Covalent Targeting of Splicing in T Cells.

Despite significant interest in therapeutic targeting of splicing, few chemical probes are available for the proteins involved in splicing. Here, we show that elaborated stereoisomeric acrylamide chemical probe EV96 and its analogues lead to a selective T cell state-dependent loss of interleukin 2-inducible T cell kinase (ITK) by targeting one of the core splicing factors SF3B1. Mechanistic investigations suggest that the state-dependency stems from a combination of differential protein turnover rates and availability of functional mRNA pools that can be depleted due to extensive alternative splicing. We further introduce a comprehensive list of proteins involved in splicing and leverage both cysteine- and protein-directed activity-based protein profiling (ABPP) data with electrophilic scout fragments to demonstrate covalent ligandability for many classes of splicing factors and splicing regulators in primary human T cells. Taken together, our findings show how chemical perturbation of splicing can lead to immune state-dependent changes in protein expression and provide evidence for the broad potential to target splicing factors with covalent chemistry.

ISGylation directly modifies hypoxia-inducible factor-2α and enhances its polysome association.

The hypoxia-inducible factors (HIF)-1α and HIF-2α are central regulators of transcriptional programmes in settings such as development and tumour expansion. HIF-2α moonlights as a cap-dependent translation factor. We provide new insights into how the interferon-stimulated gene 15 (ISG15), a ubiquitin-like modifier, and the HIFs regulate one another in hypoxia and interferon-induced cells. We show that upon ISGylation induction and HIF-α stabilization, both HIFs promote protein ISGylates through transcriptional and/or post-transcriptional pathways. We show the first evidence of HIF-2α modification by ISG15. ISGylation induces system-level alterations to the HIF transcriptional programme and increases the cytoplasmic/nuclear fraction and translation activity of HIF-2α. This work identifies ISG15 as a regulator of hypoxic mRNA translation, which has implications for immune processes and disease progression.

Synthetic efforts toward the bicyclo[3.2.1]octane fragment of rhodojaponin III.

Rhodojaponin III is a grayanane-type diterpenoid natural product with a novel chemical scaffold. It shows potent antinociceptive activity and may represent a new class of natural non-opioid analgesics with a novel mode of action. We explored the Au(I)-catalyzed Conia-ene cyclization and the Mn(III)-mediated radical cyclization of alkynyl ketones for the synthesis of the bicyclo[3.2.1]octane fragment of rhodojaponin III. These strategies will be applicable in the synthesis of rhodojaponin III and analogs for future biological studies.

Synthesis and evaluation of sulfonyl piperazine LpxH inhibitors.

The UDP-2,3-diacylglucosamine pyrophosphate hydrolase LpxH is essential in lipid A biosynthesis and has emerged as a promising target for the development of novel antibiotics against multidrug-resistant Gram-negative pathogens. Recently, we reported the crystal structure of Klebsiella pneumoniae LpxH in complex with 1 (AZ1), a sulfonyl piperazine LpxH inhibitor. The analysis of the LpxH-AZ1 co-crystal structure and ligand dynamics led to the design of 2 (JH-LPH-28) and 3 (JH-LPH-33) with enhanced LpxH inhibition. In order to harness our recent findings, we prepared and evaluated a series of sulfonyl piperazine analogs with modifications in the phenyl and N-acetyl groups of 3. Herein, we describe the synthesis and structure-activity relationship of sulfonyl piperazine LpxH inhibitors. We also report the structural analysis of an extended N-acyl chain analog 27b (JH-LPH-41) in complex with K. pneumoniae LpxH, revealing that 27b reaches an untapped polar pocket near the di-manganese cluster in the active site of K. pneumoniae LpxH. We expect that our findings will provide designing principles for new LpxH inhibitors and establish important frameworks for the future development of antibiotics against multidrug-resistant Gram-negative pathogens.

Structural basis of the UDP-diacylglucosamine pyrophosphohydrolase LpxH inhibition by sulfonyl piperazine antibiotics.

The UDP-2,3-diacylglucosamine pyrophosphate hydrolase LpxH is an essential lipid A biosynthetic enzyme that is conserved in the majority of gram-negative bacteria. It has emerged as an attractive novel antibiotic target due to the recent discovery of an LpxH-targeting sulfonyl piperazine compound (referred to as AZ1) by AstraZeneca. However, the molecular details of AZ1 inhibition have remained unresolved, stymieing further development of this class of antibiotics. Here we report the crystal structure of Klebsiella pneumoniae LpxH in complex with AZ1. We show that AZ1 fits snugly into the L-shaped acyl chain-binding chamber of LpxH with its indoline ring situating adjacent to the active site, its sulfonyl group adopting a sharp kink, and its N-CF3-phenyl substituted piperazine group reaching out to the far side of the LpxH acyl chain-binding chamber. Intriguingly, despite the observation of a single AZ1 conformation in the crystal structure, our solution NMR investigation has revealed the presence of a second ligand conformation invisible in the crystalline state. Together, these distinct ligand conformations delineate a cryptic inhibitor envelope that expands the observed footprint of AZ1 in the LpxH-bound crystal structure and enables the design of AZ1 analogs with enhanced potency in enzymatic assays. These designed compounds display striking improvement in antibiotic activity over AZ1 against wild-type K. pneumoniae, and coadministration with outer membrane permeability enhancers profoundly sensitizes Escherichia coli to designed LpxH inhibitors. Remarkably, none of the sulfonyl piperazine compounds occupies the active site of LpxH, foretelling a straightforward path for rapid optimization of this class of antibiotics.

A Review of Recent Advances in 3D Bioprinting With an Eye on Future Regenerative Therapies in Veterinary Medicine.

