Combination immunotherapy with vaccine and oncolytic HSV virotherapy is time dependent.

PURPOSE: Oncolytic virotherapy or immunovirotherapy is a strategy that utilizes viruses to selectively infect and kill tumor cells while also stimulating an immune response against the tumor. Early clinical trials in both pediatric and adult patients using oncolytic herpes simplex viruses (oHSVs) have demonstrated safety and promising efficacy; however, combinatorial strategies designed to enhance oncolysis while also promoting durable T cell responses for sustaining disease remission are likely required. We hypothesized that combining the direct tumor cell killing and innate immune stimulation by oHSV with a vaccine that promotes T cell mediated immunity may lead to more durable tumor regression. EXPERIMENTAL DESIGN: To this end, we investigated the preclinical efficacy and potential synergy of combining oHSV with a self-assembling nanoparticle vaccine co-delivering peptide antigens and Toll-like receptor-7 and -8 agonists (TLR-7/8a) (referred to as SNAPvax™), that induces robust tumor specific T cell immunity. We then assessed how timing of the treatments (i.e., vaccine before or after oHSV) impacts T cell responses, viral replication, and preclinical efficacy. RESULTS: The sequence of treatments was critical, as survival was significantly enhanced when the SNAPvax™ vaccine was given prior to oHSV. Increased clinical efficacy was associated with reduced tumour volume and increases in virus replication and tumor antigen specific CD8+ T cells. CONCLUSIONS: These findings substantiate the criticality of combination immunotherapy timing and provide preclinical support for combining SNAPvax with oHSV as a promising treatment approach for both pediatric and adult tumors.

Immune checkpoint inhibitors and pericardial disease: a systematic review.

INTRODUCTION: Despite the growing use of immune checkpoint inhibitors (ICI) in cancer treatment, data regarding ICI-associated pericardial disease are primarily derived from case reports and case series. ICI related pericardial disease can be difficult to diagnose and is associated with significant morbidity. We conducted a systematic review to further characterize the epidemiology, clinical presentation, and outcomes of this patient population. METHODS: A search of four databases resulted in 31 studies meeting inclusion criteria. Patients > 18 years old who presented with ICI mediated pericardial disease were included. Intervention was medical + surgical therapy and outcomes were development of cardiac tamponade, morbidity, and mortality. RESULTS: Thirty- eight patients across 31 cases were included. Patients were majority male (72%) with a median age of 63. Common symptoms included dyspnea (59%) and chest pain (32%), with 41% presenting with cardiac tamponade. Lung cancer (81%) was the most prevalent, and nivolumab (61%) and pembrolizumab (34%) were the most used ICIs. Pericardiocentesis was performed in 68% of patients, and 92% experienced symptom improvement upon ICI cessation. Overall mortality was 16%. DISCUSSION: This study provides the most comprehensive analysis of ICI-mediated pericardial disease to date. Patients affected were most commonly male with lung cancer treated with either Nivolumab or Pembrolizumab. Diagnosis may be challenging in the setting of occult presentation with normal EKG and physical exam as well as delayed onset from therapy initiation. ICI-associated pericardial disease demonstrates high morbidity and mortality, as evidenced by a majority of patients requiring pericardiocentesis.

A genome-wide spectrum of tandem repeat expansions in 338,963 humans.

The Genome Aggregation Database (gnomAD), widely recognized as the gold-standard reference map of human genetic variation, has largely overlooked tandem repeat (TR) expansions, despite the fact that TRs constitute ∼6% of our genome and are linked to over 50 human diseases. Here, we introduce the TR-gnomAD (https://wlcb.oit.uci.edu/TRgnomAD), a biobank-scale reference of 0.86 million TRs derived from 338,963 whole-genome sequencing (WGS) samples of diverse ancestries (39.5% non-European samples). TR-gnomAD offers critical insights into ancestry-specific disease prevalence using disparities in TR unit number frequencies among ancestries. Moreover, TR-gnomAD is able to differentiate between common, presumably benign TR expansions, which are prevalent in TR-gnomAD, from those potentially pathogenic TR expansions, which are found more frequently in disease groups than within TR-gnomAD. Together, TR-gnomAD is an invaluable resource for researchers and physicians to interpret TR expansions in individuals with genetic diseases.

