Development of High-Throughput Experimentation Approaches for Rapid Radiochemical Exploration.

Positron emission tomography is a widely used imaging platform for studying physiological processes. Despite the proliferation of modern synthetic methodologies for radiolabeling, the optimization of these reactions still primarily relies on inefficient one-factor-at-a-time approaches. High-throughput experimentation (HTE) has proven to be a powerful approach for optimizing reactions in many areas of chemical synthesis. However, to date, HTE has rarely been applied to radiochemistry. This is largely because of the short lifetime of common radioisotopes, which presents major challenges for efficient parallel reaction setup and analysis using standard equipment and workflows. Herein, we demonstrate an effective HTE workflow and apply it to the optimization of copper-mediated radiofluorination of pharmaceutically relevant boronate ester substrates. The workflow utilizes commercial equipment and allows for rapid analysis of reactions for optimizing reactions, exploring chemical space using pharmaceutically relevant aryl boronates for radiofluorinations, and constructing large radiochemistry data sets.

Zinc-Mediated Radiosynthesis of Unprotected Fluorine-18 Labelled α-Tertiary Amides.

This report describes the development of a Zn(OTf)2 -mediated method for converting α-tertiary haloamides to the corresponding fluorine-18 labelled α-tertiary fluoroamides with no-carrier-added [18 F]tetramethylammonium fluoride. 1,5,7-Triazabicyclo[4.4.0]dec-5-ene is an essential additive for achieving high radiochemical conversion. Under the optimised conditions, radiofluorination proceeds at sterically hindered tertiary sites in high radiochemical conversions, yields, and purities. This method has been successfully automated and applied to access >200 mCi (>7.4 GBq) of several model radiofluorides. Mechanistic studies led to the development of a new, nucleophilic C-H radiofluorination process using N-sulphonyloxyamide substrates.

Copper-Mediated Radiocyanation of Unprotected Amino Acids and Peptides.

This report describes a copper-mediated radiocyanation of aryl halides that is applicable to complex molecules. This transformation tolerates an exceptionally wide range of functional groups, including unprotected amino acids. As such, it enables the site-specific introduction of [11C]CN into peptides at an iodophenylalanine residue. The use of a diamine-ligated copper(I) mediator is crucial for achieving high radiochemical yield under relatively mild conditions, thus limiting racemization and competing side reactions of other amino acid side chains. The reaction has been scaled and automated to deliver radiolabeled peptides, including analogues of adrenocorticotropic hormone 1-27 (ACTH) and nociceptin (NOP). For instance, this Cu-mediated radiocyanation was leveraged to prepare >40 mCi of [11C]cyano-NOP to evaluate biodistribution in a primate using positron emission tomography. This investigation provides preliminary evidence that nociceptin crosses the blood-brain barrier and shows uptake across all brain regions (SUV > 1 at 60 min post injection), consistent with the known distribution of NOP receptors in the rhesus brain.

Multiplex qPCR assays to distinguish individual species of arbuscular mycorrhizal fungi from roots and soil.

Currently, root colonization measurements of arbuscular mycorrhizal fungi (AMF) require staining and microscopy, and species-level identification of the fungi by such observations is not possible. Here, we present novel multiplex real-time PCR assays targeting the glomalin genes of 11 different species of AMF commonly found in temperate agricultural soils, which independently detect and measure the abundance of these fungi using DNA extracts from soil and or root tissue. The availability of these tools will not only increase throughput in determining levels of root colonization but can provide species-specific levels of root colonization from a single sample. This will help to establish which AMF species, or combinations of different species, provide the most benefits to crops, and will aid in the development of AMF for use as biofertilizers.

NHC-Copper Mediated Ligand-Directed Radiofluorination of Aryl Halides.

[18F]-labeled aryl fluorides are widely used as radiotracers for positron emission tomography (PET) imaging. Aryl halides (ArX) are particularly attractive precursors to these radiotracers, as they are readily available, inexpensive, and stable. However, to date, the direct preparation of [18F]-aryl fluorides from aryl halides remains limited to SNAr reactions between highly activated ArX substrates and K18F. This report describes an aryl halide radiofluorination reaction in which the C(sp2)-18F bond is formed via a copper-mediated pathway. Copper N-heterocyclic carbene complexes serve as mediators for this transformation, using aryl halide substrates with directing groups at the ortho position. This reaction is applied to the radiofluorination of electronically diverse aryl halide derivatives, including the bioactive molecules vismodegib and PH089.

Association of Anaesthetists: anaesthesia and peri-operative care for Jehovah's Witnesses and patients who refuse blood.