3D bioprinting is a rapidly evolving industry that has been utilized for a variety of biomedical applications. It differs from traditional 3D printing in that it utilizes bioinks comprised of cells and other biomaterials to allow for the generation of complex functional tissues. Bioprinting involves computational modeling, bioink preparation, bioink deposition, and subsequent maturation of printed products; it is an intricate process where bioink composition, bioprinting approach, and bioprinter type must be considered during construct development. This technology has already found success in human studies, where a variety of functional tissues have been generated for both in vitro and in vivo applications. Although the main driving force behind innovation in 3D bioprinting has been utility in human medicine, recent efforts investigating its veterinary application have begun to emerge. To date, 3D bioprinting has been utilized to create bone, cardiovascular, cartilage, corneal and neural constructs in animal species. Furthermore, the use of animal-derived cells and various animal models in human research have provided additional information regarding its capacity for veterinary translation. While these studies have produced some promising results, technological limitations as well as ethical and regulatory challenges have impeded clinical acceptance. This article reviews the current understanding of 3D bioprinting technology and its recent advancements with a focus on recent successes and future translation in veterinary medicine.

Evaluation of the geometric accuracy of computed tomography and microcomputed tomography of the articular surface of the distal portion of the radius of cats.

OBJECTIVE: To evaluate accuracy of articular surfaces determined by use of 2 perpendicular CT orientations, micro-CT, and laser scanning. SAMPLE: 23 cat cadavers. PROCEDURES: Images of antebrachia were obtained by use of CT (voxel size, 0.6 mm) in longitudinal orientation (CTLO images) and transverse orientation (CTTO images) and by use of micro-CT (voxel size, 0.024 mm) in a longitudinal orientation. Images were reconstructed. Craniocaudal and mediolateral length, radius of curvature, and deviation of the articular surface of the distal portion of the radius of 3-D renderings for CTLO, CTTO, and micro-CT images were compared with results of 3-D renderings acquired with a laser scanner (resolution, 0.025 mm). RESULTS: Measurement of CTLO and CTTO images overestimated craniocaudal and mediolateral length of the articular surface by 4% to 10%. Measurement of micro-CT images underestimated craniocaudal and mediolateral length by 1%. Measurement of CTLO and CTTO images underestimated mediolateral radius of curvature by 15% and overestimated craniocaudal radius of curvature by > 100%; use of micro-CT images underestimated them by 3% and 5%, respectively. Mean ± SD surface deviation was 0.26 ± 0.09 mm for CTLO images, 0.30 ± 0.28 mm for CTTO images, and 0.04 ± 0.02 mm for micro-CT images. CONCLUSIONS AND CLINICAL RELEVANCE: Articular surface models derived from CT images had dimensional errors that approximately matched the voxel size. Thus, CT cannot be used to plan conforming arthroplasties in small joints and could lack precision when used to plan the correction of a limb deformity or repair of a fracture.

Pre-operative simulation of pediatric mastoid surgery with 3D-printed temporal bone models.

OBJECTIVES: As the process of additive manufacturing, or three-dimensional (3D) printing, has become more practical and affordable, a number of applications for the technology in the field of pediatric otolaryngology have been considered. One area of promise is temporal bone surgical simulation. Having previously developed a model for temporal bone surgical training using 3D printing, we sought to produce a patient-specific model for pre-operative simulation in pediatric otologic surgery. Our hypothesis was that the creation and pre-operative dissection of such a model was possible, and would demonstrate potential benefits in cases of abnormal temporal bone anatomy. METHODS: In the case presented, an 11-year-old boy underwent a planned canal-wall-down (CWD) tympano-mastoidectomy for recurrent cholesteatoma preceded by a pre-operative surgical simulation using 3D-printed models of the temporal bone. The models were based on the child's pre-operative clinical CT scan and printed using multiple materials to simulate both bone and soft tissue structures. To help confirm the models as accurate representations of the child's anatomy, distances between various anatomic landmarks were measured and compared to the temporal bone CT scan and the 3D model. RESULTS: The simulation allowed the surgical team to appreciate the child's unusual temporal bone anatomy as well as any challenges that might arise in the safety of the temporal bone laboratory, prior to actual surgery in the operating room (OR). There was minimal variability, in terms of absolute distance (mm) and relative distance (%), in measurements between anatomic landmarks obtained from the patient intra-operatively, the pre-operative CT scan and the 3D-printed models. CONCLUSIONS: Accurate 3D temporal bone models can be rapidly produced based on clinical CT scans for pre-operative simulation of specific challenging otologic cases in children, potentially reducing medical errors and improving patient safety.

Multi-material 3D Models for Temporal Bone Surgical Simulation.

HYPOTHESIS: A simulated, multicolor, multi-material temporal bone model can be created using 3-dimensional (3D) printing that will prove both safe and beneficial in training for actual temporal bone surgical cases. BACKGROUND: As the process of additive manufacturing, or 3D printing, has become more practical and affordable, a number of applications for the technology in the field of Otolaryngology-Head and Neck Surgery have been considered. One area of promise is temporal bone surgical simulation. METHODS: Three-dimensional representations of human temporal bones were created from temporal bone computed tomography (CT) scans using biomedical image processing software. Multi-material models were then printed and dissected in a temporal bone laboratory by attending and resident otolaryngologists. A 5-point Likert scale was used to grade the models for their anatomical accuracy and suitability as a simulation of cadaveric and operative temporal bone drilling. RESULTS: The models produced for this study demonstrate significant anatomic detail and a likeness to human cadaver specimens for drilling and dissection. CONCLUSION: Simulated temporal bones created by this process have potential benefit in surgical training, preoperative simulation for challenging otologic cases, and the standardized testing of temporal bone surgical skills.