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Background: Immune checkpoint inhibitors (ICIs) are effective against various advanced and metastatic cancers, but patient responses vary and can change over time, complicating treatment prediction. Therefore, better tools for patient stratification, response prediction, and response assessment are needed. This study presents the development and clinical translation of a fluorescently labelled ICI tracer pair used to perform multispectral fluorescent molecular imaging and simultaneously gain spatial and temporal insight in both programmed death ligand 1 (PD-L1) and programmed death receptor 1 (PD-1) expression. Methods: We conjugated the anti-PD-L1 antibody durvalumab to IRDye 680LT and the anti-PD-1 antibody nivolumab to IRDye 800CW. Tracers were developed and optimized for conjugation efficiency and purity to allow use in clinical trials. Stability was tested up to 12 months. An extended single-dose toxicity study in mice was performed for durvalumab-680LT and the unconjugated IRDye 680LT to demonstrate safety for first-in-human administration. Results: Durvalumab-680LT and nivolumab-800CW were successfully conjugated and purified. Conjugation optimization resulted in a robust production with labelling efficiencies of ≥88%. Long-term stability study of both tracers showed all parameters within end of shelf-life specifications for at least 12 months at 2–8 °C. No toxic effects were observed in doses up to 1000x the intended human dose for both IRDye 680LT and durvalumab-680LT, which are therefore considered safe for first-in-human use. Conclusions: We succeeded in the development and clinical translation of two novel fluorescent ICI tracers, durvalumab-680LT and nivolumab-800CW. Moreover, we demonstrated for the first time the safety of IRDye 680LT and durvalumab-680LT, enabling first-in-human use. Together, this makes durvalumab-680LT and nivolumab-800CW suitable for phase I/II clinical trials.

Real-time intraoperative guidance is essential during oncological surgery for complete and safe tumour resection. Fluorescence imaging in the near-infrared spectrum has shown potential for guiding surgeons during complex interventions. Recently, there has been a shift towards the use of fluorescence contrast agents for molecular imaging. The first targeted fluorescent agents, of which most consist of approved therapeutic antibodies conjugated to a fluorescent dye, have been evaluated in several early-phase clinical trials. Moreover, advances in protein engineering and drug design have led to the development of a variety of tracers suitable for molecular fluorescence image-guided surgery. In this Review, we discuss preclinical and clinical evidence, ongoing clinical trials, and the latest developments in the field of molecular near-infrared tracers for fluorescence-guided cancer surgery.

To meet the growing demand for intraoperative molecular imaging, the development of compatible imaging agents plays a crucial role. Given the unique requirements of surgical applications compared to diagnostics and therapy, maximizing translational potential necessitates distinctive imaging agent designs. For effective surgical guidance, exogenous signatures are essential and are achievable through a diverse range of imaging labels such as (radio)isotopes, fluorescent dyes, or combinations thereof. To achieve optimal in vivo utility a balanced molecular design of the tracer as a whole is required, which ensures a harmonious effect of the imaging label with the affinity and specificity (e.g., pharmacokinetics) of a pharmacophore/targeting moiety. This review outlines common design strategies and the effects of refinements in the molecular imaging agent design on the agent’s pharmacological profile. This includes the optimization of affinity, pharmacokinetics (including serum binding and target mediated background), biological clearance route, the achievable signal intensity, and the effect of dosing hereon.

