CRISPR-Mediated Programmable 3D Genome Positioning and Nuclear Organization.

Programmable control of spatial genome organization is a powerful approach for studying how nuclear structure affects gene regulation and cellular function. Here, we develop a versatile CRISPR-genome organization (CRISPR-GO) system that can efficiently control the spatial positioning of genomic loci relative to specific nuclear compartments, including the nuclear periphery, Cajal bodies, and promyelocytic leukemia (PML) bodies. CRISPR-GO is chemically inducible and reversible, enabling interrogation of real-time dynamics of chromatin interactions with nuclear compartments in living cells. Inducible repositioning of genomic loci to the nuclear periphery allows for dissection of mitosis-dependent and -independent relocalization events and also for interrogation of the relationship between gene position and gene expression. CRISPR-GO mediates rapid de novo formation of Cajal bodies at desired chromatin loci and causes significant repression of endogenous gene expression over long distances (30-600 kb). The CRISPR-GO system offers a programmable platform to investigate large-scale spatial genome organization and function.

Spatiotemporal dissection of the cell cycle with single-cell proteogenomics.

The cell cycle, over which cells grow and divide, is a fundamental process of life. Its dysregulation has devastating consequences, including cancer1-3. The cell cycle is driven by precise regulation of proteins in time and space, which creates variability between individual proliferating cells. To our knowledge, no systematic investigations of such cell-to-cell proteomic variability exist. Here we present a comprehensive, spatiotemporal map of human proteomic heterogeneity by integrating proteomics at subcellular resolution with single-cell transcriptomics and precise temporal measurements of individual cells in the cell cycle. We show that around one-fifth of the human proteome displays cell-to-cell variability, identify hundreds of proteins with previously unknown associations with mitosis and the cell cycle, and provide evidence that several of these proteins have oncogenic functions. Our results show that cell cycle progression explains less than half of all cell-to-cell variability, and that most cycling proteins are regulated post-translationally, rather than by transcriptomic cycling. These proteins are disproportionately phosphorylated by kinases that regulate cell fate, whereas non-cycling proteins that vary between cells are more likely to be modified by kinases that regulate metabolism. This spatially resolved proteomic map of the cell cycle is integrated into the Human Protein Atlas and will serve as a resource for accelerating molecular studies of the human cell cycle and cell proliferation.

TrackMate 7: integrating state-of-the-art segmentation algorithms into tracking pipelines.

TrackMate is an automated tracking software used to analyze bioimages and is distributed as a Fiji plugin. Here, we introduce a new version of TrackMate. TrackMate 7 is built to address the broad spectrum of modern challenges researchers face by integrating state-of-the-art segmentation algorithms into tracking pipelines. We illustrate qualitatively and quantitatively that these new capabilities function effectively across a wide range of bio-imaging experiments.

Small-molecule inhibition of the archetypal UbiB protein COQ8.

Small-molecule tools have enabled mechanistic investigations and therapeutic targeting of the protein kinase-like (PKL) superfamily. However, such tools are still lacking for many PKL members, including the highly conserved and disease-related UbiB family. Here, we sought to develop and characterize an inhibitor for the archetypal UbiB member COQ8, whose function is essential for coenzyme Q (CoQ) biosynthesis. Guided by crystallography, activity assays and cellular CoQ measurements, we repurposed the 4-anilinoquinoline scaffold to selectively inhibit human COQ8A in cells. Our chemical tool promises to lend mechanistic insights into the activities of these widespread and understudied proteins and to offer potential therapeutic strategies for human diseases connected to their dysfunction.

Building, benchmarking, and exploring perturbative maps of transcriptional and morphological data.

