Immune correlates of protection of the four-segmented Rift Valley fever virus candidate vaccine in mice.

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease caused by RVF virus (RVFV). RVFV infections in humans are usually asymptomatic or associated with mild febrile illness, although more severe cases of haemorrhagic disease and encephalitis with high mortality also occur. Currently, there are no licensed human vaccines available. The safety and efficacy of a genetically engineered four-segmented RVFV variant (hRVFV-4s) as a potential live-attenuated human vaccine has been tested successfully in mice, ruminants, and marmosets though the correlates of protection of this vaccine are still largely unknown. In the present study, we have assessed hRVFV-4s-induced humoral and cellular immunity in a mouse model of RVFV infection. Our results confirm that a single dose of hRVFV-4s is highly efficient in protecting naïve mice from developing severe disease following intraperitoneal challenge with a highly virulent RVFV strain and data show that virus neutralizing (VN) serum antibody titres in a prime-boost regimen are significantly higher compared to the single dose. Subsequently, VN antibodies from prime-boost-vaccinated recipients were shown to be protective when transferred to naïve mice. In addition, hRVFV-4s vaccination induced a significant virus-specific T cell response as shown by IFN-γ ELISpot assay, though these T cells did not provide significant protection upon passive transfer to naïve recipient mice. Collectively, this study highlights hRVFV-4s-induced VN antibodies as a major correlate of protection against lethal RVFV infection.

Circulating T cell status and molecular imaging may predict clinical benefit of neoadjuvant PD-1 blockade in oral cancer.

BACKGROUND: Addition of neoadjuvant immune checkpoint inhibition to standard-of-care interventions for locally advanced oral cancer could improve clinical outcome. METHODS: In this study, 16 evaluable patients with stage III/IV oral cancer were treated with one dose of 480 mg nivolumab 3 weeks prior to surgery. Primary objectives were safety, feasibility, and suitability of programmed death receptor ligand-1 positron emission tomography (PD-L1 PET) as a biomarker for response. Imaging included 18F-BMS-986192 (PD-L1) PET and 18F-fluorodeoxyglucose (FDG) PET before and after nivolumab treatment. Secondary objectives included clinical and pathological response, and immune profiling of peripheral blood mononuclear cells (PBMCs) for response prediction. Baseline tumor biopsies and postnivolumab resection specimens were evaluated by histopathology. RESULTS: Grade III or higher adverse events were not observed and treatment was not delayed in relation to nivolumab administration and other study procedures. Six patients (38%) had a pathological response, of whom three (19%) had a major (≥90%) pathological response (MPR). Tumor PD-L1 PET uptake (quantified using standard uptake value) was not statistically different in patients with or without MPR (median 5.3 vs 3.4). All major responders showed a significantly postnivolumab decreased signal on FDG PET. PBMC immune phenotyping showed higher levels of CD8+ T cell activation in MPR patients, evidenced by higher baseline expression levels of PD-1, TIGIT, IFNγ and lower levels of PD-L1. CONCLUSION: Together these data support that neoadjuvant treatment of advanced-stage oral cancers with nivolumab was safe and induced an MPR in a promising 19% of patients. Response was associated with decreased FDG PET uptake as well as activation status of peripheral T cell populations.

Synthesis of 18F-labelled aryl trifluoromethyl ketones with improved molar activity.

A method for the radiosynthesis of 18F-labelled aryl trifluoromethyl ketones starting from widely available Weinreb amides using [18F]fluoroform is presented. The method uses potassium hexamethyldisilazane as base and delivers products in high molar activity (up to 24 GBq µmol-1) and excellent radiochemical conversions. The applicability for PET tracer synthesis is demonstrated by the radiosynthesis of ten (hetero)aryl trifluoromethylketones, bearing electron-withdrawing and -donating substituents including a derivative of bioactive probenecid.

Porous organic crystals crosslinked by free-radical reactions.

Two hydrogen-bonded crosslinked organic frameworks (HCOFs) were synthesized via free radical reactions utilizing butadiene and isoprene as crosslinkers. These HCOFs exhibit high crystallinity, enabling detailed structural characterization via single-crystal X-ray diffraction analysis. Subsequently, one of the olefin-rich HCOFs was converted to a hydroxylated framework through hydroboration-oxidation while maintaining the high crystallinity.