Zebrafish are becoming increasingly popular as an organism in which to model human disease and to study the effects of small molecules on complex physiological and pathological processes. Since larvae are no more than a few millimetres in length, and can live in volumes as small as 100 microliters, they are particularly amenable to high-throughput and high content compound screening in 96 well plate format. There is a growing literature providing evidence that many compounds show similar pharmacological effects in zebrafish as they do in mammals, and in particular humans. However, a major question regarding their utility for small molecule screening for neurological conditions is whether a molecule will reach its target site within the central nervous system. Studies have shown that Claudin-5 and ZO-1, tight-junction proteins which are essential for blood-brain barrier (BBB) integrity in mammals, can be detected in some cerebral vessels in zebrafish from 3 days post-fertilisation (d.p.f.) onwards and this timing coincides with the retention of dyes, immunoreactive tracers and fluorescent markers within some but not all cerebral vessels. Whilst these findings demonstrate that features of a BBB are first present at 3 d.p.f., it is not clear how quickly the zebrafish BBB matures or how closely the barrier resembles that of mammals. Here, we have combined anatomical analysis by transmission electron microscopy, functional investigation using fluorescent markers and compound uptake using liquid chromatography/tandem mass spectrometry to demonstrate that maturation of the zebrafish BBB occurs between 3 d.p.f. and 10 d.p.f. and that this barrier shares both structural and functional similarities with that of mammals.

Investigating ligand-protein complexes is essential in the areas of chemical biology and drug discovery. However, detailed information on key reagents such as fluorescent tracers and associated data for the development of widely used bioluminescence resonance energy transfer (BRET) assays including NanoBRET, time-resolved Förster resonance energy transfer (TR-FRET) and fluorescence polarization (FP) assays are not easily accessible to the research community. We created tracerDB, a curated database of validated tracers. This resource provides an open access knowledge base and a unified system for tracer and assay validation. The database is freely available at https://www.tracerdb.org/ . Tracers are fluorescent protein ligands required for various displacement assays. Here, the authors announce a curated database named tracerDB, which will make essential tracer data, contributed by the worldwide research community, easily available and searchable.

Background Extracellular vesicles (EVs) have shown great potential for targeted therapy, as they have a natural ability to pass through biological barriers and, depending on their origin, can preferentially accumulate at defined sites, including tumors. Analyzing the potential of EVs to target specific cells remains challenging, considering the unspecific binding of lipophilic tracers to other proteins, the limitations of fluorescence for deep tissue imaging and the effect of external labeling strategies on their natural tropism. In this work, we determined the cell-type specific tropism of B16F10-EVs towards cancer cell and metastatic tumors by using fluorescence analysis and quantitative gold labeling measurements. Surface functionalization of plasmonic gold nanoparticles was used to promote indirect labeling of EVs without affecting size distribution, polydispersity, surface charge, protein markers, cell uptake or in vivo biodistribution. Double-labeled EVs with gold and fluorescent dyes were injected into animals developing metastatic lung nodules and analyzed by fluorescence/computer tomography imaging, quantitative neutron activation analysis and gold-enhanced optical microscopy. Results We determined that B16F10 cells preferentially take up their own EVs, when compared with colon adenocarcinoma, macrophage and kidney cell-derived EVs. In addition, we were able to detect the preferential accumulation of B16F10 EVs in small metastatic tumors located in lungs when compared with the rest of the organs, as well as their precise distribution between tumor vessels, alveolus and tumor nodules by histological analysis. Finally, we observed that tumor EVs can be used as effective vectors to increase gold nanoparticle delivery towards metastatic nodules. Conclusions Our findings provide a valuable tool to study the distribution and interaction of EVs in mice and a novel strategy to improve the targeting of gold nanoparticles to cancer cells and metastatic nodules by using the natural properties of malignant EVs.

Lipids are major constituents of food but are also highly relevant substructures of drugs and are increasingly applied for the development of lipid-based drug delivery systems. Lipids are prone to oxidative degradation, thus affecting the quality of food or medicines. Therefore, analytical methods or tools that enable the degree of lipid oxidation to be assessed are of utmost importance to guarantee food and drug safety. Herein, we report the design, synthesis and application of the first-in-class fluorogenic triacylglycerols that enable dynamic monitoring of lipid oxidation via straightforward fluorescence readout. Our fluorogenic triacylglycerols can be used in both aqueous and lipid-based environments. Furthermore, we showed that the sensitivity of our fluorescent tracers towards oxidation could be tuned by incorporating either saturated or unsaturated acyl chains in their triacylglycerol core structure. With this, we provide a first proof of principle for the applicability of fluorescently labelled triacylglycerols as tracers to monitor the dynamics of lipid oxidation, thus paving the way for novel discoveries in the area of lipid analytics.

