Genome-wide association multi-locus and multi-variate linear mixed models reveal two linked loci with major effects on partial resistance of apricot to bacterial canker.

BACKGROUND: Diseases caused by Pseudomonas syringae (Ps) are recognized as the most damaging factors in fruit trees with a significant economic and sanitary impact on crops. Among them, bacterial canker of apricot is exceedingly difficult to control due to a lack of efficient prophylactic measures. Several sources of partial resistance have been identified among genetic resources but the underlying genetic pattern has not been elucidated thus far. In this study, we phenotyped bacterial canker susceptibility in an apricot core-collection of 73 accessions over 4 years by measuring canker and superficial browning lengths issued from artificial inoculations in the orchard. In order to investigate the genetic architecture of partial resistance, we performed a genome-wide association study using best linear unbiased predictors on genetic (G) and genetic x year (G × Y) interaction effects extracted from linear mixed models. Using a set of 63,236 single-nucleotide polymorphism markers genotyped in the germplasm over the whole genome, multi-locus and multi-variate mixed models aimed at mapping the resistance while controlling for relatedness between individuals. RESULTS: We detected 11 significant associations over 7 candidate loci linked to disease resistance under the two most severe years. Colocalizations between G and G × Y terms indicated a modulation on allelic effect depending on environmental conditions. Among the candidate loci, two loci on chromosomes 5 and 6 had a high impact on both canker length and superficial browning, explaining 41 and 26% of the total phenotypic variance, respectively. We found unexpected long-range linkage disequilibrium (LD) between these two markers revealing an inter-chromosomal LD block linking the two underlying genes. This result supports the hypothesis of a co-adaptation effect due to selection through population demography. Candidate genes annotations suggest a functional pathway involving abscisic acid, a hormone mainly known for mediating abiotic stress responses but also reported as a potential factor in plant-pathogen interactions. CONCLUSIONS: Our study contributed to the first detailed characterization of the genetic determinants of partial resistance to bacterial canker in a Rosaceae species. It provided tools for fruit tree breeding by identifying progenitors with favorable haplotypes and by providing major-effect markers for a marker-assisted selection strategy.

High-Resolution SPECT Imaging of Stimuli-Responsive Soft Microrobots.

Untethered small-scale robots have great potential for biomedical applications. However, critical barriers to effective translation of these miniaturized machines into clinical practice exist. High resolution tracking and imaging in vivo is one of the barriers that limit the use of micro- and nanorobots in clinical applications. Here, the inclusion of radioactive compounds in soft thermoresponsive magnetic microrobots is investigated to enable their single-photon emission computed tomography imaging. Four microrobotic platforms differing in hydrogel structure and four 99m Tc[Tc]-based radioactive compounds are investigated in order to achieve optimal contrast agent retention and optimal imaging. Single microrobot imaging of structures as low as 100 µm in diameter, as well as tracking of shape switching from tubular to planar configurations by inclusion of 99m Tc[Tc] colloid in the hydrogel structure, is reported.

Implementation of a new separation method to produce qualitatively improved 64 Cu.

BACKGROUND: 64 Cu (T1/2  = 12.7 h) is an important radionuclide for diagnostic purposes and used for positron emission tomography (PET). A previous method utilized at Paul Scherrer Institute (PSI) proved to be unreliable and, while a method using anion exchange chromatography is a popular choice worldwide, it was felt a different approach was required to obtain a robust chemical separation method. METHODS: Enriched 64 Ni targets were created by electroplating on gold foil. The targets were irradiated with protons degraded to approximately 11 MeV at PSI's Injector 2 72 MeV research cyclotron and subsequently dissolved in HCl. The resultant solution was loaded onto AG MP-50 cation exchange resin and the 64 Cu separated from its target material and radiocobalt impurities, produced as part of the irradiation process, using various specific mixtures of HCl/acetone solution. The eluted product was evaporated and picked up in dilute HCl (0.05 M). The chemical purity of 64 Cu was determined by radiolabeling experiments at the highest possible molar activities. RESULTS: Reproducible results were obtained, yielding 3.6 to 8.3 GBq 64 Cu of high radionuclidic and radiochemical purity. The product was labeled to NODAGA-RGD, achieved at up to 500 MBq/nmol, indicating the high chemical purity. In a proof-of-concept in vivo study, 64 Cu-NODAGA-RGD was used for PET imaging of a tumor-bearing mouse. CONCLUSION: The chemical separation devised to produce high-quality 64 Cu proved to be robust and reproducible. The concept can be used at medical cyclotrons utilizing a solid target station, such that 64 Cu can be used at hospitals for PET imaging.

