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.

CD19-immunoPET for noninvasive visualization of CD19 expression in B-cell lymphoma patients.

Cell- and antibody-based CD19-directed therapies have demonstrated great potential for treating B-cell non-Hodgkin lymphoma (B-NHL). However, all these approaches suffer from limited response rates and considerable toxicity. Until now, therapy decisions have been routinely based on histopathological CD19 staining of a single lesion at initial diagnosis or relapse, disregarding heterogeneity and temporal alterations in antigen expression. To visualize in vivo CD19 expression noninvasively, we radiolabeled anti-human CD19 monoclonal antibodies with copper-64 (64Cu-αCD19) for positron emission tomography (CD19-immunoPET). 64Cu-αCD19 specifically bound to subcutaneous Daudi xenograft mouse models in vivo. Importantly, 64Cu-αCD19 did not affect the anti-lymphoma cytotoxicity of CD19 CAR-T cells in vitro. Following our preclinical validation, 64Cu-αCD19 was injected into four patients with follicular lymphoma, diffuse large B-cell lymphoma or mantle zone lymphoma. We observed varying 64Cu-αCD19 PET uptake patterns at different lymphoma sites, both within and among patients, correlating with ex vivo immunohistochemical CD19 expression. Moreover, one patient exhibited enhanced uptake in the spleen compared to that in patients with prior B-cell-depleting therapy, indicating that 64Cu-αCD19 is applicable for identifying B-cell-rich organs. In conclusion, we demonstrated the specific targeting and visualization of CD19+ B-NHL in mice and humans by CD19-immunoPET. The intra- and interindividual heterogeneous 64Cu-αCD19 uptake patterns of lymphoma lesions indicate variability in CD19 expression, suggesting the potential of CD19-immunoPET as a novel tool to guide CD19-directed therapies.

Investigating the genetic control of plant development in spring barley under speed breeding conditions.

KEY MESSAGE: This study found that the genes, PPD-H1 and ELF3, control the acceleration of plant development under speed breeding, with important implications for optimizing the delivery of climate-resilient crops. Speed breeding is a tool to accelerate breeding and research programmes. Despite its success and growing popularity with breeders, the genetic basis of plant development under speed breeding remains unknown. This study explored the developmental advancements of barley genotypes under different photoperiod regimes. A subset of the HEB-25 Nested Association Mapping population was evaluated for days to heading and maturity under two contrasting photoperiod conditions: (1) Speed breeding (SB) consisting of 22 h of light and 2 h of darkness, and (2) normal breeding (NB) consisting of 16 h of light and 8 h of darkness. GWAS revealed that developmental responses under both conditions were largely controlled by two loci: PPDH-1 and ELF3. Allelic variants at these genes determine whether plants display early flowering and maturity under both conditions. At key QTL regions, domesticated alleles were associated with late flowering and maturity in NB and early flowering and maturity in SB, whereas wild alleles were associated with early flowering under both conditions. We hypothesize that this is related to the dark-dependent repression of PPD-H1 by ELF3 which might be more prominent in NB conditions. Furthermore, by comparing development under two photoperiod regimes, we derived an estimate of plasticity for the two traits. Interestingly, plasticity in development was largely attributed to allelic variation at ELF3. Our results have important implications for our understanding and optimization of speed breeding protocols particularly for introgression breeding and the design of breeding programmes to support the delivery of climate-resilient crops.

Western diet increases brain metabolism and adaptive immune responses in a mouse model of amyloidosis.

