Accurate compound-specific 14C dating of archaeological pottery vessels.

Pottery is one of the most commonly recovered artefacts from archaeological sites. Despite more than a century of relative dating based on typology and seriation1, accurate dating of pottery using the radiocarbon dating method has proven extremely challenging owing to the limited survival of organic temper and unreliability of visible residues2-4. Here we report a method to directly date archaeological pottery based on accelerator mass spectrometry analysis of 14C in absorbed food residues using palmitic (C16:0) and stearic (C18:0) fatty acids purified by preparative gas chromatography5-8. We present accurate compound-specific radiocarbon determinations of lipids extracted from pottery vessels, which were rigorously evaluated by comparison with dendrochronological dates9,10 and inclusion in site and regional chronologies that contained previously determined radiocarbon dates on other materials11-15. Notably, the compound-specific dates from each of the C16:0 and C18:0 fatty acids in pottery vessels provide an internal quality control of the results6 and are entirely compatible with dates for other commonly dated materials. Accurate radiocarbon dating of pottery vessels can reveal: (1) the period of use of pottery; (2) the antiquity of organic residues, including when specific foodstuffs were exploited; (3) the chronology of sites in the absence of traditionally datable materials; and (4) direct verification of pottery typochronologies. Here we used the method to date the exploitation of dairy and carcass products in Neolithic vessels from Britain, Anatolia, central and western Europe, and Saharan Africa.

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.

Machine learning identifies multi-parametric functional PET/MR imaging cluster to predict radiation resistance in preclinical head and neck cancer models.

PURPOSE: Tumor hypoxia and other microenvironmental factors are key determinants of treatment resistance. Hypoxia positron emission tomography (PET) and functional magnetic resonance imaging (MRI) are established prognostic imaging modalities to identify radiation resistance in head-and-neck cancer (HNC). The aim of this preclinical study was to develop a multi-parametric imaging parameter specifically for focal radiotherapy (RT) dose escalation using HNC xenografts of different radiation sensitivities. METHODS: A total of eight human HNC xenograft models were implanted into 68 immunodeficient mice. Combined PET/MRI using dynamic [18F]-fluoromisonidazole (FMISO) hypoxia PET, diffusion-weighted (DW), and dynamic contrast-enhanced MRI was carried out before and after fractionated RT (10 × 2 Gy). Imaging data were analyzed on voxel-basis using principal component (PC) analysis for dynamic data and apparent diffusion coefficients (ADCs) for DW-MRI. A data- and hypothesis-driven machine learning model was trained to identify clusters of high-risk subvolumes (HRSs) from multi-dimensional (1-5D) pre-clinical imaging data before and after RT. The stratification potential of each 1D to 5D model with respect to radiation sensitivity was evaluated using Cohen's d-score and compared to classical features such as mean/peak/maximum standardized uptake values (SUVmean/peak/max) and tumor-to-muscle-ratios (TMRpeak/max) as well as minimum/valley/maximum/mean ADC. RESULTS: Complete 5D imaging data were available for 42 animals. The final preclinical model for HRS identification at baseline yielding the highest stratification potential was defined in 3D imaging space based on ADC and two FMISO PCs ([Formula: see text]). In 1D imaging space, only clusters of ADC revealed significant stratification potential ([Formula: see text]). Among all classical features, only ADCvalley showed significant correlation to radiation resistance ([Formula: see text]). After 2 weeks of RT, FMISO_c1 showed significant correlation to radiation resistance ([Formula: see text]). CONCLUSION: A quantitative imaging metric was described in a preclinical study indicating that radiation-resistant subvolumes in HNC may be detected by clusters of ADC and FMISO using combined PET/MRI which are potential targets for future functional image-guided RT dose-painting approaches and require clinical validation.

Linking imaging to omics utilizing image-guided tissue extraction.

