bioPROTACs as versatile modulators of intracellular therapeutic targets including proliferating cell nuclear antigen (PCNA).

Targeted degradation approaches such as proteolysis targeting chimeras (PROTACs) offer new ways to address disease through tackling challenging targets and with greater potency, efficacy, and specificity over traditional approaches. However, identification of high-affinity ligands to serve as PROTAC starting points remains challenging. As a complementary approach, we describe a class of molecules termed biological PROTACs (bioPROTACs)-engineered intracellular proteins consisting of a target-binding domain directly fused to an E3 ubiquitin ligase. Using GFP-tagged proteins as model substrates, we show that there is considerable flexibility in both the choice of substrate binders (binding positions, scaffold-class) and the E3 ligases. We then identified a highly effective bioPROTAC against an oncology target, proliferating cell nuclear antigen (PCNA) to elicit rapid and robust PCNA degradation and associated effects on DNA synthesis and cell cycle progression. Overall, bioPROTACs are powerful tools for interrogating degradation approaches, target biology, and potentially for making therapeutic impacts.

Localization of adventitious respiratory sounds.

In a recent publication by Henry and Royston [J. Acoust. Soc. Am. 142, 1774-1783 (2017)], an algorithm was introduced to calculate the acoustic response to externally introduced and endogenous respiratory sounds within a realistic, patient-specific subglottal airway tree. This work is extended using an efficient numerical boundary element (BE) approach to calculate the resulting radiated sound field from the airway tree into the lung parenchyma taking into account the surrounding chest wall. Within the BE model of the left lung parenchyma, comprised of more than 6000 triangular surface elements, more than 30 000 monopoles are used to approximate complex airway-originated acoustic sources. The chest wall is modeled as a boundary condition on the parenchymal surface. Several cases were simulated, including a bronchoconstricted lung that had an internal acoustic source introduced in a bronchiole, approximating a wheeze. An acoustic source localization algorithm coupled to the BE model estimated the wheeze source location to within a few millimeters based solely on the acoustic field at the surface. Improved noninvasive means of locating adventitious respiratory sounds may enhance an understanding of acoustic changes correlated to pathology, and potentially provide improved noninvasive tools for the diagnosis of pulmonary diseases that uniquely alter acoustics.

Blockade of Experimental Multiple Sclerosis by Inhibition of the Acid Sphingomyelinase/Ceramide System.

BACKGROUND: Multiple sclerosis (MS) is a severe and common autoimmune disorder of the central nervous system. Despite the availability of several novel treatment options, the disease is still poorly controlled, since the pathophysiological mechanisms are not fully understood. METHODS: We tested the role of the acid sphingomyelinase/ceramide system in a model of MS, i.e. experimental autoimmune encephalomyelitis (EAE). Mice were immunized with myelin-oligodendrocyte glycoprotein and the development of the disease was analyzed by histology, immunological tests and clinical assessment in wildtype and acid sphingomyelinase (Asm)-deficient mice. RESULTS: Genetic deficiency of acid sphingomyelinase (Asm) protected against clinical symptoms in EAE and markedly attenuated the characteristic detrimental neuroinflammatory response. T lymphocyte adhesion, integrity of tight junctions, blood-brain barrier disruption and subsequent intracerebral infiltration of inflammatory cells were blocked in Asm-deficient mice after immunization. This resulted in an almost complete block of the development of disease symptoms in these mice, while wildtype mice showed severe neurological symptoms typical for EAE. CONCLUSION: Activation of the Asm/ceramide system is a central step for the development of EAE. Our findings may serve to identify novel therapeutic strategies for MS patients.

A multiscale analytical model of bronchial airway acoustics.