There are approximately 8.5 million Jehovah's Witnesses and around 150,000 live in Great Britain and Ireland. Based on their beliefs and core values, Jehovah's Witnesses refuse blood component transfusion (including red cells, plasma and platelets). They regard non-consensual transfusion as a physical violation. Consent to treatment is at the heart of this guideline. Refusal of treatment by an adult with capacity is lawful. The reasons why a patient might refuse transfusion and the implications are examined. The processes and products that are deemed acceptable or unacceptable to Jehovah's Witnesses are described. When a team is faced with a patient who refuses transfusion, a thorough review of the clinical situation is advocated and all options for treatment should be explored. After discussion, a plan should then be made that is acceptable to the patient and appropriate consent obtained. When agreement cannot be reached between the doctor and the patient, referral for a second opinion should be considered. When the patient is a child, the same strategy should be used but on occasion the clinical team may have to obtain legal help.

Real-Time PCR Assays to Detect and Distinguish the Rapid 'Ohi'a Death Pathogens Ceratocystis lukuohia and C. huliohia.

Ceratocystis lukuohia and C. huliohia are recently described fungal species that cause rapid 'ohi'a death (ROD) of Metrosideros polymorpha, Hawaii's most abundant and ecologically important native species. Although the pathogens are now widespread on Hawai'i Island, a major effort is underway to study and manage affected forests, and particularly to prevent the disease from spreading to other islands in the State or throughout the Pacific. Rapid and accurate detection is critical. Molecular diagnostic real-time PCR protocols were developed to detect and distinguish the two pathogens, suitable for detection of fungal DNA from extracts of wood, soil, and insect frass. The assays detect as few as 2 to 4 or 16 spores of C. huliohia or C. lukuohia, respectively. These assays are valuable tools for monitoring disease spread and offer a significant advantage over culture-based methods for diagnostics, requiring <1 day to arrive at definitive results.

The impact of the introduction of fidaxomicin on the management of Clostridium difficile infection in seven NHS secondary care hospitals in England: a series of local service evaluations.

Clostridium difficile infection (CDI) is associated with high mortality. Reducing incidence is a priority for patients, clinicians, the National Health Service (NHS) and Public Health England alike. In June 2012, fidaxomicin (FDX) was launched for the treatment of adults with CDI. The objective of this evaluation was to collect robust real-world data to understand the effectiveness of FDX in routine practice. In seven hospitals introducing FDX between July 2012 and July 2013, data were collected retrospectively from medical records on CDI episodes occurring 12 months before/after the introduction of FDX. All hospitalised patients aged ≥18 years with primary CDI (diarrhoea with presence of toxin A/B without a previous CDI in the previous 3 months) were included. Recurrence was defined as in-patient diarrhoea re-emergence requiring treatment any time within 3 months after the first episode. Each hospital had a different protocol for the use of FDX. In hospitals A and B, where FDX was used first line for all primary and recurrent episodes, the recurrence rate reduced from 10.6 % to 3.1 % and from 16.3 % to 3.1 %, with a significant difference in 28-day mortality from 18.2 % to 3.1 % (p < 0.05) and 17.3 % to 6.3 % (p < 0.05) for hospitals A and B, respectively. In hospitals using FDX in selected patients only, the changes in recurrence rates and mortality were less marked. The pattern of adoption of FDX appears to affect its impact on CDI outcome, with maximum reduction in recurrence and all-cause mortality where it is used as first-line treatment.

Electron beam energy QA - a note on measurement tolerances.

Monthly QA is recommended to verify the constancy of high-energy electron beams generated for clinical use by linear accelerators. The tolerances are defined as 2%/2 mm in beam penetration according to AAPM task group report 142. The practical implementation is typically achieved by measuring the ratio of readings at two different depths, preferably near the depth of maximum dose and at the depth corresponding to half the dose maximum. Based on beam commissioning data, we show that the relationship between the ranges of energy ratios for different electron energies is highly nonlinear. We provide a formalism that translates measurement deviations in the reference ratios into change in beam penetration for electron energies for six Elekta (6-18 MeV) and eight Varian (6-22 MeV) electron beams. Experimental checks were conducted for each Elekta energy to compare calculated values with measurements, and it was shown that they are in agreement. For example, for a 6 MeV beam a deviation in the measured ionization ratio of ± 15% might still be acceptable (i.e., be within ± 2 mm), whereas for an 18 MeV beam the corresponding tolerance might be ± 6%. These values strongly depend on the initial ratio chosen. In summary, the relationship between differences of the ionization ratio and the corresponding beam energy are derived. The findings can be translated into acceptable tolerance values for monthly QA of electron beam energies.