Multimodal epigenetic sequencing analysis (MESA) of cell-free DNA for non-invasive colorectal cancer detection.

BACKGROUND: Detecting human cancers through cell-free DNA (cfDNA) in blood is a sensitive and non-invasive option. However, capturing multiple forms of epigenetic information remains a technical and financial challenge. METHODS: To address this, we developed multimodal epigenetic sequencing analysis (MESA), a flexible and sensitive approach to capturing and integrating a diverse range of epigenetic features in cfDNA using a single experimental assay, i.e., non-disruptive bisulfite-free methylation sequencing, such as Enzymatic Methyl-seq. MESA enables simultaneous inference of four epigenetic modalities: cfDNA methylation, nucleosome occupancy, nucleosome fuzziness, and windowed protection score for regions surrounding gene promoters and polyadenylation sites. RESULTS: When applied to 690 cfDNA samples from 3 colorectal cancer clinical cohorts, MESA's novel modalities, which include nucleosome fuzziness, and genomic features, including polyadenylation sites, improve cancer detection beyond the traditional epigenetic markers of promoter DNA methylation. CONCLUSIONS: Together, MESA stands as a major advancement in the field by utilizing comprehensive and complementary epigenetic profiles of cfDNA for effective non-invasive cancer detection.

Lite It Fly: An All-Deformable-Butterfly Network.

Most deep neural networks (DNNs) consist fundamentally of convolutional and/or fully connected layers, wherein the linear transform can be cast as the product between a filter matrix and a data matrix obtained by arranging feature tensors into columns. Recently proposed deformable butterfly (DeBut) decomposes the filter matrix into generalized, butterfly-like factors, thus achieving network compression orthogonal to the traditional ways of pruning or low-rank decomposition. This work reveals an intimate link between DeBut and a systematic hierarchy of depthwise and pointwise convolutions, which explains the empirically good performance of DeBut layers. By developing an automated DeBut chain generator, we show for the first time the viability of homogenizing a DNN into all DeBut layers, thus achieving extreme sparsity and compression. Various examples and hardware benchmarks verify the advantages of All-DeBut networks. In particular, we show it is possible to compress a PointNet to 5% parameters with 5% accuracy drop, a record not achievable by other compression schemes.

Emerging Landscape of Preclinical Models for Studying COVID-19 Neurologic Diseases.

COVID-19 (Coronavirus Disease 2019) is an infectious disease caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and has globally infected 768 million people and caused over 6 million deaths. COVID-19 primarily affects the respiratory system but increasing reports of neurologic symptoms associated with COVID-19 have been reported in the literature. The exact mechanism behind COVID-19 neurologic pathophysiology remains poorly understood due to difficulty quantifying clinical neurologic symptoms in humans and correlating them to findings in human post-mortem samples and animal models. Thus, robust preclinical experimental models for COVID-19 neurologic manifestations are urgently needed. Here, we review recent advances in in vitro, in vivo, and other models and technologies for studying COVID-19 including primary cell cultures, pluripotent stem cell-derived neurons and organoids, rodents, nonhuman primates, 3D bioprinting, artificial intelligence, and multiomics. We specifically focus our discussion on the contribution, recent advancements, and limitations these preclinical models have on furthering our understanding of COVID-19's neuropathic physiology. We also discuss these models' roles in the screening and development of therapeutics, vaccines, antiviral drugs, and herbal medicine, and on future opportunities for COVID-19 neurologic research and clinical management.

Post-Myocardial Infarction Free-Wall Rupture: Rapid Diagnosis and Management.

We present a case of post-myocardial infarction free-wall rupture in a critically ill patient presenting to the emergency department. Through our case we highlight the prompt evaluation, diagnosis, and management necessary to improve survival in a patient with this life-threatening condition. (Level of Difficulty: Beginner.).

TRPM7 transactivates the FOSL1 gene through STAT3 and enhances glioma stemness.