Diet-induced increase in body weight is a growing health concern worldwide. Often accompanied by a low-grade metabolic inflammation that changes systemic functions, diet-induced alterations may contribute to neurodegenerative disorder progression as well. This study aims to non-invasively investigate diet-induced metabolic and inflammatory effects in the brain of an APPPS1 mouse model of Alzheimer's disease. [18F]FDG, [18F]FTHA, and [18F]GE-180 were used for in vivo PET imaging in wild-type and APPPS1 mice. Ex vivo flow cytometry and histology in brains complemented the in vivo findings. 1H- magnetic resonance spectroscopy in the liver, plasma metabolomics and flow cytometry of the white adipose tissue were used to confirm metaflammatory condition in the periphery. We found disrupted glucose and fatty acid metabolism after Western diet consumption, with only small regional changes in glial-dependent neuroinflammation in the brains of APPPS1 mice. Further ex vivo investigations revealed cytotoxic T cell involvement in the brains of Western diet-fed mice and a disrupted plasma metabolome. 1H-magentic resonance spectroscopy and immunological results revealed diet-dependent inflammatory-like misbalance in livers and fatty tissue. Our multimodal imaging study highlights the role of the brain-liver-fat axis and the adaptive immune system in the disruption of brain homeostasis in amyloid models of Alzheimer's disease.

Immunocytokines with target cell-restricted IL-15 activity for treatment of B cell malignancies.

Despite the advances in cancer treatment achieved, for example, by the CD20 antibody rituximab, an urgent medical need remains to optimize the capacity of such antibodies to induce antibody-dependent cellular cytotoxicity (ADCC) that determines therapeutic efficacy. The cytokine IL-15 stimulates proliferation, activation, and cytolytic capacity of NK cells, but broad clinical use is prevented by short half-life, poor accumulation at the tumor site, and severe toxicity due to unspecific immune activation. We here report modified immunocytokines consisting of Fc-optimized CD19 and CD20 antibodies fused to an IL-15 moiety comprising an L45E-E46K double mutation (MIC+ format). The E46K mutation abrogated binding to IL-15Rα, thereby enabling substitution of physiological trans-presentation by target binding and thus conditional IL-15Rßγ stimulation, whereas the L45E mutation optimized IL-15Rßγ agonism and producibility. In vitro analysis of NK activation, anti-leukemia reactivity, and toxicity using autologous and allogeneic B cells confirmed target-dependent function of MIC+ constructs. Compared with Fc-optimized CD19 and CD20 antibodies, MIC+ constructs mediated superior target cell killing and NK cell proliferation. Mouse models using luciferase-expressing human NALM-6 lymphoma cells, patient acute lymphoblastic leukemia (ALL) cells, and murine EL-4 lymphoma cells transduced with human CD19/CD20 as targets and human and murine NK cells as effectors, respectively, confirmed superior and target-dependent anti-leukemic activity. In summary, MIC+ constructs combine the benefits of Fc-optimized antibodies and IL-15 cytokine activity and mediate superior NK cell immunity with potentially reduced side effects. They thus constitute a promising new immunotherapeutic approach shown here for B cell malignancies.

[18F]pFBC, a Covalent CLIP-Tag Radiotracer for Detection of Viral Reporter Gene Transfer in the Murine Brain.

Preclinical models of neurological diseases and gene therapy are essential for neurobiological research. However, the evaluation of such models lacks reliable reporter systems for use with noninvasive imaging methods. Here, we report the development of a reporter system based on the CLIP-tag enzyme and [18F]pFBC, an 18F-labeled covalent CLIP-tag-ligand synthesized via a DoE-optimized and fully automated process. We demonstrated its specificity using a subcutaneous xenograft model and a model of viral vector-mediated brain gene transfer by engineering HEK293 cells and striatal neurons to express membrane-tethered CLIP-tag protein. After in vitro characterization of the reporter, mice carrying either CLIP-tag expressing or control subcutaneous xenografts underwent dynamic [18F]pFBC PET imaging. The CLIP-tag expressing xenografts showed a significantly higher uptake than control xenografts (tumor-to-muscle ratio 5.0 vs 1.7, p = 0.0379). In vivo, metabolite analysis by radio-HPLC from plasma and brain homogenates showed only one radio-metabolite in plasma and none in the brain. In addition, [18F]pFBC showed fast uptake and rapid clearance from the brain in animals injected with adeno-associated virus (AAV)-CLIP in the right striatum but no right-to-left (R-L) uptake difference in the striata in the acquired PET data. In contrast, autoradiography showed a clear accumulation of radioactivity in the AAV-CLIP-injected right striatum compared to the sham-injected left striatum control. CLIP-tag expression and brain integrity were verified by immunofluorescence and light sheet microscopy. In conclusion, we established a novel reporter gene system for PET imaging of gene expression in the brain and periphery and demonstrated its potential for a wide range of applications, particularly for neurobiological research and gene therapy with viral vectors.