Sound transmission and resulting airway wall vibration in a complex multiscale viscoelastic model of the subglottal bronchial tree was calculated using a modified one-dimensional (1D) branching acoustic waveguide approach. This is an extension of previous work to enable use of complex airway trees that are partially derived from subject-specific medical images, without the need for self-similarity in the geometric structure. The approach was validated numerically for simplified airway geometries, as well as experimentally by comparison to previous studies. A comprehensive conducting airway tree with about 60 000 branches was then modified to create fibrotic, bronchoconstrictive, and pulmonary infiltrate conditions. The fibrotic case-systemic increase in soft tissue stiffness-increased the Helmholtz resonance frequency due to the increased acoustic impedance. Bronchoconstriction, with geometric changes in small conducting airways, decreased acoustic energy transmission to the peripheral airways due in part to the increased impedance mismatch between airway orders. Pulmonary infiltrate significantly altered the local acoustic field in the affected lobe. Calculation of acoustic differences between healthy versus pathologic cases can be used to enhance the understanding of vibro-acoustic changes correlated to pathology, and potentially provide improved tools for the diagnosis of pulmonary diseases that uniquely alter the acoustics of the airways.

A Review of the Clinical Subgroup Analyses From the RE-LY Trial.

Dabigatran was the first direct-acting oral anticoagulant approved by the US Food and Drug Administration for prevention of stroke and systemic embolism in people with atrial fibrillation, based on data from the Randomized Evaluation of Long-Term Anticoagulation Therapy (RE-LY) trial. Over 18,000 patients with nonvalvular atrial fibrillation and a moderate-to-high risk of thromboembolic stroke were randomized to warfarin or dabigatran. With respect to the primary endpoints for efficacy and safety, dabigatran was superior to warfarin in the prevention of stroke and thromboembolism and noninferior with respect to major bleeding. Although unified by a common arrhythmia and a similar thromboembolic stroke risk, this large patient population is also significantly heterogeneous with respect to other demographics and comorbidities that raise important questions about the efficacy and safety of dabigatran in specific patient populations. Furthermore, there were significant differences between the warfarin and dabigatran groups with respect to several important secondary endpoints. Understanding the differences in outcomes between specific patient subgroups from the RE-LY trial can better inform the practicing clinician's ability to offer the best anticoagulation options to individual patients.

Optimization of arterial spin labeling MRI for quantitative tumor perfusion in a mouse xenograft model.

Perfusion is an important biomarker of tissue function and has been associated with tumor pathophysiology such as angiogenesis and hypoxia. Arterial spin labeling (ASL) MRI allows noninvasive and quantitative imaging of perfusion; however, the application in mouse xenograft tumor models has been challenging due to the low sensitivity and high perfusion heterogeneity. In this study, flow-sensitive alternating inversion recovery (FAIR) ASL was optimized for a mouse xenograft tumor. To assess the sensitivity and reliability for measuring low perfusion, the lumbar muscle was used as a reference region. By optimizing the number of averages and inversion times, muscle perfusion as low as 32.4 ± 4.8 (mean ± standard deviation) ml/100 g/min could be measured in 20 min at 7 T with a quantification error of 14.4 ± 9.1%. Applying the optimized protocol, heterogeneous perfusion ranging from 49.5 to 211.2 ml/100 g/min in a renal carcinoma was observed. To understand the relationship with tumor pathology, global and regional tumor perfusion was compared with histological staining of blood vessels (CD34), hypoxia (CAIX) and apoptosis (TUNEL). No correlation was observed when the global tumor perfusion was compared with these pathological parameters. Regional analysis shows that areas of high perfusion had low microvessel density, which was due to larger vessel area compared with areas of low perfusion. Nonetheless, these were not correlated with hypoxia or apoptosis. The results suggest that tumor perfusion may reflect certain aspect of angiogenesis, but its relationship with other pathologies needs further investigation.

A Review of and Recommendations for the Management of Patients With Life-Threatening Dabigatran-Associated Hemorrhage: A Single-Center University Hospital Experience.

