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Itch is a protective sensation that drives scratching. Although specific cell types have been proposed to underlie itch, the neural basis for itch remains unclear. Here, we used two-photon Ca 2+ imaging of the dorsal horn to visualize neuronal populations that are activated by itch-inducing agents. We identify a convergent population of spinal interneurons recruited by diverse itch-causing stimuli that represents a subset of neurons that express the gastrin-releasing peptide receptor (GRPR). Moreover, we find that itch is conveyed to the brain via GRPR-expressing spinal output neurons that target the lateral parabrachial nuclei. We then show that the kappa opioid receptor agonist nalfurafine relieves itch by selectively inhibiting GRPR spinoparabrachial neurons. These experiments provide a population-level view of the spinal neurons that respond to pruritic stimuli, pinpoint the output neurons that convey itch to the brain, and identify the cellular target of kappa opioid receptor agonists for the inhibition of itch.

The nature of spinal output pathways that convey nociceptive information to the brain has been the subject of controversy. Here, we provide anatomical, molecular, and functional characterizations of two distinct anterolateral pathways: one, ascending in the lateral spinal cord, triggers nociceptive behaviors, and the other one, ascending in the ventral spinal cord, when inhibited, leads to sensorimotor deficits. Moreover, the lateral pathway consists of at least two subtypes. The first is a contralateral pathway that extends to the periaqueductal gray (PAG) and thalamus; the second is a bilateral pathway that projects to the bilateral parabrachial nucleus (PBN). Finally, we present evidence showing that activation of the contralateral pathway is sufficient for defensive behaviors such as running and freezing, whereas the bilateral pathway is sufficient for attending behaviors such as licking and guarding. This work offers insight into the complex organizational logic of the anterolateral system in the mouse., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Stereotactic Body Radiation Therapy (SBRT) is increasingly applied to treat non-spine bone metastases (NSBM) though data remains limited on this approach. In this retrospective study, we report outcomes and predictors of local failure (LF) and pathological fracture (PF) post-SBRT for NSBM using a mature single-institution database., Methods: Patients with NSBM treated with SBRT between 2011 and 2021 were identified. The primary objective was to assess the rates of radiographic LF. Secondary objectives were to assess the rates of in-field PF, overall survival (OS), and late grade ≥ 3 toxicity. Competing risks analysis was used to assess rates of LF and PF. Univariable regression and multivariable regression (MVR) were performed to investigate predictors of LF and PF., Results: A total of 373 patients with 505 NSBM were included in this study. Median follow-up was 26.5 months. The cumulative incidence of LF at 6, 12, and 24 months were 5.7%, 7.9%, and 12.6%, respectively. The cumulative incidence of PF at 6, 12, and 24 months were 3.8%, 6.1%, and 10.9%, respectively. Lytic NSBM (HR = 2.18; p < 0.01), a lower biologically effective dose (HR = 1.11 per 5 Gy 10 decrease; p = 0.04), and a PTV ≥ 54 cc (HR = 4.32; p < 0.01) predicted for a higher risk of LF on MVR. Lytic NSBM (HR = 3.43; p < 0.01), mixed (lytic/sclerotic) lesions (HR = 2.70; p = 0.04), and rib metastases (HR = 2.68; p < 0.01) predicted for a higher risk of PF on MVR., Conclusion: SBRT is an effective modality to treat NSBM with high rates of radiographic local control with an acceptable rate of PF. We identify predictors of both LF and PF that can serve to inform practice and trial design., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Type 2 diabetes (T2D) is associated with increased risk of atherosclerotic vascular disease due to excessive vascular smooth muscle cell (VSMC) proliferation. Here, we investigated the role of mitochondrial dysfunction and Ca2+ levels in VSMC proliferation in T2D. VSMCs were isolated from normoglycemic and T2D-like mice induced by diet. The effects of mitochondrial Ca2+ uptake were studied using mice with selectively inhibited mitochondrial Ca2+/calmodulin-dependent kinase II (mtCaMKII) in VSMCs. Mitochondrial transition pore (mPTP) was blocked using ER-000444793. VSMCs from T2D compared to normoglycemic mice exhibited increased proliferation and baseline cytosolic Ca2+ levels ([Ca2+]cyto). T2D cells displayed lower endoplasmic reticulum Ca2+ levels, reduced mitochondrial Ca2+ entry, and increased Ca2+ leakage through the mPTP. Mitochondrial and cytosolic Ca2+ transients were diminished in T2D cells upon platelet-derived growth factor (PDGF) administration. Inhibiting mitochondrial Ca2+ uptake or the mPTP reduced VSMC proliferation in T2D, but had contrasting effects on [Ca2+]cyto. In T2D VSMCs, enhanced activation of Erk1/2 and its upstream regulators was observed, driven by elevated [Ca2+]cyto. Inhibiting mtCaMKII worsened the Ca2+ imbalance by blocking mitochondrial Ca2+ entry, leading to further increases in [Ca2+]cyto and Erk1/2 hyperactivation. Under these conditions, PDGF had no effect on VSMC proliferation. Inhibiting Ca2+-dependent signaling in the cytosol reduced excessive Erk1/2 activation and VSMC proliferation. Our findings suggest that altered Ca2+ handling drives enhanced VSMC proliferation in T2D, with mitochondrial dysfunction contributing to this process.