Performance evaluation of the Verigene® (Nanosphere) and FilmArray® (BioFire®) molecular assays for identification of causative organisms in bacterial bloodstream infections.

Molecular assays designed to provide bacterial identification and detection of resistance genes directly from positive blood cultures can significantly reduce the time to definitive results. This has the potential to improve patient management and antimicrobial stewardship. However, the extent of such an impact is yet to be fully assessed. We tested two such assays, the Verigene® System Bloodstream Infection Tests (Nanosphere, Inc., Northbrook, IL, USA) (both Gram-positive and Gram-negative cartridges) and the FilmArray® Blood Culture Identification Panel (BioFire® Diagnostics, Inc., Salt Lake City, UT, USA). We compared their accuracy and speed of organism and resistance gene identification to conventional culture-based methods for 173 positive blood cultures. We also retrospectively determined, for organisms deemed not to be contaminants, the potential impact on antimicrobial prescribing. Both the Verigene® and FilmArray® assays accurately identified organisms, on average, 27.95 and 29.17 h earlier than conventional methods, respectively. There were a significant number of false-positives for Pseudomonas aeruginosa with the FilmArray® assay, which may have been related to contamination of the bioMérieux BacT standard anaerobic blood culture bottles, which the manufacturer has acknowledged. Both panels provided results significantly faster than conventional methods. In our setting, the extent of the potential positive impact on antimicrobial prescribing was modest (9 out of 173 samples). However, this may be an underestimation, since probable contaminants were not included in this analysis. In conclusion, both panels gave accurate results with significantly improved turnaround times.

RIP1, a member of an Arabidopsis protein family, interacts with the protein RARE1 and broadly affects RNA editing.

Transcripts of plant organelle genes are modified by cytidine-to-uridine (C-to-U) RNA editing, often changing the encoded amino acid predicted from the DNA sequence. Members of the PLS subclass of the pentatricopeptide repeat (PPR) motif-containing family are site-specific recognition factors for either chloroplast or mitochondrial C targets of editing. However, other than PPR proteins and the cis-elements on the organelle transcripts, no other components of the editing machinery in either organelle have previously been identified. The Arabidopsis chloroplast PPR protein Required for AccD RNA Editing 1 (RARE1) specifies editing of a C in the accD transcript. RARE1 was detected in a complex of >200 kDa. We immunoprecipitated epitope-tagged RARE1, and tandem MS/MS analysis identified a protein of unknown function lacking PPR motifs; we named it RNA-editing factor interacting protein 1 (RIP1). Yeast two-hybrid analysis confirmed RIP1 interaction with RARE1, and RIP1-GFP fusions were found in both chloroplasts and mitochondria. Editing assays for all 34 known Arabidopsis chloroplast targets in a rip1 mutant revealed altered efficiency of 14 editing events. In mitochondria, 266 editing events were found to have reduced efficiency, with major loss of editing at 108 C targets. Virus-induced gene silencing of RIP1 confirmed the altered editing efficiency. Transient introduction of a WT RIP1 allele into rip1 improved the defective RNA editing. The presence of RIP1 in a protein complex along with chloroplast editing factor RARE1 indicates that RIP1 is an important component of the RNA editing apparatus that acts on many chloroplast and mitochondrial C targets.

Inoculation of black turtle beans (Phaseolus vulgaris) with mycorrhizal fungi increases the nutritional quality of seeds.

The use of arbuscular mycorrhizal fungi (AMF) as biofertilizers has proven successful in boosting the yield and nutritional quality of a variety of crops. AMF associate with plant roots and exchange soil nutrients for photosynthetically derived C in the form of sugars and lipids. Past research has shown that not all AMF species are equal in their benefit to nutrient uptake and crop health, and that the most beneficial AMF species appear to vary by host species. Although an important human food staple, especially in developing regions where nutrient deficiency is a prevalent threat to public health, little work has been done to test the effectiveness of AMF in enhancing the nutritional quality of common bean (Phaseolus vulgaris L.). Therefore, our objective was to determine the most beneficial AMF species for inoculation of this important crop. We inoculated black beans (Phaseolus vulgaris black turtle beans) with eight individual AMF species and one mixed species inoculum in an outdoor pot trial over 3 months and assessed the extent to which they altered yield, mineral nutrient and anthocyanin concentration of seeds and leaf tissues. Despite seeing no yield effects from inoculation, we found that across treatments percent root length colonized by AMF was positively correlated with plant tissue P, Cu, and Zn concentration. Underlying these broad benefits, seeds from plants inoculated with three AMF species, Claroideoglomus claroideum (+15%), Funneliformis mosseae (+13%), and Gigaspora rosea (+11%) had higher P concentration than non-mycorrhizal plants. C. claroideum also increased seed potassium (K) and copper (Cu), as well as leaf aluminum (Al) concentration making it a promising candidate to further test the benefit of individual AMF species on black bean growth in field trials.