INTRODUCTION: We previously reported that TRPM7 regulates glioma cells' stemness through STAT3. In addition, we demonstrated that FOSL1 is a response gene for TRPM7, and the FOSL1 gene serves as an oncogene to promote glioma proliferation and invasion. METHODS: In the present study, we determined the effects of FOSL1 on glioma stem cell (GSC) markers CD133 and ALDH1 by flow cytometry, and the maintenance of stem cell activity by extreme limiting dilution assays (ELDA). To further gain insight into the mechanism by which TRPM7 activates transcription of the FOSL1 gene to contribute to glioma stemness, we constructed a FOSL1 promoter and its GAS mutants followed by luciferase reporter assays and ChIP-qPCR in a glioma cell line and glioma patient-derived xenoline. We further examined GSC markers ALDH1 and TRPM7 as well as FOSL1 by immunohistochemistry staining (IHC) in brain tissue microarray (TMA) of glioma patients. RESULTS: We revealed that FOSL1 knockdown reduces the expression of GSC markers CD133 and ALDH1, and FOSL1 is required to maintain stem cell activity in glioma cells. The experiments also showed that mutations of - 328 to - 336 and - 378 to - 386 GAS elements markedly reduced FOSL1 promoter activity. Constitutively active STAT3 increased while dominant-negative STAT3 decreased FOSL1 promoter activity. Furthermore, overexpression of TRPM7 enhanced while silencing of TRPM7 reduced FOSL1 promoter activity. ChIP-qPCR assays revealed that STAT3, present in nuclear lysates of glioma cells stimulated by constitutively activated STAT3, can bind to two GAS elements, respectively. We demonstrated that deacetylation of FOSL1 at the Lys-116 residue located within its DNA binding domain led to an increase in FOSL1 transcriptional activity. We found that the expression of TRPM7, ALDH1, and FOSL1 protein is associated with grades of malignant glioma, and TRPM7 protein expression correlates to the expression of ALDH1 and FOSL1 in glioma patients. CONCLUSIONS: These combined results demonstrated that TRPM7 induced FOSL1 transcriptional activation, which is mediated by the action of STAT3, a mechanism shown to be important in glioma stemness. These results indicated that FOSL1, similar to GSC markers ALDH1 and TRPM7, is a diagnostic marker and potential drug target for glioma patients.

Cardiovascular Protection by Metformin: Latest Advances in Basic and Clinical Research.

BACKGROUND: Metformin is among the most frequently prescribed antidiabetic drugs worldwide and remains the first-line therapy for type 2 diabetes due to its well-established glucose-lowering efficacy and favorable safety profile. SUMMARY: Studies over the past decades show that metformin also exerts many other beneficial effects independent of its glucose-lowering effect both in experimental models and human subjects. Among them, the most notable is its cardiovascular protective effect. In this review, we discuss the latest cutting-edge research findings on metformin's cardiovascular protection from both preclinical studies and randomized clinical trials. We focus on describing novel basic research discoveries reported in influential journals and discussing their implications in the context of latest clinical trial findings related to common cardiovascular and metabolic disorders, including atherosclerosis and dyslipidemia, myocardial injury, and heart failure. KEY MESSAGES: While substantial preclinical and clinical evidence suggests metformin as a potential cardiovascular protectant, large-scale randomized controlled trials are warranted to establish its clinical efficacy in treating patients with atherosclerotic cardiovascular disease and heart failure.

An allosteric pan-TEAD inhibitor blocks oncogenic YAP/TAZ signaling and overcomes KRAS G12C inhibitor resistance.

The Hippo pathway is a key growth control pathway that is conserved across species. The downstream effectors of the Hippo pathway, YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif), are frequently activated in cancers to drive proliferation and survival. Based on the premise that sustained interactions between YAP/TAZ and TEADs (transcriptional enhanced associate domain) are central to their transcriptional activities, we discovered a potent small-molecule inhibitor (SMI), GNE-7883, that allosterically blocks the interactions between YAP/TAZ and all human TEAD paralogs through binding to the TEAD lipid pocket. GNE-7883 effectively reduces chromatin accessibility specifically at TEAD motifs, suppresses cell proliferation in a variety of cell line models and achieves strong antitumor efficacy in vivo. Furthermore, we uncovered that GNE-7883 effectively overcomes both intrinsic and acquired resistance to KRAS (Kirsten rat sarcoma viral oncogene homolog) G12C inhibitors in diverse preclinical models through the inhibition of YAP/TAZ activation. Taken together, this work demonstrates the activities of TEAD SMIs in YAP/TAZ-dependent cancers and highlights their potential broad applications in precision oncology and therapy resistance.

