Extensive exploration of structure activity relationships for the SARS-CoV-2 macrodomain from shape-based fragment merging and active learning.

The macrodomain contained in the SARS-CoV-2 non-structural protein 3 (NSP3) is required for viral pathogenesis and lethality. Inhibitors that block the macrodomain could be a new therapeutic strategy for viral suppression. We previously performed a large-scale X-ray crystallography-based fragment screen and discovered a sub-micromolar inhibitor by fragment linking. However, this carboxylic acid-containing lead had poor membrane permeability and other liabilities that made optimization difficult. Here, we developed a shape-based virtual screening pipeline - FrankenROCS - to identify new macrodomain inhibitors using fragment X-ray crystal structures. We used FrankenROCS to exhaustively screen the Enamine high-throughput screening (HTS) collection of 2.1 million compounds and selected 39 compounds for testing, with the most potent compound having an IC50 value equal to 130 µM. We then paired FrankenROCS with an active learning algorithm (Thompson sampling) to efficiently search the Enamine REAL database of 22 billion molecules, testing 32 compounds with the most potent having an IC50 equal to 220 µM. Further optimization led to analogs with IC50 values better than 10 µM, with X-ray crystal structures revealing diverse binding modes despite conserved chemical features. These analogs represent a new lead series with improved membrane permeability that is poised for optimization. In addition, the collection of 137 X-ray crystal structures with associated binding data will serve as a resource for the development of structure-based drug discovery methods. FrankenROCS may be a scalable method for fragment linking to exploit ever-growing synthesis-on-demand libraries.

Nicotine-induced ER Stress and ASM Cell Proliferation is Mediated by α7nAChR and Chaperones-RIC-3 and TMEM35.

Nicotine exposure in the context of smoking or vaping worsens airway function. Although commonly thought to exert effects through the peripheral nervous system, we previously showed airway smooth muscle (ASM) expresses nicotinic acetylcholine receptors (nAChRs), particularly alpha7 subtype (α7nAChR) with functional effects on contractility and metabolism. However, the mechanisms of nAChR regulation and downstream effects in ASM are not fully understood. Using human ASM cells from non-asthmatics vs. mild-moderate asthmatics, we tested the hypothesis that nAChR-specific ER chaperones RIC-3 and TMEM35 promote cell surface localization of α7nAChR with downstream influence on its functionality: effects exacerbated by inflammation. We found that mild-moderate asthma and exposure to pro-inflammatory cytokines relevant to asthma promote chaperone and α7nAChR expression in ASM. Downstream, ER stress was linked to nicotine/α7nAChR signaling, where RIC-3 and TMEM35 regulate nicotine-induced ER stress, Ca2+ regulation and ASM cell proliferation. Overall, our data highlights the importance α7nAChR chaperones in mediating and modulating nicotine effects in ASM towards airway contractility and remodeling.

Healthcare transition in pediatric neurosurgery: lessons learned from a pilot program for patients with hydrocephalus and spina bifida.

OBJECTIVE: The pediatric neurosurgical community has increasingly recognized the importance of healthcare transition, the process of moving a patient from a pediatric to an adult model of care. However, surveys of pediatric neurosurgeons have revealed that few institutions have formal transition programs. Here, the authors share their preliminary experience with the development of a formal transition pilot program for patients with spina bifida and/or hydrocephalus. METHODS: Patients 18 years of age or older with a diagnosis of spina bifida and/or hydrocephalus who were followed by a pediatric neurosurgeon at Connecticut Children's from January 2017 to December 2023 and were recommended to transition to an adult neurosurgeon were retrospectively reviewed. Patients in the informal transition program (ITP) cohort (i.e., the recommendation to transition was made before the formal transition program [FTP] was developed in early 2020) were compared with those in the FTP cohort. RESULTS: Twenty-two patients met inclusion criteria with 7 (31.8%) in the ITP cohort and 15 (68.2%) in the FTP cohort. The median age at the time of the recommendation to transition was similar in both ITP and FTP cohorts (24 [IQR 20-35] years vs 25 [IQR 24-27] years, respectively). Four (57.1%) patients in the ITP cohort had a confirmed visit with an adult neurosurgeon, compared with 13 (86.7%) patients in the FTP cohort (p = 0.274). One patient in the ITP cohort with a failed transition returned to pediatric neurosurgical care, and 1 patient in the FTP cohort required a shunt revision by an adult neurosurgeon within 1 year of the recommendation to transition. CONCLUSIONS: Healthcare transition is recognized as a priority within pediatric neurosurgery, but structured, formal transition programs remain underdeveloped. The authors' preliminary experience with a pilot transition program demonstrated that patients who underwent a formal transition were more likely to successfully establish care with an adult neurosurgeon and trended toward less resource utilization.