Phenotypic heterogeneity is commonly observed in diseased tissue, specifically in tumors. Multimodal imaging technologies can reveal tissue heterogeneity noninvasively in vivo, enabling imaging-based profiling of receptors, metabolism, morphology, or function on a macroscopic scale. In contrast, in vitro multiomics, immunohistochemistry, or histology techniques accurately characterize these heterogeneities in the cellular and subcellular scales in a more comprehensive but ex vivo manner. The complementary in vivo and ex vivo information would provide an enormous potential to better characterize a disease. However, this requires spatially accurate coregistration of these data by image-driven sampling as well as fast sample-preparation methods. Here, a unique image-guided milling machine and workflow for precise extraction of tissue samples from small laboratory animals or excised organs has been developed and evaluated. The samples can be delineated on tomographic images as volumes of interest and can be extracted with a spatial accuracy better than 0.25 mm. The samples remain cooled throughout the procedure to ensure metabolic stability, a precondition for accurate in vitro analysis.

The garlic compound ajoene targets protein folding in the endoplasmic reticulum of cancer cells.

Ajoene is a natural allylsulfur compound found in crushed garlic that arrests growth and induces apoptosis in cancer cells. To gain mechanistic insights into the cytotoxicity of ajoene in cancer cells, two fluorescently labelled ajoene analogs with dansyl- (DP) and fluorescein- (FOX) tags were synthesized. The tagged ajoenes were found to retain their activity at inhibiting proliferation and inducing apoptosis in MDA-MB-231 human breast-cancer and WHCO1 human esophageal-cancer cells. Both tagged ajoenes localized to the endoplasmic reticulum (ER) in MDA-MB-231 cells as observed by live cell confocal laser scanning microscopy (CLSM) and confirmed by generating an MDA-MB-231 cell line expressing yellow fluorescent protein (YFP) in the ER. DP appears to S-thiolate multiple protein targets in MDA-MB-231 cells as observed by immunoblotting under non-reducing conditions only; and a competition assay demonstrated that DP and Z-ajoene in fact share the same target. Ajoene S-thiolation interfered with protein folding and led to an accumulation of misfolded protein aggregates and activated the unfolded protein response (UPR). Consistent with this mechanism, increased levels of GRP78 and total ubiquitinated proteins were observed; and an ER-folded protein, type-1 collagen, was tracked to the proteasome following ajoene treatment. The intracellular protein aggregates were observed by CLSM and transmission electron microscopy (TEM). This is the first time that ajoene has been shown to target protein folding in the ER of cancer cells. © 2015 Wiley Periodicals, Inc.

Advances in PET imaging of cancer.

Molecular imaging has experienced enormous advancements in the areas of imaging technology, imaging probe and contrast development, and data quality, as well as machine learning-based data analysis. Positron emission tomography (PET) and its combination with computed tomography (CT) or magnetic resonance imaging (MRI) as a multimodality PET-CT or PET-MRI system offer a wealth of molecular, functional and morphological data with a single patient scan. Despite the recent technical advances and the availability of dozens of disease-specific contrast and imaging probes, only a few parameters, such as tumour size or the mean tracer uptake, are used for the evaluation of images in clinical practice. Multiparametric in vivo imaging data not only are highly quantitative but also can provide invaluable information about pathophysiology, receptor expression, metabolism, or morphological and functional features of tumours, such as pH, oxygenation or tissue density, as well as pharmacodynamic properties of drugs, to measure drug response with a contrast agent. It can further quantitatively map and spatially resolve the intertumoural and intratumoural heterogeneity, providing insights into tumour vulnerabilities for target-specific therapeutic interventions. Failure to exploit and integrate the full potential of such powerful imaging data may lead to a lost opportunity in which patients do not receive the best possible care. With the desire to implement personalized medicine in the cancer clinic, the full comprehensive diagnostic power of multiplexed imaging should be utilized.

Intratumoral in vivo staging of breast cancer by multi-tracer PET and advanced analysis.

