Prehospital recognition of stroke is associated with a lower risk of death.

OBJECTIVE: Among patients assessed by the emergency medical service (EMS) and hospitalized with a final diagnosis of stroke, to describe delays, patient characteristics, actions taken and outcome in relation to the early recognition of stroke by the EMS clinician. METHODS: Patients admitted to any of six stroke units in Region Västra Götaland, Sweden, with a final diagnosis of stroke from 1 January 2013 to 31 December 2015 were included. Data on follow-up were retrieved from the Swedish Stroke Register. RESULTS: In all, 5467 patients were included. Stroke was recognized by the EMS clinician in 4396 cases (80.4%). The mean difference in the time from dialling 112 until arrival at the stroke unit was 556 min shorter when stroke was recognized, while the mean difference in the time from dialling 112 until a preliminary report from a computed tomography (CT) scan was 219 min shorter as compared with the patients in whom stroke was not recognized. After adjustment for age, sex, neurological deficits and coma, a lack of suspicion of stroke on EMS arrival was associated with an increased risk of death during three months of follow-up (odds ratio 1.66; 95% confidence interval 1.19-2.32; p = .003). CONCLUSION: Among patients with a stroke, more than 80% were recognized by the EMS clinician. Early recognition of stroke was associated with a markedly shorter time until arrival at the stroke unit and until the preliminary report of a CT scan. A lack of early stroke recognition was associated with an increased risk of death.

The Effects of Visual Field Loss from Stroke on Performance in a Driving Simulator.

SIGNIFICANCE: Visual field loss is a common consequence of stroke and often precludes driving. However, legal visual requirements for drivers' licenses are largely without scientific basis. PURPOSE: This study aimed to examine the effects of different types of homonymous visual field loss after stroke on simulated driving. METHODS: Data on performance and safety from a traffic simulator test for 153 participants with withdrawn drivers' licenses due to visual field loss from stroke were retrospectively compared with data from 83 healthy individuals without visual deficits in a cross-sectional study. The 93 individuals in the stroke group who regained their driving licenses after a successful simulator test were then followed in a national accident database. RESULTS: Sixty-five percent of the stroke participants passed the simulator test (95% confidence interval, 57 to 72%). Younger patients were more successful than older. However, classification by neither type of homonymous visual field loss nor side of visual field loss was predictive of driver safety. Participants with hemianopia had their lateral lane position dislocated to the nonaffected side of the visual field. None of the participants with a regained license were involved in motor vehicle accidents 3 to 6 years after the test. CONCLUSIONS: In this large cohort, driver safety could not be predicted from the type of homonymous visual field loss. Even individuals with severe visual field loss might be safe drivers. Therefore, it seems reasonable to provide an opportunity for individualized assessments of practical fitness to drive in circumstances of licensing issues. This study demonstrates the potential of using a standardized driving simulator test for such assessments of fitness to drive.

The Effects of Visual Field Loss from Optic Disc Drusen on Performance in a Driving Simulator.

The purpose of this study was to compare the driving simulator performance of participants with visual field loss (VFL) from optic disc drusen (ODD) with a normally sighted control group and a group of individuals with glaucoma. Data on performance and safety from a traffic simulator test for five participants with VFL from ODD were retrospectively compared with data from 49 male individuals without visual deficits in a cross-sectional study. VFL of the ODD group was also compared with a group of 20 male glaucoma participants who had failed the same simulator test. Four individuals with ODD regained their driving licences after a successful simulator test and were then followed in a national accident database. All participants with ODD passed the test. No significant differences in safety or performance measures were detected between the normally sighted participants and the ODD group despite severe concentric visual field constrictions. Compared with failed glaucoma male participants, the ODD group had even lower mean sensitivity in the peripheral and peripheral inferior field of vision. None of the four participants with a regained licence were involved in a motor vehicle accident during a 3-year follow-up period after the simulator test. Despite having severe VFL, participants with ODD had no worse performance or safety than controls. As even individuals with severe VFL might drive safely, there is a need for individual practical assessments on licencing issues.

The intensity of pain in the prehospital setting is most strongly reflected in the respiratory rate among physiological parameters.