Dual, Site-Specific Modification of Antibodies by Using Solid-Phase Immobilized Microbial Transglutaminase.

Microbial transglutaminase (MTG) was stably solid-phase immobilized on glass microbeads by using a second-generation dendronized polymer. Immobilized MTG enabled the efficient generation of site-specifically conjugated proteins, including antibody fragments, as well as whole antibodies through distinct glutamines and, unprecedentedly, also through lysines with various bifunctional substrates with defined stoichiometries. With this method, we generated dual, site-specifically modified antibodies comprising a fluorescent probe and a metal chelator for radiolabeling-a strategy anticipated to design antibodies for imaging and simultaneous therapy. Furthermore, we provide evidence that immobilized MTG features higher siteselectivity than soluble MTG.

Radiolabeled 111In-FGF-2 Is Suitable for In Vitro/Ex Vivo Evaluations and In Vivo Imaging.

Fibroblast growth factor-2 (FGF-2) is a potent modulator of cell growth and regulation, with improper FGF-2 signaling being involved in impaired responses to injury or even cancer. Therefore, the exploitation of FGF-2 as a therapeutic drives the prerequisite for effective insight into drug disposition kinetics. In this article, we present an 111In-radiolabeled FGF-2 derivative for noninvasive imaging in small animals deploying single photon emission tomography (SPECT). 111In-FGF-2 is equally well suitable for in vitro and ex vivo investigations as 125I-FGF-2. Furthermore, 111In-FGF-2 permits the performance of in vivo imaging, for example for the analysis of FGF-2 containing pharmaceutical formulations in developmental or preclinical stages. 111In-FGF-2 had affinity for the low-molecular-weight heparin enoxaparin identical to that of unlabeled FGF-2 (Kd: 0.6 ± 0.07 µM and 0.33 ± 0.03 µM, respectively) as assessed by isothermal titration calorimetry. The binding of 111In-FGF-2 to heparan sulfate proteoglycans (HPSGs) and the biological activity were comparable to those of unlabeled FGF-2, with EC50 values of 12 ± 2 pM and 25 ± 6 pM, respectively. In vivo biodistribution in healthy nude mice indicated a predominant accumulation of 111In-FGF-2 in filtering organs and minor uptake in the retina and the salivary and pituitary glands, which was confirmed by SPECT imaging. Therefore, 111In-FGF-2 is a valid tracer for future noninvasive animal imaging of FGF-2 in pharmaceutical development.

Organochlorine pesticides, polychlorinated biphenyls, seafood consumption, and time-to-pregnancy.