Automated Radiosynthesis of [18F]FluoFAPI and Its Dosimetry and Single Acute Dose Toxicological Evaluation.

BACKGROUND: Cancer-associated fibroblasts have become a new target for therapy. Fibroblasts present within malignancies express the fibroblast activation protein (FAP). Inhibitors to FAP (FAPI) are small molecules recently developed as a theranostic agents for imaging and radiotherapy. All currently used FAPI rely on a linker-chelator complex attached to the 'inhibitor'. We describe a new automated method of the direct attachment of the radioisotope to the inhibitor, resulting in a >50% MW reduction with the hope of an improved tumor-to-background ratio and tumor uptake. METHODS: [18F]FluroFAPI was developed from a Sn precursor. This allowed for subsequent automated radioflourination. We obtained the biodistribution of [18F]FluroFAPI in rats, performed estimated human radiation dosimetry, and performed a 100× expected single dose toxicology analysis for eventual first-in-human experiments. RESULTS: The synthesis of the Sn precursor for FluorFAPI and the automated synthesis of [18F]FluroFAPI was demonstrated. [18F]FluroFAPI had favorable estimated human radiation dosimetry, and demonstrated no adverse effects when injected at a dose of 100× that planned for [18F]FluroFAPI. CONCLUSIONS: With the successful development of an automated synthesis of [18F]FluroFAPI, first-in-human testing can be planned with the hope of an improved tumor-to-background performance compared to other FAPI agents.

3AcFNP-59 for Positron Emission Tomography Imaging of Cholesterol Trafficking and Utilization.

Cholesteryl ester analogues of [18F]FNP-59 have the ability to provide information on cholesterol trafficking and utilization at earlier time points than those of [18F]FNP-59 or [131I]NP-59. It is well-known that free cholesterol and cholesteryl esters have differing distribution profiles and that they can be interconverted enzymatically. Substitution of the ester influences the rate of cholesterol ester hydrolysis and the subsequent mixing of cholesterol esters with the lipid pool in the body. This can be utilized by preparing esters that are more readily taken up by lipoprotein, are quickly hydrolyzed and mixed with the endogenous lipid pool and delivered to tissues of interest more quickly than free cholesterol analogues that require esterification for lipoprotein association. The acetyl ester of FNP-59 demonstrated the preferred uptake properties and response to adrenal cortical manipulation, indicating its ability to image hormone production. Finally, dosimetry studies were conducted in preparation for the clinical translation of [18F]3AcFNP-59.

Automated radiosynthesis and preclinical imaging of a novel [18F]fluorolidocaine analogue via sequential C-H radiolabelling.

The most prominent myocardial voltage-gated sodium channel, NaV1.5, is a major drug target for treating cardiovascular disease. However, treatment determination and therapeutic development are complicated partly by an inadequate understanding of how the density of SCN5A, the gene that encodes NaV1.5, relates to treatment response and disease prognosis. To address these challenges, imaging agents derived from NaV1.5 blocking therapeutics have been employed in positron emission tomography (PET) imaging to infer how SCN5A expression relates to human disease in vivo. Herein, we describe the preparation of a novel fluorine-18 labelled analogue of lidocaine, a known NaV1.5 inhibitor, and compare this agent to a previously described analogue. Evidence from rodent and non-human primate PET imaging experiments suggests that the imaging utility of these agents may be limited by rapid metabolism and clearance.

Immunometabolic cues recompose and reprogram the microenvironment around implanted biomaterials.

Circulating monocytes infiltrate and coordinate immune responses in tissues surrounding implanted biomaterials and in other inflamed tissues. Here we show that immunometabolic cues in the biomaterial microenvironment govern the trafficking of immune cells, including neutrophils and monocytes, in a manner dependent on the chemokine receptor 2 (CCR2) and the C-X3-C motif chemokine receptor 1 (CX3CR1). This affects the composition and activation states of macrophage and dendritic cell populations, ultimately orchestrating the relative composition of pro-inflammatory, transitory and anti-inflammatory CCR2+, CX3CR1+ and CCR2+ CX3CR1+ immune cell populations. In amorphous polylactide implants, modifying immunometabolism by glycolytic inhibition drives a pro-regenerative microenvironment principally by myeloid cells. In crystalline polylactide implants, together with arginase-1-expressing myeloid cells, T helper 2 cells and γδ+ T cells producing interleukin-4 substantially contribute to shaping the metabolically reprogrammed pro-regenerative microenvironment. Our findings inform the premise that local metabolic states regulate inflammatory processes in the biomaterial microenvironment.