Unilateral versus bilateral pedicle screw fixation with anterior lumbar interbody fusion: a comparison of postoperative outcomes.

PURPOSE: To determine of the impact of ALIF with minimally invasive unilateral pedicle screw fixation (UPSF) versus bilateral pedicle screw fixation (BPSF) on perioperative outcomes, radiographic outcomes, and the rates of fusion, subsidence, and adjacent segment stenosis. METHODS: All adult patients who underwent one-level ALIF with UPSF or BPSF at an academic institution between 2015 and 2022 were retrospectively identified. Postoperative outcomes including length of hospital stay (LOS), wound complications, readmissions, and revisions were determined. The rates of fusion, screw loosening, adjacent segment stenosis, and subsidence were assessed on one-year postoperative CT. Lumbar alignment including lumbar lordosis, L4-S1 lordosis, regional lordosis, pelvic tilt, pelvic incidence, and sacral slope were assessed on standing x-rays at preoperative, immediate postoperative, and final postoperative follow-up. Univariate and multivariate analysis compared outcomes across posterior fixation groups. RESULTS: A total of 60 patients were included (27 UPSF, 33 BPSF). Patients with UPSF were significantly younger (p = 0.011). Operative time was significantly greater in the BPSF group in univariate (p < 0.001) and multivariate analysis (ß=104.1, p < 0.001). Intraoperative blood loss, LOS, lordosis, pelvic parameters, fusion rate, subsidence, screw loosening, adjacent segment stenosis, and revision rate did not differ significantly between fixation groups. Though sacral slope (p = 0.037) was significantly greater in the BPSF group, fixation type was not a significant predictor on regression. CONCLUSIONS: ALIF with UPSF relative to BPSF predicted decreased operative time but was not a significant predictor of postoperative outcomes. ALIF with UPSF can be considered to increase operative efficiency without compromising construct stability.

The history and potential future of monoclonal antibody therapeutics development and manufacturing in four eras.

Therapeutic monoclonal antibody (mAb) development and the processes for manufacturing drug substance have evolved since the first approval of the mAb in 1986. As the past is often the prologue to the future, the history of these technologies has been classified here into three eras, leading to speculation about what the next era may hold with regard to development and manufacturing strategies, as well as the potential impacts to patients. The substantial increase in production culture titers and bioreactor production volumes and the availability of large-scale contract manufacturing facilities could translate into improved global access for these therapies and an expansion of indications for therapeutic antibodies.

Targeting of nanoparticles to the cerebral vasculature after traumatic brain injury.

Traumatic brain injury has faced numerous challenges in drug development, primarily due to the difficulty of effectively delivering drugs to the brain. However, there is a potential solution in targeted drug delivery methods involving antibody-drug conjugates or nanocarriers conjugated with targeting antibodies. Following a TBI, the blood-brain barrier (BBB) becomes permeable, which can last for years and allow the leakage of harmful plasma proteins. Consequently, an appealing approach for TBI treatment involves using drug delivery systems that utilize targeting antibodies and nanocarriers to help restore BBB integrity. In our investigation of this strategy, we examined the efficacy of free antibodies and nanocarriers targeting a specific endothelial surface marker called vascular cell adhesion molecule-1 (VCAM-1), which is known to be upregulated during inflammation. In a mouse model of TBI utilizing central fluid percussion injury, free VCAM-1 antibody did not demonstrate superior targeting when comparing sham vs. TBI brain. However, the administration of VCAM-1-targeted nanocarriers (liposomes) exhibited a 10-fold higher targeting specificity in TBI brain than in sham control. Flow cytometry and confocal microscopy analysis confirmed that VCAM-1 liposomes were primarily taken up by brain endothelial cells post-TBI. Consequently, VCAM-1 liposomes represent a promising platform for the targeted delivery of therapeutics to the brain following traumatic brain injury.

