A Genome-Wide CRISPR Screen Identifies Sortilin as the Receptor Responsible for Galectin-1 Lysosomal Trafficking.

Galectins are a family of mammalian glycan-binding proteins that have been implicated as regulators of myriad cellular processes including cell migration, apoptosis, and immune modulation. Several members of this family, such as galectin-1, exhibit both cell-surface and intracellular functions. Interestingly, galectin-1 can be found in the endomembrane system, nucleus, or cytosol, as well as on the cell surface. The mechanisms by which galectin-1 traffics between cellular compartments, including its unconventional secretion and internalization processes, are poorly understood. Here, we determined the pathways by which exogenous galectin-1 enters cells and explored its capacity as a delivery vehicle for protein and siRNA therapeutics. We used a galectin-1-toxin conjugate, modelled on antibody-drug conjugates, as a selection tool in a genome-wide CRISPR screen. We discovered that galectin-1 interacts with the endosome-lysosome trafficking receptor sortilin in a glycan-dependent manner, which regulates galectin-1 trafficking to the lysosome. Further, we show that this pathway can be exploited for delivery of a functional siRNA. This study sheds light on the mechanisms by which galectin-1 is internalized by cells and suggests a new strategy for intracellular drug delivery via galectin-1 conjugation.

Promoting appropriate utilisation of laboratory tests for inflammation at an academic medical centre.

Erythrocyte sedimentation rate (ESR) and C reactive protein (CRP) are commonly ordered in clinical practice to evaluate for inflammation. CRP is a more sensitive and specific test for detecting acute phase inflammation, and the American Society for Clinical Pathology recommends ordering CRP rather than ESR to detect acute phase inflammation in patients with undiagnosed conditions. We sought to understand CRP and ESR ordering practices and reduce unnecessary use of ESR testing at our academic medical centre. We surveyed physician leaders in clinical areas with high utilisation of ESR testing to understand the drivers of potential overutilisation of these tests. Based on survey responses, we designed an intervention focused on education, clinical decision support within the electronic medical record and quarterly audit and feedback. We evaluated appropriateness of ESR ordering before and after the intervention via structured chart audit. Comparison of monthly rates of ESR tests during the preintervention and postintervention periods was conducted using interrupted time series analysis. Clinical habit and ease of test ordering were identified as key drivers of ESR overuse. Compared with the preintervention period, we observed a 33% reduction in the number of ESR tests per month and a 25% reduction in combined CRP and ESR tests per month during the postintervention period. This reduction corresponded to an annual avoidance of 2633 ESR tests with a corresponding estimated direct cost avoidance of $23 701 annually. Although the rate of ESR testing decreased, there was no significant improvement in the clinical appropriateness of residual ESR test ordering following the intervention. A multifaceted intervention was associated with significant decreases in unnecessary ESR tests and concurrent ESR and CRP tests at our academic medical centre. Despite these reductions, there are continued opportunities to reduce inappropriate ESR testing.

Versatile Peptide Macrocyclization with Diels-Alder Cycloadditions.

Macrocyclization can improve bioactive peptide ligands through preorganization of molecular topology, leading to improvement of pharmacologic properties like binding affinity, cell permeability, and metabolic stability. Here we demonstrate that Diels-Alder [4 + 2] cycloadditions can be harnessed for peptide macrocyclization and stabilization within a range of peptide scaffolds and chemical environments. Diels-Alder cyclization of diverse diene-dienophile reactive pairs proceeds rapidly, in high yield and with tunable stereochemical preferences on solid-phase or in aqueous solution. This reaction can be applied alone or in concert with other stabilization chemistries, such as ring-closing olefin metathesis, to stabilize loop, turn, and α-helical secondary structural motifs. NMR and molecular dynamics studies of model loop peptides confirmed preferential formation of endo cycloadduct stereochemistry, imparting significant structural rigidity to the peptide backbone that resulted in augmented protease resistance and increased biological activity of a Diels-Alder cyclized (DAC) RGD peptide. Separately, we demonstrated the stabilization of DAC α-helical peptides derived from the ERα-binding protein SRC2. We solved a 2.25 Å cocrystal structure of one DAC helical peptide bound to ERα, which unequivocally corroborated endo stereochemistry of the resulting Diels-Alder adduct, and confirmed that the unique architecture of stabilizing motifs formed with this chemistry can directly contribute to target binding. These data establish Diels-Alder cyclization as a versatile approach to stabilize diverse protein structural motifs under a range of chemical environments.

Adding value to daily chest X-rays in the ICU through education, restricted daily orders and indication-based prompting.

