Indirect Labeling Research Articles

Novel synthesis of an [18F]aryl boronic acid ester as a reagent for 18F-labeling via Suzuki coupling

Indirect 18F labeling methods using 18F-containing compounds such as N-succinimidyl-4-[18F]fluorobenzoate and 4-[18F]fluoroiodobenzene as labeling reagents have been reported because direct 18F labeling has difficulty in labeling aromatic compounds. In this study, we synthesized the 18F-labeling reagent 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) [18F]fluorobenzene ([18F]TDBFB) using a resonant-type microwave reactor in the presence of (2,2,6,6-tetramethylpiperidine 1-oxyl and a copper catalyst. Compared with a previous report on [18F]fluorophenylboronic acid, [18F]TDBFB was synthesized simply. Moreover, we applied [18F]TDBFB for the Suzuki coupling with triflate and bromide precursors. The Suzuki coupling of [18F]TDBFB and precursors using resonant-type microwave reactor yielded 4-[18F]fluorobiphenyl and the [18F]pitavastatin derivative as the coupling products. These results show the potential of [18F]TDBFB obtained using rapid synthesis as an indirect 18F-labeling reagent.

α1B‐Adrenoceptor is Required for Chemokine (C‐X‐C Motif) Receptor 4 Mediated Chemotaxis in THP1 Cells

IntroductionChemokine (C‐X‐C motif) receptor 4 (CXCR4) is a key regulator of leukocyte trafficking under physiological conditions and in numerous inflammatory disease processes. Previously, we demonstrated that CXCR4 hetero‐oligomerizes with α1B‐adrenoceptors (ARs), through which the receptors cross‐talk. The existence and function of these hetero‐oligomers in leukocytes, however, is unknown. Thus, we tested whether such heteromeric complexes are detectable in the human monocytic cell line THP1 and in freshly isolated human monocytes, and evaluated how α1B‐AR knockdown by CRISPR/Cas9 gene editing in THP1 cells affects CXCR4‐mediated chemotaxis.MethodsPeripheral blood mononuclear cells were isolated by density gradient centrifugation from human whole blood; monocytes were then isolated via negative selection using MACS LS, an indirect magnetic labeling system. Proximity ligation assays (PLA) were used to visualize individual receptors and receptor‐receptor interactions in THP1 cells and freshly isolated monocytes. CRISPR/Cas9 gene editing was utilized to create a α1B‐AR knockout strain (ADRA1BKO); wild type THP1 cells were used as a negative control (ADRA1Bwt). Cell migration toward the cognate CXCR4 agonist chemokine (C‐X‐C motif) ligand 12 (CXCL12) was tested employing a 96 well Boyden chamber. Transmigrated cells were counted automatically utilizing high contrast bright field and post‐imaging particle analyses. The chemotactic index (ChI) was calculated as the ratio of cells that transmigrated in the presence versus the absence of CXCL12. Data were analyzed with ANOVA/Tukey’s post‐hoc. Data are mean ± SE from n=4 experiments performed on different days. A 2‐tailed p<0.05 was considered significant.ResultsPLA confirmed the presence of endogenously and constitutively expressed CXCR4:α1B‐AR heteromers in freshly isolated monocytes and THP1 cells. PLA signals for α1B‐AR and CXCR4:α1B‐AR heteromers were not detectable in ADRA1BKO cells. CXCR4 signals were not affected by CRISPR/Cas9 gene knockout. ADRA1Bwtcells showed a bell‐shaped chemotactic dose response to CXCL12 with a maximal ChI of 9.1±1.1 at 100 nM. CRISPR/Cas9 knockout of α1B‐AR reduced the chemotactic response to CXCL12 by 89±52% (p<0.05). ADRA1BKO results were replicated in an additional α1B‐AR knockout strain to rule out clonal effects.ConclusionOur observations suggest that CXCR4 heteromerizes with α1B‐AR in human monocytes and that CXCL12‐mediated chemotaxis depends on the formation of CXCR4:α1B‐AR heteromers. These data provide evidence for a novel molecular mechanism by which CXCR4‐mediated chemotaxis in monocytes/leukocytes is controlled.