BACKGROUND: People in developed countries are widely exposed to low levels of organochlorine pesticides, polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (PBDEs). Seafood is a major contributor to PCB exposure. Toxicity of those various pollutants to reproductive and endocrine functions raises questions about possible effects on fertility. We explored whether serum levels of these pollutants and seafood consumption were associated with the fertility of couples enrolled in a French birth cohort (PELAGIE). METHODS: Time-to-pregnancy was investigated in 3,421 pregnant women by asking how many months they had taken to conceive. Levels of 14 organochlorine pesticides, 12 PCBs, and 10 PBDE compounds were measured in cord blood serum from a random subcohort (n = 394). Mercury concentrations measured in maternal hair were considered as a potential coexposure. Fecundability odds ratios (ORs) were estimated from multivariate discrete-time Cox proportional hazard models. RESULTS: Shellfish consumption was associated with longer time-to-pregnancy (fecundability OR ≥twice/week vs. 0.410 µg/L vs. <0.266 µg/L, fecundability OR = 0.46 [0.32-0.66]). In multiple sensitivity analyses, reduced fecundability was most consistently associated with shellfish consumption, p,p'-DDE, total PCBs, PCB153, and PCB187. Models that simultaneously included multiple coexposure factors led to similar conclusions. CONCLUSIONS: Our findings were robust in sensitivity analyses, including analysis restricted to primiparous women. These results suggest that PCBs, p,p'-DDE, and other shellfish contaminants may impair human fertility.

Signaling Network Response to α-Particle-Targeted Therapy with the 225Ac-Labeled Minigastrin Analog 225Ac-PP-F11N Reveals the Radiosensitizing Potential of Histone Deacetylase Inhibitors.

α-particle emitters have recently been explored as valuable therapeutic radionuclides. Yet, toxicity to healthy organs and cancer radioresistance limit the efficacy of targeted α-particle therapy (TAT). Identification of the radiation-activated mechanisms that drive cancer cell survival provides opportunities to develop new points for therapeutic interference to improve the efficacy and safety of TAT. Methods: Quantitative phosphoproteomics and matching proteomics followed by the bioinformatics analysis were used to identify alterations in the signaling networks in response to TAT with the 225Ac-labeled minigastrin analog 225Ac-PP-F11N (DOTA-(dGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe) in A431 cells, which overexpress cholecystokinin B receptor (CCKBR). Western blot analysis and microscopy verified the activation of the selected signaling pathways. Small-molecule inhibitors were used to validate the potential of the radiosensitizing combinatory treatments both in vitro and in A431/CCKBR tumor-bearing nude mice. Results: TAT-induced alterations were involved in DNA damage response, cell cycle regulation, and signal transduction, as well as RNA transcription and processing, cell morphology, and transport. Western blot analysis and microscopy confirmed increased phosphorylations of the key proteins involved in DNA damage response and carcinogenesis, including p53, p53 binding protein 1 (p53BP1), histone deacetylases (HDACs), and H2AX. Inhibition of HDAC class II, ataxia-telangiectasia mutated (ATM), and p38 kinases by TMP269, AZD1390, and SB202190, respectively, sensitized A431/CCKBR cells to 225Ac-PP-F11N. As compared with the control and monotherapies, the combination of 225Ac-PP-F11N with the HDAC inhibitor vorinostat (suberoylanilide hydroxamic acid, SAHA) significantly reduced the viability and increased the DNA damage of A431/CCKBR cells, led to the most pronounced tumor growth inhibition, and extended the mean survival of A431/CCKBR xenografted nude mice. Conclusion: Our study revealed the cellular responses to TAT and demonstrated the radiosensitizing potential of HDAC inhibitors to 225Ac-PP-F11N in CCKBR-positive tumors. This proof-of-concept study recommends development of novel radiosensitizing strategies by targeting TAT-activated and survival-promoting signaling pathways.

Comparative analysis of cancer cell responses to targeted radionuclide therapy (TRT) and external beam radiotherapy (EBRT).