Artificial intelligence for clinical decision support for monitoring patients in cardiovascular ICUs: A systematic review.

Background: Artificial intelligence (AI) and machine learning (ML) models continue to evolve the clinical decision support systems (CDSS). However, challenges arise when it comes to the integration of AI/ML into clinical scenarios. In this systematic review, we followed the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA), the population, intervention, comparator, outcome, and study design (PICOS), and the medical AI life cycle guidelines to investigate studies and tools which address AI/ML-based approaches towards clinical decision support (CDS) for monitoring cardiovascular patients in intensive care units (ICUs). We further discuss recent advances, pitfalls, and future perspectives towards effective integration of AI into routine practices as were identified and elaborated over an extensive selection process for state-of-the-art manuscripts. Methods: Studies with available English full text from PubMed and Google Scholar in the period from January 2018 to August 2022 were considered. The manuscripts were fetched through a combination of the search keywords including AI, ML, reinforcement learning (RL), deep learning, clinical decision support, and cardiovascular critical care and patients monitoring. The manuscripts were analyzed and filtered based on qualitative and quantitative criteria such as target population, proper study design, cross-validation, and risk of bias. Results: More than 100 queries over two medical search engines and subjective literature research were developed which identified 89 studies. After extensive assessments of the studies both technically and medically, 21 studies were selected for the final qualitative assessment. Discussion: Clinical time series and electronic health records (EHR) data were the most common input modalities, while methods such as gradient boosting, recurrent neural networks (RNNs) and RL were mostly used for the analysis. Seventy-five percent of the selected papers lacked validation against external datasets highlighting the generalizability issue. Also, interpretability of the AI decisions was identified as a central issue towards effective integration of AI in healthcare.

Coronavirus disease 2019 (COVID-19) outbreak on an in-patient medical unit associated with unrecognized exposures in common areas-Epidemiological and whole-genome sequencing investigation.

OBJECTIVE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hospital outbreaks have been common and devastating during the coronavirus disease 2019 (COVID-19) pandemic. Understanding SARS-CoV-2 transmission in these environments is critical for preventing and managing outbreaks. DESIGN: Outbreak investigation through epidemiological mapping and whole-genome sequencing phylogeny. SETTING: Hospital in-patient medical unit outbreak in Toronto, Canada, from November 2020 to January 2021. PARTICIPANTS: The outbreak involved 8 patients and 10 staff and was associated with 3 patient deaths. RESULTS: Patients being cared for in geriatric chairs at the nursing station were at high risk for both acquiring and transmitting SARS-CoV-2 to other patients and staff. Furthermore, given the informal nature of these transmissions, they were not initially recognized, which led to further transmission and missing the opportunity for preventative COVID-19 therapies. CONCLUSIONS: During outbreak prevention and management, the risk of informal patient care settings, such as geriatric chairs, should be considered. During high-risk periods or during outbreaks, efforts should be made to care for patients in their rooms when possible.

BRCA1 and BRCA2 carriers with breast, ovarian and prostate cancer demonstrate a different pattern of metastatic disease compared with non-carriers: results from a rapid autopsy programme.

AIM: To catalogue and compare the pattern of metastatic disease in germline BRCA1/2 pathogenic mutation carriers and non-carriers with breast, ovarian and prostate cancer from a rapid autopsy programme. METHODS AND RESULTS: The number of metastases in the major body systems and the proportion of participants with metastases were documented in 50 participants (19 germline mutation carriers). Analysis was conducted on the participants' pattern of disease for the different cancers and mutation subgroups. The four commonly affected organ systems were the digestive (liver only) (82%), respiratory (76%), gastrointestinal (65%) and reticuloendothelial (42%). There were significant differences in the pattern of metastatic breast cancer in BRCA1/2 germline carriers compared with non-carriers. Breast cancer carriers had significantly fewer organ systems involved (median n = 3, range = 1-3) compared with non-carriers (median n = 9, range = 1-7) (P = 0.03). BRCA1/2 carriers with ovarian carcinomas had significantly more organ systems with metastatic carcinoma (median n = 10, range = 3-8) than non-carriers (median n = 5, range = 3-5) (P < 0.001). There were no significant differences in the number of involved systems in BRCA2 carriers compared with non-carriers with prostate cancer (P = 1.0). There was an absence of locoregional disease (6.5%) compared with distant disease (93.5%) among the three cancer subtypes (P < 0.001). The majority of metastatic deposits (97%) collected during the autopsy were identified by recent diagnostic imaging. CONCLUSION: Even though a major limitation of this study is that our numbers are small, especially in the breast cancer carrier group, the metastatic patterns of breast and ovarian cancers may be impacted by BRCA1/2 carrier status, suggesting that tumours derived from patients with these mutations use different mechanisms of dissemination. The findings may focus clinical diagnostic imaging for monitoring metastases where whole-body imaging resources are scant.