Sex differences in the extent of acute axonal pathologies after experimental concussion.

Although human females appear be at a higher risk of concussion and suffer worse outcomes than males, underlying mechanisms remain unclear. With increasing recognition that damage to white matter axons is a key pathologic substrate of concussion, we used a clinically relevant swine model of concussion to explore potential sex differences in the extent of axonal pathologies. At 24 h post-injury, female swine displayed a greater number of swollen axonal profiles and more widespread loss of axonal sodium channels than males. Axon degeneration for both sexes appeared to be related to individual axon architecture, reflected by a selective loss of small caliber axons after concussion. However, female brains had a higher percentage of small caliber axons, leading to more extensive axon loss after injury compared to males. Accordingly, sexual dimorphism in axonal size is associated with more extensive axonal pathology in females after concussion, which may contribute to worse outcomes.

"Toward Breast Reinnervation- What is our Endpoint" A systematic review of normal breast sensibility.

BACKGROUND: To restore breast sensibility, some centers are offering nerve reconstruction as a component of implant and flap-based breast reconstruction. To interpret and contextualize the results of these procedures, it is necessary to understand the normal range of breast sensibility, the factors that affect it, and the best methods for its objective measurement. METHODS: We conducted systematic and comprehensive searches across PubMed, Web of Science, and Cochrane Library databases using keywords and controlled vocabulary for the concepts of the breast, nipple, areola, and measurement. The search results were imported into Rayyan QCRI for a blinded screening of titles and abstracts. Studies were evaluated for bias using RevMan 5 software. The results of sensory measurements were pooled, and a quantitative summary of breast sensibility was generated. RESULTS: A total of 36 articles were identified, including retrospective, cross-sectional, and prospective studies. Although there were some consistent findings across studies, such that breast sensibility is inversely related to breast volume, there was wide variability in the following parameters: population, breast condition, measurement modality, anatomic areas of measurement, and sensibility findings. This heterogeneity precluded the generation of normative breast sensibility measurements. Furthermore, we detected a high degree of bias in most studies, due to self-selection of participants and failure to record patient characteristics that may alter sensibility. CONCLUSIONS: The literature lacks consistent data delineating normative values for breast sensibility. Standardized measurements of healthy volunteers with various breast characteristics are necessary to elucidate normative values and interpret efforts to restore sensibility in breast reconstruction.

Exploring the combinatorial explosion of amine-acid reaction space via graph editing.

Amines and carboxylic acids are abundant chemical feedstocks that are nearly exclusively united via the amide coupling reaction. The disproportionate use of the amide coupling leaves a large section of unexplored reaction space between amines and acids: two of the most common chemical building blocks. Herein we conduct a thorough exploration of amine-acid reaction space via systematic enumeration of reactions involving a simple amine-carboxylic acid pair. This approach to chemical space exploration investigates the coarse and fine modulation of physicochemical properties and molecular shapes. With the invention of reaction methods becoming increasingly automated and bringing conceptual reactions into reality, our map provides an entirely new axis of chemical space exploration for rational property design.

To flip or not: Case series of coronary angioplasty in patients with right-sided heart.

Coronary angioplasty in patients with a right-sided heart may be difficult due to challenges in engaging the coronary arteries, interpreting angiogram, and further delivering intracoronary therapies. We present our experience of percutaneous coronary intervention in two cases and propose a practical algorithm to approach cardiac catheterization in these patients.