The vast majority of our knowledge regarding cancer radiobiology and the activation of radioresistance mechanisms emerged from studies using external beam radiation therapy (EBRT). Yet, less is known about the cancer response to internal targeted radionuclide therapy (TRT). Our comparative phosphoproteomics analyzed cellular responses to TRT with lutetium-177-labeled minigastrin analogue [177Lu]Lu-PP-F11N (ß-emitter) and EBRT (É£-rays) in CCKBR-positive cancer cells. Activation of DNA damage response by p53 was induced by both types of radiotherapy, whereas TRT robustly increased activation of signaling pathways including epidermal growth factor receptor (EGFR), mitogen-activated protein kinases (MAPKs) or integrin receptor. Inhibition of EGFR or integrin signaling sensitized cancer cells to radiolabeled minigastrin. In vivo, EGFR inhibitor erlotinib increased therapeutic response to [177Lu]Lu-PP-F11N and median survival of A431/CCKBR-tumor bearing nude mice. In summary, our study explores a complex scenario of cancer responses to different types of irradiation and pinpoints the radiosensitizing strategy, based on the targeting survival pathways, which are activated by TRT.

Therapeutic Response of CCKBR-Positive Tumors to Combinatory Treatment with Everolimus and the Radiolabeled Minigastrin Analogue [177Lu]Lu-PP-F11N.

The inhibition of the mammalian target of rapamycin complex 1 (mTORC1) by everolimus (RAD001) was recently shown to enhance the tumor uptake of radiolabeled minigastrin. In this paper, we investigate if this finding can improve the in vivo therapeutic response to [177Lu]Lu-PP-F11N treatment. The N-terminal DOTA-conjugated gastrin analogue PP-F11N (DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe) was used to evaluate treatment efficacy in the human A431/CCKBR xenograft nude mouse model in combination with RAD001. Both RAD001 and [177Lu]Lu-PP-F11N single treatments as well as their combination inhibited tumor growth and increased survival. In concomitantly treated mice, the average tumor size and median survival time were significantly reduced and extended, respectively, as compared to the monotherapies. The histological analysis of kidney and stomach dissected after treatment with RAD001 and [177Lu]Lu-PP-F11N did not indicate significant adverse effects. In conclusion, our study data demonstrate the potential of mTORC1 inhibition to substantially improve the therapeutic efficacy of radiolabeled minigastrin analogues in CCKBR-positive cancers.

ATP Impedes the Inhibitory Effect of Hsp90 on Aß40 Fibrillation.

Heat shock protein 90 (Hsp90) is a molecular chaperone that assists protein folding in an Adenosine triphosphate (ATP)-dependent way. Hsp90 has been reported to interact with Alzheimers disease associated amyloid-ß (Aß) peptides and to suppress toxic oligomer- and fibril formation. However, the mechanism remains largely unclear. Here we use a combination of atomic force microscopy (AFM) imaging, circular dichroism (CD) spectroscopy and biochemical analysis to quantify this interaction and put forward a microscopic picture including rate constants for the different transitions towards fibrillation. We show that Hsp90 binds to Aß40 monomers weakly but inhibits Aß40 from growing into fibrils at substoichiometric concentrations. ATP impedes this interaction, presumably by modulating Hsp90's conformational dynamics and reducing its hydrophobic surface. Altogether, these results might indicate alternative ways to prevent Aß40 fibrillation by manipulating chaperones that are already abundant in the brain.

In Vivo Imaging of Local Inflammation: Monitoring LPS-Induced CD80/CD86 Upregulation by PET.

PURPOSE: The co-stimulatory molecules CD80 and CD86 are upregulated on activated antigen-presenting cells (APC). We investigated whether local APC activation, induced by subcutaneous (s.c.) inoculation of lipopolysaccharides (LPS), can be imaged by positron emission tomography (PET) with CD80/CD86-targeting 64Cu-labelled abatacept. PROCEDURES: Mice were inoculated s.c. with extracellular-matrix gel containing either LPS or vehicle (PBS). Immune cell populations were analysed by flow cytometry and marker expression by RT-qPCR. 64Cu-NODAGA-abatacept distribution was analysed using PET/CT and ex vivo biodistribution. RESULTS: The number of CD80+ and CD86+ immune cells at the LPS inoculation site significantly increased a few days after inoculation. CD68 and CD86 expression were higher at the LPS than the PBS inoculation site, and CD80 was only increased at the LPS inoculation site. CTLA-4 was highest 10 days after LPS inoculation, when CD80/CD86 decreased again. A few days after inoculation, 64Cu-NODAGA-abatacept distribution to the inoculation site was significantly higher for LPS than PBS (4.2-fold). Co-administration of unlabelled abatacept or human immunoglobulin reduced tracer uptake. The latter reduced the number of CD86+ immune cells at the LPS inoculation site. CONCLUSIONS: CD80 and CD86 are upregulated in an LPS-induced local inflammation, indicating invasion of activated APCs. 64Cu-NODAGA-abatacept PET allowed following APC activation over time.