The continued scaling of genetic perturbation technologies combined with high-dimensional assays such as cellular microscopy and RNA-sequencing has enabled genome-scale reverse-genetics experiments that go beyond single-endpoint measurements of growth or lethality. Datasets emerging from these experiments can be combined to construct perturbative "maps of biology", in which readouts from various manipulations (e.g., CRISPR-Cas9 knockout, CRISPRi knockdown, compound treatment) are placed in unified, relatable embedding spaces allowing for the generation of genome-scale sets of pairwise comparisons. These maps of biology capture known biological relationships and uncover new associations which can be used for downstream discovery tasks. Construction of these maps involves many technical choices in both experimental and computational protocols, motivating the design of benchmark procedures to evaluate map quality in a systematic, unbiased manner. Here, we (1) establish a standardized terminology for the steps involved in perturbative map building, (2) introduce key classes of benchmarks to assess the quality of such maps, (3) construct 18 maps from four genome-scale datasets employing different cell types, perturbation technologies, and data readout modalities, (4) generate benchmark metrics for the constructed maps and investigate the reasons for performance variations, and (5) demonstrate utility of these maps to discover new biology by suggesting roles for two largely uncharacterized genes.

The human subthalamic nucleus transiently inhibits active attentional processes.

The subthalamic nucleus (STN) of the basal ganglia is key to the inhibitory control of movement. Consequently, it is a primary target for the neurosurgical treatment of movement disorders like Parkinson's disease, where modulating the STN via deep brain stimulation (DBS) can release excess inhibition of thalamocortical motor circuits. However, the STN is also anatomically connected to other thalamocortical circuits, including those underlying cognitive processes like attention. Notably, STN-DBS can also affect these processes. This suggests that the STN may also contribute to the inhibition of non-motor activity and that STN-DBS may cause changes to this inhibition. Here we tested this hypothesis in humans. We used a novel, wireless outpatient method to record intracranial local field potentials (LFP) from STN DBS implants during a visual attention task (Experiment 1, n = 12). These outpatient measurements allowed the simultaneous recording of high-density EEG, which we used to derive the steady state visual evoked potential (SSVEP), a well established neural index of visual attentional engagement. By relating STN activity to this neural marker of attention (instead of overt behaviour), we avoided possible confounds resulting from STN's motor role. We aimed to test whether the STN contributes to the momentary inhibition of the SSVEP caused by unexpected, distracting sounds. Furthermore, we causally tested this association in a second experiment, where we modulated STN via DBS across two sessions of the task, spaced at least 1 week apart (n = 21, no sample overlap with Experiment 1). The LFP recordings in Experiment 1 showed that reductions of the SSVEP after distracting sounds were preceded by sound-related γ-frequency (>60 Hz) activity in the STN. Trial-to-trial modelling further showed that this STN activity statistically mediated the sounds' suppressive effect on the SSVEP. In Experiment 2, modulating STN activity via DBS significantly reduced these sound-related SSVEP reductions. This provides causal evidence for the role of the STN in the surprise-related inhibition of attention. These findings suggest that the human STN contributes to the inhibition of attention, a non-motor process. This supports a domain-general view of the inhibitory role of the STN. Furthermore, these findings also suggest a potential mechanism underlying some of the known cognitive side effects of STN-DBS treatment, especially on attentional processes. Finally, our newly established outpatient LFP recording technique facilitates the testing of the role of subcortical nuclei in complex cognitive tasks, alongside recordings from the rest of the brain, and in much shorter time than peri-surgical recordings.

Inappropriate prescribing of opioids for patients undergoing surgery.

In response to the opioid epidemic in the United States, states have passed policies aimed at regulating how opioids are prescribed by physicians. For such policies to be effective, however, opioids must be prescribed to the patients for whom they are intended. Whether opioid prescriptions are written for those who are not intended to consume them is empirically difficult to show. In a commercially insured population, we examined opioid prescriptions written for and filled by spouses of patients undergoing outpatient surgery on the day of a patient's surgery compared with the surrounding days. Because patients may be unable to fill prescriptions themselves immediately after surgery, surgeons may prescribe opioids to a patient's spouse, which would be clinically inappropriate. Among 450,125 opioid-naïve couples studied, for patients who did not fill perioperative opioid prescriptions themselves, the rate of spousal fills on the day of surgery (DOS) was 2.39 fills per 1,000 surgeries compared with 0.44 fills on all other perioperative days (adjusted odds ratio (aOR), 5.5, 95% CI, 4.6-6.5). Increases in spousal opioid fills were not present for patients that filled opioid prescriptions themselves. These findings suggest intentional, clinically inappropriate prescribing of opioids.