BACKGROUND: In order to treat pain optimally, the Emergency Medical Service (EMS) clinician needs to be able to make a reasonable estimation of the severity of the pain. It is hypothesised that various physiological parameters will change as a response to pain. AIM: In a cohort of patients who were seen by EMS clinicians, to relate the patients' estimated intensity of pain to various physiological parameters. METHODS: Patients who called for EMS due to pain in a part of western Sweden were included. The intensity of pain was assessed according to the visual analogue scale (VAS) or the Numerical Rating Scale (NRS). The following were assessed the same time as pain on EMS arrival: heart rate, systolic and diastolic blood pressure, respiratory rate, moist skin and paleness. RESULTS: In all, 19,908 patients (≥18 years), were studied (51% women). There were significant associations between intensity of pain and the respiratory rate (r = 0.198; p < 0.0001), heart rate (r = 0.037; p < 0.0001), systolic blood pressure (r = -0.029; p < 0.0001), moist skin (r = 0.143; p < 0.0001) and paleness (r = 0.171; p < 0.0001). The strongest association was found with respiratory rate among patients aged 18-64 years (r = 0.258; p < 0.0001). CONCLUSION: In the prehospital setting, there were significant but weak correlations between intensity of pain and physiological parameters. The most clinically relevant association was found with an increased respiratory rate and presence of pale and moist skin among patients aged < 65 years. Among younger patients, respiratory rate may support in the clinical evaluation of pain.

Peptides Derived from Histone 3 and Modified at Position 18 Inhibit Histone Demethylase KDM6 Enzymes.

The KDM6 subfamily of histone lysine demethylases has recently been implicated as a putative target in the treatment of a number of diseases; this makes the availability of potent and selective inhibitors important. Due to high sequence similarity of the catalytic domain of Jumonji C histone demethylases, the development of small-molecule, family-specific inhibitors has, however, proven challenging. One approach to achieve the selective inhibition of these enzymes is the use of peptides derived from the substrate, the histone 3 C terminus. Here we used computational methods to optimize such inhibitors of the KDM6 family. Through natural amino acid substitution, it is shown that a K18I variant of a histone H3 derived peptide significantly increases affinity towards the KDM6 enzymes. The crystal structure of KDM6B in complex with a histone 3 derived K18I peptide reveals a tighter fit of the isoleucine side chain, compared with that of the arginine. As a consequence, the peptide R17 residue also has increased hydrophilic interactions. These interactions of the optimized peptide are likely to be responsible for the increased affinity to the KDM6 enzymes.

Impact of intratumoral heterogeneity of breast cancer tissue on quantitative metabolomics using high-resolution magic angle spinning 1 H NMR spectroscopy.

High-resolution magic angle spinning (HR MAS) nuclear magnetic resonance (NMR) spectroscopy is increasingly being used to study metabolite levels in human breast cancer tissue, assessing, for instance, correlations with prognostic factors, survival outcome or therapeutic response. However, the impact of intratumoral heterogeneity on metabolite levels in breast tumor tissue has not been studied comprehensively. More specifically, when biopsy material is analyzed, it remains questionable whether one biopsy is representative of the entire tumor. Therefore, multi-core sampling (n = 6) of tumor tissue from three patients with breast cancer, followed by lipid (0.9- and 1.3-ppm signals) and metabolite quantification using HR MAS 1 H NMR, was performed, resulting in the quantification of 32 metabolites. The mean relative standard deviation across all metabolites for the six tumor cores sampled from each of the three tumors ranged from 0.48 to 0.74. This was considerably higher when compared with a morphologically more homogeneous tissue type, here represented by murine liver (0.16-0.20). Despite the seemingly high variability observed within the tumor tissue, a random forest classifier trained on the original sample set (training set) was, with one exception, able to correctly predict the tumor identity of an independent series of cores (test set) that were additionally sampled from the same three tumors and analyzed blindly. Moreover, significant differences between the tumors were identified using one-way analysis of variance (ANOVA), indicating that the intertumoral differences for many metabolites were larger than the intratumoral differences for these three tumors. That intertumoral differences, on average, were larger than intratumoral differences was further supported by the analysis of duplicate tissue cores from 15 additional breast tumors. In summary, despite the observed intratumoral variability, the results of the present study suggest that the analysis of one, or a few, replicates per tumor may be acceptable, and supports the feasibility of performing reliable analyses of patient tissue.

Studying the fragmentation mechanism of selected components present in crude oil by collision-induced dissociation mass spectrometry.

RATIONALE: Structural characterization of individual compounds in very complex mixtures is difficult to achieve. One important step in structural elucidation is understanding the mass spectrometric fragmentation mechanisms of the compounds present in such mixtures. Here, different individual compounds presumed to be present in a complex crude oil mixture have been synthesized and structurally characterized by tandem mass spectrometry (MS/MS) studies. METHODS: Model compounds with different aromatic cores and various substitutents were synthesized. Major effort has been put into producing isomerically pure compounds to better understand the fragmentation pattern. Each synthesized compound has been subjected to MSn studies using either a triple quadrupole or a linear ion trap mass spectrometer with electrospray or atmospheric pressure photoionization. The results are used to analyze individual compounds from a complex vacuum gas oil (VGO). RESULTS: The synthesized compounds and a chromatographically simplified vacuum gas oil were used for structural analysis. The major fragmentation mechanism is the benzylic cleavage of the aliphatic side chain. Each side chain can be separately removed from the aromatic core by using MSn methods. At the end of a series of fragmentations, the base aromatic core structure remains and can be chararcterized. CONCLUSIONS: By defining the fragmentation mechanism in complex oil samples it was possible to structurally characterize individual compounds present in a chromatographically simplified VGO. The compounds consist of an aromatic core with aliphatic side chains. Cleavage of all side chains can be achieved by MSn measurements, allowing characterization of the remaining core structure.