Dynamic Phytomeric Growth Contributes to Local Adaptation in Barley.

Vascular plants have segmented body axes with iterative nodes and internodes. Appropriate node initiation and internode elongation are fundamental to plant fitness and crop yield; however, how these events are spatiotemporally coordinated remains elusive. We show that in barley (Hordeum vulgare L.), selections during domestication have extended the apical meristematic phase to promote node initiation, but constrained subsequent internode elongation. In both vegetative and reproductive phases, internode elongation displays a dynamic proximal-distal gradient, and among subpopulations of domesticated barleys worldwide, node initiation and proximal internode elongation are associated with latitudinal and longitudinal gradients, respectively. Genetic and functional analyses suggest that, in addition to their converging roles in node initiation, flowering-time genes have been repurposed to specify the timing and duration of internode elongation. Our study provides an integrated view of barley node initiation and internode elongation and suggests that plant architecture should be recognized as a collection of dynamic phytomeric units in the context of crop adaptive evolution.

Open-source flow setup for rapid and efficient [18 F]fluoride drying for automation of PET tracer syntheses.

One of the key strategies for radiochemical research facilities is the automation of synthesis processes. Unnecessary manual operations increase the radiation exposure of personnel, while simultaneously threatening the reliability of syntheses. We have previously reported an affordable open-source system comprising 3D-printed continuous flow reactors, a custom syringe pump, and a pressure regulator that can be used to perform radiofluorinations. In this paper, we address additional essential processes that are needed for radiotracer development and synthesis, with the aim of making laboratory work safer and research more efficient. We have designed and evaluated a fully automated system for rapidly and effectively processing and drying aqueous [18 F]fluoride that can be directly connected to the cyclotron. This process relies on triflyl fluoride gas generation and allows nucleophilic [18 F]fluoride to be prepared safely in a hotcell within 10 min and an activity recovery of 91.7 ± 1.6% (n = 5). Owing to the need for convenient radiofluorinated prosthetic ligands, we have adapted our continuous flow system to produce [18 F]fluoroethyl tosylate (FEOTs) and [18 F]fluoroethyl triflate (FEOTf), prosthetic groups that are widely used for late-stage fluoroethylation of PET tracers. The processes as well as the radiolabeling of different groups are compared and comprehensively discussed. Having a method providing [18 F]fluoroethyl tosylate (FEOTs) as well as [18 F]fluoroethyl triflate (FEOTf) quickly and highly efficiently is beneficial for radiochemical research.

Acidosis-mediated increase in IFN-γ-induced PD-L1 expression on cancer cells as an immune escape mechanism in solid tumors.

Immune checkpoint inhibitors have revolutionized cancer therapy, yet the efficacy of these treatments is often limited by the heterogeneous and hypoxic tumor microenvironment (TME) of solid tumors. In the TME, programmed death-ligand 1 (PD-L1) expression on cancer cells is mainly regulated by Interferon-gamma (IFN-γ), which induces T cell exhaustion and enables tumor immune evasion. In this study, we demonstrate that acidosis, a common characteristic of solid tumors, significantly increases IFN-γ-induced PD-L1 expression on aggressive cancer cells, thus promoting immune escape. Using preclinical models, we found that acidosis enhances the genomic expression and phosphorylation of signal transducer and activator of transcription 1 (STAT1), and the translation of STAT1 mRNA by eukaryotic initiation factor 4F (elF4F), resulting in an increased PD-L1 expression. We observed this effect in murine and human anti-PD-L1-responsive tumor cell lines, but not in anti-PD-L1-nonresponsive tumor cell lines. In vivo studies fully validated our in vitro findings and revealed that neutralizing the acidic extracellular tumor pH by sodium bicarbonate treatment suppresses IFN-γ-induced PD-L1 expression and promotes immune cell infiltration in responsive tumors and thus reduces tumor growth. However, this effect was not observed in anti-PD-L1-nonresponsive tumors. In vivo experiments in tumor-bearing IFN-γ-/- mice validated the dependency on immune cell-derived IFN-γ for acidosis-mediated cancer cell PD-L1 induction and tumor immune escape. Thus, acidosis and IFN-γ-induced elevation of PD-L1 expression on cancer cells represent a previously unknown immune escape mechanism that may serve as a novel biomarker for anti-PD-L1/PD-1 treatment response. These findings have important implications for the development of new strategies to enhance the efficacy of immunotherapy in cancer patients.