Dabigatran is an oral direct thrombin inhibitor that is approved for the prevention of stroke and systemic embolism in nonvalvular atrial fibrillation. Dabigatran has several advantages over warfarin including predictable pharmacokinetics and pharmacodynamics which eliminates the need for routine laboratory monitoring, superiority over warfarin in preventing stroke, or systemic embolism without having an increased risk of bleeding. However, as with any anticoagulant, there remains a real chance of bleeding, including major or life-threatening hemorrhage. Many physicians feel comfortable managing bleeding complications on older anticoagulants like warfarin and heparin, due to extensive experience with the medications along with antidotes to reverse their effects as well as established protocols for treating anticoagulant-associated hemorrhage. However, most physicians have limited clinical experience with dabigatran, there is no specific antidote for dabigatran reversal and there is a paucity of protocols, guidelines, and recommendations for how to manage dabigatran-associated hemorrhage. In this review, we present a case series of patients admitted to our institution for management of bleeding while receiving dabigatran. We retrospectively reviewed these cases to evaluate the efficacy and rationale of the various anticoagulation reversal strategies employed in the context of the existing evidence found in the literature. Specific focus is placed on the therapies utilized and the coagulation studies used to manage these patients.

A comprehensive computational model of sound transmission through the porcine lung.

A comprehensive computational simulation model of sound transmission through the porcine lung is introduced and experimentally evaluated. This "subject-specific" model utilizes parenchymal and major airway geometry derived from x-ray CT images. The lung parenchyma is modeled as a poroviscoelastic material using Biot theory. A finite element (FE) mesh of the lung that includes airway detail is created and used in comsol FE software to simulate the vibroacoustic response of the lung to sound input at the trachea. The FE simulation model is validated by comparing simulation results to experimental measurements using scanning laser Doppler vibrometry on the surface of an excised, preserved lung. The FE model can also be used to calculate and visualize vibroacoustic pressure and motion inside the lung and its airways caused by the acoustic input. The effect of diffuse lung fibrosis and of a local tumor on the lung acoustic response is simulated and visualized using the FE model. In the future, this type of visualization can be compared and matched with experimentally obtained elastographic images to better quantify regional lung material properties to noninvasively diagnose and stage disease and response to treatment.

mRNAid, an open-source platform for therapeutic mRNA design and optimization strategies.

Recent COVID-19 vaccines unleashed the potential of mRNA-based therapeutics. A common bottleneck across mRNA-based therapeutic approaches is the rapid design of mRNA sequences that are translationally efficient, long-lived and non-immunogenic. Currently, an accessible software tool to aid in the design of such high-quality mRNA is lacking. Here, we present mRNAid, an open-source platform for therapeutic mRNA optimization, design and visualization that offers a variety of optimization strategies for sequence and structural features, allowing one to customize desired properties into their mRNA sequence. We experimentally demonstrate that transcripts optimized by mRNAid have characteristics comparable with commercially available sequences. To encompass additional aspects of mRNA design, we experimentally show that incorporation of certain uridine analogs and untranslated regions can further enhance stability, boost protein output and mitigate undesired immunogenicity effects. Finally, this study provides a roadmap for rational design of therapeutic mRNA transcripts.

Design-rules for stapled peptides with in vivo activity and their application to Mdm2/X antagonists.

Although stapled α-helical peptides can address challenging targets, their advancement is impeded by poor understandings for making them cell permeable while avoiding off-target toxicities. By synthesizing >350 molecules, we present workflows for identifying stapled peptides against Mdm2(X) with in vivo activity and no off-target effects. Key insights include a clear correlation between lipophilicity and permeability, removal of positive charge to avoid off-target toxicities, judicious anionic residue placement to enhance solubility/behavior, optimization of C-terminal length/helicity to enhance potency, and optimization of staple type/number to avoid polypharmacology. Workflow application gives peptides with >292x improved cell proliferation potencies and no off-target cell proliferation effects ( > 3800x on-target index). Application of these 'design rules' to a distinct Mdm2(X) peptide series improves ( > 150x) cellular potencies and removes off-target toxicities. The outlined workflow should facilitate therapeutic impacts, especially for those targets such as Mdm2(X) that have hydrophobic interfaces and are targetable with a helical motif.