A modified TurboID approach identifies tissue-specific centriolar components in C. elegans.

Proximity-dependent labeling approaches such as BioID have been a great boon to studies of protein-protein interactions in the context of cytoskeletal structures such as centrosomes which are poorly amenable to traditional biochemical approaches like immunoprecipitation and tandem affinity purification. Yet, these methods have so far not been applied extensively to invertebrate experimental models such as C. elegans given the long labeling times required for the original promiscuous biotin ligase variant BirA*. Here, we show that the recently developed variant TurboID successfully probes the interactomes of both stably associated (SPD-5) and dynamically localized (PLK-1) centrosomal components. We further develop an indirect proximity labeling method employing a GFP nanobody-TurboID fusion, which allows the identification of protein interactors in a tissue-specific manner in the context of the whole animal. Critically, this approach utilizes available endogenous GFP fusions, avoiding the need to generate multiple additional strains for each target protein and the potential complications associated with overexpressing the protein from transgenes. Using this method, we identify homologs of two highly conserved centriolar components, Cep97 and BLD10/Cep135, which are present in various somatic tissues of the worm. Surprisingly, neither protein is expressed in early embryos, likely explaining why these proteins have escaped attention until now. Our work expands the experimental repertoire for C. elegans and opens the door for further studies of tissue-specific variation in centrosome architecture.

Tumor-targeted fluorescence labeling systems for cancer diagnosis and treatment.

Conventional imaging techniques are available for clinical identification of tumor sites. However, detecting metastatic tumor cells that are spreading from primary tumor sites using conventional imaging techniques remains difficult. In contrast, fluorescence‐based labeling systems are useful tools for detecting tumor cells at the single‐cell level in cancer research. The ability to detect fluorescent‐labeled tumor cells enables investigations of the biodistribution of tumor cells for the diagnosis and treatment of cancer. For example, the presence of fluorescent tumor cells in the peripheral blood of cancer patients is a predictive biomarker for early diagnosis of distant metastasis. The elimination of fluorescent tumor cells without damaging normal tissues is ideal for minimally invasive treatment of cancer. To capture fluorescent tumor cells within normal tissues, however, tumor‐specific activated target molecules are needed. This review focuses on recent advances in tumor‐targeted fluorescence labeling systems, in which indirect reporter labeling using tumor‐specific promoters is applied to fluorescence labeling of tumor cells for the diagnosis and treatment of cancer. Telomerase promoter‐dependent fluorescence labeling using replication‐competent viral vectors produces fluorescent proteins that can be used to detect and eliminate telomerase‐positive tumor cells. Tissue‐specific promoter‐dependent fluorescence labeling enables identification of specific tumor cells. Vimentin promoter‐dependent fluorescence labeling is a useful tool for identifying tumor cells that undergo epithelial–mesenchymal transition (EMT). The evaluation of tumor cells undergoing EMT is important for accurately assessing metastatic potential. Thus, tumor‐targeted fluorescence labeling systems represent novel platforms that enable the capture of tumor cells for the diagnosis and treatment of cancer.

Label-free Raman and fluorescence imaging of amyloid plaques in human Alzheimer’s disease brain tissue reveal carotenoid accumulations