The design of functional proteins using tensorized energy calculations.

In protein design, the energy associated with a huge number of sequence-conformer perturbations has to be routinely estimated. Hence, enhancing the throughput and accuracy of these energy calculations can profoundly improve design success rates and enable tackling more complex design problems. In this work, we explore the possibility of tensorizing the energy calculations and apply them in a protein design framework. We use this framework to design enhanced proteins with anti-cancer and radio-tracing functions. Particularly, we designed multispecific binders against ligands of the epidermal growth factor receptor (EGFR), where the tested design could inhibit EGFR activity in vitro and in vivo. We also used this method to design high-affinity Cu2+ binders that were stable in serum and could be readily loaded with copper-64 radionuclide. The resulting molecules show superior functional properties for their respective applications and demonstrate the generalizable potential of the described protein design approach.

A Fluorescent Probe as a Lead Compound for a Selective α-Synuclein PET Tracer: Development of a Library of 2-Styrylbenzothiazoles and Biological Evaluation of [18F]PFSB and [18F]MFSB.

A method to detect and quantify aggregated α-synuclein (αSYN) fibrils in vivo would drastically impact the current understanding of multiple neurodegenerative diseases, revolutionizing their diagnosis and treatment. Several efforts have produced promising scaffolds, but a notable challenge has hampered the establishment of a clinically successful αSYN positron emission tomography (PET) tracer: the requirement of high selectivity over the other misfolded proteins amyloid ß (Aß) and tau. By designing and screening a library of 2-styrylbenzothiazoles based on the selective fluorescent probe RB1, this study aimed at developing a selective αSYN PET tracer. [3H]PiB competition binding assays identified PFSB (Ki = 25.4 ± 2.3 nM) and its less lipophilic analogue MFSB, which exhibited enhanced affinity to αSYN (Ki = 10.3 ± 4.7 nM) and preserved selectivity over Aß. The two lead compounds were labeled with fluorine-18 and evaluated using in vitro autoradiography on human brain slices, where they demonstrated up to 4-fold increased specific binding in MSA cases compared to the corresponding control, reasonably reflecting selective binding to αSYN pathology. In vivo PET imaging showed [18F]MFSB successfully crosses the blood-brain barrier (BBB) and is taken up in the brain (SUV = 1.79 ± 0.02). Although its pharmacokinetic profile raises the need for additional structural optimization, [18F]MFSB represents a critical step forward in the development of a successful αSYN PET tracer by overcoming the major challenge of αSYN/Aß selectivity.

The Structural Combination of SIL and MODAG Scaffolds Fails to Enhance Binding to α-Synuclein but Reveals Promising Affinity to Amyloid ß.