SpectiCal: m/z Calibration of MS2 Peptide Spectra Using Known Low Mass Ions.

Most tandem mass spectrometry fragmentation spectra have small calibration errors that can lead to suboptimal interpretation and annotation. We developed SpectiCal, a software tool that can read mzML files from data-dependent acquisition proteomics experiments in parallel, compute m/z calibrations for each file prior to identification analysis based on known low-mass ions, and produce information about frequently observed peaks and their explanations. Using calibration coefficients, the data can be corrected to generate new calibrated mzML files. SpectiCal was tested using five public data sets, creating a table of commonly observed low-mass ions and their identifications. Information about the calibration and individual peaks is written in PDF and TSV files. This includes information for each peak, such as the number of runs in which it appears, the percentage of spectra in which it appears, and a plot of the aggregated region surrounding each peak. SpectiCal can be used to compute MS run calibrations, examine MS runs for artifacts that might hinder downstream analysis, and generate tables of detected low-mass ions for further analysis. SpectiCal is freely available at https://github.com/PlantProteomes/SpectiCal.

Aim18p and Aim46p are chalcone isomerase domain-containing mitochondrial hemoproteins in Saccharomyces cerevisiae.

Chalcone isomerases (CHIs) have well-established roles in the biosynthesis of plant flavonoid metabolites. Saccharomyces cerevisiae possesses two predicted CHI-like proteins, Aim18p (encoded by YHR198C) and Aim46p (YHR199C), but it lacks other enzymes of the flavonoid pathway, suggesting that Aim18p and Aim46p employ the CHI fold for distinct purposes. Here, we demonstrate using proteinase K protection assays, sodium carbonate extractions, and crystallography that Aim18p and Aim46p reside on the mitochondrial inner membrane and adopt CHI folds, but they lack select active site residues and possess an extra fungal-specific loop. Consistent with these differences, Aim18p and Aim46p lack CHI activity and also the fatty acid-binding capabilities of other CHI-like proteins, but instead bind heme. We further show that diverse fungal homologs also bind heme and that Aim18p and Aim46p possess structural homology to a bacterial hemoprotein. Collectively, our work reveals a distinct function and cellular localization for two CHI-like proteins, introduces a new variation of a hemoprotein fold, and suggests that ancestral CHI-like proteins were hemoproteins.

Safety and efficacy of ibrutinib in combination with rituximab and lenalidomide in previously untreated follicular and marginal zone lymphoma: An open label, phase 2 study.

BACKGROUND: Follicular lymphoma (FL) and marginal zone lymphoma (MZL) are indolent non-Hodgkin lymphomas (iNHL). Median survival for iNHL is approximately 20 years. Because standard treatments are not curative, patients often receive multiple lines of therapy with associated toxicity-rationally designed, combination therapies with curative potential are needed. The immunomodulatory drug lenalidomide was evaluated in combination with rituximab for the frontline treatment of FL in the phase 3 RELEVANCE study. Ibrutinib, an oral Bruton tyrosine kinase inhibitor, is active in NHL and was evaluated in combination with lenalidomide, rituximab, and ibrutinib (IRR) in a phase 1 study. METHODS: The authors conducted an open-label, phase 2 clinical trial of IRR for previously untreated FL and MZL. The primary end point was progression-free survival (PFS) at 24 months. RESULTS: This study included 48 participants with previously untreated FL grade 1-3a (N = 38), or MZL (N = 10). Participants received 12, 28-day cycles of lenalidomide (15 mg, days 1-21 cycle 1; 20 mg, cycles 2-12), rituximab (375 mg/m2 weekly in cycle 1; day 1 cycles 2-12), and ibrutinib 560 mg daily. With a median follow-up of 65.3 months, the estimated PFS at 24 months was 78.8% (95% confidence interval [CI], 68.0%-91.4%) and 60-month PFS was 59.7% (95% CI, 46.6%-76.4%). One death occurred unrelated to disease progression. Grade 3-4 adverse events were observed in 64.6%, including 50% with grade 3-4 rash. CONCLUSIONS: IRR is highly active as frontline therapy for FL and MZL. Compared to historical results with lenalidomide and rituximab, PFS is similar with higher grade 3-4 toxicity, particularly rash. The study was registered with ClinicalTrials.gov (NCT02532257).