Addressing the Physical and Mental Impacts of Postsurgical Scarring Among Transgender and Gender Diverse People.

Few studies have considered the diverse mental and physical health impacts of scarring among transgender and gender diverse (TGD) patients after medically necessary gender-affirming surgery (GAS). For some TGD patients, post-GAS scarring may exacerbate gender dysphoria. For others, it is a physical representation of authenticity. The dearth of research or validated instruments capturing the diversity of priorities and concerns pre- and post-GAS hinders providers' ability to deliver optimal clinical care throughout the gender-affirmation process and impedes progress for evidence-based policy change regarding post-GAS scar treatment. This article provides suggestions for future research directions to address post-GAS scar-related health needs.

Alternative polyadenylation transcriptome-wide association study identifies APA-linked susceptibility genes in brain disorders.

Alternative polyadenylation (APA) plays an essential role in brain development; however, current transcriptome-wide association studies (TWAS) largely overlook APA in nominating susceptibility genes. Here, we performed a 3' untranslated region (3'UTR) APA TWAS (3'aTWAS) for 11 brain disorders by combining their genome-wide association studies data with 17,300 RNA-seq samples across 2,937 individuals. We identified 354 3'aTWAS-significant genes, including known APA-linked risk genes, such as SNCA in Parkinson's disease. Among these 354 genes, ~57% are not significant in traditional expression- and splicing-TWAS studies, since APA may regulate the translation, localization and protein-protein interaction of the target genes independent of mRNA level expression or splicing. Furthermore, we discovered ATXN3 as a 3'aTWAS-significant gene for amyotrophic lateral sclerosis, and its modulation substantially impacted pathological hallmarks of amyotrophic lateral sclerosis in vitro. Together, 3'aTWAS is a powerful strategy to nominate important APA-linked brain disorder susceptibility genes, most of which are largely overlooked by conventional expression and splicing analyses.

In Vitro and In Vivo Characterization of 89Zirconium-Labeled Lintuzumab Molecule.

Objective: Positron emission tomography (PET) imaging is a powerful non-invasive method to determine the in vivo behavior of biomolecules. Determining biodistribution and pharmacokinetic (PK) properties of targeted therapeutics can enable a better understanding of in vivo drug mechanisms such as tumor uptake, off target accumulation and clearance. Zirconium-89 (89Zr) is a readily available tetravalent PET-enabling radiometal that has been used to evaluate the biodistribution and PK of monoclonal antibodies. In the current study, we performed in vitro and in vivo characterization of 89Zr-lintuzumab, a radiolabeled anti-CD33 antibody, as a model to evaluate the in vivo binding properties in preclinical models of AML. Methods: Lintuzumab was conjugated to p-SCN-Bn-deferoxamine (DFO) and labeled with 89Zr using a 5:1 µCi:µg specific activity at 37 °C for 1h. The biological activity of 89Zr-lintuzumab was evaluated in a panel of CD33 positive cells using flow cytometry. Fox Chase SCID mice were injected with 2 × 106 OCI-AML3 cells into the right flank. After 12 days, a cohort of mice (n = 4) were injected with 89Zr-lintuzumab via tail vein. PET/CT scans of mice were acquired on days 1, 2, 3 and 7 post 89Zr-lintuzumab injection. To demonstrate 89Zr-lintuzumab specific binding to CD33 expressing tumors in vivo, a blocking study was performed. This cohort of mice (n = 4) was injected with native lintuzumab and 24 h later 89Zr-lintuzumab was administered. This group was imaged 3 and 7 days after injection of 89Zr-lintuzumab. A full ex vivo biodistribution study on both cohorts was performed on day 7. The results from the PET image and ex vivo biodistribution studies were compared. Results: Lintuzumab was successfully radiolabeled with 89Zr resulting in a 99% radiochemical yield. The 89Zr-lintuzumab radioconjugate specifically binds CD33 positive cells in a similar manner to native lintuzumab as observed by flow cytometry. PET imaging revealed high accumulation of 89Zr-lintuzumab in OCI-AML3 tumors within 24h post-injection of the radioconjugate. The 89Zr-lintuzumab high tumor uptake remains for up to 7 days. Tumor analysis of the PET data using volume of interest (VOI) showed significant blocking of 89Zr-lintuzumab in the group pre-treated with native lintuzumab (pre-blocked group), thus indicating specific targeting of CD33 on OCI-AML3 cells in vivo. The tumor uptake findings from the PET imaging study are in agreement with those from the ex vivo biodistribution results. Conclusions: PET imaging of 89Zr-lintuzumab shows high specific uptake in CD33 positive human OCI-AML3 tumors. The results from the image study agree with the observations from the ex vivo biodistribution study. Our findings collectively suggest that PET imaging using 89Zr-lintuzumab could be a powerful drug development tool to evaluate binding properties of anti-CD33 monoclonal antibodies in preclinical cancer models.