Design, Expression, Purification, and Characterization of a YFP-Tagged 2019-nCoV Spike Receptor-Binding Domain Construct.

2019-nCoV is the causative agent of the serious, still ongoing, worldwide coronavirus disease (COVID-19) pandemic. High quality recombinant virus proteins are required for research related to the development of vaccines and improved assays, and to the general understanding of virus action. The receptor-binding domain (RBD) of the 2019-nCoV spike (S) protein contains disulfide bonds and N-linked glycosylations, therefore, it is typically produced by secretion. Here, we describe a construct and protocol for the expression and purification of yellow fluorescent protein (YFP) labeled 2019-nCoV spike RBD. The fusion protein, in the vector pcDNA 4/TO, comprises an N-terminal interferon alpha 2 (IFNα2) signal peptide, an eYFP, a FLAG-tag, a human rhinovirus 3C protease (HRV3C) cleavage site, the RBD of the 2019-nCoV spike protein and a C-terminal 8x His-tag. We stably transfected HEK 293 cells. Following expansion of the cells, the fusion protein was secreted from adherent cells into serum-free medium. Ni-NTA immobilized metal ion affinity chromatography (IMAC) purification resulted in very high protein purity, based on analysis by SDS-PAGE. The fusion protein was soluble and monodisperse, as confirmed by size-exclusion chromatography (SEC) and negative staining electron microscopy. Deglycosylation experiments confirmed the presence of N-linked glycosylations in the secreted protein. Complex formation with the peptidase domain of human angiotensin-converting enzyme 2 (ACE2), the receptor for the 2019-nCoV spike RBD, was confirmed by SEC, both for the YFP-fused spike RBD and for spike RBD alone, after removal of YFP by proteolytic cleavage. Possible applications for the fusion protein include binding studies on cells or in vitro, fluorescent labeling of potential virus-binding sites on cells, the use as an antigen for immunization studies or as a tool for the development of novel virus- or antibody-detection assays.

Evaluation of Actinium-225 Labeled Minigastrin Analogue [225Ac]Ac-DOTA-PP-F11N for Targeted Alpha Particle Therapy.

The overexpression of cholecystokinin B receptor (CCKBR) in human cancers led to the development of radiolabeled minigastrin analogues for targeted radionuclide therapy, which aims to deliver cytotoxic radiation specifically to cancer cells. Alpha emitters (e.g., actinium-225) possess high potency in cancer cell-killing and hold promise for the treatment of malignant tumors. In these preclinical studies, we developed and evaluated CCKBR-targeted alpha particle therapy. The cellular uptake and cytotoxic effect of actinium-225 labeled and HPLC-purified minigastrin analogue [225Ac]Ac-PP-F11N were characterized in the human squamous cancer A431 cells transfected with CCKBR. Nude mice bearing A431/CCKBR tumors were used for biodistribution and therapy studies followed by histological analysis and SPECT/CT imaging. In vitro, [225Ac]Ac-PP-F11N showed CCKBR-specific and efficient internalization rate and potent cytotoxicity. The biodistribution studies of [225Ac]Ac-PP-F11N revealed CCKBR-specific uptake in tumors, whereas the therapeutic studies demonstrated dose-dependent inhibition of tumor growth and extended mean survival time, without apparent toxicity. The histological analysis of kidney and stomach indicated no severe adverse effects after [225Ac]Ac-PP-F11N administration. The post-therapy SPECT-CT images with [111In]In-PP-F11N confirmed no CCKBR-positive tumor left in the mice with complete remission. In conclusion, our study demonstrates therapeutic efficacy of [225Ac]Ac-PP-F11N without acute radiotoxicity in CCKBR-positive cancer model.