Risk assessment with low-pass whole-genome sequencing of cell-free DNA before CD19 CAR T-cell therapy for large B-cell lymphoma.

Patients with relapsed or refractory large B-cell lymphomas (rrLBCL) can achieve long-term remission after CD19 chimeric antigen receptor T-cell therapy (CART19). However, more than half of recipients will experience treatment failure. Thus, approaches are needed to identify high-risk patients who may benefit from alternative or consolidative therapy. We evaluated low-pass whole-genome sequencing (lpWGS) of cell-free DNA (cfDNA) before CART19 as a new approach for risk stratification. We performed lpWGS on pretreatment plasma samples from 122 patients at time of leukapheresis who received standard-of-care CART19 for rrLBCL to define DNA copy number alterations (CNAs). In multivariable selection, high focal CNA score (FCS) denoting genomic instability was the most significant pretreatment variable associated with inferior 3-month complete response rates (28% vs 56%, P = .0029), progression-free survival (PFS; P = .0007; hazard ratio, 2.11), and overall survival (OS; P = .0026; hazard ratio, 2.10). We identified 34 unique focal CNAs in 108 (89%) patients; of these, deletion 10q23.3 leading to loss of FAS death receptor was the most highly associated with poor outcomes, leading to inferior PFS (P < .0001; hazard ratio, 3.49) and OS (P = .0027; hazard ratio, 2.68). By combining FCS with traditional markers of increased tumor bulk (elevated lactate dehydrogenase and >1 extranodal site), we built a simple risk model that could reliably risk stratify patients. Thus, lpWGS of cfDNA is a minimally invasive assay that could rapidly identify high-risk patients and may guide patient selection for and targeted therapies to evaluate in future clinical trials.

Multidimensional mass profiles increase confidence in bacterial identification when using low-resolution mass spectrometers.

Field-forward analytical technologies, such as portable mass spectrometry (MS), enable essential capabilities for real-time monitoring and point-of-care diagnostic applications. Significant and recent investments improving the features of miniaturized mass spectrometers enable various new applications outside of small molecule detection. Most notably, the addition of tandem mass spectrometry scans (MS/MS) allows the instrument to isolate and fragment ions and increase the analytical specificity by measuring unique chemical signatures for ions of interest. Notwithstanding these technological advancements, low-cost, portable systems still struggle to confidently identify clinically significant organisms of interest, such as bacteria, viruses, and proteinaceous toxins, due to the limitations in resolving power. To overcome these limitations, we developed a novel multidimensional mass fingerprinting technique that uses tandem mass spectrometry to increase the chemical specificity for low-resolution mass spectral profiles. We demonstrated the method's capabilities for differentiating four different bacteria, including attentuated strains of Yersinia pestis. This approach allowed for the accurate (>92%) identification of each organism at the strain level using de-resolved matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) data to mimic the performance characteristics of miniaturized mass spectrometers. This work demonstrates that low-resolution mass spectrometers, equipped with tandem MS acquisition modes, can accurately identify clinically relevant bacteria. These findings support the future application of these technologies for field-forward and point-of-care applications where high-performance mass spectrometers would be cost-prohibitive or otherwise impractical.

The Long-Term Impact of the Tips From Former Smokers® Campaign on Calls to 1-800-QUIT-NOW, 2012-2023.