Alzheimer’s disease (AD) is a neurodegenerative disease, characterized by the presence of extracellular deposits (plaques) of amyloid-beta peptide and intracellular aggregates of phosphorylated tau. In general, these hallmarks are studied by techniques requiring chemical pre-treatment and indirect labeling. Imaging techniques that require no labeling and could be performed on tissue in its native form could contribute to a better understanding of the disease. In this article a combination of label-free and non-invasive techniques is presented to study the biomolecular composition of AD human brain tissue. We build on previous research that already revealed the autofluorescence property of plaque, and the presence of carotenoids in cored plaques. Here, we present further results on cored plaques: showing blue and green autofluorescence emission coming from the same plaque location. Raman microscopy was used to confirm the presence of carotenoids in the plaque areas, with clear peaks around 1150 and 1514 cm−1. Carotenoid reference spectra were recorded in hexane solution, but also adsorbed on aggregated Aβ42 peptides; the latter agreed better with the Raman spectra observed in plaques. From the six single carotenoids measured, lycopene matched closest with the peak positions observed in the cored plaques. Lastly, stimulated Raman scattering (SRS) microscopy measurements were performed, targeting the shift of the beta-sheet Amide I peak observed in plaques. Employing SRS in the C–H stretch region we also looked for the presence of a lipid halo around plaque, as reported in the literature for transgenic AD mice, but such a halo was not observed in these human AD brain samples.

Adeno-Associated Viral Vector Immobilization and Local Delivery from Bare Metal Surfaces.

Spatially and temporally controlled delivery of biologicals, including gene vectors, represents an unmet need for regenerative medicine and gene therapy applications. Here we describe a method of reversible attachment of serotype 2 adeno-associated viral vectors (AAV2) to metal surfaces. This technique enables localized delivery of the vector to the target cell population in vitro and in vivo with the subsequent effective transduction of cells adjacent to the metal substrate. The underlying bioengineering approach employs coordination chemistry between the bisphosphonic groups of polyallylamine bisphosphonates and the metal atoms on the surface of metallic samples. Formation of a stable polybisphosphonate monolayer with plentiful allyl-derived amines allows for further chemical modification to consecutively append thiol-modified protein G, an anti-AAV2 antibody, and AAV2 particles. Herein we present a detailed protocols for the metal substrate modification, for the visualization of the metal surface-immobilized vector using direct and indirect fluorescent AAV2 labeling and scanning electron microscopy, for quantification of the surface-immobilized vector load with RT-PCR, and for the localized vector transduction in vitro and in vivo.

Preparation and preclinical characterization of 111In-DTPA-Anti-MUC1 as a radioimmunoconjugate for diagnosis of breast cancer by single-photon emission computed tomography.

Targeting MUC1 antigens which are overexpressed in 80% of breast cancers can be widely used in the field of radioimmunoscintigraphy (RIS) of breast cancer. The aim of this study was to develop a new diagnostic labeled compound for breast cancer RIS. In this study, an efficient indirect labeling method of PR81 with Indium-111 was developed and preliminary preclinical qualifications were reported. 111In-DTPA-PR81 was prepared and its radiochemical purity and stabilities in human serum and in phosphate-buffered saline (PBS) buffer were surveyed. Furthermore, cellular studies including complex reactivity, binding specificity, cell toxicity, etc., were examined. Finally, biodistribution and scintigraphy of the complex were studied in normal and tumoral animals. Statistical analyses were performed using SPSS 10.0. 111In-DTPA-PR81 was prepared with a radiochemical purity of >99% at optimized conditions. Stability studies showed the radiochemical purity of >90% in PBS buffer after 96 h, while the stability in human serum showed decrement to 81% after 96 h. Reactivity of the complex with MUC1 was significantly (P < 0.005) higher than bovine serum albumin (BSA) (about 7-8 times), even though BSA concentration was about twice the MUC1. The binding specificity of the complex to the MUC1 antigen was confirmed by means of immunoreactivity assay. Cell toxicity examination showed no significant lethal effect of the radiolabeled compound on the cells. Biodistribution studies of the complex in normal rats were consistent with the biodistribution of antibodies and high accumulation was observed in the tissues expressing MUC1 antigen. The results of 111In-DTPA-PR81 scintigraphy in tumoral female BALB/c mice at 24 and 48 h after injection showed an increasement of the accumulation in the tumor site. 111In-DTPA-PR81 can be considered as a potential agent for imaging of the MUC1 +breast tumors.

Age-dependent cerebrospinal fluid-tissue water exchange detected by magnetization transfer indirect spin labeling MRI.