Mi-2 complex couples DNA methylation to chromatin remodelling and histone deacetylation.

Methylation of DNA at the dinucleotide CpG is essential for mammalian development and is correlated with stable transcriptional silencing. This transcriptional silencing has recently been linked at a molecular level to histone deacetylation through the demonstration of a physical association between histone deacetylases and the methyl CpG-binding protein MeCP2 (refs 4,5). We previously purified a histone deacetylase complex from Xenopus laevis egg extracts that consists of six subunits, including an Rpd3-like deacetylase, the RbA p48/p46 histone-binding protein and the nucleosome-stimulated ATPase Mi-2 (ref. 6). Similar species were subsequently isolated from human cell lines, implying functional conservation across evolution. This complex represents the most abundant form of deacetylase in amphibian eggs and cultured mammalian cells. Here we identify the remaining three subunits of this enzyme complex. One of them binds specifically to methylated DNA in vitro and molecular cloning reveals a similarity to a known methyl CpG-binding protein. Our data substantiate the mechanistic link between DNA methylation, histone deacetylation and transcriptional silencing.

DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters.

Methylation of CpG islands is associated with transcriptional silencing and the formation of nuclease-resistant chromatin structures enriched in hypoacetylated histones. Methyl-CpG-binding proteins, such as MeCP2, provide a link between methylated DNA and hypoacetylated histones by recruiting histone deacetylase, but the mechanisms establishing the methylation patterns themselves are unknown. Whether DNA methylation is always causal for the assembly of repressive chromatin or whether features of transcriptionally silent chromatin might target methyltransferase remains unresolved. Mammalian DNA methyltransferases show little sequence specificity in vitro, yet methylation can be targeted in vivo within chromosomes to repetitive elements, centromeres and imprinted loci. This targeting is frequently disrupted in tumour cells, resulting in the improper silencing of tumour-suppressor genes associated with CpG islands. Here we show that the predominant mammalian DNA methyltransferase, DNMT1, co-purifies with the retinoblastoma (Rb) tumour suppressor gene product, E2F1, and HDAC1 and that DNMT1 cooperates with Rb to repress transcription from promoters containing E2F-binding sites. These results establish a link between DNA methylation, histone deacetylase and sequence-specific DNA binding activity, as well as a growth-regulatory pathway that is disrupted in nearly all cancer cells.

Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription.

CpG methylation in vertebrates correlates with alterations in chromatin structure and gene silencing. Differences in DNA-methylation status are associated with imprinting phenomena and carcinogenesis. In Xenopus laevis oocytes, DNA methylation dominantly silences transcription through the assembly of a repressive nucleosomal array. Methylated DNA assembled into chromatin binds the transcriptional repressor MeCP2 which cofractionates with Sin3 and histone deacetylase. Silencing conferred by MeCP2 and methylated DNA can be relieved by inhibition of histone deacetylase, facilitating the remodelling of chromatin and transcriptional activation. These results establish a direct causal relationship between DNA methylation-dependent transcriptional silencing and the modification of chromatin.

Automated production of [11C]butyrate for keto body PET imaging.

The report describes an updated, fully automated method for the production of [11C]butyrate, validated for use in clinical studies. A commercially available GE Tracerlab FXM synthesis module was reconfigured to allow for air-free introduction of n-propyl magnesium chloride and to incorporate Sep-Pak cartridges to simplify and shorten the purification process, as compared to purifying the product using traditional HPLC. The method takes 20 min from end-of-bombardment and reliably produces injectable doses of [11C]butyrate (8029 ± 1628 MBq (217 ± 44 mCi), 14 % radiochemical yield based on [11C]CO2, non-decay corrected) in high radiochemical purity (>97 %), n = 3. With radiotracer in hand, PET scans of rats confirmed uptake of the radiopharmaceutical in the brain. Rat biodistribution data was obtained and used in conjunction with OLINDA software to determine an estimated human total body effective dose of 3.20 × 10-3 mSv/MBq (1.19 × 10-2 rem/mCi), along with preliminary rodent PET imaging that confirmed brain uptake. Lastly, our first human [11C]butyrate PET studies using a dynamic bolus injection technique (n = 5), with a graphical Logan analysis using a white matter reference region, confirmed good radiotracer uptake in the brain and with relatively more prominent uptake in the cerebellar hemispheres, vermis, cingulum cortex and the thalami.