Metabolic profiling of ob/ob mouse fatty liver using HR-MAS 1H-NMR combined with gene expression analysis reveals alterations in betaine metabolism and the transsulfuration pathway.

Metabolic perturbations resulting from excessive hepatic fat accumulation are poorly understood. Thus, in this study, leptin-deficient ob/ob mice, a mouse model of fatty liver disease, were used to investigate metabolic alterations in more detail. Metabolites were quantified in intact liver tissues of ob/ob (n = 8) and control (n = 8) mice using high-resolution magic angle spinning (HR-MAS) 1H-NMR. In addition, after demonstrating that HR-MAS 1H-NMR does not affect RNA integrity, transcriptional changes were measured by quantitative real-time PCR on RNA extracted from the same specimens after HR-MAS 1H-NMR measurements. Importantly, the gene expression changes obtained agreed with those observed by Affymetrix microarray analysis performed on RNA isolated directly from fresh-frozen tissue. In total, 40 metabolites could be assigned in the spectra and subsequently quantified. Quantification of lactate was also possible after applying a lactate-editing pulse sequence that suppresses the lipid signal, which superimposes the lactate methyl resonance at 1.3 ppm. Significant differences were detected for creatinine, glutamate, glycine, glycolate, trimethylamine-N-oxide, dimethylglycine, ADP, AMP, betaine, phenylalanine, and uridine. Furthermore, alterations in one-carbon metabolism, supported by both metabolic and transcriptional changes, were observed. These included reduced demethylation of betaine to dimethylglycine and the reduced expression of genes coding for transsulfuration pathway enzymes, which appears to preserve methionine levels, but may limit glutathione synthesis. Overall, the combined approach is advantageous as it identifies changes not only at the single gene or metabolite level but also deregulated pathways, thus providing critical insight into changes accompanying fatty liver disease. Graphical abstract A Evaluation of RNA integrity before and after HR-MAS 1H-NMR of intact mouse liver tissue. B Metabolite concentrations and gene expression levels assessed in ob/ob (steatotic) and ob/+ (control) mice using HR-MAS 1H-NMR and qRT-PCR, respectively.

The genome of Spironucleus salmonicida highlights a fish pathogen adapted to fluctuating environments.

Spironucleus salmonicida causes systemic infections in salmonid fish. It belongs to the group diplomonads, binucleated heterotrophic flagellates adapted to micro-aerobic environments. Recently we identified energy-producing hydrogenosomes in S. salmonicida. Here we present a genome analysis of the fish parasite with a focus on the comparison to the more studied diplomonad Giardia intestinalis. We annotated 8067 protein coding genes in the ∼12.9 Mbp S. salmonicida genome. Unlike G. intestinalis, promoter-like motifs were found upstream of genes which are correlated with gene expression, suggesting a more elaborate transcriptional regulation. S. salmonicida can utilise more carbohydrates as energy sources, has an extended amino acid and sulfur metabolism, and more enzymes involved in scavenging of reactive oxygen species compared to G. intestinalis. Both genomes have large families of cysteine-rich membrane proteins. A cluster analysis indicated large divergence of these families in the two diplomonads. Nevertheless, one of S. salmonicida cysteine-rich proteins was localised to the plasma membrane similar to G. intestinalis variant-surface proteins. We identified S. salmonicida homologs to cyst wall proteins and showed that one of these is functional when expressed in Giardia. This suggests that the fish parasite is transmitted as a cyst between hosts. The extended metabolic repertoire and more extensive gene regulation compared to G. intestinalis suggest that the fish parasite is more adapted to cope with environmental fluctuations. Our genome analyses indicate that S. salmonicida is a well-adapted pathogen that can colonize different sites in the host.

On the reversibility of parasitism: adaptation to a free-living lifestyle via gene acquisitions in the diplomonad Trepomonas sp. PC1.