Mechanosensitive Channels in Lung Health and Disease.

The lung is an inherently mechanosensitive organ, where cells of the airway and parenchyma experience a range of mechanical forces throughout life including shear, stretch, and compression, in both health and disease. In this regard, pediatric and adult lung diseases such as wheezing and asthma, bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis (PF) all involve macroscopic and cellular changes to the mechanical properties of the bronchial airways and/or parenchyma to varying extents. Accordingly, understanding how mechanical forces are sensed in the lung, and the responses of cells and tissues in the context of normal development and health versus disease conditions becomes highly relevant. There is increasing recognition that transduction of mechanical forces into cellular responses involves a number of channels, some of which are inherently mechanosensitive. Such channels trigger mechanotransduction pathways that may further mediate cellular remodeling, inflammation, and other pathophysiologic mechanisms in response to stretch, stiffness, and inflammatory cascades. Two particularly important channel families have emerged in pulmonary pathophysiology: the transient receptor potential vanilloid family with focus on member TRPV4 and the recently identified Piezo (PZ) channels. Here, we explore current understanding of the contributions of TRPV4 and PZ channels in lung health and disease states, focusing on the interactions between these mechanosensitive channels and their local environment including immune cells, the extracellular matrix, and cellular cytoskeletal elements. We further discuss potential areas for future research to better understand the impact of mechanical channels on pulmonary health and disease. © 2023 American Physiological Society. Compr Physiol 13:5157-5178, 2023.

Piezo channels in stretch effects on developing human airway smooth muscle.

The use of respiratory support strategies such as continuous positive airway pressure in premature infants can substantially stretch highly compliant perinatal airways, leading to airway hyperreactivity and remodeling in the long term. The mechanisms by which stretch detrimentally affects the airway are unknown. Airway smooth muscle cells play a critical role in contractility and remodeling. Using 18-22-wk gestation human fetal airway smooth muscle (fASM) as an in vitro model, we tested the hypothesis that mechanosensitive Piezo (PZ) channels contribute to stretch effects. We found that PZ1 and PZ2 channels are expressed in the smooth muscle of developing airways and that their expression is influenced by stretch. PZ activation via agonist Yoda1 or stretch results in significant [Ca2+]i responses as well as increased extracellular matrix production. These data suggest that functional PZ channels may play a role in detrimental stretch-induced airway changes in the context of prematurity.NEW & NOTEWORTHY Piezo channels were first described just over a decade ago and their function in the lung is largely unknown. We found that piezo channels are present and functional in the developing airway and contribute to intracellular calcium responses and extracellular matrix remodeling in the setting of stretch. This may improve our understanding of the mechanisms behind development of chronic airway diseases, such as asthma, in former preterm infants exposed to respiratory support, such as continuous positive airway pressure (CPAP).

Functional α7 nicotinic receptors in human airway smooth muscle increase intracellular calcium concentration and contractility in asthmatics.

Although nicotinic acetylcholine receptors (nAChRs) are commonly associated with neurons in the brain and periphery, recent data indicate that they are also expressed in non-neuronal tissues. We recently found the alpha7 (α7nAChR) subunit is highly expressed in human airway smooth muscle (hASM) with substantial increase in asthmatics, but their functionality remains unknown. We investigated the location and functional role of α7nAChRs in hASM cells from normal versus mild-moderate asthmatic patients. Immunostaining and protein analyses showed α7nAChR in the plasma membrane including in asthmatics. In asthmatic hASM, patch-clamp recordings revealed significantly higher functional homomeric α7nAChR channels. Real-time fluorescence imaging showed nicotine, via α7nAChR, increases intracellular Ca2+ ([Ca2+]i) independent of ACh effects, particularly in asthmatic hASM, while cellular traction force microscopy showed nicotine-induced contractility including in asthmatics. These results indicate functional homomeric and heteromeric nAChRs that are increased in asthmatic hASM, with pharmacology that likely differ owing to different subunit interfaces that form the orthosteric sites. nAChRs may represent a novel target in alleviating airway hyperresponsiveness in asthma.NEW & NOTEWORTHY Cigarette smoking and vaping exacerbate asthma. Understanding the mechanisms of nicotine effects in asthmatic airways is important. This study demonstrates that functional alpha7 nicotinic acetylcholine receptors (α7nAChRs) are expressed in human airway smooth muscle, including from asthmatics, and enhance intracellular calcium and contractility. Although a7nAChRs are associated with neuronal pathways, α7nAChR in smooth muscle suggests inhaled nicotine (e.g., vaping) can directly influence airway contractility. Targeting α7nAChR may represent a novel approach to alleviating airway hyperresponsiveness in asthma.