The staging and local management of breast cancer involves the evaluation of the extent and completeness of excision of both the invasive carcinoma component and also the intraductal component or ductal carcinoma in situ. When both invasive ductal carcinoma and coincident ductal carcinoma in situ are present, assessment of the extent and localization of both components is required for optimal therapeutic planning. We have used a mouse model of breast cancer to evaluate the feasibility of applying molecular imaging to assess the local status of cancers in vivo. Multi-tracer positron emission tomography (PET) and magnetic resonance imaging (MRI) characterize the transition from premalignancy to invasive carcinoma. PET tracers for glucose consumption, membrane synthesis, and neoangiogenesis in combination with a Gaussian mixture model-based analysis reveal image-derived thresholds to separate the different stages within the whole-lesion. Autoradiography, histology, and quantitative image analysis of immunohistochemistry further corroborate our in vivo findings. Finally, clinical data further support our conclusions and demonstrate translational potential. In summary, this preclinical model provides a platform for characterizing multistep tumor progression and provides proof of concept that supports the utilization of advanced protocols for PET/MRI in clinical breast cancer imaging.

Synthesis and Biological Evaluation of a Radiolabeled PET (Positron Emission Tomography) Probe for Visualization of In Vivo α-Fucosidase Expression.

The acidic hydrolase α-fucosidase (AF) is a biomarker for maladies such as cancer and inflammation. The most advanced probes for α-fucosidase are unfortunately constrained to ex vivo or in vitro applications. The in vivo detection and quantification of AF using positron emission tomography would allow for better discovery and diagnosis of disease as well as provide better understanding of disease progression. We synthesized, characterized, and evaluated a radiolabeled small molecule inhibitor of AF based on a known molecule. The radiosynthesis involved the 11C methylation of a phenoxide, which was generated in situ by ultrasonification of the precursor with sodium hydride. The tracer was produced with a decay corrected yield of 41.7 ± 16.5% and had a molar activity of 65.4 ± 30.3 GBq/µmol. The tracer was shown to be stable in mouse serum at 60 min. To test the new tracer, HCT116 colorectal carcinoma cells were engineered to overexpress human AF. In vitro evaluation revealed 3.5-fold higher uptake in HCT116AF cells compared to HCT116 controls (26.4 ± 7.8 vs. 7.5 ± 1.0 kBq/106 cells). Static PET scans 50 min post injection revealed 2.5-fold higher tracer uptake in the HCT116AF tumors (3.0 ± 0.8%ID/cc (n = 6)) compared with the controls (1.2 ± 0.8 (n = 5)). Dynamic scans showed higher uptake in the HCT116AF tumors at all time-points (n = 2). Ex vivo analysis of the tumors, utilizing fluorescent DDK2 antibodies, confirmed the expression of human AF in the HCT116AF xenografts. We have developed a novel PET tracer to image AF in vivo and will now apply this to relevant disease models.

Covalent 18F-Radiotracers for SNAPTag: A New Toolbox for Reporter Gene Imaging.

There is a need for versatile in vivo nuclear imaging reporter systems to foster preclinical and clinical research. We explore the applicability of the SNAPTag and novel radiolabeled small-molecule ligands as a versatile reporter gene system for in vivo nuclear imaging. SNAPTag is a high-affinity protein tag used in a variety of biochemical research areas and based on the suicide DNA repair enzyme O6-methylguanine methyl transferase (MGMT). Its ligands are well suited for reporter gene imaging as the benzyl guanine core scaffold can be derivatized with fluorescent or radiolabeled moieties for various applications. Three guanine-based SNAPTag ligands ([18F]FBBG, [18F]pFBG and [18F]mFBG) were synthesized in high yields and were (radio)chemically characterized. HEK293 cells were engineered to express the SNAPTag on the cell surface and served as cell model to assess target affinity by radiotracer uptake assays, Western blotting and SDS-PAGE autoradiography. A subcutaneous HEK293-SNAPTag xenograft model in immunodeficient mice was used for in vivo evaluation of [18F]FBBG and [18F]pFBG while the biodistribution of [18F]mFBG was characterized in naïve animals. The results were validated by ex vivo biodistribution studies and immunofluorescence staining of the xenografts. All three radiotracers were produced in high radiochemical purity, molar activity and good yields. Western blot analysis revealed successful SNAPTag expression by the transfected HEK293 cells. In vitro testing revealed high target affinity of all three tracers with an up to 191-fold higher signal in the HEK293-SNAPTag cells compared to untransfected cells. This was further supported by a prominent radioactive protein band at the expected size in the SDS-PAGE autoradiograph of cells incubated with [18F]FBBG or [18F]pFBG. The in vivo studies demonstrated high uptake in HEK293-SNAP xenografts compared to HEK293 xenografts with excellent tumor-to-muscle ratios (7.5 ± 4.2 for [18F]FBBG and 10.6 ± 6.2 for [18F]pFBG). In contrast to [18F]pFBG and its chemical analogue [18F]mFBG, [18F]FBBG showed no signs of unspecific bone uptake and defluorination in vivo. Radiolabeled SNAPTag ligands bear great potential for clinical applications such as in vivo tracking of cell populations, antibody fragments and targeted radiotherapy. With excellent target affinity, good stability, and low non-specific binding, [18F]FBBG is a highly promising candidate for further preclinical evaluation.