Inhibition of mutant IDH1 promotes cycling of acute myeloid leukemia stem cells.

Approximately 20% of acute myeloid leukemia (AML) patients carry mutations in IDH1 or IDH2 that result in over-production of the oncometabolite D-2-hydroxyglutarate (2-HG). Small molecule inhibitors that block 2-HG synthesis can induce complete morphological remission; however, almost all patients eventually acquire drug resistance and relapse. Using a multi-allelic mouse model of IDH1-mutant AML, we demonstrate that the clinical IDH1 inhibitor AG-120 (ivosidenib) exerts cell-type-dependent effects on leukemic cells, promoting delayed disease regression. Although single-agent AG-120 treatment does not fully eradicate the disease, it increases cycling of rare leukemia stem cells and triggers transcriptional upregulation of the pyrimidine salvage pathway. Accordingly, AG-120 sensitizes IDH1-mutant AML to azacitidine, with the combination of AG-120 and azacitidine showing vastly improved efficacy in vivo. Our data highlight the impact of non-genetic heterogeneity on treatment response and provide a mechanistic rationale for the observed combinatorial effect of AG-120 and azacitidine in patients.

Effect of Incorporating 1 Avocado Per Day Versus Habitual Diet on Visceral Adiposity: A Randomized Trial.

Background Excess visceral adiposity is associated with increased risk of cardiometabolic disorders. Short-term well-controlled clinical trials suggest that regular avocado consumption favorably affects body weight, visceral adiposity, and satiety. Methods and Results The HAT Trial (Habitual Diet and Avocado Trial) was a multicenter, randomized, controlled parallel-arm trial designed to test whether consuming 1 large avocado per day for 6 months in a diverse group of free-living individuals (N=1008) with an elevated waist circumference compared with a habitual diet would decrease visceral adiposity as measured by magnetic resonance imaging. Secondary and additional end points related to risk factors associated with cardiometabolic disorders were assessed. The primary outcome, change in visceral adipose tissue volume during the intervention period, was not significantly different between the Avocado Supplemented and Habitual Diet Groups (estimated mean difference (0.017 L [-0.024 L, 0.058 L], P=0.405). No significant group differences were observed for the secondary outcomes of hepatic fat fraction, hsCRP (high-sensitivity C-reactive protein), and components of the metabolic syndrome. Of the additional outcome measures, modest but nominally significant reductions in total and low-density lipoprotein cholesterol were observed in the Avocado Supplemented compared with the Habitual Diet Group. Changes in the other additional and post hoc measures (body weight, body mass index, insulin, very low-density lipoprotein concentrations, and total cholesterol:high-density lipoprotein cholesterol ratio) were similar between the 2 groups. Conclusions Addition of 1 avocado per day to the habitual diet for 6 months in free-living individuals with elevated waist circumference did not reduce visceral adipose tissue volume and had minimal effect on risk factors associated with cardiometabolic disorders. Registration URL: https://clinicaltrials.gov; Unique identifier: NCT03528031.