Proteomic discovery of chemical probes that perturb protein complexes in human cells.

Most human proteins lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such "binding-first" assays affect protein function, nonetheless, often remains unclear. Here, we describe a "function-first" proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based protein profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disrupt the PA28 proteasome regulatory complex and stabilize a dynamic state of the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells.

Targeted Protein Degradation by Electrophilic PROTACs that Stereoselectively and Site-Specifically Engage DCAF1.

Targeted protein degradation induced by heterobifunctional compounds and molecular glues presents an exciting avenue for chemical probe and drug discovery. To date, small-molecule ligands have been discovered for only a limited number of E3 ligases, which is an important limiting factor for realizing the full potential of targeted protein degradation. We report herein the discovery by chemical proteomics of azetidine acrylamides that stereoselectively and site-specifically react with a cysteine (C1113) in the E3 ligase substrate receptor DCAF1. We demonstrate that the azetidine acrylamide ligands for DCAF1 can be developed into electrophilic proteolysis-targeting chimeras (PROTACs) that mediated targeted protein degradation in human cells. We show that this process is stereoselective and does not occur in cells expressing a C1113A mutant of DCAF1. Mechanistic studies indicate that only low fractional engagement of DCAF1 is required to support protein degradation by electrophilic PROTACs. These findings, taken together, demonstrate how the chemical proteomic analysis of stereochemically defined electrophilic compound sets can uncover ligandable sites on E3 ligases that support targeted protein degradation.

Selective inhibitors of SARM1 targeting an allosteric cysteine in the autoregulatory ARM domain.

The nicotinamide adenine dinucleotide hydrolase (NADase) sterile alpha toll/interleukin receptor motif containing-1 (SARM1) acts as a central executioner of programmed axon death and is a possible therapeutic target for neurodegenerative disorders. While orthosteric inhibitors of SARM1 have been described, this multidomain enzyme is also subject to intricate forms of autoregulation, suggesting the potential for allosteric modes of inhibition. Previous studies have identified multiple cysteine residues that support SARM1 activation and catalysis, but which of these cysteines, if any, might be selectively targetable by electrophilic small molecules remains unknown. Here, we describe the chemical proteomic discovery of a series of tryptoline acrylamides that site-specifically and stereoselectively modify cysteine-311 (C311) in the noncatalytic, autoregulatory armadillo repeat (ARM) domain of SARM1. These covalent compounds inhibit the NADase activity of WT-SARM1, but not C311A or C311S SARM1 mutants, show a high degree of proteome-wide selectivity for SARM1_C311 and stereoselectively block vincristine- and vacor-induced neurite degeneration in primary rodent dorsal root ganglion neurons. Our findings describe selective, covalent inhibitors of SARM1 targeting an allosteric cysteine, pointing to a potentially attractive therapeutic strategy for axon degeneration-dependent forms of neurological disease.

A study on safe forceps grip force for the intestinal tract using haptic technology.

PURPOSE: The present study used haptic technology to determine the safe forceps grip force for preventing organ damage when handling the intestinal tract. MATERIAL AND METHODS: The small intestines of ten male beagle dogs (weighing 9.5-10 kg) were grasped with the entire forceps for one minute; the small intestines were then pulled out of the forceps and evaluated for damage. The force at which the shaft inside the forceps was pulled to close the tip of the forceps was defined as the grip force. Small intestine damage was classified into macroscopic (serosal defects, hemorrhage, hematomas, grip marks) and microscopic (damage layer to the mucosa, submucosa/muscularis mucosa, inner orbicularis muscle, external longitudinal muscle, serosa/subserosa). Grip marks and damage layer to the serosa/subserosa have been considered as acceptable safety margins when grasping the small intestines of beagle dogs. RESULTS: The macroscopic findings showed that the maximum grip force that produced a 0% incidence of hemorrhage and hematoma was 15 N. At the microscopic level, the maximum grip force that produced a 0% incidence of external longitudinal muscle injury was 15 N, respectively. CONCLUSIONS: A grip force of 15 N does not damage the small intestines of beagle dogs.

3D-Printed Micro-Tweezers with a Compliant Mechanism Designed Using Topology Optimization.

The development of handling technology for microscopic biological samples such as cells and spheroids has been required for the advancement of regenerative medicine and tissue engineering. In this study, we developed micro-tweezers with a compliant mechanism to manipulate organoids. The proposed method combines high-resolution microstereolithography that uses a blue laser and topology optimization for shape optimization of micro-tweezers. An actuation system was constructed using a linear motor stage with a force control system to operate the micro-tweezers. The deformation of the topology-optimized micro-tweezers was examined analytically and experimentally. The results verified that the displacement of the tweezer tip was proportional to the applied load; furthermore, the displacement was sufficient to grasp biological samples with an approximate diameter of several hundred micrometers. We experimentally demonstrated the manipulation of an organoid with a diameter of approximately 360 µm using the proposed micro-tweezers. Thus, combining microstereolithography and topology optimization to fabricate micro-tweezers can be potentially used in modifying tools capable of handling various biological samples.

An Activity-Guided Map of Electrophile-Cysteine Interactions in Primary Human T Cells.

