Effects of vancomycin and tobramycin on compressive and tensile strengths of antibiotic bone cement: A biomechanical study.

Purpose: This study aims to compare the compressive and tensile strengths of bone cement mixed with various concentrations of vancomycin, tobramycin, and combinations of the two. Methods: 12 mm × 6 mm antibiotic bone cement samples were created by vacuum mixing 0-4 g of vancomycin, tobramycin, and combinations of the two in 0.5 g increments per one pouch (40 g) of Palacos LV cement. An Instron 3369 Universal Testing System was used to determine the compressive and tensile strengths. Results: Compressive and tensile strengths of the bone cement without antibiotics were 118 ± 4 MPa and 30.3 ± 12 MPa, respectively. 4 g of vancomycin alone decreased the compressive strength to 108 ± 4 MPa (p-value 0.001) and decreased the tensile strength beginning at 2 g which yielded a strength of 28.1 ± 12 MPa (p-value 0.016). Tobramycin alone decreased the tensile strength beginning at 1.5 g yielding a strength of 27.7 ± 7 MPa (p-value 0.003). Although it decreased compressive strength at 1 g to 117 ± 7 MPa (p-value 0.002), it demonstrated variable effects with increasing concentrations. A combination of vancomycin and tobramycin decreased both the compressive (111 ± 5 MPa, p-value 0.014) and tensile (27.9 ± 8 MPa, p-value 0.007) strengths beginning at 1 g each. Conclusions: Various combinations of vancomycin and tobramycin affect the compressive and tensile strengths of bone cement. Clinicians should be diligent when mixing these antibiotics in bone cement to prevent possible failure of the constructs.

Time-resolved cryo-EM (TR-EM) analysis of substrate polyubiquitination by the RING E3 anaphase-promoting complex/cyclosome (APC/C).

Substrate polyubiquitination drives a myriad of cellular processes, including the cell cycle, apoptosis and immune responses. Polyubiquitination is highly dynamic, and obtaining mechanistic insight has thus far required artificially trapped structures to stabilize specific steps along the enzymatic process. So far, how any ubiquitin ligase builds a proteasomal degradation signal, which is canonically regarded as four or more ubiquitins, remains unclear. Here we present time-resolved cryogenic electron microscopy studies of the 1.2 MDa E3 ubiquitin ligase, known as the anaphase-promoting complex/cyclosome (APC/C), and its E2 co-enzymes (UBE2C/UBCH10 and UBE2S) during substrate polyubiquitination. Using cryoDRGN (Deep Reconstructing Generative Networks), a neural network-based approach, we reconstruct the conformational changes undergone by the human APC/C during polyubiquitination, directly visualize an active E3-E2 pair modifying its substrate, and identify unexpected interactions between multiple ubiquitins with parts of the APC/C machinery, including its coactivator CDH1. Together, we demonstrate how modification of substrates with nascent ubiquitin chains helps to potentiate processive substrate polyubiquitination, allowing us to model how a ubiquitin ligase builds a proteasomal degradation signal.

Functional conservation and divergence of the helix-turn-helix motif of E2 ubiquitin-conjugating enzymes.

Polyubiquitination by E2 and E3 enzymes is crucial to cell cycle control, epigenetic regulation, and development. The hallmark of the E2 family is the ubiquitin (Ub)-conjugating (UBC) domain that forms a dynamic thioester conjugate with ubiquitin (E2~Ub). Numerous studies have focused on E2 surfaces, such as the N-terminal and crossover helices, that directly interact with an E3 or the conjugated ubiquitin to stabilize the active, "closed" state of the E2~Ub. However, it remains unclear how other E2 surfaces regulate ubiquitin transfer. Here, we demonstrate the helix-turn-helix (HTH) motif of the UBC tunes the intrinsic polyubiquitination activity through distinct functions in different E2s. Interestingly, the E2HTH motif is repurposed in UBE2S and UBE2R2 to interact with the conjugated or acceptor ubiquitin, respectively, modulating ubiquitin transfer. Furthermore, we propose that Anaphase-Promoting Complex/Cyclosome binding to the UBE2SHTH reduces the conformational space of the flexible E2~Ub, demonstrating an atypical E3-dependent activation mechanism. Altogether, we postulate the E2HTH motif evolved to provide new functionalities that can be harnessed by E3s and permits additional regulation to facilitate specific E2-E3-mediated polyubiquitination.

Identification of disease-linked hyperactivating mutations in UBE3A through large-scale functional variant analysis.