Advancing Parkinson's Disease Diagnostics: The Potential of Arylpyrazolethiazole Derivatives for Imaging α-Synuclein Aggregates.

The development of positron emission tomography (PET) tracers capable of detecting α-synuclein (α-syn) aggregates in vivo would represent a breakthrough for advancing the understanding and enabling the early diagnosis of Parkinson's disease and related disorders. It also holds the potential to assess the efficacy of therapeutic interventions. However, this remains challenging due to different structures of α-syn aggregates, the need for selectivity over other structurally similar amyloid proteins, like amyloid-ß (Aß), which frequently coexist with α-syn pathology, and the low abundance of the target in the brain that requires the development of a high-affinity ligand. To develop a successful PET tracer for the central nervous system (CNS), stringent criteria in terms of polarity and molecular size must also be considered, as the tracer must penetrate the blood-brain barrier and have low nonspecific binding to brain tissue. Here, we report a series of arylpyrazolethiazole (APT) derivatives, rationally designed from a structure-activity relationship study centered on existing ligands for α-syn fibrils, with a particular focus on the selectivity toward α-syn fibrils and control of physicochemical properties suitable for a CNS PET tracer. In vitro competition binding assays performed against [3H]MODAG-001 using recombinant α-syn and Aß1-42 fibrils revealed APT-13 with an inhibition constant of 27.8 ± 9.7 nM and a selectivity of more than 3.3 fold over Aß. Radiolabeled [11C]APT-13 demonstrated excellent brain penetration in healthy mice with a peak standardized uptake value of 1.94 ± 0.29 and fast washout from the brain (t 1/2 = 9 ± 1 min). This study highlights the potential of APT-13 as a lead compound for developing PET tracers to detect α-syn aggregates in vivo.

Navigating the landscape of PD-1/PD-L1 imaging tracers: from challenges to opportunities.

Immunotherapy targeted to immune checkpoint inhibitors, such as the program cell death receptor (PD-1) and its ligand (PD-L1), has revolutionized cancer treatment. However, it is now well-known that PD-1/PD-L1 immunotherapy response is inconsistent among patients. The current challenge is to customize treatment regimens per patient, which could be possible if the PD-1/PD-L1 expression and dynamic landscape are known. With positron emission tomography (PET) imaging, it is possible to image these immune targets non-invasively and system-wide during therapy. A successful PET imaging tracer should meet specific criteria concerning target affinity, specificity, clearance rate and target-specific uptake, to name a few. The structural profile of such a tracer will define its properties and can be used to optimize tracers in development and design new ones. Currently, a range of PD-1/PD-L1-targeting PET tracers are available from different molecular categories that have shown impressive preclinical and clinical results, each with its own advantages and disadvantages. This review will provide an overview of current PET tracers targeting the PD-1/PD-L1 axis. Antibody, peptide, and antibody fragment tracers will be discussed with respect to their molecular characteristics and binding properties and ways to optimize them.

Relations Between Perceptions of Parental Messages Supporting Fighting and Nonviolence and Adolescents' Physical Aggression: Beliefs as Mediators.

The purpose of this study was to examine adolescents' beliefs about fighting as mediators of longitudinal relations between perceptions of parental support for fighting and nonviolence and changes in adolescents' physical aggression. Participants were 2,575 middle school students (Mage = 12.20, SD = 1.02; 52% female; 83% African American) from the southeastern U.S. attending schools in communities with high rates of violence. Participants completed four waves of assessments every 3 months (i.e., fall, winter, spring, and summer). Each belief subscale mediated relations between perceptions of parental support for fighting and nonviolence and changes in aggression. Parental support for nonviolence was negatively associated with beliefs supporting reactive aggression and positively associated with beliefs against fighting. Parental support for retaliation was positively associated with beliefs supporting reactive and proactive aggression, and negatively associated with beliefs against fighting. Parental support for fighting as sometimes necessary was positively associated with beliefs supporting reactive aggression and beliefs that fighting is sometimes necessary. Beliefs supporting reactive and proactive aggression and beliefs that fighting is sometimes necessary were positively associated with aggression, whereas beliefs against fighting was negatively associated with aggression. Parents' support for fighting and for nonviolence may directly and indirectly reduce adolescents' physical aggression by influencing beliefs about the appropriateness of using aggression for self-defense and to attain a goal. This highlights the importance of jointly investigating multiple types of parental messages and types of beliefs about fighting.