Purpose: Stereotactic body radiotherapy (SBRT) has proven to be a highly effective treatment for selected patients with spinal metastases. Randomized evidence shows improvements in complete pain response rates and local control with lower retreatment rates favoring SBRT, compared to conventional external beam radiotherapy (cEBRT). While there are several reported dose-fractionation schemes for spine SBRT, 24 Gy in 2 fractions has emerged with Level 1 evidence providing an excellent balance between minimizing treatment toxicity while respecting patient convenience and financial strain., Methods: We provide an overview of the 24 Gy in 2 SBRT fraction regimen for spine metastases, which was developed at the University of Toronto and tested in an international Phase 2/3 randomized controlled trial., Results: The literature summarizing global experience with 24 Gy in 2 SBRT fractions suggests 1-year local control rates ranging from 83-93.9%, and 1-year rates of vertebral compression fracture ranging from 5.4-22%. Reirradiation of spine metastases that failed prior cEBRT is also feasible with 24 Gy in 2 fractions, and 1-year local control rates range from 72-86%. Post-operative spine SBRT data are limited but do support the use of 24 Gy in 2 fractions with reported 1-year local control rates ranging from 70-84%. Typically, the rates of plexopathy, radiculopathy and myositis are under 5% in those series reporting mature follow up, with no cases of radiation myelopathy (RM) reported in the de novo setting when the spinal cord avoidance structure is limited to 17 Gy in 2 fractions. However, re-irradiation RM has been observed following 2 fraction SBRT. More recently, 2-fraction dose escalation with 28 Gy, with a higher dose constraint to the critical neural tissues, has been reported suggesting improved rates of local control. This regimen may be important in those patients with radioresistant histologies, high grade epidural disease, and/or paraspinal disease., Conclusion: The dose-fractionation of 24 Gy in 2 fractions is well-supported by published literature and is an ideal starting point for centers looking to establish a spine SBRT program., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The sandwich format immunoassay is generally more sensitive and specific than more common assay formats, including direct, indirect, or competitive. A sandwich assay, however, requires two receptors to bind non-competitively to the target analyte. Typically, pairs of antibodies (Abs) or antibody fragments (Fabs) that are capable of forming a sandwiching with the target are identified through a slow, guess-and-check method with panels of candidate binding partners. Additionally, sandwich assays that are reliant on commercial antibodies can suffer from changes to reagent quality outside the researchers' control. This report presents a reimagined and simplified phage display selection protocol that directly identifies sandwich binding peptides and Fabs. The approach yielded two sandwich pairs, one peptide-peptide and one Fab-peptide sandwich for the cancer and Parkinson's disease biomarker DJ-1. Requiring just a few weeks to identify, the sandwich pairs delivered apparent affinity that is comparable to other commercial peptide and antibody sandwiches. The results reported here could expand the availability of sandwich binding partners for a wide range of clinical biomarker assays.