Pharmacological inhibition of mTORC1 increases CCKBR-specific tumor uptake of radiolabeled minigastrin analogue [177Lu]Lu-PP-F11N.

Rationale: A high tumor-to-healthy-tissue uptake ratio of radiolabeled ligands is an essential prerequisite for safe and effective peptide receptor radionuclide therapy (PRRT). In the present study, we searched for novel opportunities to increase tumor-specific uptake of the radiolabeled minigastrin analogue [177Lu]Lu-DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 ([177Lu]Lu-PP-F11N), that targets the cholecystokinin B receptor (CCKBR) in human cancers. Methods: A kinase inhibitor library screen followed by proliferation and internalization assays were employed to identify compounds which can increase uptake of [177Lu]Lu-PP-F11N in CCKBR-transfected human epidermoid carcinoma A431 cells and natural CCKBR-expressing rat pancreatic acinar AR42J cells. Western blot (WB) analysis verified the inhibition of the signaling pathways and the CCKBR level, whereas the cell-based assay analyzed arrestin recruitment. Biodistribution and SPECT imaging of the A431/CCKBR xenograft mouse model as well as histological analysis of the dissected tumors were used for in vivo validation. Results: Our screen identified the inhibitors of mammalian target of rapamycin complex 1 (mTORC1), which increased cell uptake of [177Lu]Lu-PP-F11N. Pharmacological mTORC1 inhibition by RAD001 and metformin increased internalization of [177Lu]Lu-PP-F11N in A431/CCKBR and in AR42J cells. Analysis of protein lysates from RAD001-treated cells revealed increased levels of CCKBR (2.2-fold) and inhibition of S6 phosphorylation. PP-F11N induced recruitment of ß-arrestin1/2 and ERK1/2 phosphorylation. In A431/CCKBR-tumor bearing nude mice, 3 or 5 days of RAD001 pretreatment significantly enhanced tumor-specific uptake of [177Lu]Lu-PP-F11N (ratio [RAD001/Control] of 1.56 or 1.79, respectively), whereas metformin treatment did not show a significant difference. Quantification of SPECT/CT images confirmed higher uptake of [177Lu]Lu-PP-F11N in RAD001-treated tumors with ratios [RAD001/Control] of average and maximum concentration reaching 3.11 and 3.17, respectively. HE staining and IHC of RAD001-treated tumors showed a significant increase in necrosis (1.4% control vs.10.6% of necrotic area) and the reduction of proliferative (80% control vs. 61% of Ki67 positive cells) and mitotically active cells (1.08% control vs. 0.75% of mitotic figures). No significant difference in the tumor vascularization was observed after five-day RAD001 or metformin treatment. Conclusions: Our data demonstrates, that increased CCKBR protein level by RAD001 pretreatment has the potential to improve tumor uptake of [177Lu]Lu-PP-F11N and provides proof-of-concept for the development of molecular strategies aimed at enhancing the level of the targeted receptor, to increase the efficacy of PRRT and nuclear imaging.

Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization.