A technique to image α-synuclein (αSYN) fibrils in vivo is an unmet scientific and clinical need that would represent a transformative tool in the understanding, diagnosis, and treatment of various neurodegenerative diseases. Several classes of compounds have shown promising results as potential PET tracers, but no candidate has yet exhibited the affinity and selectivity required to reach clinical application. We hypothesized that the application of the rational drug design technique of molecular hybridization to two promising lead scaffolds could enhance the binding to αSYN up to the fulfillment of those requirements. By combining the structures of SIL and MODAG tracers, we developed a library of diarylpyrazoles (DAPs). In vitro evaluation through competition assays against [3H]SIL26 and [3H]MODAG-001 showed the novel hybrid scaffold to have preferential binding affinity for amyloid ß (Aß) over αSYN fibrils. A ring-opening modification on the phenothiazine building block to produce analogs with increased three-dimensional flexibility did not result in an improved αSYN binding but a complete loss of competition, as well as a significant reduction in Aß affinity. The combination of the phenothiazine and the 3,5-diphenylpyrazole scaffolds into DAP hybrids did not generate an enhanced αSYN PET tracer lead compound. Instead, these efforts identified a scaffold for promising Aß ligands that may be relevant to the treatment and monitoring of Alzheimer's disease (AD).

Novel exotic alleles of EARLY FLOWERING 3 determine plant development in barley.

EARLY FLOWERING 3 (ELF3) is an important regulator of various physiological and developmental processes and hence may serve to improve plant adaptation which will be essential for future plant breeding. To expand the limited knowledge on barley ELF3 in determining agronomic traits, we conducted field studies with heterogeneous inbred families (HIFs) derived from selected lines of the wild barley nested association mapping population HEB-25. During two growing seasons, phenotypes of nearly isogenic HIF sister lines, segregating for exotic and cultivated alleles at the ELF3 locus, were compared for 10 developmental and yield-related traits. We determine novel exotic ELF3 alleles and show that HIF lines, carrying the exotic ELF3 allele, accelerated plant development compared with the cultivated ELF3 allele, depending on the genetic background. Remarkably, the most extreme effects on phenology could be attributed to one exotic ELF3 allele differing from the cultivated Barke ELF3 allele in only one single nucleotide polymorphism (SNP). This SNP causes an amino acid substitution (W669G), which as predicted has an impact on the protein structure of ELF3. Consequently, it may affect phase separation behaviour and nano-compartment formation of ELF3 and, potentially, also its local cellular interactions causing significant trait differences between HIF sister lines.

Identifying genomic regions determining shoot and root traits related to nitrogen uptake efficiency in a multiparent advanced generation intercross (MAGIC) winter wheat population in a high-throughput phenotyping facility.

In the context of a continuously increasing human population that needs to be fed, with environmental protection in mind, nitrogen use efficiency (NUE) improvement is becoming very important. To understand the natural variation of traits linked to nitrogen uptake efficiency (UPE), one component of NUE, the multiparent advanced generation intercross (MAGIC) winter wheat population WM-800 was phenotyped under two contrasting nitrogen (N) levels in a high-throughput phenotyping facility for six weeks. Three biomass-related, three root-related, and two reflectance-related traits were measured weekly under each treatment. Subsequently, the population was genetically analysed using a total of 13,060 polymorphic haplotypes and singular SNPs for a genome-wide association study (GWAS). In total, we detected 543 quantitative trait loci (QTL) across all time points and traits, which were pooled into 42 stable QTL (sQTL; present in at least three of the six weeks). Besides Rht-B1 and Rht-D1, candidate genes playing a role in gibberellic acid-regulated growth and nitrate transporter genes from the NPF gene family, like NRT 1.1, were linked to sQTL. Two novel sQTL on chromosomes 5 A and 6D showed pleiotropic effects on several traits. The high number of N-specific sQTL indicates that selection for UPE is useful specifically under N-limited conditions.

Radiolabeled GPVI-Fc for PET Imaging of Multiple Extracellular Matrix Fibers: A New Look into Pulmonary Fibrosis Progression.