Challenges in Evaluating a Community-Level Intervention to Address Root Causes of Youth Violence.

Violence disproportionately impacts Black American youth, representing a major health disparity. Addressing the possible root causes of structural inequities to reduce violence may increase the impact of prevention strategies. However, efforts to evaluate the impact of such interventions pose numerous methodological challenges, particularly around selecting an effective evaluation design to detect change at the community level, with adequate power and sampling, and appropriate constructs and measurement strategies. We propose a multiple baseline experimental design to evaluate the impact of a community-level youth violence and suicidality prevention strategy. A multiple baseline experimental design with multiple community units balances the need for scientific rigor with practical and values-based considerations. It includes randomization and plausible counterfactuals without requiring large samples or placing some communities in the position of not receiving the intervention. Considerations related to the conceptualization of the logic model, mechanisms of change, and health disparity outcomes informed the development of the measurement strategy. The strengths and weaknesses of a multiple baseline experimental design are discussed in comparison to versions of randomized clinical trials. Future health disparity intervention evaluation research will benefit from (1) building a shared sense of urgent public need to promote health; (2) respecting the validity of values- and partnership-based decision-making; and (3) promoting community-based and systems-level partnerships in scientific grant funding. The described study has been registered prospectively at clinicaltrials.gov, Protocol Record 21-454.

Challenges in Pharmacokinetic Modelling of [18F]fluoro-PEG-folate PET/CT Imaging in Epithelial Ovarian Cancer Patients.

PURPOSE: To describe the pharmacokinetic properties of the [18F]fluoro-polyethylene glycol(PEG)-folate radiotracer in PET/CT imaging of patients with advanced stage epithelial ovarian cancer (EOC). PROCEDURES: In five patients with advanced EOC (FIGO stage IIIB/IIIC, Fédération Internationale de Gynécologie et d'Obstétrique), a 90-min dynamic PET acquisition of the pelvis was performed directly after i.v. administration of 185 MBq [18F]fluoro-PEG6-folate. Arterial blood samples collected at nineteen timepoints were used to determine the plasma input function. A static volume of interest (VOI) for included tumor lesions was drawn manually on the PET images. Modelling was performed using PMOD software. Three different models (a 1-tissue compartment model (1T2k) and two 2-tissue compartment models, irreversible (2T3k) and reversible (2T4k)) were compared in goodness of fit with the time activity curves by means of the Akaike information criterion. RESULTS: The pharmacokinetic analysis in the pelvic area has proven to be much more challenging than expected. Only four out of 22 tumor lesions in five patients were considered suitable to perform modelling on. The remaining tumor lesions were inapt due to either low tracer uptake, small size, proximity to other [18F]fluoro-PEG6-folate -avid structures and/or displacement by abdominal organ motion in the dynamic scan. Data from the four analyzed tumor lesions suggest that the irreversible 2T3k may best describe the pharmacokinetics. All 22 lesions were immunohistochemically stained positive for the folate receptor alpha (FRα) after resection. CONCLUSION: Performing pharmacokinetic analysis in the abdominal pelvic region is very challenging. This brief article describes the challenges and pitfalls in pharmacokinetic analysis of a tracer with high physiological accumulation in the intestines, in case of lesions of limited size in the abdominal pelvic area.

Recent advances and impending challenges for the radiopharmaceutical sciences in oncology.

This paper is the first of a Series on theranostics that summarises the current landscape of the radiopharmaceutical sciences as they pertain to oncology. In this Series paper, we describe exciting developments in radiochemistry and the production of radionuclides, the development and translation of theranostics, and the application of artificial intelligence to our field. These developments are catalysing growth in the use of radiopharmaceuticals to the benefit of patients worldwide. We also highlight some of the key issues to be addressed in the coming years to realise the full potential of radiopharmaceuticals to treat cancer.

