Receptor-regulated cellular signaling often is mediated by formation of transient, heterogeneous protein complexes of undefined structure. We used single and two-color photoactivated-localization microscopy (PALM) to study complexes downstream of the T cell antigen receptor (TCR) in single molecule detail at the plasma membrane of intact T cells. The kinase ZAP-70 distributed completely with the TCRζ chain and both partially mixed with the adapter LAT in activated cells thus showing localized activation of LAT by TCR-coupled ZAP-70. In resting and activated cells LAT primarily resided in nanoscale clusters as small as dimers whose formation depended on protein-protein and protein-lipid interactions. Surprisingly, the adapter SLP-76 localized to the periphery of LAT clusters. This nanoscale structure depended on polymerized actin and its disruption affected TCR-dependent cell function. These results extend our understanding of the mechanism of T cell activation and the formation and organization of TCR-mediated signaling complexes, findings also relevant to other receptor systems.

Study Design. A cross-sectional database study. Objective. The aim of this study was to train and validate machine learning models to identify risk factors for complications following posterior lumbar spine fusion. Summary of Background Data. Machine learning models such as artificial neural networks (ANNs) are valuable tools for analyzing and interpreting large and complex datasets. ANNs have yet to be used for risk factor analysis in orthopedic surgery. Methods. The American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) database was queried for patients who underwent posterior lumbar spine fusion. This query returned 22,629 patients, 70% of whom were used to train our models, and 30% were used to evaluate the models. The predictive variables used included sex, age, ethnicity, diabetes, smoking, steroid use, coagulopathy, functional status, American Society for Anesthesiology (ASA) class ≥3, body mass index (BMI), pulmonary comorbidities, and cardiac comorbidities. The models were used to predict cardiac complications, wound complications, venous thromboembolism (VTE), and mortality. Using ASA class as a benchmark for prediction, area under receiver operating curves (AUC) was used to determine the accuracy of our machine learning models. Results. On the basis of AUC values, ANN and LR both outperformed ASA class for predicting all four types of complications. ANN was the most accurate for predicting cardiac complications, and LR was most accurate for predicting wound complications, VTE, and mortality, though ANN and LR had comparable AUC values for predicting all types of complications. ANN had greater sensitivity than LR for detecting wound complications and mortality. Conclusion. Machine learning in the form of logistic regression and ANNs were more accurate than benchmark ASA scores for identifying risk factors of developing complications following posterior lumbar spine fusion, suggesting they are potentially great tools for risk factor analysis in spine surgery.

Thymocytes must transit at least two distinct developmental checkpoints, governed by signals that emanate from either the pre-T cell receptor (pre-TCR) or the TCR to the small G protein Ras before emerging as functional T lymphocytes. Recent studies have shown a role for the Ras guanine exchange factor (RasGEF) Sos1 at the pre-TCR checkpoint. At the second checkpoint, the quality of signaling through the TCR is interrogated to ensure the production of an appropriate T cell repertoire. Although Ras-GRP1 is the only confirmed RasGEF required at the TCR checkpoint, current models suggest that the intensity and character of Ras activation, facilitated by both Sos and RasGRP1, will govern the boundary between survival (positive selection) and death (negative selection) at this stage. Using mouse models, we have assessed the independent and combined roles for the RasGEFs Sos1, Sos2, and RasGRP1 during thymocyte development. Although Sos1 was the dominant RasGEF at the pre-TCR checkpoint, combined Sos1/RasGRP1 deletion was required to effectively block development at this stage. Conversely, while RasGRP1 deletion efficiently blocked positive selection, combined RasGRP1/Sos1 deletion was required to block negative selection. This functional redundancy in RasGEFs during negative selection may act as a failsafe mechanism ensuring appropriate central tolerance. Tcell development is initiated when immature precursor cells emigrate from the fetal liver or adult bone marrow to the thymus. In the thymus, these cells undergo a receptor-driven differentiation program, passing through at least two distinct developmental checkpoints before emerging as functional T lymphocytes (2). At the first checkpoint, a properly rearranged T cell receptor ␤ (TCR␤) chain pairs with a pre-TCR␣ chain to form a pre-T cell receptor (pre-TCR). The pre-TCR signals in a ligand-independent manner to promote proliferation and drive differentiation from the CD4 Ϫ CD8 Ϫ double-negative (DN) to the CD4 ϩ CD8 ϩ double-positive (DP) stage of thymocyte development. At the second checkpoint, the strength and quality of signaling through the mature TCR is interrogated. Cells that fail to signal through the TCR die by neglect, cells expressing a TCR that binds self-antigen in the context of the major histocompatibility complex (MHC) with strong affinity generate strong TCR signals and die via TCR-dependent apoptotic pathways (negative selection), whereas cells expressing a TCR that has weak affinity for self antigen-MHC complexes signal weakly and selectively survive (positive selection) (8). Genetic studies have shown that signaling from either the pre-TCR or the TCR, through the adaptors LAT and Slp-76, to the small G protein Ras and the downstream extracellular signal-regulated kinase (ERK) cascade is required for thymocyte development at both checkpoints (1,7,14,17,22,(24)(25)(26). However, an understanding of how Ras signals are generated to drive thymocyte development has remained enigmatic, despite intense study over the past 20 years.Ras is thought to be...