Purpose: The goal of this study was to assess the potential real-world effect of the recently reported SC.24 trial on spine stereotactic body radiation therapy (SBRT) utilization. We estimated the proportion of patients treated with conventional radiation therapy (CRT) who would have been eligible for spine SBRT per trial inclusion criteria and analyzed the potential estimated increased costs to our institution., Methods and Materials: This was a retrospective review of patients who received spine CRT at our institution between August and October 2020. Data abstracted included demographics, SC.24 eligibility criteria, provider-reported pain response, and survival. A cost analysis and time survey was performed using institutional and provincial data., Results: Of 73 patients reviewed, 24 patients (33%) were eligible. The most common exclusion factors included irradiation of ≥3 consecutive spinal segments (n = 32, 44%), Eastern Cooperative Oncology Group performance status >2 (n = 17, 23%), and symptomatic spinal cord compression (n = 13, 18%). Of eligible patients, the mean age was 68.92 years, median spinal instability in neoplasia score was 8 (interquartile range, 7-9), and median Eastern Cooperative Oncology Group performance status was 2 (interquartile range, 1-2). The most common primary cancer types among eligible patients were lung (n = 10) and breast (n = 4). The median survival of eligible patients was 10 months (95% confidence interval, 4 months to not reached) with 58% surviving longer than 3 months. Of patients who had subjective pain documented after CRT, 54% had at least some response. The cost of spine SBRT was estimated at CA$4764.80 compared with $3589.10 for CRT, and tasks for spine SBRT took roughly 3 times as long as those for CRT., Conclusions: One-third of patients who received palliative spine CRT met eligibility criteria for SC.24. This possible expanded indication for spine SBRT can have a substantial effect on resource utilization. These data may be useful in guiding resource planning at institutions looking to commence a spine SBRT program., (© 2023 The Authors.)

Purpose: With the increasing use of stereotactic body radiation therapy (SBRT) for primary and metastatic cancer, use of multitarget thoracic (MTT) SBRT is rising. Given the limited safety and efficacy data, we report the experience of this strategy from a large academic center., Methods and Materials: Between 2012 and 2021, patients who received SBRT for ≥2 thoracic targets separated by ≤1 year were retrospectively reviewed. The primary endpoint was clinically significant radiation pneumonitis (CSRP) requiring steroids, oxygen, or intubation. Secondary endpoints included local failure (LF), initiation or change of systemic therapy (ICST), progression-free survival, and overall survival. Competing risk analysis was used to evaluate the cumulative incidence of CSRP, LF, and ICST. Univariate and multivariable analyses were performed to look for clinical and dosimetric predictive factors of CSRP and LF., Results: One hundred ninety patients (481 lesions) were treated with MTT SBRT with a median follow-up of 19.7 months. Indications for SBRT were oligometastases (n = 70; 36.8%), oligoprogression (n = 62; 32.6%), curative intent in patients with primary lung cancer (n = 37; 19.5%), and control of dominant areas of metastatic progression (n = 21; 11.0%). The number of irradiated tumors ranged from 2 to 7 and the majority of SBRT courses were delivered simultaneously (88.2%). Overall, 14 patients (7.4%) had CSRP, with 5 cases requiring oxygen. The cumulative incidence of CSRP at 6 and 12 months was 5.3% and 7.6%, respectively. The cumulative incidence of LF at 2 years was 10.5%. The cumulative incidence of ICST at 2 years was 41.1%. Median progression-free survival was 11.8 months and median overall survival was 51.3 months. On multivariable analysis, a higher lung V35 Gy (hazard ratio, 2.59; P = .02) was a statistically significant predictor of CSRP and colorectal histology predicted for higher LF (hazard ratio, 2.12; P = .02)., Conclusions: In one of the largest institutional series of MTT SBRT, rates of CSRP and LF were low. Optimizing plans to lower the lung V35 Gy may decrease the risk of CSRP., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Purpose: With the integration of immunotherapy (IO) agents in the management of metastatic renal cell carcinoma (mRCC), there has been interest in the combined use with radiation therapy (RT). However, real world data are limited. The purpose of this study was to evaluate outcomes in patients with mRCC receiving both RT and IO compared with IO alone., Methods and Materials: Data were collected from Canadian Kidney Cancer Information System from January 2011 to September 2019 across 14 academic centers. Patients with mRCC who received IO as first- or second-line therapy were included. RT was categorized as radical dose or palliative dose. Kaplan-Meier estimates were reported for overall survival (OS) and time to treatment failure. Cox proportional hazard models were used adjusted for age and International Metastatic RCC Database Consortium risk categories., Results: In total, 505 patients were included in the study: 179 received RT + IO and 326 received IO alone. Two-year OS for the RT + IO group was 55.0% compared with 66.4% in the IO alone cohort (adjusted hazard ratio [aHR], 1.38; P  = .07). At 2 years, 12.2% of the RT + IO patients remained on therapy versus 30.9% in the IO alone group (aHR, 1.30; P  = .02). For patients receiving first-line therapy, 2-year OS in the RT + IO group was 56.4% versus 78.4% in the IO alone arm, though this difference was not statistically significant (aHR, 1.23; P  = .56). For patients receiving radical dose and palliative dose, 2-year OS was 57.0% and 53.9%, respectively (aHR, 0.86; P  = .63)., Conclusions: In this descriptive analysis, more than one-third of patients with mRCC received RT and demonstrated inferior outcomes compared with IO alone. Potential explanations include greater presence of adverse metastatic sites in those receiving RT. Prospective clinical trials evaluating potential benefits of RT in an IO era remain an important need., (© 2022 The Author(s).)