A non-invasive magnetization transfer indirect spin labeling (MISL) MRI method is developed to quantify the water exchange between cerebrospinal fluid (CSF) and other tissues in the brain and to examine the age-dependence of water exchange. In the pulsed MISL, we implemented a short selective pulse followed by a post-labeling delay before an MRI acquisition with a long echo time; in the continuous MISL, a train of saturation pulses was applied. MISL signal (∆Z) was obtained by the subtraction of the label MRI at -3.5 ppm from the control MRI at 200 ppm. CSF was extracted from the mouse ventricles for the MISL optimization and validation. Comparison between wild type (WT) and aquaporin-4 knockout (AQP4-/- ) mice was performed to examine the contributions of CSF water exchange, whereas its age-dependence was investigated by comparing the adult and young WT mice. The pulsed MISL method observed that the MISL signal reached the maximum at 1.5 s. The continuous MISL method showed the highest MISL signal in the fourth ventricle (∆Z=13.5%±1.4%), whereas the third ventricle and the lateral ventricles had similar MISL ∆Z values (∆Z=12.0%±1.8%). Additionally, significantly lower ∆Z (9.3%-18.7% reduction) was found in all ventricles for the adult mice than those of the young mice (p<0.02). For the AQP4-/- mice, the ∆Z values were 5.9%-8.3% smaller than those of the age-matched WT mice in the lateral and fourth ventricles, but were not significant. The MISL method has a great potential to study CSF water exchange with the surrounding tissues in brain.

695 Upregulated monocyte expression of PLIN2 is associated with plaque instability in coronary artery disease

Abstract Aims Perilipin 2 (PLIN2), a protein associated with intracellular lipid droplets (LDs), is involved in lipid metabolism of macrophages resident in atherosclerotic plaques and its up-regulation leads to LDs accumulation. LDs enlargement results in the macrophage transformation into foam cells, a key step for the onset of atherosclerosis. In the present study, we investigated the role of PLIN2 and its regulation mechanisms in atherosclerosis and plaque instability in patients with a diagnosis of ST-elevation myocardial infarction (STEMI) and stable chronic angina (SA). Methods and results We enrolled 120 patients with a diagnosis of STEMI and 42 SA patients with symptoms of stable effort angina lasting more than 12 months. Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA whole blood samples through standard gradient centrifugation over Ficoll-Hypaque. Monocytes were purified through indirect magnetic labelling of PBMCs. PLIN2 mRNA expression was investigated by Real Time-PCR and PLIN2 protein level was analysed in CD14+ monocytes by flow cytometry. Proteasome activity was assayed using AMC-tagged peptide substrate (Succ-LLVY-AMC), which releases free highly fluorescent AMC (Ex/Em 350/440 nm) in the presence of proteolytic activity. In CD14+ monocyte, PLIN2 protein expression was significantly increased in STEMI as compared to SA patients (P &amp;lt; 0.001), while PLIN2 mRNA level was not different in the two groups (P = n.s.). Despite proteasome activity was higher in STEMI as compared to SA patients (P &amp;lt; 0.001), significant inverse correlations were evident between PLIN2 levels and proteasome activity in the two groups (P = 0.05). Conclusions CD14+ monocyte PLIN2 protein expression was higher in STEMI as compared to SA patients suggesting an involvement in plaque instability. Despite proteasome activity was higher in STEMI patients, probably due to the elevated inflammatory burden, PLIN2 could escape proteasome degradation in a more efficient manner in STEMI as compared to SA patients.

Fluorescent Immunohistochemistry.

Immunofluorescence is an important immunochemical technique that utilizes fluorescence-labeled antibodies to detect specific target antigens. It is used widely in both scientific research and clinical laboratories. Immunofluorescence allows for excellent sensitivity and amplification of signal in comparison to immunohistochemistry. However, analysis of samples labeled with fluorescence-labeled antibodies has to be performed using a fluorescence microscope or other type of fluorescence imaging. There are two methods available: direct (primary) and indirect (secondary) immunofluorescence. Here, we describe the principle of immunofluorescence methods as well as the preparation of fresh-frozen and formalin-fixed, paraffin embedded tissues for both direct and indirect immunofluorescence labeling.