The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice of detergent is critical, because it affects both the stability and monodispersity of the complex. We recently determined the crystal structure of an active state of bovine rhodopsin coupled to an engineered G protein, mini-Go, at 3.1 Å resolution. Here, we detail the procedure for optimizing the preparation of the rhodopsin-mini-Go complex. Dark-state rhodopsin was prepared in classical and neopentyl glycol (NPG) detergents, followed by complex formation with mini-Go under light exposure. The stability of the rhodopsin was assessed by ultraviolet-visible (UV-VIS) spectroscopy, which monitors the reconstitution into rhodopsin of the light-sensitive ligand, 9-cis retinal. Automated size-exclusion chromatography (SEC) was used to characterize the monodispersity of rhodopsin and the rhodopsin-mini-Go complex. SDS-polyacrylamide electrophoresis (SDS-PAGE) confirmed the formation of the complex by identifying a 1:1 molar ratio between rhodopsin and mini-Go after staining the gel with Coomassie blue. After cross-validating all this analytical data, we eliminated unsuitable detergents and continued with the best candidate detergent for large-scale preparation and crystallization. An additional problem arose from the heterogeneity of N-glycosylation. Heterologously-expressed rhodopsin was observed on SDS-PAGE to have two different N-glycosylated populations, which would probably have hindered crystallogenesis. Therefore, different deglycosylation enzymes were tested, and endoglycosidase F1 (EndoF1) produced rhodopsin with a single species of N-glycosylation. With this strategic pipeline for characterizing protein quality, preparation of the rhodopsin-mini-Go complex was optimized to deliver the crystal structure. This was only the third crystal structure of a GPCR-G protein signaling complex. This approach can also be generalized for other membrane proteins and their complexes to facilitate sample preparation and structure determination.

A stable neurotensin-based radiopharmaceutical for targeted imaging and therapy of neurotensin receptor-positive tumours.

PURPOSE: Neurotensin (NT) and its high affinity receptor (NTR1) are involved in several neoplastic processes. Thus, NT-based radiopharmaceuticals are potential tracers for targeted diagnosis and therapy of NTR-positive tumours. A new analogue based on NT(8-13), NT-XIX, with the three enzymatic cleavage sites stabilised, was synthesised and tested. METHODS: The synthesis was performed by Boc strategy. Labelling with (99m)Tc/(188)Re was performed using the tricarbonyl technique. Metabolic stability was tested in vitro and in vivo. NT-XIX was further characterised in vitro in HT-29 cells and in vivo in nude mice with HT-29 xenografts. RESULTS: NT-XIX showed much longer half-lives than non-stabilised analogues. Binding to NTR1 was highly specific, although the affinity was lower than that of natural NT. Bound activity rapidly internalised into HT-29 cells and 50% remained trapped after 24 h. In the time-course biodistribution, the highest uptake was found in the tumour at all p.i. times. In vivo uptake was specific, and accumulation of activity in the kidneys was low. Radioactivity clearance from healthy organs was faster than that from the tumour, resulting in improved tumour-to-tissue ratios and good SPECT/CT imaging. Treatment with (188)Re-NT-XIX (30 MBq, in three or four fractions) decreased tumour growth by 50% after 3 weeks. CONCLUSION: The high in vivo stability and the favourable in vivo behaviour makes NT-XIX an excellent candidate for the imaging and therapy of NTR1-positive tumours.

Chemical and biological characterization of new Re(CO)3/[99mTc](CO)3 bombesin analogues.

INTRODUCTION: Bombesin, a neuropeptide with potential for breast and prostate tumor targeting, is rapidly metabolized in vivo, and as a result, uptake in tumor xenografts in mice is poor. An improvement can be expected from the introduction of nonnatural amino acids and spacers. Leu13 was replaced by cyclohexylalanine and Met14 by norleucine. Two spacers, -betaAla-betaAla- and 3,6-dioxa-8-aminooctanoic acid, were inserted between the receptor-binding amino acid sequence (7-14) of bombesin (BBS) and the retroN(alpha)-carboxymethyl histidine chelator used for labeling with the [99mTc](CO)3 core and the rhenium (Re) congener. METHODS: The biological characterization of the new compounds was performed both in vitro on prostate carcinoma PC-3 cells (binding affinity, internalization/externalization) and in vivo (biodistribution in nude mice with tumor xenografts). The stability was also investigated in human plasma. The Re analogues were prepared for chemical characterization. RESULTS: The nonnatural amino acids led to markedly slower degradation in human plasma and PC-3 cell cultures. The receptor affinity of the new technetium 99m ([99mTc])-labeled BBS analogues was similar to the unmodified compound with Kd<1 nM. Uptake in the pancreas and in PC-3 tumor xenografts in nude mice was blocked by unlabeled BBS. The best target-to-nontarget uptake ratio was clearly due to the presence of the more polar spacer, -betaAla-betaAla-. CONCLUSIONS: The different spacers did not have a significant effect on stability or receptor affinity but had a clear influence on the uptake in healthy organs and tumors. Uptake in the kidneys was lower than in the liver, which is likely to be due to the lipophilicity of the compounds. A specific, high uptake was also observed in the gastrin-releasing peptide receptor-rich pancreas. Thus, with the introduction of spacers the in vivo properties of the compounds can be improved while leaving the affinity unaffected.