BACKGROUND: Chest X-rays (CXRs) are traditionally obtained daily in all patients on invasive mechanical ventilation (IMV) in the intensive care unit (ICU). We sought to reduce overutilisation of CXRs obtained in the ICU, using a multifaceted intervention to eliminate automated daily studies. METHODS: We first educated ICU staff about the low diagnostic yield of automated daily CXRs, then removed the 'daily' option from the electronic health records-based ordering system, and added a query (CXR indicated or not indicated) to the ICU daily rounding checklist to prompt a CXR order when clinically warranted. We built a report from billing codes, focusing on all CXRs obtained on IMV census days in the medical (MICU) and surgical (SICU) ICUs, excluding the day of admission and days that a procedure warranting CXR was performed. This generated the number of CXRs obtained every 1000 'included' ventilator days (IVDs), the latter defined as not having an 'absolute' clinical indication for CXR. RESULTS: The average monthly number of CXRs on an IVD decreased from 919±90 (95% CI 877 to 963) to 330±87 (95% CI 295 to 354) per 1000 IVDs in the MICU, and from 995±69 (95% CI 947 to 1055) to 649±133 (95% CI 593 to 697) in the SICU. This yielded an estimated 1830 to 2066 CXRs avoided over 2 years and an estimated annual savings of $191 600 to $224 200. There was no increase in reported adverse events. CONCLUSION: ICUs can safely transition to a higher value strategy of indication-based chest imaging by educating staff, eliminating the 'daily' order option and adding a simplified prompt to avoid missing clinically indicated CXRs.

Mode-based analysis of silicon nanohole arrays for photovoltaic applications.

We investigate the optical properties of silicon nanohole arrays for application in photovoltaic cells in terms of the modes within the structure. We highlight three types of modes: fundamental modes, important at long wavelengths; guided resonance modes, which enhance absorption for wavelengths where the intrinsic absorption of silicon is low; and channeling modes, which suppress front-surface reflection. We use this understanding to explain why the parameters of optimized nanohole arrays occur in specific ranges even as the thickness is varied.

Improved spatial resolution and electrogram wave direction independence with the use of an orthogonal electrode configuration.

To improve spatial resolution in recordings of intra-cardiac electrograms we characterized the utility of a novel configuration of two recording electrodes arranged perpendicularly to the endocardial surface. We hypothesized that this configuration denoted as orthogonal close unipolar (OCU) would combine advantages of conventional unipolar and contact bipolar (CBP) configurations. Electrical excitation was simulated in a computational model as arising from dipole current or from multi-wavelet reentry sources. Recordings were calculated for electrode tips 1 mm above the plane of the heart. Analogous recordings were obtained from swine hearts. Electrograms recorded with CBP showed strong dependence on orientation of the electrode pair with respect to the direction of spread of tissue excitation. By contrast, OCU recordings exhibited no directional dependence. OCU was significantly superior to CBP with respect to avoidance of far-field confounding of local tissue activity; the average far-field/near-field ratios for CBP and OCU were 0.09 and 0.05, respectively, for the simulated dipole current sources. In the swine hearts the ratios of ventricular to atrial signals for CBP and OCU were 0.15 ± 0.07 and 0.08 ± 0.09, respectively (p < 0.001). The difference between the actual dominant frequency in the tissue and that recorded by the electrodes was 0.44 ± 0.33 Hz for OCU, 0.58 ± 0.40 Hz for unipolar, and 0.62 ± 0.46 Hz for CBP. OCU confers improved spatial resolution compared with both unipolar and CBP electrode configurations. Unlike the case with CBP, OCU recordings are independent of excitation wave-front direction.

Ablation of multi-wavelet re-entry: general principles and in silico analyses.

AIMS: Catheter ablation strategies for treatment of cardiac arrhythmias are quite successful when targeting spatially constrained substrates. Complex, dynamic, and spatially varying substrates, however, pose a significant challenge for ablation, which delivers spatially fixed lesions. We describe tissue excitation using concepts of surface topology which provides a framework for addressing this challenge. The aim of this study was to test the efficacy of mechanism-based ablation strategies in the setting of complex dynamic substrates. METHODS AND RESULTS: We used a computational model of propagation through electrically excitable tissue to test the effects of ablation on excitation patterns of progressively greater complexity, from fixed rotors to multi-wavelet re-entry. Our results indicate that (i) focal ablation at a spiral-wave core does not result in termination; (ii) termination requires linear lesions from the tissue edge to the spiral-wave core; (iii) meandering spiral-waves terminate upon collision with a boundary (linear lesion or tissue edge); (iv) the probability of terminating multi-wavelet re-entry is proportional to the ratio of total boundary length to tissue area; (v) the efficacy of linear lesions varies directly with the regional density of spiral-waves. CONCLUSION: We establish a theoretical framework for re-entrant arrhythmias that explains the requirements for their successful treatment. We demonstrate the inadequacy of focal ablation for spatially fixed spiral-waves. Mechanistically guided principles for ablating multi-wavelet re-entry are provided. The potential to capitalize upon regional heterogeneity of spiral-wave density for improved ablation efficacy is described.

Effects of electrode size and spacing on the resolution of intracardiac electrograms.

