Fluorescein-Based SynNotch Adaptors for Regulating Gene Expression Responses to Diverse Extracellular Cues.

We introduce an adaptor-based strategy for regulating fluorescein-binding synthetic Notch (SynNotch) receptors using ligands based on conjugates of fluorescein isomers and analogs. To develop a versatile system, we evaluated the surface expression and activities of multiple constructs containing distinct extracellular fluorescein-binding domains. Using an optimized receptor, we devised ways to regulate signaling via fluorescein-based chemical transformations, including an approach based on a bio-orthogonal chemical ligation and a spatially controllable strategy via the photo-patterned uncaging of an o -nitrobenzyl-caged fluorescein conjugate. We further demonstrate that fluorescein-conjugated extracellular matrix (ECM)-binding peptides can regulate SynNotch activity depending on the folding state of collagen-based ECM networks. Treatment with these conjugates enabled cells to distinguish between folded versus denatured collagen proteins and enact dose-dependent gene expression responses depending on the nature of the signaling adaptors presented. To demonstrate the utility of these tools, we applied them to control the myogenic conversion of fibroblasts into myocytes with spatial and temporal precision and in response to denatured collagen-I, a biomarker of multiple pathological states. Overall, we introduce an optimized fluorescein-binding SynNotch as a versatile tool for regulating transcriptional responses to extracellular ligands based on the widely used and clinically-approved fluorescein dye.

Biomechanical Comparison of Anterior Cervical Plate Fixation Versus Integrated Fixation Cage for Anterior Cervical Discectomy and Fusion.

STUDY DESIGN: Cadaveric, biomechanic study. OBJECTIVE: To compare the range of motion profiles of the cervical spine following one-level anterior cervical discectomy and fusion (ACDF) constructs instrumented with either an interbody cage and anterior plate or integrated fixation cage in a cadaveric model. SUMMARY OF BACKGROUND DATA: While anterior plates with interbody cages are the most common construct of fixation in ACDF, newer integrated cage-plate devices seek to provide similar stability with a decreased implant profile. However, differences in postoperative cervical range of motion between the 2 constructs remain unclear. METHODS: Six cadaveric spines were segmented into 2 functional spine units (FSUs): C2-C5 and C6-T2. Each FSU was nondestructively bent in flexion-extension (FE), right-left lateral bending (LB), and right-left axial rotation (AR) at a rate of 0.5°/s under a constant axial load until a limit of 2-Nm was reached to evaluate baseline range of motion (ROM). Matched pairs were then randomly assigned to undergo instrumentation with either the standard anterior cage and plate (CP) or the integrated fixation cage (IF). Following instrumentation, ROM was then remeasured as previously described. RESULTS: For CP fixation, ROM increased by 61.2±31.7% for FE, 36.3±20.4% for LB, and 31.7±19.1% for AR. For IF fixation, ROM increased by 64.2±15.5% for FE, 56.7±39.8% for LB, and 94.5±65.1% for AR. There was no significant difference in motion between each group across FE, LB, and AR. CONCLUSION: This biomechanical study demonstrated increased motion in both the CP and IF groups relative to the intact, un-instrumented state. However, our model showed no differences in ROM between CP and IF constructs in any direction of motion. These results suggest that either method of instrumentation is a suitable option for ACDF with respect to constructing stiffness at time zero.

Feasibility of Using Bacteriophage Therapy to Reduce Morbidity and Mortality Associated with Spinal Epidural Abscesses.

BACKGROUND: The aim of this study was to determine the feasibility of using bacteriophage therapeutics in spinal epidural abscess (SEA) by reviewing the causes and outcomes of SEA at a single institution and testing a bacteriophage for activity against preserved SEA clinical isolates. MATERIALS AND METHODS: Medical records were reviewed of patients that received incision and drainage for SEA at a single medical center. Causative organisms, incidence of coinciding bacteremia and outcomes were recorded. A subset of SEA patients (N = 11), that had preserved clinical isolates, were assessed to evaluate if a bacteriophage therapeutic had ample activity to those isolates as seen with spot tests and growth inhibition assays. RESULTS: Staphylococcus aureus was the predominate bacterial cause (71%) and bacteremia was associated with 96% of S. aureus SEA. Over 50% of the patients either died within three months, had recurrence of their infection, required repeat debridement, or had long term sequalae. A single bacteriophage had positive spot tests for all the S. aureus clinical isolates and inhibited bacterial growth for more than 24 hours for 9 of the 11 (82%) clinical isolates. CONCLUSION: SEA is associated with significant mortality and morbidity making this a potential indication for adjuvant bacteriophage therapeutics. Since S. aureus is the predominate cause of SEA and most cases are associated bacteremia this creates a potential screening and treatment platform for Staphylococcal bacteriophages therapeutics, allowing for potential pilot studies to be devised.

Tension-tuned receptors for synthetic mechanotransduction and intercellular force detection.

Cells interpret mechanical stimuli from their environments and neighbors, but the ability to engineer customized mechanosensing capabilities has remained a synthetic and mechanobiology challenge. Here we introduce tension-tuned synthetic Notch (SynNotch) receptors to convert extracellular and intercellular forces into specifiable gene expression changes. By elevating the tension requirements of SynNotch activation, in combination with structure-guided mutagenesis, we designed a set of receptors with mechanical sensitivities spanning the physiologically relevant picoNewton range. Cells expressing these receptors can distinguish between varying tensile forces and respond by enacting customizable transcriptional programs. We applied these tools to design a decision-making circuit, through which fibroblasts differentiate into myoblasts upon stimulation with distinct tension magnitudes. We also characterize cell-generated forces transmitted between cells during Notch signaling. Overall, this work provides insight into how mechanically induced changes in protein structure can be used to transduce physical forces into biochemical signals. The system should facilitate the further programming and dissection of force-related phenomena in biological systems.

Surgical Intervention vs. Radiation Therapy: The Shifting Paradigm in Treating Metastatic Spinal Disease.

The spine is one of the most common sites to which metastatic cancer is likely to spread and is one of the leading causes of morbidity and mortality in cancer patients. While no medical treatments have been definitively shown to extend the life expectancy of patients with spinal metastasis, interventional options may be the only viable option in improving outcomes. Currently, two main options exist: surgical resection and radiotherapy, with radiotherapy being the primary treatment modality. In this review, we discuss the research comparing the efficacy and outcomes of radiotherapy and surgical resection in treating spinal metastasis. We conclude that while radiosurgery will continue to remain a major treatment modality, surgical intervention has proven to have equal to or superior outcomes at improving function, symptoms, and life expectancy for patients with metastatic spinal disease and should be considered a primary modality in an expanding subset of patients.

Toward drug repurposing in epigenetics: olsalazine as a hypomethylating compound active in a cellular context.

DNA hypomethylating drugs that act on DNA methyltransferase (DNMT) isoforms are promising anticancer agents. By using a well-characterized live-cell system to measure DNA methylation revisions (imprints), we characterize olsalazine, an approved anti-inflammatory drug, as a novel DNA hypomethylating agent. The cell-based screen used in this work is highly tractable, internally controlled, and well-suited for a drug repurposing strategy in epigenetics. Olsalazine very closely mimics the action of 5-aza-2'-deoxycytidine, a known hypomethylating drug, with minimal cytotoxicity at the concentrations tested. Olsalazine was identified by a rapid computer-guided similarity search of a database of approved drugs to a previously identified inhibitor of DNMTs.