Running times and memory requirements are also discussed., Conclusions: This reference summary of scATAC-seq methods offers recommendations for best practices with consideration for both the non-expert user and the methods developer. Despite variation across methods and datasets, SnapATAC, Cusanovich2018, and cisTopic outperform other methods in separating cell populations of different coverages and noise levels in both synthetic and real datasets. Notably, SnapATAC is the only method able to analyze a large dataset (> 80,000 cells).

Recent technological developments have enabled the characterization of the epigenetic landscape of single cells across a range of tissues in normal and diseased states and under various biological and chemical perturbations. While analysis of these profiles resembles methods from single-cell transcriptomic studies, unique challenges are associated with bioinformatics processing of single-cell epigenetic data, including a much larger (10-1,000×) feature set and significantly greater sparsity, requiring customized solutions. Here, we discuss the essentials of the computational methodology required for analyzing common single-cell epigenomic measurements for DNA methylation using bisulfite sequencing and open chromatin using ATAC-Seq.

We introduce an approach to identify disease-relevant tissues and cell types by analyzing gene expression data together with genome-wide association study (GWAS) summary statistics. Our approach uses stratified linkage disequilibrium (LD) score regression to test whether disease heritability is enriched in regions surrounding genes with the highest specific expression in a given tissue. We applied our approach to gene expression data from several sources together with GWAS summary statistics for 48 diseases and traits (average N = 169,331) and found significant tissue-specific enrichments (false discovery rate (FDR) < 5%) for 34 traits. In our analysis of multiple tissues, we detected a broad range of enrichments that recapitulated known biology. In our brain-specific analysis, significant enrichments included an enrichment of inhibitory over excitatory neurons for bipolar disorder, and excitatory over inhibitory neurons for schizophrenia and body mass index. Our results demonstrate that our polygenic approach is a powerful way to leverage gene expression data for interpreting GWAS signals.

Response to antidepressant treatment in major depressive disorder (MDD) cannot be predicted currently, leading to uncertainty in medication selection, increasing costs, and prolonged suffering for many patients. Despite tremendous efforts in identifying response-associated genes in large genome-wide association studies, the results have been fairly modest, underlining the need to establish conceptually novel strategies. For the identification of transcriptome signatures that can distinguish between treatment responders and nonresponders, we herein submit a novel animal experimental approach focusing on extreme phenotypes. We utilized the large variance in response to antidepressant treatment occurring in DBA/2J mice, enabling sample stratification into subpopulations of good and poor treatment responders to delineate response-associated signature transcript profiles in peripheral blood samples. As a proof of concept, we translated our murine data to the transcriptome data of a clinically relevant human cohort. A cluster of 259 differentially regulated genes was identified when peripheral transcriptome profiles of good and poor treatment responders were compared in the murine model. Differences in expression profiles from baseline to week 12 of the human orthologues selected on the basis of the murine transcript signature allowed prediction of response status with an accuracy of 76% in the patient population. Finally, we show that glucocorticoid receptor (GR)-regulated genes are significantly enriched in this cluster of antidepressant-response genes. Our findings point to the involvement of GR sensitivity as a potential key mechanism shaping response to antidepressant treatment and support the hypothesis that antidepressants could stimulate resilience-promoting molecular mechanisms. Our data highlight the suitability of an appropriate animal experimental approach for the discovery of treatment response-associated pathways across species.

Recent advances in single-cell, transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. In this study, we used massively parallel, single-cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow, constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner, our approach was useful in characterizing immune-cell deficiencies within DNA-protein kinase catalytic subunit ( prkdc ), interleukin-2 receptor γ a ( il2rga ), and double-homozygous-mutant fish, identifying blood cell losses in T, B, and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types, including two classes of natural killer immune cells, classically defined and erythroid-primed hematopoietic stem and progenitor cells, mucin-secreting kidney cells, and kidney stem/progenitor cells. In total, our work provides the first, comprehensive, single-cell, transcriptomic analysis of kidney and marrow cells in the adult zebrafish., (© 2017 Tang et al.)