Introduction: Hypofractionated stereotactic radiotherapy (hSRT) has emerged as an alternative to single-fraction stereotactic radiosurgery (SRS) and conventionally fractionated radiotherapy for the treatment of intracranial meningiomas (ICMs). However, there is a need for data showing long-term efficacy and complication rates, particularly for larger tumors in sensitive locations., Methods: A retrospective review was conducted on adult patients with ICMs seen at a tertiary care center. Eligible patients were treated with the CyberKnife platform and had a planned treatment course of 3-5 fractions from 2011-2020. The local control was assessed based on radiographic stability and the late toxicity/radionecrosis rates were recorded. Radiographic progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method., Results: In total, 62 patients (age 26-87) with 67 treated tumors were included in this study with a median follow-up of 64.7 months. RT was delivered as the primary treatment in 62.7% of cases and for recurrence in 37.3%. The most common tumor locations were the convexity of the brain and the base of the skull. The tumor sizes ranged from 0.1-51.8 cc and the median planning target volume was 4.9 cc. The most common treatment schedule was 18 Gy in 3 fractions. The five-year PFS and OS were 85.2% and 91.0%, respectively. The late grade III/IV toxicity rate was 3.2% and the radionecrosis rate was 4.8%., Conclusions: Based on our data, hSRT remains an effective modality to treat low-grade ICMs with acceptable long-term toxicity and radionecrosis rates. hSRT should be offered to patients who are not ideal candidates for SRS while preserving the benefits of hypofractionation.

Background: Stereotactic Body Radiotherapy (SBRT) has shown effectiveness in treating bone metastases to alleviate pain. The benefit of SBRT may be further harnessed especially when radiating disease from primary malignancies with low alpha-beta ratios in order to maximize the magnitude and durability of pain relief. However, such an approach has not been studied in a prospective trial. We look to assess single-fraction SBRT for painful non-spinal bone metastases from radioresistant primaries., Methods: Forty patients will be enrolled on an open label, phase II single arm trial to receive a single fraction of SBRT (15-20 Gray) to all sites of bone metastases requiring treatment for pain relief. Eligible patients will include those with primary malignancies consisting of prostate cancer, breast cancer, renal cell carcinoma, or melanoma. The primary endpoint is pain response at 3 months post-treatment using the Brief Pain Inventory. Secondary endpoints include pain response at 1 month and 6 months post-treatment, toxicity, patient-reported quality of life, re-irradiation or salvage surgery, and local control., Discussion: This study will evaluate the efficacy of single-fraction SBRT on painful bone metastases from primary cancers with low alpha-beta ratios. These data will be valuable to promote future randomized trials and support clinical implementation. Trial registration Clinicaltrials.gov, NCT04177056. Date of registration: November 26, 2019. https://clinicaltrials.gov/ct2/show/NCT04177056., (© 2021. The Author(s).)