Hemodynamic Doppler echocardiographic evaluation of permanent His bundle and biventricular pacing after AV nodal ablation.

placing after atrioventricular (AV) nodal ablation for permanent atrial fibrillation (AF) may include cardiac resynchronization therapy (CRT) with either His bundle pacing (HBP) or biventricular pacing (BVP), or conventional single site right ventricular apical pacing (RVAP). To determine the relationship between pacing method and hemodynamic outcome, we used Doppler echocardiographic methods to evaluate left ventricular (LV) hemodynamics after AV nodal ablation and either HBP, BVP, or RVAP. Method: 20 patients were evaluated > 6 months after AV nodal ablation, 10 each with chronic HBP or BVP, and all with RVAP lead. Doppler echocardiography was used to measure 3 parameters indicative of CRT: 1) LV dP/dt, 2) the LV pre-ejection interval, and 3) myocardial performance index, relative to intra-patient RVAP. Results: Primary endpoint of LV dP/dt on average improved by > 17% with both HBP and BVP, compared to RVAP. HBP but not BVP, had improvement across all three parameters. Conclusion: HBP provides LV electromechanical synchrony across multiple echo Doppler parameters. Both HBP and BVP were hemodynamically superior to RVAP following AV nodal ablation.

Monoclonal antibody therapies for COVID-19: lessons learned and implications for the development of future products.

Several companies were authorized to treat COVID-19 patients with monoclonal antibodies within 1-2 years of the start of the pandemic. These products were discovered, developed, manufactured, clinically tested, and approved under emergency-use authorization at unprecedented speed. Pandemic urgency led to novel development approaches that reduced the time to clinical trials by 75% or more without creating unacceptable patient or product-safety risks. Hundreds of thousands of patients now benefit from these therapeutics that have reduced the rates of hospitalization and death. The chemistry, manufacturing, and control development strategies set a new precedent of speed, safety, and demonstrated clinical benefit and will likely have a lasting impact on the development of future monoclonal antibody therapies for not only infectious diseases but also for oncology, inflammation, and rare diseases.

Traumatic brain injury recapitulates developmental changes of axons.

During development, half of brain white matter axons are maintained for growth, while the remainder undergo developmental axon degeneration. After traumatic brain injury (TBI), injured axons also appear to follow pathways leading to either degeneration or repair. These observations raise the intriguing, but unexamined possibility that TBI recapitulates developmental axonal programs. Here, we examined axonal changes in the developing brain in young rats and after TBI in adult rat. Multiple shared changes in axonal microtubule (MT) through tubulin post-translational modifications and MT associated proteins (MAPs), tau and MAP6, were found in both development and TBI. Specifically, degenerating axons in both development and TBI underwent phosphorylation of tau and excessive tubulin tyrosination, suggesting MT instability and depolyermization. Conversely, nearby axons without degenerating morphologies, had increased MAP6 expression and maintenance of tubulin acetylation, suggesting enhanced MT stabilization, thereby supporting survival or repair. Quantitative proteomics revealed similar signaling pathways of axon degeneration and growth/repair, including protein clusters and networks. This comparison approach demonstrates how focused evaluation of developmental processes may provide insight into pathways initiated by TBI. In particular, the data suggest that TBI may reawaken dormant axonal programs that direct axons towards either degeneration or growth/repair, supporting further study in this area.