Investigation of Fluorine-18 Labelled Peptides for Binding to Cholecystokinin-2 Receptors with High Affinity

Many cancers of neuroendocrine origin overexpress cholecystokinin-2 receptors (CCK-2R) including medullary thyroid cancer, small cell lung cancer and other lung carcinoids. Fluorine-18 labelled peptides targeting CCK-2R enable direct visualization and quantification of this receptor in vivo using positron emission tomography imaging. CP04 1 and MG11 2 are two previously described truncated peptides derived from the native CCK-2R hormone ligand, gastrin. The N-terminus of the MG11 2 octopeptide was chemically modified with various fluorine containing aromatic (4-fluorobenzoate), heterocyclic (6-fluoronicotinate) and aliphatic (2-fluoropropionate) moieties. To assess the impact these modifications had on CCK-2R binding, ligand-binding assays were conducted using A431 cells overexpressing human CCK-2R. MG11 2 modified by 4-fluorobenzoate (FB-MG11 3) demonstrated the highest binding affinity (0.20 nM) followed by MG11 2 modified by 6-fluoronicotinate (FNic-MG11 4; 0.74 nM) and 2-fluoropropionate (FP-MG11 5; 1.80 nM), respectively. Whilst indirect labelling of MG11 2 using fluorine-18 labelled activated esters of fluorobenzoate and 6-fluoronicotinate was unsuccessful, direct fluorine-18 labelling at the N-terminus modified with 6-nitronicotinate afforded a 47.6% radiochemical yield of [18F]FNic-MG11. Unfortunately, [18F]FNic-MG11 4 was chemically unstable, decomposing slowly through defluorination, thereby impeding any further work with this radiotracer.

Synthesis, stability, and cellular uptake of 131I-estradiol against MCF7 and T-47D human cell lines as a radioligand for binding assay

Estradiol is a steroid hormone that works as an agonist estrogen receptor (ER). This compound is widely used as a ligand and bind specifically to the ERα. Radioligand binding assay is an in vitro method for drug development from natural products by synthesizing estradiol through radiolabeling using the radioiodination method. Synthesis of 131I-estradiol was perfomed by direct method using chloramine T as an oxidizer and by indirect labeling using 131I-histamine. The purity of chemical was determined by thin-layer chromatography and paper electrophoresis, as well as its stability for 30 days of storage in refrigerator, freezer and room temperature. The cellular uptake test of the radioligands from both methods was carried out with MCF7 and T-47D cell lines at 60 min. The results exhibited that 131I-estradiol was succesfully obtained with radiochemical purity greater than 95% and more stable in the refrigerator until 21 days than freezer and room temperature. 131I-estradiol and 131I-his-estradiol were internalized higher in T-47D cells than MCF7 cells (44.34 ± 5.93% vs. 17.27 ± 1.71% and 45.34 ± 6.42% vs. 4.92 ± 1.59%, respectively). Furthermore, the radioligands can be used to binding assay in determining the agonist or antagonist to ER of new drugs development.

Nanobody-Based Theranostic Agents for HER2-Positive Breast Cancer: Radiolabeling Strategies.

The overexpression of human epidermal growth factor 2 (HER2) in breast cancer (BC) has been associated with a more aggressive tumor subtype, poorer prognosis and shorter overall survival. In this context, the development of HER2-targeted radiotracers is crucial to provide a non-invasive assessment of HER2 expression to select patients for HER2-targeted therapies, monitor response and identify those who become resistant. Antibodies represent ideal candidates for this purpose, as they provide high contrast images for diagnosis and low toxicity in the therapeutic setting. Of those, nanobodies (Nb) are of particular interest considering their favorable kinetics, crossing of relevant biological membranes and intratumoral distribution. The purpose of this review is to highlight the unique characteristics and advantages of Nb-based radiotracers in BC imaging and therapy. Additionally, radiolabeling methods for Nb including direct labeling, indirect labeling via prosthetic group and indirect labeling via complexation will be discussed, reporting advantages and drawbacks. Furthermore, the preclinical to clinical translation of radiolabeled Nbs as promising theranostic agents will be reported.