Leptomeningeal disease (LMD) is a poor prognosis pattern of disease progression in patients with metastatic malignancy with limited treatment options. Patients may be asymptomatic or present with non-specific neurologic deficits, therefore gadolinium-enhanced magnetic resonance imaging of the brain and spine is critical for establishing a diagnosis. Although the treatment intent is palliative in the context of LMD, a multidisciplinary approach is still important to ensure patients receive a timely diagnosis and appropriate treatment to maximize symptom control and preserve quality of life. Radiotherapy is typically delivered to the whole brain or focal spinal regions for the purposes of treating bulky disease, stabilizing symptoms, or relieving cerebrospinal fluid obstruction. Whole craniospinal irradiation (CSI) is generally avoided given its toxicity profile and should only be considered in carefully selected patients where the potential benefit may outweigh the adverse effects. CSI with proton radiotherapy (oppose to conventional photon radiotherapy) has shown promise with improved toxicity for patients with primary CNS tumors. This may be a preferred option for patients being considered for CSI at centres with the proton therapy capabilities. Focal hypofractionated stereotactic radiotherapy (SRT) to intracranial targets is an emerging approach to LMD that may be useful in select patients with limited disease particularly in the setting of reirradiation. Chemotherapies may be delivered intrathecally, although the evidence supporting its efficacy is limited and heterogeneous in regards to the tumor sites examined. Finally, targeted therapy and novel applications of immune checkpoint inhibitors are promising; however, further research is required to guide the use of these agents.

The lateral parabrachial nucleus (lPBN) is a major target of spinal projection neurons conveying nociceptive input into supraspinal structures. However, the functional role of distinct lPBN efferents in diverse nocifensive responses have remained largely uncharacterized. Here we show that that the lPBN is required for escape behaviors and aversive learning to noxious stimulation. In addition, we find that two populations of efferent neurons from different regions of the lPBN collateralize to distinct targets. Activation of efferent projections to the ventromedial hypothalamus (VMH) or lateral periaqueductal gray (lPAG) drives escape behaviors, whereas activation of lPBN efferents to the bed nucleus stria terminalis (BNST) or central amygdala (CEA) generates an aversive memory. Finally, we provide evidence that dynorphin-expressing neurons, which span cytoarchitecturally distinct domains of the lPBN, are required for aversive learning., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020. Published by Elsevier Inc.)

Introduction: Although radical cystectomy is considered the standard of care for muscle-invasive bladder cancer (MIBC), recent data has suggested comparable survival outcomes for bladder-sparing trimodality therapy (TMT). We conducted a retrospective, single-institution analysis of MIBC patients to evaluate the efficacy of TMT as an alternative, curative approach to surgical intervention., Methods: We conducted a retrospective analysis of MIBC patients assessed by a multidisciplinary team at the Juravinski Cancer Centre from 2010-2016. Patients underwent transurethral resection of bladder tumor (TURBT) followed by radiotherapy with or without concurrent chemotherapy. Patients could receive neoadjuvant treatment. Clinical data and response rates were summarized, and overall survival (OS) and disease-free survival (DFS) were estimated using the Kaplan-Meier method., Results: Our analytic cohort included 115 patients, of whom 53 underwent TMT and 62 underwent radiotherapy alone following TURBT. Median age at diagnosis was 79 years and median followup was 21 months. Complete response rates in those receiving TMT and radiation without chemotherapy were 84.4% and 66.7%, respectively. For TMT patients, three-year OS and DFS were 68.5% and 49.6%, respectively. Patients who received TMT had reduction in risk of mortality (hazard ratio [HR] 0.49; p=0.026) and disease recurrence (HR 0.55; p=0.017) compared to those who had radiation without chemotherapy. Overall, four patients had grade 3 or higher late toxicity., Conclusions: In this single-institution analysis, TMT appears to be a safe and effective approach in the short-term management of MIBC in appropriately selected patients. Extended followup and analysis are necessary to validate these results.