Developing a Mechanistic Approach to Sudden Death Prevention in Mitral Valve Prolapse.

Sudden cardiac death (SCD) from ventricular fibrillation (VF) can occur in mitral valve prolapse (MVP) in the absence of other comorbidities including mitral regurgitation, heart failure or coronary disease. Although only a small proportion with MVP are at risk, it can affect young, otherwise healthy adults, most commonly premenopausal women, often as the first presentation of MVP. In this review, we discuss arrhythmic mechanisms in MVP and mechanistic approaches for sudden death risk assessment and prevention. We define arrhythmogenic or arrhythmic MVP (AMVP) as MVP associated with complex and frequent ventricular ectopy, and malignant MVP (MMVP) as MVP with high risk of SCD. Factors predisposing to AMVP are myxomatous, bileaflet MVP and mitral annular disjunction (MAD). Data from autopsy, cardiac imaging and electrophysiological studies suggest that ectopy in AMVP is due to inflammation, fibrosis and scarring within the left ventricular (LV) base, LV papillary muscles and Purkinje tissue. Postulated mechanisms include repetitive injury to these regions from systolic papillary muscle stretch and abrupt mitral annular dysmotility (excursion and curling) and diastolic endocardial interaction of redundant mitral leaflets and chordae. Whereas AMVP is seen relatively commonly (up to 30%) in those with MVP, MVP-related SCD is rare (2-4%). However, the proportion at risk (i.e., with MMVP) is unknown. The clustering of cardiac morphological and electrophysiological characteristics similar to AMVP in otherwise idiopathic SCD suggests that MMVP arises when specific arrhythmia modulators allow for VF initiation and perpetuation through action potential prolongation, repolarization heterogeneity and Purkinje triggering. Adequately powered prospective studies are needed to assess strategies for identifying MMVP and the primary prevention of SCD, including ICD implantation, sympathetic modulation and early surgical mitral valve repair. Given the low event rate, a collaborative multicenter approach is essential.

Translational PET Imaging of Spinal Cord Injury with the Serotonin Transporter Tracer [11C]AFM.

PURPOSE: The descending raphespinal serotonin (5-HT) system contributes to neural activities required for locomotion. The presynaptic serotonin transporter (SERT) is a marker of 5-HT innervation. In this study, we explored the use of PET imaging with the SERT radioligand [11C]AFM as a biomarker of 5-HT axon damage after spinal cord injury (SCI) in a rodent model and its translation to imaging SCI in humans. PROCEDURES: PET imaging with [11C]AFM was performed in healthy rats under baseline and citalopram blocking conditions and a mid-thoracic transection rat model of SCI. The lumbar-to-cervical activity (L/C) ratio was calculated for the healthy and SCI animals to assess SERT binding decrease after SCI. Finally, translation of [11C]AFM PET was attempted to explore its potential to image SCI in humans. RESULTS: Intense uptake in the brain and intact spinal cord was observed at 30-60 min post-injection of [11C]AFM in healthy rats. About 65% of [11C]AFM uptake in the spinal cord was blocked by citalopram. In the SCI rat model, the cervical uptake of [11C]AFM was similar to that in healthy rats, but the lumbar uptake was dramatically reduced, resulting in about half the L/C ratio in SCI rats compared to healthy rats. In contrast, [11C]AFM uptake in the human spinal cord showed no obvious decrease after treatment with citalopram. In the human subjects with SCI, decreases in [11C]AFM uptake were also not obvious in the section of spinal cord caudal to the injury point. CONCLUSION: [11C]AFM PET imaging of SERT provides a useful preclinical method to non-invasively visualize the rodent spinal cord and detect SERT changes in SCI rodent models. However, there appears to be little detectable specific binding signal for [11C]AFM in the human spinal cord. An SERT tracer with higher affinity and lower non-specific binding signal is needed to image the spinal cord in humans and to assess the axonal status in SCI patients.