Nuclear imaging for immune cell tracking in vivo – Comparison of various cell labeling methods and their application

• SPECT and PET are of great importance for in vivo tracking of immune cells. • Cell tracking technique depends on the type of labeled cell and targeted pathology. • Various radiotracers are used in direct and indirect cell labeling methods. • In the indirect labeling methods, applied radiotracers depend on reporter genes. • The most crucial radiotracers, reporter genes and cell labeling methods are described. There is currently great interest in the development of cell-based therapies, particularly those focused on the idea of exploiting systemically administered immune cells for therapeutic purposes in cancer ( e.g. , CAR-T and CAR-NK cells). The ability to monitor the fate of cells in vivo after administration is vital for the assessment of therapeutic outcomes of immune cell-based therapies both in preclinical research and clinical practice. In this context, imaging techniques, which allow for noninvasive and real-time monitoring of the distribution and long-term viability of the adoptively transferred immune cells in the tissue of interest, are of paramount importance. Among clinically relevant in vivo imaging modalities, the nuclear imaging techniques, comprising planar scintigraphy, SPECT, and PET, are considered to be of pivotal importance to in vivo cell tracking and designing optimal treatment strategies for cell-based therapies. This review paper focuses on nuclear imaging and its role in the evaluation of immune cell-based therapies’ effectiveness - tracking cells and their ability to home to the target tissue. We compare different types of radiotracers, outline various ways of immune cell labeling, and provide the latest examples for the use of nuclear imaging techniques both in preclinical studies and clinical settings.

Analyses of consumers' preferences and of the correspondence between direct and indirect label claims and the fatty acid profile of milk in large retail chains in northern Italy

Combined results from 2 survey studies were used to obtain information useful for the industries and retailers involved in the milk production and selling chain in North Italy. The first survey identified different clusters of fluid milk purchasers by examining their preferences and attitudes toward 12 intrinsic-extrinsic and credence milk attributes, by applying best-worst scaling methodology, whereas the second survey characterized the fatty acid (FA) profiles of commercial milk sold by large-scale retailers to verify the correspondence between the actual FA profile and the direct and indirect claims on the labels. To summarize information about the FA profile of milk, which may be considered an advanced attribute of milk quality, the milk FA index (MFAI) was calculated for each milk sample. A total of 130 milk samples (around 85% of the labels in northern Italy) and a total of 502 participants who answered a face-to-face questionnaire were considered in the 2 surveys. The milk samples were 13.1% organic, 9.2% certified as being of mountain origin, and over 50% noncertified but linked to cow grazing or to a mountain environment on their labels. The FA profiles showed a wide range of variation, with saturated FA ranging from 63.4 to 71.8, and polyunsaturated FA from 2.76 to 5.85. The FA profile and MFAI index of certified milk (organic or mountain-derived) were significantly different from the profiles of noncertified milk, whereas no correspondence was observed between the retail price and milk quality. When ranked on the basis of MFAI, which proved to be a good discriminating tool, the certified milks presented a bimodal distribution, indicating that certification does not always guarantee a real difference. The consumers chose milk considering the origin of the product, brand, expiration date, and process certification as the most important attributes, whereas they rated price and organic certification as the least important attributes. The study showed that about 20% of the consumers had a high propensity to buy milk on the basis of its quality. However, this attribute is often incorrectly indicated or not indicated at all on the milk label, with misleading images or claims that do not correspond to the actual FA quality of the milk. Having a clear index that offers information about the FA profile could thus be an interesting tool to improve the awareness of buyers and to valorize and differentiate milk products.