INTRODUCTION: There is substantial evidence that mass media campaigns increase calls to quitlines and smoking cessation. In 2012, the Centers for Disease Control and Prevention launched the first federally funded national tobacco education campaign, Tips From Former Smokers® (i.e. Tips), which has since aired television advertisements annually. To date, no studies have examined the long-term effect of a national smoking cessation campaign on quitline calls. This study examined the long-term impact of Tips television ads on calls to 1-800-QUIT-NOW from 2012 through 2023. METHODS: Exposure to the Tips campaign was measured using weekly television gross rating points (GRPs) in each U.S. designated market area. We obtained data on calls to 1-800-QUIT-NOW from the National Cancer Institute and used linear regression to model calls to 1-800-QUIT-NOW, from 2012 through 2023, as a function of weekly media market-level GRPs for Tips television ads. Using the regression model results, we calculated predicted values of calls to 1-800-QUIT-NOW across observed GRP values to determine the total additional calls to 1-800-QUIT-NOW that were attributable to the Tips campaign during 2012-2023. RESULTS: Tips GRPs were positively and significantly associated with calls to 1-800-QUIT-NOW across all years (b = 39.94, p < 0.001). Based on this association, we estimate the Tips campaign generated nearly 2.1 million additional calls to 1-800-QUIT-NOW during 2012-2023. CONCLUSIONS: Exposure to the Tips campaign has consistently and significantly increased calls to tobacco quitlines. IMPLICATIONS: Quitlines provide evidence-based support to help people quit smoking. They have been shown to increase the likelihood of successfully quitting. Mass media campaigns have promoted quitline services, and quitline calls have increased significantly with media promotion. The long-term effect of campaigns - like the Centers for Disease Control and Prevention's Tips From Former Smokers® (i.e. Tips) - on quitline calls has not been determined. From 2012 through 2023, exposure to the Tips campaign is estimated to have generated nearly 2.1 million additional calls to 1-800-QUIT-NOW. This study supports continued use of mass media to promote quitlines.

Formation of Products of Incomplete Destruction (PID) from the Thermal Oxidative Decomposition of Perfluorooctanoic Acid (PFOA): Measurement, Modeling, and Reaction Pathways.

The thermal decomposition of perfluorooctanoic acid (PFOA) under oxidative conditions was investigated using air (O2) and N2O as oxidants over temperatures ranging from 400 to 1000 °C in an α-alumina reactor. In the presence of air, PFOA was found to decompose into perfluorohept-1-ene (C7F14) and perfluoroheptanoyl fluoride (C7F14O) in addition to HF, CO, and CO2. At temperatures above 800 °C, both C7F14 and C7F14O were no longer detected. A comprehensive analysis of the reaction mechanisms through quantum chemical analysis and kinetic modeling in combination with experimental observations was utilized to identify key reaction pathways. Quantum chemical analysis led to the conclusion that oxygen atoms are crucial in decomposing perfluoroalk-1-enes, especially the stable perfluorohept-1-ene (C7F14). Under oxidative conditions, it was found that significant quantities of C2F6 and CF4 were formed. Further quantum chemical analysis suggests that the O atoms facilitate the formation of volatile fluorinated compounds (VFCs) such as tetrafluoromethane (CF4) and hexafluoroethane (C2F6), particularly at higher temperatures. By elucidating these key reactions, an improved understanding of the potential formation products of incomplete combustion (PICs) or products of incomplete destruction (PIDs) is made.

Why adolescents conform to high-status peers: Associations among conformity, identity alignment, and self-esteem.

This study examined whether conformity to high- but not low-status e-confederates was associated with increases in identification with popular peers and subsequent increases in self-esteem. A sample of 250 adolescents (55.1% male; Mage = 12.70 years; 40.3% White, 28.2% Black, 23.4% Hispanic/Latino, and 7.7% multiracial/other) participated in a well-established experimental chat room paradigm where they were exposed to norms communicated by high- and low-status e-confederates. Results revealed that for boys in the high-status condition only, but not girls, the positive relation between conformity and self-esteem was mediated by greater response alignment with popular peers. These findings bolster prior research by suggesting that conformity to popular peers may be partly motivated by drives for self-esteem and alignment with a valued reference group.