Invariably fatal and with a particularly fast progression, pulmonary fibrosis (PF) is currently devoid of curative treatment options. Routine clinical diagnosis relies on breathing tests and visualizing the changes in lung structure by CT, but anatomic information is often not sufficient to identify early signs of progressive PF. For more efficient diagnosis, additional imaging techniques were investigated in combination with CT, such as 18F-FDG PET, although with limited success because of lack of disease specificity. Therefore, novel molecular targets enabling specific diagnosis are investigated, in particular for molecular imaging techniques. Methods: In this study, we used a 64Cu-radiolabeled platelet glycoprotein VI fusion protein (64Cu-GPVI-Fc) targeting extracellular matrix (ECM) fibers as a PET tracer to observe longitudinal ECM remodeling in a bleomycin-induced PF mouse model. Results: 64Cu-GPVI-Fc showed significant uptake in fibrotic lungs, matching histology results. Contrary to 18F-FDG PET measurements, 64Cu-GPVI-Fc uptake was linked entirely to the fibrotic activity of tissue and not was susceptible to inflammation. Conclusion: Our study highlights 64Cu-GPVI-Fc as a specific tracer for ECM remodeling in PF, with clear therapy-monitoring and clinical translation potential.

An exotic allele of barley EARLY FLOWERING 3 contributes to developmental plasticity at elevated temperatures.

Increase in ambient temperatures caused by climate change affects various morphological and developmental traits of plants, threatening crop yield stability. In the model plant Arabidopsis thaliana, EARLY FLOWERING 3 (ELF3) plays prominent roles in temperature sensing and thermomorphogenesis signal transduction. However, how crop species respond to elevated temperatures is poorly understood. Here, we show that the barley ortholog of AtELF3 interacts with high temperature to control growth and development. We used heterogeneous inbred family (HIF) pairs generated from a segregating mapping population and systematically studied the role of exotic ELF3 variants in barley temperature responses. An exotic ELF3 allele of Syrian origin promoted elongation growth in barley at elevated temperatures, whereas plant area and estimated biomass were drastically reduced, resulting in an open canopy architecture. The same allele accelerated inflorescence development at high temperature, which correlated with early transcriptional induction of MADS-box floral identity genes BM3 and BM8. Consequently, barley plants carrying the exotic ELF3 allele displayed stable total grain number at elevated temperatures. Our findings therefore demonstrate that exotic ELF3 variants can contribute to phenotypic and developmental acclimation to elevated temperatures, providing a stimulus for breeding of climate-resilient crops.

Neurovascular Uncoupling: Multimodal Imaging Delineates the Acute Effects of 3,4-Methylenedioxymethamphetamine.

Psychedelic compounds such as 3,4-methylenedioxymethamphetamine (MDMA) have attracted increasing interest in recent years because of their therapeutic potential in psychiatric disorders. To understand the acute effects of psychedelic drugs in vivo, blood-oxygenation-level-dependent (BOLD) functional MRI (fMRI) has been widely used. In particular, fMRI studies have suggested that MDMA leads to inhibition of brain activity, challenging previous hypotheses indicating mainly excitatory effects based, among others, on increased metabolism shown by 18F-FDG functional PET (fPET). However, interpretation of hemodynamic changes induced by psychedelics is difficult because of their potent vascular effects. Methods: We aimed to delineate the acute effects of MDMA using simultaneous PET/fMRI in rats. For this purpose, hemodynamic changes measured by BOLD fMRI were related to alterations in glucose utilization and serotonin transporter (SERT) occupancy using 18F-FDG fPET/fMRI and 11C-DASB PET/fMRI. Results: We show that MDMA induces localized increases in glucose metabolism in limbic projection areas involved in emotional processing. The increased glucose metabolism was accompanied by global cerebral and extracerebral hemodynamic decreases. We further demonstrated a strong correlation between SERT occupancies and regional BOLD reductions after acute MDMA administration. Conclusion: Our data indicate that hemodynamic decreases after acute MDMA administration are of a nonneuronal nature and initiate peripherally. Within the brain, MDMA triggers neuronal activation in limbic projection areas, whereas increased serotonin levels induced by SERT blockage cause neurovascular uncoupling through direct vascular effects. Correct understanding of the in vivo mechanism of MDMA not only supports ongoing research but also warrants a reassessment of previous studies on neuronal effects of psychedelics relying on neurovascular coupling and recommends 18F-FDG fPET as a potentially more robust measure for pharmacologic research.