Acetuino-A Handy Open-Source Radiochemistry Module for the Preparation of [1-11C]Acetate.

Radiosynthesis of [1-11C]acetate is well described in literature, but all syntheses either require adaptations in complex commercial synthesizers or rely on closed-source hardware and software control. Arduino microcontrollers are ideal for the compact, flexible, and inexpensive control of low-complexity hardware, making them particularly suited for radiochemistry where operation in space-limited shielded hot cells is mandatory. We established a [1-11C]acetate radiosynthesis module for combination with a [11C]MeI module available in almost every lab working with 11C. Its small footprint even enables back-to-back production in a hot cell already occupied by other modules. Using this setup, we achieved a reliable and flexible supply of this tracer, with radiochemical yields of 51.4 ± 28.2% and radiochemical purities (RCPs) of 94.4 ± 6.7% ( n = 9) in a synthesis time of 15 minutes. Positron emission tomography (PET) and biodistribution analysis demonstrated low background uptake in healthy mice, with highest uptake in liver and kidneys. Arduino microcontrollers have become valuable and versatile tools in our lab for the automatization of low-complexity procedures not requiring full-blown commercial radiochemistry synthesizers, as showcased here for the production of [1-11C]acetate.

A comparative analysis of isopropyl alcohol and ondansetron in the treatment of postoperative nausea and vomiting from the hospital setting to the home.

We compared the efficacy of inhaled isopropyl alcohol (IPA) with ondansetron for the control of postoperative nausea and vomiting (PONV) during a 24-hour period in 100 ASA class I-III women undergoing laparoscopic surgery. Nausea was measured postoperatively using a 0 to 10 verbal numeric rating scale (VNRS). The control group received ondansetron, 4 mg intravenously, and the experimental group inhaled IPA vapors. Breakthrough PONV was treated with 25-mg promethazine suppositories. Demographic and anesthesia characteristics were similar between groups. There was a significant difference between groups in mean +/- SD time to alleviation of PONV symptoms: for a 50% reduction in VNRS scores, 15.00 +/- 10.6 vs. 33.88 +/- 23.2 minutes was required in the experimental vs. the control group (P = .001). A total of 21 subjects (10 control; 11 experimental) reported PONV symptoms following discharge to home. The IPA treatment was successful in alleviating PONV symptoms in the home in 91% of the experimental group. We determined that using IPA after discharge from the postanesthesia care unit is a valuable method to control PONV in the hospital and at home. The results of this study suggest that IPA is much faster than ondansetron for 50% relief of nausea.

Biomechanical Analysis of Cement Augmentation Techniques on Pedicle Screw Fixation in Osteopenic Bone: A Cadaveric Study.