The glow of the night: The tapetum lucidum as a co-adaptation for the inverted retina.

The vertebrate retina is said to be inverted because the photoreceptors are oriented in the posterior direction and are thus unable to maximize photodetection under conditions of low illumination. The tapetum lucidum is a photoreflective structure located posterior to the photoreceptors in the eyes of some fish and terrestrial animals. The tapetum reflects light forward, giving incident photons a "second chance" to collide with a photoreceptor, substantially enhancing retinal photosensitivity in dim light. Across vertebrates (and arthropods), there are a wide variety of tapeta that vary in structure, chemical composition, and even tissue architecture, indicating repeated convergent evolution. To date, the tapetum has not been observed in any cephalopod, however, which also possess a camera-like eye, but with the retinal photoreceptors oriented in the anterior direction. We therefore hypothesize that the tapetum lucidum is a compensatory adaptation for the suboptimal design of the inverted retina of vertebrates.

Transesophageal echocardiographic acquisition of the venous excess ultrasound exam-a case series and technical description.

PURPOSE: Obtaining an objective, reproducible, and accurate assessment of volume status is one of the more difficult tasks in the perioperative arena. Since its advent in 2020, the Venous Excess Ultrasound (VExUS) score has gained popularity in the minimally invasive assessment of venous congestion. The VExUS exam has been well described as an additional series of images (hepatic vein, portal vein, and intrarenal vein) obtained with a phased-array probe during a transthoracic echocardiogram. Nevertheless, there are no descriptions of comprehensive VExUS exams performed using transesophageal echocardiography (TEE)-a modality that is routinely employed in patients undergoing cardiac surgery. CLINICAL FEATURES: We describe techniques to acquire and interpret a comprehensive TEE-supported VexUS exam, which may be used to optimize the perioperative care of cardiac surgical patients. CONCLUSION: Given the risks of fluid overload in critically ill cardiac surgery patients, TEE-supported VExUS examination may be a way to reduce morbidity in this population.

RéSUMé: OBJECTIF: L'obtention d'une évaluation objective, reproductible et précise du statut volémique est l'une des tâches les plus difficiles dans l'arène périopératoire. Depuis son introduction en 2020, le score VExUS (pour Venous Excess Ultrasound, soit échographie de l'excès veineux) a gagné en popularité dans l'évaluation minimalement invasive de la congestion veineuse. L'examen échographique VExUS a été bien décrit en tant que série supplémentaire d'images (veine hépatique, veine porte et veine intrarénale) obtenues à l'aide d'une sonde type « phased-array ¼ lors d'un échocardiogramme transthoracique. Néanmoins, il n'existe aucune description d'examens VExUS complets réalisés à l'aide d'une sonde d'ETO (échocardiographie transœsophagienne), une modalité couramment utilisée chez les patient·es bénéficiant d'une chirurgie cardiaque. CARACTéRISTIQUES CLINIQUES: Nous décrivons des techniques permettant d'acquérir et d'interpréter un examen VexUS complet par ETO, qui peut être utilisé pour optimiser les soins périopératoires de la patientèle en chirurgie cardiaque. CONCLUSION: Compte tenu des risques de surcharge hydrique chez la patientèle gravement malade en chirurgie cardiaque, l'examen VExUS basé sur l'ETO peut être un moyen de réduire la morbidité dans cette population.

The Year in Cardiothoracic and Vascular Anesthesia: Selected Highlights From 2023.