Although many diseases and traits show large heritability, few genetic variants have been found to strongly separate phenotype groups by genotype. Complex regulatory networks of variants and expression of multiple genes lead to small individual-variant effects and difficulty replicating the effect of any single variant in an affected pathway. Interaction network modeling of GWAS identifies effects ignored by univariate models, but population differences may still cause specific genes to not replicate. Integrative network models may help detect indirect effects of variants in the underlying biological pathway. In this study, we used gene-level functional interaction information from the Integrative Multi-species Prediction (IMP) tool to reveal important genes associated with a complex phenotype through evidence from epistasis networks and pathway enrichment. We test this method for augmenting variant-based network analyses with functional interactions by applying it to a smallpox vaccine immune response GWAS. The integrative analysis spotlights the role of genes related to retinoid X receptor alpha (RXRA), which has been implicated in a previous epistasis network analysis of smallpox vaccine.

Management of posterior malleolus fractures continues to be controversial, with respect to both need for fixation and fixation methods. Fixation methods include an open posterior approach to the ankle as well as percutaneous reduction and fixation with or without arthroscopy for visualization of the articular surface. Plain radiographs are unreliable in identifying fracture pattern and intraoperative reduction, making arthroscopy a valuable adjunct to posterior malleolus fracture management. In this article, we report a case of tibialis posterior tendon entrapment within a posterior malleolus fracture, as identified by arthroscopy and managed with open reduction. Tibialis posterior tendon entrapment within a posterior malleolus has not been previously reported. Ankle arthroscopy for posterior malleolus fractures provides an opportunity to identify soft-tissue or tendinous entrapment, articular surface reduction, and articular cartilage injuries unlikely to be identified with fluoroscopy alone and should be considered in reduction and fixation of posterior malleolus fractures.

Background: Supination external rotation (SER) injuries are commonly fixed with a one third tubular neutralization plate. This study investigated if a combination locked plate with additional fixation options was biomechanically superior in osteoporotic bone and comminuted fracture models., Methods: Using an osteoporotic and a comminuted Sawbones model, SER injuries were fixed with a lag screw for simple oblique fibula fractures, and either a one third tubular neutralization plate or a locking plate. Samples were tested in stiffness, peak torque, displacement at failure, and torsion fatigue., Results: There was no statistically significant difference in biomechanical testing for fractures treated with a lag screw and plate. For comminuted fractures, locked plating demonstrated statistically significant stiffer fixation., Conclusion: A combination locked plate is biomechanically superior to a standard one third tubular plate in comminuted SER ankle fractures. There was no biomechanical superiority between locked and one third tubular plates when the fracture was amenable to a lag screw., (Copyright © 2013 European Foot and Ankle Society. Published by Elsevier Ltd. All rights reserved.)

No consensus exists in the literature on the optimal operative treatment method for distal biceps tendon ruptures. It is our hypothesis that a single-incision technique with a poly-L-lactide Bio-Tenodesis screw (Arthrex, Inc, Naples, Florida) is a safe and effective method for operative management of distal biceps tendon ruptures, with success and complication rates comparable to previous reports in the literature. This article describes a prospective case series of 29 consecutive patients (30 operations) managed by the same surgeon over 34 months. Average follow-up was 19.6 months. Two patients were lost to follow-up. Elbow range of motion (ROM) and strength; Disabilities of the Arm, Shoulder, and Hand (DASH) score; and SF-12 score were measured at various time points up to 2 years. All patients had full extension and supination. Supination and flexion strength was at least 4/5 in all patients. Mean DASH, SF-12 Physical Component (PCS), and SF-12 Mental Component (MCS) scores were 5.86 (range, 0-16.67), 50.35 (range, 30.4-60.1), and 57.15 (range, 41.7-64.4), respectively. These scores are comparable to normative values reported by the American Academy of Orthopaedic Surgeons. Complication rates were similar to those previously reported in the literature. This type of fixation allowed for early mobilization of the operative arm. Our study demonstrates that use of a tenodesis screw for distal biceps repair results in DASH and SF-12 scores comparable to the norm for the general population with complications similar to those seen in past studies. In addition, biomechanical studies suggest that the repair is strong enough to allow early ROM, and the fixation technique may allow for more anatomic positioning of the distal biceps along the ulnar border of the bicipital tuberosity., (Copyright 2010, SLACK Incorporated.)

The Worcester Heart Attack Study is an ongoing population-based investigation examining changes over time in the incidence and case-fatality rates of acute myocardial infarction (AMI) in residents of metropolitan Worcester, Massachusetts. Using this large population-based database of patients with independently validated AMI, we examined the impact of the September 11th tragedy on the occurrence rates of AMI in residents of greater Worcester presenting to all area hospitals during 2001 compared with rates in the previous 10 years.