The mechanisms that underlie the extensive phenotypic diversity in genetic disorders are poorly understood. Here, we develop a large-scale assay to characterize the functional valence (gain or loss-of-function) of missense variants identified in UBE3A, the gene whose loss-of-function causes the neurodevelopmental disorder Angelman syndrome. We identify numerous gain-of-function variants including a hyperactivating Q588E mutation that strikingly increases UBE3A activity above wild-type UBE3A levels. Mice carrying the Q588E mutation exhibit aberrant early-life motor and communication deficits, and individuals possessing hyperactivating UBE3A variants exhibit affected phenotypes that are distinguishable from Angelman syndrome. Additional structure-function analysis reveals that Q588 forms a regulatory site in UBE3A that is conserved among HECT domain ubiquitin ligases and perturbed in various neurodevelopmental disorders. Together, our study indicates that excessive UBE3A activity increases the risk for neurodevelopmental pathology and suggests that functional variant analysis can help delineate mechanistic subtypes in monogenic disorders.

Mechanically transduced immunosorbent assay to measure protein-protein interactions.

Measuring protein-protein interaction (PPI) affinities is fundamental to biochemistry. Yet, conventional methods rely upon the law of mass action and cannot measure many PPIs due to a scarcity of reagents and limitations in the measurable affinity ranges. Here, we present a novel technique that leverages the fundamental concept of friction to produce a mechanical signal that correlates to binding potential. The mechanically transduced immunosorbent (METRIS) assay utilizes rolling magnetic probes to measure PPI interaction affinities. METRIS measures the translational displacement of protein-coated particles on a protein-functionalized substrate. The translational displacement scales with the effective friction induced by a PPI, thus producing a mechanical signal when a binding event occurs. The METRIS assay uses as little as 20 pmols of reagents to measure a wide range of affinities while exhibiting a high resolution and sensitivity. We use METRIS to measure several PPIs that were previously inaccessible using traditional methods, providing new insights into epigenetic recognition.

Aggregation dynamics of active rotating particles in dense passive media.

Active matter systems are able to exhibit emergent non-equilibrium behavior due to activity-induced effective interactions between the active particles. Here we study the aggregation and dynamical behavior of active rotating particles, spinners, embedded in 2D passive colloidal monolayers. Using both experiments and simulations we observe aggregation of active particles or spinners whose behavior resembles classical 2D Cahn-Hilliard coarsening. The aggregation behavior and spinner attraction depend on the mechanical properties of the passive monolayer and the activity of spinners. Spinner aggregation only occurs when the passive monolayer behaves elastically and when the spinner activity exceeds a minimum activity threshold. Interestingly, for the spinner concentrations investigated here, the spinner concentration does not seem to change the dynamics of the aggregation behavior. There is a characteristic cluster size which maximizes spinner aggregation by minimizing the drag through the passive monolayer and maximizing the stress applied on the passive medium. We also show a ternary mixture of passive particles and co-rotating and counter-rotating spinners that aggregate into clusters of co and counter-rotating spinners respectively.

Emergent ultra-long-range interactions between active particles in hybrid active-inactive systems.

Particle-particle interactions determine the state of a system. Control over the range of such interactions as well as their magnitude has been an active area of research for decades due to the fundamental challenges it poses in science and technology. Very recently, effective interactions between active particles have gathered much attention as they can lead to out-of-equilibrium cooperative states such as flocking. Inspired by nature, where active living cells coexist with lifeless objects and structures, here we study the effective interactions that appear in systems composed of active and passive mixtures of colloids. Our systems are 2D colloidal monolayers composed primarily of passive (inactive) colloids, and a very small fraction of active (spinning) ferromagnetic colloids. We find an emergent ultra-long-range attractive interaction induced by the activity of the spinning particles and mediated by the elasticity of the passive medium. Interestingly, the appearance of such interaction depends on the spinning protocol and has a minimum actuation timescale below which no attraction is observed. Overall, these results clearly show that, in the presence of elastic components, active particles can interact across very long distances without any chemical modification of the environment. Such a mechanism might potentially be important for some biological systems and can be harnessed for newer developments in synthetic active soft materials.

Artificial tribotactic microscopic walkers: walking based on friction gradients.

Friction, the resistive force between two surfaces sliding past each other, is at the core of a wide diversity of locomotion schemes. While such schemes are well described for homogeneous environments, locomotion based on friction in inhomogeneous environments has not received much attention. Here we introduce and demonstrate the concept of tribotaxis, a motion that is guided by gradients in the friction coefficient. Our system is composed of microwalkers that undergo an effective frictional interaction with biological receptors on the substrate, which is regulated by the density of such receptors. When actuated stochastically, microwalkers migrate to regions of higher friction, much like a chemotactic cell migrates to regions of higher chemoattractant concentration. Simulations and theory based on biased random walks are in excellent agreement with experiments. We foresee important implications for tribotaxis in artificial and natural locomotion in biological environments.