This special article is the 16th in an annual series for the Journal of Cardiothoracic and Vascular Anesthesia. The authors thank the editor-in-chief, Dr. Kaplan, and the editorial board for the opportunity to continue this series, namely the research highlights of the past year in the specialty of cardiothoracic and vascular anesthesiology. The major themes selected for 2023 are outlined in this introduction, and each highlight is reviewed in detail in the main article. The literature highlights in the specialty for 2023 begin with an update on perioperative rehabilitation in cardiothoracic surgery, with a focus on novel methods to best assess patients in the preoperative and postoperative periods, and the impact of rehabilitation on outcomes. The second major theme is focused on cardiac surgery, with the authors discussing new insights into inhaled pulmonary vasodilators, coronary revascularization surgery, and discussion of causes of coronary graft failure after surgery. The third theme is focused on cardiothoracic transplantation, with discussions focusing on bridge-to-transplantation strategies. The fourth theme is focused on mechanical circulatory support, with discussions focusing on both temporary and durable support. The fifth and final theme is an update on medical cardiology, with a focus on outcomes of invasive approaches to heart disease. The themes selected for this article are only a few of the diverse advances in the specialty during 2023. These highlights will inform the reader of key updates on various topics, leading to improved perioperative outcomes for patients with cardiothoracic and vascular disease.

The Year in Graduate Medical Education: Selected Highlights from 2023.

This special article is the third in an annual series of the Journal of Cardiothoracic and Vascular Anesthesia that highlights significant literature from the world of graduate medical education published over the past year. Major themes addressed in this review include the potential uses and pitfalls of artificial intelligence in graduate medical education, trainee well-being and the rise of unionized house staff, the effect of gender and race/ethnicity on residency application and attrition rates, and the adoption of novel technologies in medical simulation and education. The authors thank the editorial board for again allowing us to draw attention to some of the more interesting work published in the field of graduate medical education during 2023. We hope that the readers find these highlights thought-provoking and informative as we all strive to successfully educate the next generation of anesthesiologists.

The Number of Patients Lost to Follow-Up May Exceed the Fragility Index of a Randomized Controlled Trial Without Reversing Statistical Significance: A Systematic Review and Statistical Model.

PURPOSE: To (1) analyze trends in the publishing of statistical fragility index (FI)-based systematic reviews in the orthopaedic literature, including the prevalence of misleading or inaccurate statements related to the statistical fragility of randomized controlled trials (RCTs) and patients lost to follow-up (LTF), and (2) determine whether RCTs with relatively "low" FIs are truly as sensitive to patients LTF as previously portrayed in the literature. METHODS: All FI-based studies published in the orthopaedic literature were identified using the Cochrane Database of Systematic Reviews, Web of Science Core Collection, PubMed, and MEDLINE databases. All articles involving application of the FI or reverse FI to study the statistical fragility of studies in orthopaedics were eligible for inclusion in the study. Study characteristics, median FIs and sample sizes, and misleading or inaccurate statements related to the FI and patients LTF were recorded. Misleading or inaccurate statements-defined as those basing conclusions of trial fragility on the false assumption that adding patients LTF back to a trial has the same statistical effect as existing patients in a trial experiencing the opposite outcome-were determined by 2 authors. A theoretical RCT with a sample size of 100, P = .006, and FI of 4 was used to evaluate the difference in effect on statistical significance between flipping outcome events of patients already included in the trial (FI) and adding patients LTF back to the trial to show the true sensitivity of RCTs to patients LTF. RESULTS: Of the 39 FI-based studies, 37 (95%) directly compared the FI with the number of patients LTF. Of these 37 studies, 22 (59%) included a statement regarding the FI and patients LTF that was determined to be inaccurate or misleading. In the theoretical RCT, a reversal of significance was not observed until 7 patients LTF (nearly twice the FI) were added to the trial in the distribution of maximal significance reversal. CONCLUSIONS: The claim that any RCT in which the number of patients LTF exceeds the FI could potentially have its significance reversed simply by maintaining study follow-ups is commonly inaccurate and prevalent in orthopaedic studies applying the FI. Patients LTF and the FI are not equivalent. The minimum number of patients LTF required to flip the significance of a typical RCT was shown to be greater than the FI, suggesting that RCTs with relatively low FIs may not be as sensitive to patients LTF as previously portrayed in the literature; however, only a holistic approach that considers the context in which the trial was conducted, potential biases, and study results can determine the merits of any particular RCT. CLINICAL RELEVANCE: Surgeons may benefit from re-examining their interpretation of prior FI reviews that have made claims of substantial RCT fragility based on comparisons between the FI and patients LTF; it is possible the results are more robust than previously believed.