Study Design:Meta-analysis.Objective:Proximal junctional kyphosis (PJK) is a complication of surgical management for adult spinal deformity with a multifactorial etiology. Many risk factors are controversial and their relative importance are not fully understood. We aimed to identify the surgical, radiographic, and patient-related risk factors associated with PJK and proximal junctional failure (PJF).Methods:A systematic literature search was performed using PubMed, Cochrane Database of Systematic Reviews, and EMBASE. The inclusion criteria included prospective randomized control trials and prospective/retrospective cohort studies of adult patients with radiographic evidence of PJK, which was defined as a proximal junctional sagittal Cobb angle ≥10° and at least 10° greater than the preoperative measurement. Studies required a minimum of 10 patients and 12 months of follow-up.Results:A total of 14 unique studies, including 1908 patients were included. The pooled analysis showed significant differences between the PJK and non-PJK groups in age (weighted mean difference [WMD] −3.80; P = .03), prevalence of osteopenia/osteoporosis (odds ratio [OR] 1.99; P = .0004), preoperative sagittal vertical axis (SVA) (WMD −17.52; P = .02), preoperative lumbar lordosis (LL) (WMD −1.22; P = .002), pedicle screw instrumentation at the upper instrumented vertebra (UIV) (OR 1.67; P = .02), change in SVA (WMD −11.87; P = .01), fusion to sacrum/pelvis/ilium (OR 2.14; P < .00 001), change in LL (WMD −5.61; P = .01), and postoperative SVA (WMD −7.79; P = .008).Conclusions:Our meta-analysis suggests that age, osteopenia/osteoporosis, high preoperative SVA, high postoperative SVA, low preoperative LL, use of pedicle screws at the UIV, SVA change/correction, LL change/correction, and fusion to sacrum/pelvis/iliac region are risk factors for PJK.

Defects in the annulus fibrosus (AF) of intervertebral discs allow nucleus pulposus tissue to herniate causing painful disability. Microdiscectomy procedures remove herniated tissue fragments, but unrepaired defects remain allowing reherniation or progressive degeneration. Cell therapies show promise to enhance repair, but methods are undeveloped and carriers are required to prevent cell leakage. To address this challenge, this study developed and evaluated genipin-crosslinked fibrin (FibGen) as an adhesive cell carrier optimized for AF repair that can deliver cells, match AF material properties, and have low risk of extrusion during loading. Part 1 determined that feasibility of bovine AF cells encapsulated in high concentration FibGen (F140G6: 140 mg/mL fibrinogen; 6 mg/mL genipin) for 7 weeks could maintain high viability, but had little proliferation or matrix deposition. Part 2 screened tissue mechanics and in situ failure testing of nine FibGen formulations (fibrin: 35-140 mg/mL; genipin: 1-6 mg/mL). F140G6 formulation matched AF shear and compressive properties and significantly improved failure strength in situ. Formulations with reduced genipin also exhibited satisfactory material properties and failure behaviors warranting further biological screening. Part 3 screened AF cells encapsulated in four FibGen formulations for 1 week and found that reduced genipin concentrations increased cell viability and glycosaminoglycan production. F70G1 (70 mg/mL fibrinogen; 1 mg/mL genipin) demonstrated balanced biological and biomechanical performance warranting further testing. We conclude that FibGen has potential to serve as an adhesive cell carrier to repair AF defects with formulations that can be tuned to enhance biomechanical and biological performance; future studies are required to develop strategies to enhance matrix production.