Two birds with one stone: human SIRPα nanobodies for functional modulation and in vivo imaging of myeloid cells.

Signal-regulatory protein α (SIRPα) expressed by myeloid cells is of particular interest for therapeutic strategies targeting the interaction between SIRPα and the "don't eat me" ligand CD47 and as a marker to monitor macrophage infiltration into tumor lesions. To address both approaches, we developed a set of novel human SIRPα (hSIRPα)-specific nanobodies (Nbs). We identified high-affinity Nbs targeting the hSIRPα/hCD47 interface, thereby enhancing antibody-dependent cellular phagocytosis. For non-invasive in vivo imaging, we chose S36 Nb as a non-modulating binder. By quantitative positron emission tomography in novel hSIRPα/hCD47 knock-in mice, we demonstrated the applicability of 64Cu-hSIRPα-S36 Nb to visualize tumor infiltration of myeloid cells. We envision that the hSIRPα-Nbs presented in this study have potential as versatile theranostic probes, including novel myeloid-specific checkpoint inhibitors for combinatorial treatment approaches and for in vivo stratification and monitoring of individual responses during cancer immunotherapies.

A Haplotype-Based GWAS Identified Trait-Improving QTL Alleles Controlling Agronomic Traits under Contrasting Nitrogen Fertilization Treatments in the MAGIC Wheat Population WM-800.

The multi-parent-advanced-generation-intercross (MAGIC) population WM-800 was developed by intercrossing eight modern winter wheat cultivars to enhance the genetic diversity present in breeding populations. We cultivated WM-800 during two seasons in seven environments under two contrasting nitrogen fertilization treatments. WM-800 lines exhibited highly significant differences between treatments, as well as high heritabilities among the seven agronomic traits studied. The highest-yielding WM-line achieved an average yield increase of 4.40 dt/ha (5.2%) compared to the best founder cultivar Tobak. The subsequent genome-wide-association-study (GWAS), which was based on haplotypes, located QTL for seven agronomic traits including grain yield. In total, 40, 51, and 46 QTL were detected under low, high, and across nitrogen treatments, respectively. For example, the effect of QYLD_3A could be associated with the haplotype allele of cultivar Julius increasing yield by an average of 4.47 dt/ha (5.2%). A novel QTL on chromosome 2B exhibited pleiotropic effects, acting simultaneously on three-grain yield components (ears-per-square-meter, grains-per-ear, and thousand-grain-weight) and plant-height. These effects may be explained by a member of the nitrate-transporter-1 (NRT1)/peptide-family, TaNPF5.34, located 1.05 Mb apart. The WM-800 lines and favorable QTL haplotypes, associated with yield improvements, are currently implemented in wheat breeding programs to develop advanced nitrogen-use efficient wheat cultivars.

Unraveling Genomic Regions Controlling Root Traits as a Function of Nitrogen Availability in the MAGIC Wheat Population WM-800.

An ever-growing world population demands to be fed in the future and environmental protection and climate change need to be taken into account. An important factor here is nitrogen uptake efficiency (NUpE), which is influenced by the root system (the interface between plant and soil). To understand the natural variation of root system architecture (RSA) as a function of nitrogen (N) availability, a subset of the multiparent advanced generation intercross (MAGIC) winter wheat population WM-800 was phenotyped under two contrasting N treatments in a high-throughput phenotyping system at the seedling stage. Fourteen root and shoot traits were measured. Subsequently, these traits were genetically analyzed using 13,060 polymorphic haplotypes and SNPs in a genome-wide association study (GWAS). In total, 64 quantitative trait loci (QTL) were detected; 60 of them were N treatment specific. Candidate genes for the detected QTL included NRT1.1 and genes involved in stress signaling under N-, whereas candidate genes under N+ were more associated with general growth, such as mei2 and TaWOX11b. This finding may indicate (i) a disparity of the genetic control of root development under low and high N supply and, furthermore, (ii) the need for an N specific selection of genes and genotypes in breeding new wheat cultivars with improved NUpE.