In Vivo Cell Tracking Using PET: Opportunities and Challenges for Clinical Translation in Oncology.

Simple SummaryTracking therapeutic cells with non-invasive imaging methods has the potential to provide important information on the efficacy of cell therapies. In oncology, for example, monitoring the spatial distribution of chimeric antigen receptor (CAR) T-cells or tumour-infiltrating lymphocytes (TILs) could be used to monitor the efficiency of cellular trafficking to target sites within a patient. This review covers different cell labelling approaches for the non-invasive detection of therapeutic cells using positron emission tomography (PET). The potential for the clinical translation of these approaches and first-in-human studies is examined, as well as the translational challenges involved and how imaging can help overcome some of these challenges.Cell therapy is a rapidly evolving field involving a wide spectrum of therapeutic cells for personalised medicine in cancer. In vivo imaging and tracking of cells can provide useful information for improving the accuracy, efficacy, and safety of cell therapies. This review focuses on radiopharmaceuticals for the non-invasive detection and tracking of therapeutic cells using positron emission tomography (PET). A range of approaches for imaging therapeutic cells is discussed: Direct ex vivo labelling of cells, in vivo indirect labelling of cells by utilising gene reporters, and detection of specific antigens expressed on the target cells using antibody-based radiopharmaceuticals (immuno-PET). This review examines the evaluation of PET imaging methods for therapeutic cell tracking in preclinical cancer models, their role in the translation into patients, first-in-human studies, as well as the translational challenges involved and how they can be overcome.

Ultrafast Photoclick Reaction for Selective 18F-Positron Emission Tomography Tracer Synthesis in Flow.

The development of very fast, clean, and selective methods for indirect labeling in PET tracer synthesis is an ongoing challenge. Here we present the development of an ultrafast photoclick method for the synthesis of short-lived 18F-PET tracers based on the photocycloaddition reaction of 9,10-phenanthrenequinones with electron-rich alkenes. The respective precursors are synthetically easily accessible and can be functionalized with various target groups. Using a flow photo-microreactor, the photoclick reaction can be performed in 60 s, and clinically relevant tracers for prostate cancer and bacterial infection imaging were prepared to demonstrate practicality of the method.

FC 103PROTEOME WIDE OXIDATIVE STRESS PROFILING IN MESOTHELIAL CELLS INDUCED BY PERITONEAL DIALYSIS FLUID

Abstract Background and Aims Reactive oxygen species (ROS) in the peritoneal cavity may result both from CKD and the specific composition of peritoneal dialysis fluids (PDF). Elevated cellular oxidative stress is defined as a cellular oxidant/antioxidant imbalance which impairs not only peritoneal cell viability but also contributes to progression of local and systemic PD-related pathomechanisms. So far only single targets or mediators of oxidative stress were investigated in mesothelial cells exposed to PD fluids. Here, we aim to analyze the broad impact and also identify individual targets of ROS during PD. Using the developed technique the anti-oxidative effect of alanyl-glutamine (AlaGln) supplementation of PDF was characterized on the proteome level. Method To establish a redox-proteomics workflow for studying oxidative stress in peritoneal mesothelial cells we used a gold-standard model of redox-stress (100 µm hydrogen peroxide (H2O2)) and PD-fluid induced stress. Levels of oxidative stress were first evaluated by intracellular ROS levels and superoxide dismutase activity. Oxidative stress levels induced by PDF were titrated to comparable levels of H2O2 treatment to be able to characterize redox modifications and the effect of addition of 8 mM AlaGln. To detect alterations of the redox proteome we adapted and refined an approach combining redox-sensitive isobaric mass tags and high-performance liquid chromatography coupled to mass spectrometry (LC/MS). We used a sequential combination of direct and indirect labeling of redox-sensitive cysteine residues. Results Exposure to PDF increased intracellular ROS production and accumulation as well as cell damage assessed by LDH-release compared to control cells. Cells exposed to AlaGln supplemented PDF showed less cell damage compared to PDF alone. Addition of AlaGln not only reduced the overall redox status (intracellular ROS and superoxide dismutase activity) but also led to different proteins being affected by redox modifications. The carefully optimized highly sensitive LC/MS-based redox proteomics workflow allowed identification of 5537 proteins of which 2614 contained a labeled cysteine. H2O2 treatment resulted in a shift of median oxidation from 11% under control conditions to 36%. While PDF alone increased the oxidation level to 31%, AlaGln supplemented PDF only led to 15% oxidation. Pathway analysis of proteins that changed their oxidation level &amp;gt;50% following the treatment were subjected to molecular pathway analysis revealing distinct differences. PDF exposure leads to regulation of general cell processes like regulation of glucokinase, RNA-binding and SUMOylation, addition of AlaGln regulated more specific signaling pathways for example fibrosis related pathways like TGF-ß and SMAD signaling. Conclusion Redox proteomics of peritoneal cells could represent a novel tool for the identification of mediators of uraemia and PD-induced pathomechanisms, and also to evaluate anti-oxidant pharmacological interventions to improve PD outcomes.