Electrophilic compounds originating from nature or chemical synthesis have profound effects on immune cells. These compounds are thought to act by cysteine modification to alter the functions of immune-relevant proteins; however, our understanding of electrophile-sensitive cysteines in the human immune proteome remains limited. Here, we present a global map of cysteines in primary human T cells that are susceptible to covalent modification by electrophilic small molecules. More than 3,000 covalently liganded cysteines were found on functionally and structurally diverse proteins, including many that play fundamental roles in immunology. We further show that electrophilic compounds can impair T cell activation by distinct mechanisms involving the direct functional perturbation and/or degradation of proteins. Our findings reveal a rich content of ligandable cysteines in human T cells and point to electrophilic small molecules as a fertile source for chemical probes and ultimately therapeutics that modulate immunological processes and their associated disorders.

Treatment of an intraarticular comminuted fracture of the base of the proximal phalanx in a ring finger using the Ichi-Fixator external fixator system: A case report.

INTRODUCTION: Comminuted fractures involving the articular surface of the base of the proximal phalanx are relatively rare. We treated a patient with this type of fracture by open reduction and internal fixation with a locked-wire-type external fixator (Ichi-Fixator System). PRESENTATION OF CASE: A 45-year-old man was injured because his ring finger was kicked during a Futsal game. Radiographs and computed tomography revealed a comminuted intraarticular fracture of the proximal phalanx of this ring finger. We treated the fracture with open reduction and K-wires and external fixation. We removed the K-wire and external fixator 5 weeks postoperatively and initiated range of motion exercises. Five months postoperatively, his finger motion was fully recovered without restriction. DISCUSSION: Comminuted intraarticular fractures of the base of the proximal phalanx are usually treated with plating. Complications such as interference with excursion of the central slip and lateral bands, extensor tendon rupture, and plate prominence have been reported in these fractures. In our patient, the Ichi-Fixator System was useful as a distraction apparatus for metacarpophalangeal joint fixation. CONCLUSION: A comminuted intra-articular fracture of the base of the proximal phalanx was treated successfully using the Ichi-Fixator system.

Masquelet technique for the treatment of acute osteomyelitis of the PIP joint caused by clenched-fist human bite injury: A case report.

INTRODUCTION: The Masquelet technique is a well-known and efficient procedure for lower limb soft tissue reconstruction after severe osteomyelitis requiring bone excision. However, this technique is rarely used in the hand. PRESENTATION OF CASE: The patient was 38-year-old man. We used this technique to reconstruct a proximal interphalangeal (PIP) joint osteochondral defect after osteomyelitis caused by clenched-fist human bite injury. The pathogen was Prevotella intermedia, which is an anaerobic pathogenic bacterium involved in periodontal infections and is a black-pigmented periodontal pathogen. Following completion of the Masquelet method, the bone remodeled at an angle at the PIP joint. DISCUSSION: Prevotella intermedia is known as Bacteroides melaninogenicus subsp. intermedius. When the infection site is black-pigmented, this pathogen is highly suspected. The Masquelet technique is rarely used in the hand, and when used, it has been in a straight fashion in the hand. We were able to reconstruct a more anatomical, bent PIP joint, and the fixed angle of the PIP joint at 40° of flexion using Masquelet technique. CONCLUSION: The angled joint resulting from this technique created a relatively normal permanently bent PIP joint.

Survival and clinical results of a modified "crosse de hockey" procedure for chronic isolated patellofemoral joint osteoarthritis: mid-term follow-up.

BACKGROUND: The optimum treatment for isolated patellofemoral joint osteoarthritis (PFJ-OA) remains controversial. The aim of this study was to assess the mid-term clinical results of a modified crosse de hockey procedure for the treatment of isolated PFJ-OA. MATERIALS AND METHODS: We assessed 37 knees in 31 patients treated by a modified crosse de hockey procedure. The mean age was 57.6 years (range, 46-75 years) and mean follow-up was 90.1 months (range, 24-216 months). We evaluated clinical and radiographic outcomes, as well as complication rates at the mid-term follow-up. RESULTS: The Kujala score (mean improvement of 46.7, P < 0.001) and the Fulkerson score (mean improvement of 19, P = 0.001) were significantly higher compared to preoperative values. Overall clinical results rated excellent in 24.3 %, very good in 21.6 %, good in 35.1 %, fair in 13.5 %, and poor in 5.4 % of knees. Patellar tilting (P = 0.015) and congruence angle (P = 0.018) significantly improved postoperatively. On the other hand, the Insall-Salvati index decreased at the time of follow-up, although it remained in the physiologic range. Postoperatively, consecutive disease progression in the tibiofemoral joint and patellofemoral joint osteoarthritis were 18.9 and 5.4 %, respectively. The operative complication rate was 5.4 % in this case series. These percentages were lower than those of alternative tibial tuberosity osteotomy techniques. CONCLUSION: In most patients with chronic isolated PFJ-OA, tibial tuberosity osteotomy by modified crosse de hockey is a reliable procedure that provides good/excellent mid-term clinical results. LEVEL OF EVIDENCE: Level IV.