Background: The availability of image guidance and intensity modulation has led to the increasing use of hypofractionated stereotactic radiotherapy (hSRT) as an alternative to conventionally fractionated radiotherapy or radiosurgery for intracranial meningiomas (ICMs). As the safety and efficacy of this approach is not well characterized, we conducted a systematic review of the literature to assess the clinical outcomes of hSRT in the setting of ICMs., Methods: A systematic review of Medline and EMBASE databases was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Included studies were retrospective or prospective series that examined an ICM population of ≥10 patients, delivered >1 fraction of photon hSRT (≥2.5 Gy per fraction), and had a median follow-up of ≥2 years. Descriptive statistics were generated for included studies., Results: Of 1480 initial studies, 14 met eligibility criteria for inclusion, reporting on 630 patients (age range, 18-90) treated for 638 tumors. Primary radiotherapy was delivered in 37% of patients, 36% had radiation following surgery, and surgical details were unavailable for 27%. In 474 tumors assessed for radiologic response, 78% remained stable, 18% decreased in size, and 4% increased in size. Crude local control was 90%-100% as reported in 10 studies. The median late toxicity rate was 10%. The most common significant late toxicities were decreased visual acuity and new cranial neuropathy., Conclusions: With limited follow-up, the available literature suggests hSRT for ICMs has local control and toxicity profiles comparable to other radiotherapy approaches. Confirmation in larger patient cohorts with a longer duration of follow-up is required.

Mitochondria regulate major aspects of cell function by producing ATP, contributing to Ca 2+ signaling, influencing redox potential, and controlling levels of reactive oxygen species. In this review, we will discuss recent findings that illustrate how mitochondrial respiration, Ca 2+ handling, and production of reactive oxygen species affect vascular smooth muscle cell function during neointima formation. We will review mitochondrial fission/fusion as fundamental mechanisms for smooth muscle proliferation, migration, and metabolism and examine the role of mitochondrial mobility in cell migration. In addition, we will summarize novel aspects by which mitochondria regulate apoptosis.

The role of the mitochondrial Ca 2+ uniporter (MCU) in physiologic cell proliferation remains to be defined. Here, we demonstrated that the MCU was required to match mitochondrial function to metabolic demands during the cell cycle. During the G 1 -S transition (the cycle phase with the highest mitochondrial ATP output), mitochondrial fusion, oxygen consumption, and Ca 2+ uptake increased in wild-type cells but not in cells lacking MCU. In proliferating wild-type control cells, the addition of the growth factors promoted the activation of the Ca 2+ /calmodulin-dependent kinase II (CaMKII) and the phosphorylation of the mitochondrial fission factor Drp1 at Ser 616 The lack of the MCU was associated with baseline activation of CaMKII, mitochondrial fragmentation due to increased Drp1 phosphorylation, and impaired mitochondrial respiration and glycolysis. The mitochondrial fission/fusion ratio and proliferation in MCU-deficient cells recovered after MCU restoration or inhibition of mitochondrial fragmentation or of CaMKII in the cytosol. Our data highlight a key function for the MCU in mitochondrial adaptation to the metabolic demands during cell cycle progression. Cytosolic CaMKII and the MCU participate in a regulatory circuit, whereby mitochondrial Ca 2+ uptake affects cell proliferation through Drp1., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Purpose: Randomized clinical trials (RCTs) are essential to evidence-based medicine, yet a significant proportion fail to be completed. In radiation oncology, factors contributing to trial failure are not well understood. We sought to compare completed and incomplete clinical trials involving radiation therapy (RT) to identify predictors of trial failure., Methods and Materials: We undertook a review of ClinicalTrials.gov to identify RCTs involving RT. Eligible trials mandated external beam RT in ≥1 arm of the study and were registered between September 27, 2007, and December 31, 2010. Univariate and multivariate logistic regression analyses were performed to determine factors predictive of trial failure., Results: We included 134 eligible studies, of which 94 (70.1%) were successful and 40 (29.9%) failed. The reasons for trial failure were categorized as follows: lack of accrual (57.5%), inadequate funding (15.0%), drug unavailability (7.5%), interim data-monitoring report recommendations (7.5%), and other (12.5%). Over time, significantly more trials were failing to be completed (P = .010), with rates increasing from 11.8% (before 2007) to 34.0% (2007-2008) to 39.5% (2009-2012). On univariate analysis, predictors of failure were trials with a surgical comparator (odds ratio [OR], 8.12; P = .013), government sponsorship (vs non-government; OR, 3.67; P = .025), inclusion of a safety endpoint (OR, 2.85; P = .022), and studies starting after 2006 (P = .033). On multivariate analysis, surgical trials were strongly predictive of failure (OR, 12.30; P = .025) while behavioral trials were associated with success (OR, 0.11; P = .045)., Conclusions: RT RCTs involving ≥1 surgical arms are at a very high risk of failure, with 75% failing to be completed. In contrast, behavioral studies are associated with study completion, with 94% of studies being successful. Future RT trials involving surgical interventions should consider novel methods to reduce the risk of trial failure., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: The main objective of this study is to define the mechanisms by which mitochondria control vascular smooth muscle cell (VSMC) migration and impact neointimal hyperplasia., Approach and Results: The multifunctional CaMKII (Ca 2+ /calmodulin-dependent kinase II) in the mitochondrial matrix of VSMC drove a feed-forward circuit with the mitochondrial Ca 2+ uniporter (MCU) to promote matrix Ca 2+ influx. MCU was necessary for the activation of mitochondrial CaMKII (mtCaMKII), whereas mtCaMKII phosphorylated MCU at the regulatory site S92 that promotes Ca 2+ entry. mtCaMKII was necessary and sufficient for platelet-derived growth factor-induced mitochondrial Ca 2+ uptake. This effect was dependent on MCU. mtCaMKII and MCU inhibition abrogated VSMC migration and mitochondrial translocation to the leading edge. Overexpression of wild-type MCU, but not MCU S92A, mutant in MCU - /- VSMC rescued migration and mitochondrial mobility. Inhibition of microtubule, but not of actin assembly, blocked mitochondrial mobility. The outer mitochondrial membrane GTPase Miro-1 promotes mitochondrial mobility via microtubule transport but arrests it in subcellular domains of high Ca 2+ concentrations. In Miro-1 -/- VSMC, mitochondrial mobility and VSMC migration were abolished, and overexpression of mtCaMKII or a CaMKII inhibitory peptide in mitochondria (mtCaMKIIN) had no effect. Consistently, inhibition of mtCaMKII increased and prolonged cytosolic Ca 2+ transients. mtCaMKII inhibition diminished phosphorylation of focal adhesion kinase and myosin light chain, leading to reduced focal adhesion turnover and cytoskeletal remodeling. In a transgenic model of selective mitochondrial CaMKII inhibition in VSMC, neointimal hyperplasia was significantly reduced after vascular injury., Conclusions: These findings identify mitochondrial CaMKII as a key regulator of mitochondrial Ca 2+ uptake via MCU, thereby controlling mitochondrial translocation and VSMC migration after vascular injury., (© 2018 American Heart Association, Inc.)