Trends in nuclear medicine and the radiopharmaceutical sciences in oncology: workforce challenges and training in the age of theranostics.

Although the promise of radionuclides for the diagnosis and treatment of disease was recognised soon after the discovery of radioactivity in the late 19th century, the systematic use of radionuclides in medicine only gradually increased over the subsequent hundred years. The past two decades, however, has seen a remarkable surge in the clinical application of diagnostic and therapeutic radiopharmaceuticals, particularly in oncology. This development is an exciting time for the use of theranostics in oncology, but the rapid growth of this area of nuclear medicine has created challenges as well. In particular, the infrastructure for the manufacturing and distribution of radiopharmaceuticals remains in development, and regulatory bodies are still optimising guidelines for this new class of drug. One issue of paramount importance for achieving equitable access to theranostics is building a sufficiently trained workforce in high-income, middle-income, and low-income countries. Here, we discuss the key challenges and opportunities that face the field as it seeks to build its workforce for the 21st century.

Subclinical Atherosclerosis to Guide Treatment in Dyslipidemia and Diabetes Mellitus.

PURPOSE OF REVIEW: Dyslipidemia and type 2 diabetes mellitus are two common conditions that are associated with an increased risk of atherosclerotic cardiovascular disease (ASCVD). In this review, we aimed to provide an in-depth and contemporary review of non-invasive approaches to assess subclinical atherosclerotic burden, predict cardiovascular risk, and guide appropriate treatment strategies. We focused this paper on two main imaging modalities: coronary artery calcium (CAC) score and computed tomography coronary angiography. RECENT FINDINGS: Recent longitudinal studies have provided stronger evidence on the relationship between increased CAC, thoracic aorta calcification, and risk of cardiovascular events among those with primary hypercholesterolemia, highlighting the beneficial role of statin therapy. Interestingly, resilient profiles of individuals not exhibiting atherosclerosis despite dyslipidemia have been described. Non-conventional markers of dyslipidemia have also been associated with increased subclinical atherosclerosis presence and burden, highlighting the contribution of apolipoprotein B-100 (apoB)-rich lipoprotein particles, such as remnant cholesterol and lipoprotein(a), to the residual risk of individuals on-target for low-density lipoprotein cholesterol (LDL-C) goals. Regarding type 2 diabetes mellitus, variability in atherosclerotic burden has also been found, and CAC testing has shown significant predictive value in stratifying cardiovascular risk. Non-invasive assessment of subclinical atherosclerosis can help reveal the continuum of ASCVD risk in those with dyslipidemia and diabetes mellitus and can inform personalized strategies for cardiovascular disease prevention in the primary prevention setting.

Synthesis of 18F-labeled Aryl Trifluoromethyl Sulfones, -Sulfoxides, and -Sulfides for Positron Emission Tomography.

Positron emission tomography (PET) is becoming increasingly important in nuclear medicine and drug discovery. To date, the development of many potential PET tracers is hampered by the lack of suitable synthetic pathways for their preparation. This is particularly true for the highly desired radiolabeling of compounds bearing [18F]CF3-groups. For instance, S(O)nCF3-groups (n=0, 1, 2) serve as structural motif in a range of biologically active compounds, but their radiosynthesis remains largely unprecedented (for n=1, 2). Herein, we describe general methods for the radiosynthesis of 18F-labeled aryl trifluoromethyl sulfones, -sulfoxides, and -sulfides. All three methods are operationally straightforward, start from widely available precursors, i.e., sulfonyl fluorides and thiophenols, and make use of the recently established [18F]Ruppert-Prakash reagent. Further, the syntheses display good functional group tolerance as demonstrated by the 18F-labeling of more than 40 compounds. The applicability of the new method is demonstrated by the radiolabeling of three bioactive molecules, optionally to be used as PET tracers. In a broader context, this work presents a substantial expansion of the chemical space of radiofluorinated structural motifs to be used for the development of new PET tracers.

Timeless noncoding DNA contains cell-type preferential enhancers important for proper Drosophila circadian regulation.