STUDY DESIGN: Three techniques for cement injection into osteopenic pedicles for screw augmentation were compared in a cadaver model. OBJECTIVES: To compare the safety and efficacy of 3 techniques for cement augmentation of pedicle screws in a cadaver model. BACKGROUND: Cement augmentation of pedicle screws improves fixation in osteopenic spines. Some investigators have used kyphoplasty osteo introducers to inject viscous cement into the pedicle. In addition, a novel fenestrated tapping system was studied that leaves a threaded cement tract for final pedicle screw insertion. METHODS: This 3-phase biomechanical study compared cement augmentation effects on the cephalocaudal toggle of pedicle screws within 34 fresh osteopenic human cadaveric vertebrae. Phase 1 compared injection through a fenestrated tap to a direct injection. Phase 2 evaluated the fenestrated tap versus injection of more viscous cement using a kyphoplasty osteo inducer. Phase 3 compared kyphoplasty and direct injection techniques. Each vertebral body was prepared with each of the 2 techniques. The pedicle screws were subjected to 10,000 cycles of cephalocaudal toggling, and total vertical displacement of each screw head was measured. RESULTS: In a combined analysis, overall displacement was 2.26 ± 0.57 mm for the direct injection, 2.88 ± 0.56 mm for the kyphoplasty technique, and 3.74 ± 0.59 mm for the fenestrated tap (p < .01). In Phase 1, extravasation of cement occurred in 5% of the fenestrated tap, 86% of the direct injection. Phase 2 results showed that extravasation occurred in 0% for the fenestrated tap group and 18% for the kyphoplasty group. In Phase 3, extravasation of cement was 54% of the direct injection, compared with 31% of the kyphoplasty group. CONCLUSIONS: The novel fenestrated tap system provided less resistance to toggle than either of the other 2 techniques but provided a lower incidence of cement extravasation. More viscous cement injected using kyphoplasty technique provides a combination of safety and efficacy.

Structure-activity studies on the anti-proliferation activity of ajoene analogues in WHCO1 oesophageal cancer cells.

The organosulfur compound ajoene derived from the rearrangement of allicin found in crushed garlic can inhibit the proliferation of tumour cells by inducing G(2)/M cell cycle arrest and apoptosis. We report on the application of a concise four-step synthesis (Hunter et al., 2008 [1]) that allows access to ajoene analogues with the end allyl groups substituted. A library of twelve such derivatives tested for their anti-proliferation activity against WHCO1 oesophageal cancer cells has identified a derivative containing p-methoxybenzyl (PMB)-substituted end groups that is twelve times more active than Z-ajoene, with an IC(50) of 2.1µM (Kaschula et al., 2011 [2]). Structure-activity studies involving modification of the sulfoxide and vinyl disulfide groups of this lead have revealed that the disulfide is the ajoene pharmacophore responsible for inhibiting WHCO1 cell growth, inducing G(2)/M cell cycle arrest and apoptosis by caspase-3 activation, and that the vinyl group serves to enhance the anti-proliferation activity a further eightfold. Reaction of the lead with cysteine in refluxing THF as a model reaction for ajoene's mechanism of action based on a thiol/disulfide exchange reveals that the allylic sulfur of the vinyl disulfide is the site of thiol attack in the exchange.

Anti-proliferation activity of synthetic ajoene analogues on cancer cell-lines.

The ability of garlic preparations to inhibit cancer cell-growth has been attributed to a group of structurally-related organosulfur compounds found in the crushed clove. Historically, interest has centred on three such compounds as allicin, diallyl disulfide and diallyl trisulfide, with less interest on E- and Z-ajoene. A recently developed synthetic route from our laboratory for preparing ajoene analogues allows access to derivatives containing the sulfoxide / vinyl disulfide core whilst varying the terminal end-group functionality. A small library has been synthesized and an advanced lead with p-methoxybenzyl end groups (8) identified. Data on the in vitro anti-proliferation activity of compound (8) is presented here against six cancer cell-lines in comparison with that of Z- and E-ajoene to reveal an enhancement in activity of up to twelvefold. In addition, a modest selectivity is observed for tumour over normal cell-lines of up to threefold. Data on ajoene and its derivatives is presented in the context of chemosensitization in drug-resistance, and ideas on ajoene's mode of action at the molecular level are presented and discussed.