Foliar water uptake (FWU) is a common water acquisition mechanism for plants inhabiting temperate fog-affected ecosystems, but the prevalence and consequences of this process for the water and carbon balance of tropical cloud forest species are unknown. We performed a series of experiments under field and glasshouse conditions using a combination of methods (sap flow, fluorescent apoplastic tracers and stable isotopes) to trace fog water movement from foliage to belowground components of Drimys brasiliensis. In addition, we measured leaf water potential, leaf gas exchange, leaf water repellency and growth of plants under contrasting soil water availabilities and fog exposure in glasshouse experiments to evaluate FWU effects on the water and carbon balance of D. brasiliensis saplings. Fog water diffused directly through leaf cuticles and contributed up to 42% of total foliar water content. FWU caused reversals in sap flow in stems and roots of up to 26% of daily maximum transpiration. Fog water transported through the xylem reached belowground pools and enhanced leaf water potential, photosynthesis, stomatal conductance and growth relative to plants sheltered from fog. Foliar uptake of fog water is an important water acquisition mechanism that can mitigate the deleterious effects of soil water deficits for D. brasiliensis.

Herpes simplex virus type 1 (HSV-1) has great potential to be applied as a viral tool for gene delivery or oncolysis. The broad infection tropism of HSV-1 makes it a suitable tool for targeting many different cell types, and its 150 kb double-stranded DNA genome provides great capacity for exogenous genes. Moreover, the features of neuron infection and neuron-to-neuron spread also offer special value to neuroscience. HSV-1 strain H129, with its predominant anterograde transneuronal transmission, represents one of the most promising anterograde neuronal circuit tracers to map output neuronal pathways. Decades of development have greatly expanded the H129-derived anterograde tracing toolbox, including polysynaptic and monosynaptic tracers with various fluorescent protein labeling. These tracers have been applied to neuroanatomical studies, and have contributed to revealing multiple important neuronal circuits. However, current H129-derived tracers retain intrinsic drawbacks that limit their broad application, such as yet-to-be improved labeling intensity, potential nonspecific retrograde labeling, and high toxicity. The biological complexity of HSV-1 and its insufficiently characterized virological properties have caused difficulties in its improvement and optimization as a viral tool. In this review, we focus on the current H129-derived viral tracers and highlight strategies in which future technological development can advance its use as a tool.

The presence of plasmodesmata (PD) constitutes the structural basis for information exchange between cells, and symplasmic communication is involved in the regulation of cell differentiation and plant development. Most recent studies concerning an analysis of symplasmic communication between seed compartments and the embryo have been predominantly performed on Arabidopsis thaliana. The results presented in this paper describe the analysis of symplasmic communication on the example of Sedum acre seeds, because the ultrastructure of the seed compartments and the embryo proper, including the PD, have already been described, and this species represents an embryonic type of development different to Arabidopsis. Moreover, in this species, an unusual electron-dense dome associated with plasmodesmata on the border between the basal cell/chalazal suspensor cells and the basal cell/the endosperm has been described. This prompted the question as to whether these plasmodesmata are functional. Thus, the aim of this study was to describe the movement of symplasmic transport fluorochromes between different Sedum seed compartments, with particular emphasis on the movement between the basal cell and the embryo proper and endosperm, to answer the following questions: (1) are seeds divided into symplasmic domains; (2) if so, are they stable or do they change with the development? The results have shown that symplasmic tracers movement: (a) from the external integument to internal integument is restricted; (b) from the basal cell to the other part of the embryo proper and from the basal cell to the endosperm is also restricted; (c) the embryo is a single symplasmic domain with respect to molecules of a molecular weight below 0.5 kDa.