BACKGROUND: Electrogram fractionation can result when multiple groups of cardiac cells are excited asynchronously within the recording region of a mapping electrode. The spatial resolution of an electrode thus plays an important role in mapping complex rhythms. METHODS: We used a computational model, validated against experimental measurements in vitro, to determine how spatial resolution is affected by electrode diameter, electrode length, interelectrode distance (in the case of bipolar recordings), and height of the electrode above a dipole current source. RESULTS: We found that increases in all these quantities caused progressive degradation in two independent measures of spatial resolution, with the strongest effect being due to changes in height above the tissue. CONCLUSION: Our calculations suggest that if electrodes could be constructed to have negligible dimensions compared with those in use today, we would increase resolution by about one order of magnitude at most.

Electrogram fractionation: the relationship between spatiotemporal variation of tissue excitation and electrode spatial resolution.

BACKGROUND: Fractionated electrograms are used by some as targets for ablation in atrial and ventricular arrhythmias. Fractionation has been demonstrated to result when there is repetitive or asynchronous activation of separate groups of cells within the recording region of a mapping electrode(s). METHODS AND RESULTS: Using a computer model, we generated tissue activation patterns with increasing spatiotemporal variation and calculated virtual electrograms from electrodes with decreasing resolution. We then quantified electrogram fractionation. In addition, we recorded unipolar electrograms during atrial fibrillation in 20 patients undergoing atrial fibrillation ablation. From these we constructed bipolar electrograms with increasing interelectrode spacing and quantified fractionation. During modeling of spatiotemporal variation, fractionation varied directly with electrode length, diameter, height, and interelectrode spacing. When resolution was held constant, fractionation increased with increasing spatiotemporal variation. In the absence of spatial variation, fractionation was independent of resolution and proportional to excitation frequency. In patients with atrial fibrillation, fractionation increased as interelectrode spacing increased. CONCLUSIONS: We created a model for distinguishing the roles of spatial and temporal electric variation and electrode resolution in producing electrogram fractionation. Spatial resolution affects fractionation attributable to spatiotemporal variation but not temporal variation alone. Electrogram fractionation was directly proportional to spatiotemporal variation and inversely proportional to spatial resolution. Spatial resolution limits the ability to distinguish high-frequency excitation from overcounting. In patients with atrial fibrillation, complex fractionated atrial electrogram detection varies with spatial resolution. Electrode resolution must therefore be considered when interpreting and comparing studies of fractionation.

In vivo remodeling of intervertebral discs in response to short- and long-term dynamic compression.

This study evaluated how dynamic compression induced changes in gene expression, tissue composition, and structural properties of the intervertebral disc using a rat tail model. We hypothesized that daily exposure to dynamic compression for short durations would result in anabolic remodeling with increased matrix protein expression and proteoglycan content, and that increased daily load exposure time and experiment duration would retain these changes but also accumulate changes representative of mild degeneration. Sprague-Dawley rats (n = 100) were instrumented with an Ilizarov-type device and divided into three dynamic compression (2 week-1.5 h/day, 2 week-8 h/day, 8 week-8 h/day at 1 MPa and 1 Hz) and two sham (2 week, 8 week) groups. Dynamic compression resulted in anabolic remodeling with increased matrix mRNA expression, minimal changes in catabolic genes or disc structure and stiffness, and increased glysosaminoglycans (GAG) content in the nucleus pulposus. Some accumulation of mild degeneration with 8 week-8 h included loss of annulus fibrosus GAG and disc height although 8-week shams also had loss of disc height, water content, and minor structural alterations. We conclude that dynamic compression is consistent with a notion of "healthy" loading that is able to maintain or promote matrix biosynthesis without substantially disrupting disc structural integrity. A slow accumulation of changes similar to human disc degeneration occurred when dynamic compression was applied for excessive durations, but this degenerative shift was mild when compared to static compression, bending, or other interventions that create greater structural disruption.

A Removable Precision Device for In-Vivo Mechanical Compression of Rat Tail Intervertebral Discs.

The rat tail intervertebral disc has emerged as an important model to examine the mechanisms for mechanically induced degeneration and remodeling. Previous methods used to apply high precision axial compressive loading to a rat tail intervertebral disc in vivo either required anesthesia, or the permanent mounting of a loading device to the animal, and were not well described in the literature. Therefore, a new device to apply compressive loading to the rat tail intervertebral disc was developed and validated. The rat tail compressive loading system utilized a pneumatically driven device weighing 18 g, and was capable of delivering a 12.6 N sinusoidal or square waveform at frequencies up to 1.0 Hz. The system improved on previous methods in its modular construction, relative ease of fabrication, compatibility with existing tail model technology and overall cost effectiveness. The removable system eliminated the need for anesthesia and through a modular, cost effective, design allowed for the simultaneous loading of multiple animals. This system expanded the ability to accurately, ethically, and efficiently apply dynamic compressive loads to the rat tail intervertebral disc for extended periods of time in order to address questions related to disc mechanobiology.