Activation of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and reactive oxygen species (ROS) promote neointimal hyperplasia after vascular injury. CaMKII can be directly activated by ROS through oxidation. In this study, we determined whether abolishing the oxidative activation site of CaMKII alters vascular smooth muscle cell (VCMC) proliferation, migration and apoptosis in vitro and neointimal formation in vivo. VSMC isolated from a knock-in mouse with oxidation-resistant CaMKIIδ (CaMKII M2V) displayed similar proliferation but decreased migration and apoptosis. Surprisingly, ROS production and expression of the NADPH oxidase subunits p47 and p22 were decreased in M2V VSMC, whereas superoxide dismutase 2 protein expression was upregulated. In vivo, after carotid artery ligation, no differences in neointimal size or remodeling were observed. In contrast to VSMC, CaMKII expression and autonomous activity were significantly higher in M2V compared to WT carotid arteries, suggesting that an autoregulatory mechanism determines CaMKII activity in vivo. Our findings demonstrate that preventing oxidative activation of CaMKII decreases migration and apoptosis in vitro and suggest that CaMKII regulates ROS production. Our study presents novel evidence that CaMKII expression in vivo is regulated by a negative feedback loop following oxidative activation., (Published by Elsevier Inc.)

Human alpha-defensins are evolutionarily conserved effectors of the innate immune response with broadly acting antibacterial activity. Their role in antiviral immunity is less well understood. We previously showed that these antimicrobial peptides are potent inhibitors of human adenovirus infection. Based on biochemical studies and indirect evidence from confocal microscopy, we proposed that defensins bind to and stabilize the virus capsid and neutralize infection by preventing the release of the endosomalytic protein VI. To determine whether defensin action also restricts exposure of the viral genome, we developed a system to evaluate adenovirus uncoating during cell entry by monitoring the exposure of BrdU-labeled viral genomes. This assay allowed us to determine the kinetics of uncoating of virus particles in single cells. Using this assay, we now provide direct evidence that human alpha-defensins block adenovirus infection by preventing uncoating during cell entry.