Double-stabilized neurotensin analogues as potential radiopharmaceuticals for NTR-positive tumors.

INTRODUCTION: Overexpression of neurotensin (NT) receptors in exocrine pancreatic cancer and other neuroendocrine cancers make them interesting targets for tumor imaging and therapy. Modifications at the cleavage bonds 8-9 and 11-12 led to the synthesis of NT-XII, NT-XIII and NT-XVIII, three new stabilized analogues. (NalphaHis)Ac was coupled to the N-terminus for labeling with [(99m)Tc]-tricarbonyl. METHODS: Stability was tested in vitro in human plasma and HT-29 cells. Binding to NT1 receptors and internalization/efflux were analyzed in intact HT-29 cells. Biodistribution studies were performed in nude mice bearing HT-29 xenografts. RESULTS: All analogues were very stable in human plasma, with half-lives of 20-21 days. Degradation in HT-29 cells was more rapid (t(1/2) of 6.5, 5 and 2.5 h for NT-XII, NT-XIII and NT-XVIII, respectively). They also showed high affinity and specificity for NT1 receptors. Bound activity was rapidly internalized at 37 degrees C. The pattern of externalization was different. NT-XII was released more slowly than NT-XIII and NT-XVIII (half of the activity still inside the cells after 24 h). Bigger differences were found in the biodistribution studies. NT-XII showed the highest tumor uptake as well as the best tumor to nontumor ratios. CONCLUSION: The modifications introduced in NT(8-13) increased plasma stability, maintaining unaffected the in vitro binding properties. The best biodistribution corresponded to NT-XII, which shows to be a good candidate for NT1 receptors overexpressing tumors. First clinical trials are ongoing.

Disruptions of ultradian and circadian organization of core temperature in a rat model of African trypanosomiasis using periodogram techniques on detrended data.

Periodogram techniques on detrended data were used to determine the incidence of Trypanosoma brucei brucei infection on the distribution of the core temperature of rats and the expression of temperature rhythms. In such an animal model, sudden episodic hypothermic bouts were described. These episodes of hypothermia are used here as temporal marks for the purpose of performing punctual comparisons on temperature organization. The experiment was conducted on 10 infected and 3 control Sprague-Dawley rats reared under a 24 h light-dark cycle. Core temperature was recorded continuously throughout the experiment, until the animals' death. Temperature distributions, analyzed longitudinally across the full duration of the experiment, exhibited a progressive shift from a bimodal to unimodal pattern, suggesting a weakening of the day/night core temperature differences. After hypothermic events, the robustness of the circadian rhythm substantially weakened, also affecting the ultradian components. The ultradian periods were reduced, suggesting fragmentation of temperature generation. Moreover, differences between daytime and nighttime ultradian patterns decreased during illness, confirming the weakening of the circadian component. The results of the experiments show that both core temperature distribution and temperature rhythm were disrupted during the infection. These disruptions worsened after each episode of hypothermia, suggesting an alteration of the temperature regulatory system.