To address the contribution of transcriptional regulation to Drosophila clock gene expression and to behavior, we generated a series of CRISPR-mediated deletions within two regions of the circadian gene timeless (tim), an intronic E-box region and an upstream E-box region that are both recognized by the key transcription factor Clock (Clk) and its heterodimeric partner Cycle. The upstream deletions but not an intronic deletion dramatically impact tim expression in fly heads; the biggest upstream deletion reduces peak RNA levels and tim RNA cycling amplitude to about 15% of normal, and there are similar effects on tim protein (TIM). The cycling amplitude of other clock genes is also strongly reduced, in these cases due to increases in trough levels. These data underscore the important contribution of the upstream E-box enhancer region to tim expression and of TIM to clock gene transcriptional repression in fly heads. Surprisingly, tim expression in clock neurons is only modestly affected by the biggest upstream deletion and is similarly affected by a deletion of the intronic E-box region. This distinction between clock neurons and glia is paralleled by a dramatically enhanced accessibility of the intronic enhancer region within clock neurons. This distinctive feature of tim chromatin was revealed by ATAC-seq (assay for transposase-accessible chromatin with sequencing) assays of purified neurons and glia as well as of fly heads. The enhanced cell type-specific accessibility of the intronic enhancer region explains the resilience of clock neuron tim expression and circadian behavior to deletion of the otherwise more prominent upstream tim E-box region.

Structural basis of ligand recognition and design of antihistamines targeting histamine H4 receptor.

The histamine H4 receptor (H4R) plays key role in immune cell function and is a highly valued target for treating allergic and inflammatory diseases. However, structural information of H4R remains elusive. Here, we report four cryo-EM structures of H4R/Gi complexes, with either histamine or synthetic agonists clobenpropit, VUF6884 and clozapine bound. Combined with mutagenesis, ligand binding and functional assays, the structural data reveal a distinct ligand binding mode where D943.32 and a π-π network determine the orientation of the positively charged group of ligands, while E1825.46, located at the opposite end of the ligand binding pocket, plays a key role in regulating receptor activity. The structural insight into H4R ligand binding allows us to identify mutants at E1825.46 for which the agonist clobenpropit acts as an inverse agonist and to correctly predict inverse agonism of a closely related analog with nanomolar potency. Together with the findings regarding receptor activation and Gi engagement, we establish a framework for understanding H4R signaling and provide a rational basis for designing novel antihistamines targeting H4R.

Validation of image-derived input function using a long axial field of view PET/CT scanner for two different tracers.

BACKGROUND: Accurate image-derived input function (IDIF) from highly sensitive large axial field of view (LAFOV) PET/CT scanners could avoid the need of invasive blood sampling for kinetic modelling. The aim is to validate the use of IDIF for two kinds of tracers, 3 different IDIF locations and 9 different reconstruction settings. METHODS: Eight [18F]FDG and 10 [18F]DPA-714 scans were acquired respectively during 70 and 60 min on the Vision Quadra PET/CT system. PET images were reconstructed using various reconstruction settings. IDIFs were taken from ascending aorta (AA), descending aorta (DA), and left ventricular cavity (LV). The calibration factor (CF) extracted from the comparison between the IDIFs and the manual blood samples as reference was used for IDIFs accuracy and precision assessment. To illustrate the effect of various calibrated-IDIFs on Patlak linearization for [18F]FDG and Logan linearization for [18F]DPA-714, the same target time-activity curves were applied for each calibrated-IDIF. RESULTS: For [18F]FDG, the accuracy and precision of the IDIFs were high (mean CF ≥ 0.82, SD ≤ 0.06). Compared to the striatum influx (Ki) extracted using calibrated AA IDIF with the updated European Association of Nuclear Medicine Research Ltd. standard reconstruction (EARL2), Ki mean differences were < 2% using the other calibrated IDIFs. For [18F]DPA714, high accuracy of the IDIFs was observed (mean CF ≥ 0.86) except using absolute scatter correction, DA and LV (respectively mean CF = 0.68, 0.47 and 0.44). However, the precision of the AA IDIFs was low (SD ≥ 0.10). Compared to the distribution volume (VT) in a frontal region obtained using calibrated continuous arterial sampler input function as reference, VT mean differences were small using calibrated AA IDIFs (for example VT mean difference = -5.3% using EARL2), but higher using calibrated DA and LV IDIFs (respectively + 12.5% and + 19.1%). CONCLUSIONS: For [18F]FDG, IDIF do not need calibration against manual blood samples. For [18F]DPA-714, AA IDIF can replace continuous arterial sampling for simplified kinetic quantification but only with calibration against arterial blood samples. The accuracy and precision of IDIF from LAFOV PET/CT system depend on tracer, reconstruction settings and IDIF VOI locations, warranting careful optimization.