Homophilic and heterophilic cadherin bond rupture forces in homo- or hetero-cellular systems measured by AFM-based single-cell force spectroscopy

Cadherins enable intercellular adherens junctions to withstand tensile forces in tissues, e.g. generated by intracellular actomyosin contraction. In-vitro single molecule force spectroscopy experiments can reveal cadherin–cadherin extracellular region binding dynamics such as bond formation and strength. However, characterization of cadherin-presenting cell homophilic and heterophilic binding in the proteins’ native conformational and functional states in living cells has rarely been done. Here, we used atomic force microscopy (AFM) based single-cell force spectroscopy (SCFS) to measure rupture forces of homophilic and heterophilic bond formation of N- (neural), OB- (osteoblast) and E- (epithelial) cadherins in living fibroblast and epithelial cells in homo- and hetero-cellular arrangements, i.e., between cells and cadherins of the same and different types. In addition, we used indirect immunofluorescence labelling to study and correlate the expression of these cadherins in intercellular adherens junctions. We showed that N/N and E/E-cadherin homophilic binding events are stronger than N/OB heterophilic binding events. Disassembly of intracellular actin filaments affects the cadherin bond rupture forces suggesting a contribution of actin filaments in cadherin extracellular binding. Inactivation of myosin did not affect the cadherin rupture force in both homo- and hetero-cellular arrangements, but particularly strengthened the N/OB heterophilic bond and reinforced the other cadherins’ homophilic bonds.

Multimodal, label-free fluorescence and Raman imaging of amyloid deposits in snap-frozen Alzheimer\u2019s disease human brain tissue

Alzheimer’s disease (AD) neuropathology is characterized by hyperphosphorylated tau containing neurofibrillary tangles and amyloid-beta (Aβ) plaques. Normally these hallmarks are studied by (immuno-) histological techniques requiring chemical pretreatment and indirect labelling. Label-free imaging enables one to visualize normal tissue and pathology in its native form. Therefore, these techniques could contribute to a better understanding of the disease. Here, we present a comprehensive study of high-resolution fluorescence imaging (before and after staining) and spectroscopic modalities (Raman mapping under pre-resonance conditions and stimulated Raman scattering (SRS)) of amyloid deposits in snap-frozen AD human brain tissue. We performed fluorescence and spectroscopic imaging and subsequent thioflavin-S staining of the same tissue slices to provide direct confirmation of plaque location and correlation of spectroscopic biomarkers with plaque morphology; differences were observed between cored and fibrillar plaques. The SRS results showed a protein peak shift towards the β-sheet structure in cored amyloid deposits. In the Raman maps recorded with 532 nm excitation we identified the presence of carotenoids as a unique marker to differentiate between a cored amyloid plaque area versus a non-plaque area without prior knowledge of their location. The observed presence of carotenoids suggests a distinct neuroinflammatory response to misfolded protein accumulations.