Filamentous fungus-produced human monoclonal antibody provides protection against SARS-CoV-2 in hamster and non-human primate models.

Monoclonal antibodies are an increasingly important tool for prophylaxis and treatment of acute virus infections like SARS-CoV-2 infection. However, their use is often restricted due to the time required for development, variable yields and high production costs, as well as the need for adaptation to newly emerging virus variants. Here we use the genetically modified filamentous fungus expression system Thermothelomyces heterothallica (C1), which has a naturally high biosynthesis capacity for secretory enzymes and other proteins, to produce a human monoclonal IgG1 antibody (HuMab 87G7) that neutralises the SARS-CoV-2 variants of concern (VOCs) Alpha, Beta, Gamma, Delta, and Omicron. Both the mammalian cell and C1 produced HuMab 87G7 broadly neutralise SARS-CoV-2 VOCs in vitro and also provide protection against VOC Omicron in hamsters. The C1 produced HuMab 87G7 is also able to protect against the Delta VOC in non-human primates. In summary, these findings show that the C1 expression system is a promising technology platform for the development of HuMabs in preventive and therapeutic medicine.

Synthesis and preclinical evaluation of [11C]EAI045 as a PET tracer for imaging tumors expressing mutated epidermal growth factor receptor.

BACKGROUND: Mutations in the epidermal growth factor receptor (EGFR) kinase domain are common in non-small cell lung cancer. Conventional tyrosine kinase inhibitors target the mutation site in the ATP binding pocket, thereby inhibiting the receptor's function. However, subsequent treatment resistance mutations in the ATP binding site are common. The EGFR allosteric inhibitor, EAI045, is proposed to have an alternative mechanism of action, disrupting receptor signaling independent of the ATP-binding site. The antibody cetuximab is hypothesized to increase the number of accessible allosteric pockets for EAI045, thus increasing the potency of the inhibitor. This work aimed to gain further knowledge on pharmacokinetics, the EGFR mutation-targeting potential, and the influence of cetuximab on the uptake by radiolabeling EAI045 with carbon-11 and tritium. RESULTS: 2-(5-fluoro-2-hydroxyphenyl)-2-((2-iodobenzyl)amino)-N-(thiazol-2-yl)acetamide and 2-(5-fluoro-2-hydroxyphenyl)-N-(5-iodothiazol-2-yl)-2-(1-oxoisoindolin-2-yl)acetamide were synthesized as precursors for the carbon-11 and tritium labeling of EAI045, respectively. [11C]EAI045 was synthesized using [11C]CO in a palladium-catalyzed ring closure in a 10 ± 1% radiochemical yield (decay corrected to end of [11C]CO2 production), > 97% radiochemical purity and 26 ± 1 GBq/µmol molar activity (determined at end of synthesis) in 51 min. [3H]EAI045 was synthesized by a tritium-halogen exchange in a 0.2% radiochemical yield, 98% radiochemical purity, and 763 kBq/nmol molar activity. The ability of [11C]EAI045 to differentiate between L858R/T790M mutated EGFR expressing H1975 xenografts and wild-type EGFR expressing A549 xenografts was evaluated in female nu/nu mice. The uptake was statistically significantly higher in H1975 xenografts compared to A549 xenografts (0.45 ± 0.07%ID/g vs. 0.31 ± 0.10%ID/g, P = 0.0166). The synergy in inhibition between EAI045 and cetuximab was evaluated in vivo and in vitro. While there was some indication that cetuximab influenced the uptake of [3H]EAI045 in vitro, this could not be confirmed in vivo when tumor-bearing mice were administered cetuximab (0.5 mg), 24 h prior to injection of [11C]EAI045. CONCLUSIONS: EAI045 was successfully labeled with tritium and carbon-11, and the in vivo results indicated [11C]EAI045 may be able to distinguish between mutated and non-mutated EGFR in non-small cell lung cancer mouse models. Cetuximab was hypothesized to increase EAI045 uptake; however, no significant effect was observed on the uptake of [11C]EAI045 in vivo or [3H]EAI045 in vitro in H1975 xenografts and cells.

Pharmacological Characterization and Radiolabeling of VUF15485, a High-Affinity Small-Molecule Agonist for the Atypical Chemokine Receptor ACKR3.

Atypical chemokine receptor 3 (ACKR3), formerly referred to as CXCR7, is considered to be an interesting drug target. In this study, we report on the synthesis, pharmacological characterization and radiolabeling of VUF15485, a new ACKR3 small-molecule agonist, that will serve as an important new tool to study this ß-arrestin-biased chemokine receptor. VUF15485 binds with nanomolar affinity (pIC50 = 8.3) to human ACKR3, as measured in [125I]CXCL12 competition binding experiments. Moreover, in a bioluminescence resonance energy transfer-based ß-arrestin2 recruitment assay VUF15485 acts as a potent ACKR3 agonist (pEC50 = 7.6) and shows a similar extent of receptor activation compared with CXCL12 when using a newly developed, fluorescence resonance energy transfer-based ACKR3 conformational sensor. Moreover, the ACKR3 agonist VUF15485, tested against a (atypical) chemokine receptor panel (agonist and antagonist mode), proves to be selective for ACKR3. VUF15485 labeled with tritium at one of its methoxy groups ([3H]VUF15485), binds ACKR3 saturably and with high affinity (K d = 8.2 nM). Additionally, [3H]VUF15485 shows rapid binding kinetics and consequently a short residence time (<2 minutes) for binding to ACKR3. The selectivity of [3H]VUF15485 for ACKR3, was confirmed by binding studies, whereupon CXCR3, CXCR4, and ACKR3 small-molecule ligands were competed for binding against the radiolabeled agonist. Interestingly, the chemokine ligands CXCL11 and CXCL12 are not able to displace the binding of [3H]VUF15485 to ACKR3. The radiolabeled VUF15485 was subsequently used to evaluate its binding pocket. Site-directed mutagenesis and docking studies using a recently solved cryo-EM structure propose that VUF15485 binds in the major and the minor binding pocket of ACKR3. SIGNIFICANCE STATEMENT: The atypical chemokine receptor atypical chemokine receptor 3 (ACKR3) is considered an interesting drug target in relation to cancer and multiple sclerosis. The study reports on new chemical biology tools for ACKR3, i.e., a new agonist that can also be radiolabeled and a new ACKR3 conformational sensor, that both can be used to directly study the interaction of ACKR3 ligands with the G protein-coupled receptor.

Concurrent Detection of a Papillomatous Lesion and Sequence Reads Corresponding to a Member of the Family Adintoviridae in a Bell's Hinge-Back Tortoise (Kinixys belliana).

An adult male Bell's hinge-back tortoise (Kinixys belliana) was admitted to a veterinary clinic due to a swelling in the oral cavity. Physical examination revealed an approximately 2.5 × 1.5 cm sized, irregularly shaped tissue mass with villiform projections extending from its surface located in the oropharyngeal cavity. An initial biopsy was performed, and the lesion was diagnosed as squamous papilloma. Swabs taken for virological examination tested negative with specific PCRs for papillomavirus and herpesvirus. Further analysis of the oropharyngeal mass via metagenomic sequencing revealed sequence reads corresponding to a member of the family Adintoviridae. The tissue mass was removed one week after the initial examination. The oral cavity remained unsuspicious in follow-up examinations performed after one, five and twenty weeks. However, a regrowth of the tissue was determined 23 months after the initial presentation. The resampled biopsy tested negative for sequence reads of Adintoviridae. Conclusively, this report presents the diagnostic testing and therapy of an oral cavity lesion of unknown origin. The significance of concurrent metagenomic determination of adintovirus sequence reads within the tissue lesion is discussed.