BACKGROUND: It is generally thought that the evolutionary transition to parasitism is irreversible because it is associated with the loss of functions needed for a free-living lifestyle. Nevertheless, free-living taxa are sometimes nested within parasite clades in phylogenetic trees, which could indicate that they are secondarily free-living. Herein, we test this hypothesis by studying the genomic basis for evolutionary transitions between lifestyles in diplomonads, a group of anaerobic eukaryotes. Most described diplomonads are intestinal parasites or commensals of various animals, but there are also free-living diplomonads found in oxygen-poor environments such as marine and freshwater sediments. All these nest well within groups of parasitic diplomonads in phylogenetic trees, suggesting that they could be secondarily free-living. RESULTS: We present a transcriptome study of Trepomonas sp. PC1, a diplomonad isolated from marine sediment. Analysis of the metabolic genes revealed a number of proteins involved in degradation of the bacterial membrane and cell wall, as well as an extended set of enzymes involved in carbohydrate degradation and nucleotide metabolism. Phylogenetic analyses showed that most of the differences in metabolic capacity between free-living Trepomonas and the parasitic diplomonads are due to recent acquisitions of bacterial genes via gene transfer. Interestingly, one of the acquired genes encodes a ribonucleotide reductase, which frees Trepomonas from the need to scavenge deoxyribonucleosides. The transcriptome included a gene encoding squalene-tetrahymanol cyclase. This enzyme synthesizes the sterol substitute tetrahymanol in the absence of oxygen, potentially allowing Trepomonas to thrive under anaerobic conditions as a free-living bacterivore, without depending on sterols from other eukaryotes. CONCLUSIONS: Our findings are consistent with the phylogenetic evidence that the last common ancestor of diplomonads was dependent on a host and that Trepomonas has adapted secondarily to a free-living lifestyle. We believe that similar studies of other groups where free-living taxa are nested within parasites could reveal more examples of secondarily free-living eukaryotes.

Comparative genomic analyses of freshly isolated Giardia intestinalis assemblage A isolates.

BACKGROUND: The diarrhea-causing protozoan Giardia intestinalis makes up a species complex of eight different assemblages (A-H), where assemblage A and B infect humans. Comparative whole-genome analyses of three of these assemblages have shown that there is significant divergence at the inter-assemblage level, however little is currently known regarding variation at the intra-assemblage level. We have performed whole genome sequencing of two sub-assemblage AII isolates, recently axenized from symptomatic human patients, to study the biological and genetic diversity within assemblage A isolates. RESULTS: Several biological differences between the new and earlier characterized assemblage A isolates were identified, including a difference in growth medium preference. The two AII isolates were of different sub-assemblage types (AII-1 [AS175] and AII-2 [AS98]) and showed size differences in the smallest chromosomes. The amount of genetic diversity was characterized in relation to the genome of the Giardia reference isolate WB, an assemblage AI isolate. Our analyses indicate that the divergence between AI and AII is approximately 1 %, represented by ~100,000 single nucleotide polymorphisms (SNP) distributed over the chromosomes with enrichment in variable genomic regions containing surface antigens. The level of allelic sequence heterozygosity (ASH) in the two AII isolates was found to be 0.25-0.35 %, which is 25-30 fold higher than in the WB isolate and 10 fold higher than the assemblage AII isolate DH (0.037 %). 35 protein-encoding genes, not found in the WB genome, were identified in the two AII genomes. The large gene families of variant-specific surface proteins (VSPs) and high cysteine membrane proteins (HCMPs) showed isolate-specific divergences of the gene repertoires. Certain genes, often in small gene families with 2 to 8 members, localize to the variable regions of the genomes and show high sequence diversity between the assemblage A isolates. One of the families, Bactericidal/Permeability Increasing-like protein (BPIL), with eight members was characterized further and the proteins were shown to localize to the ER in trophozoites. CONCLUSIONS: Giardia genomes are modular with highly conserved core regions mixed up by variable regions containing high levels of ASH, SNPs and variable surface antigens. There are significant genomic variations in assemblage A isolates, in terms of chromosome size, gene content, surface protein repertoire and gene polymorphisms and these differences mainly localize to the variable regions of the genomes. The large genetic differences within one assemblage of G. intestinalis strengthen the argument that the assemblages represent different Giardia species.

Pulmonary tuberculosis patients with a vitamin D deficiency demonstrate low local expression of the antimicrobial peptide LL-37 but enhanced FoxP3+ regulatory T cells and IgG-secreting cells.

Control of human tuberculosis (TB) requires induction and maintenance of both macrophage and T cell effector functions. We demonstrate that pulmonary TB patients with a vitamin D deficiency had significantly reduced local levels of the vitamin D-inducible antimicrobial peptide LL-37 in granulomatous lesions compared to distal parenchyma from the infected lung. Instead, TB lesions were abundant in CD3(+) T cells and FoxP3(+) regulatory T cells as well as IgG-secreting CD20(+) B cells, particularly in sputum-smear positive patients with cavitary TB. Mycobacteria-specific serum IgG titers were also elevated in patients with active TB. An up-regulation of the B cell stimulatory cytokine IL-21 correlated with mRNA expression of CD20, total IgG and also IL-10 in the TB lesions. Altogether, vitamin D-deficient TB patients expressed a weak antimicrobial response but an IL-21 associated expansion of IgG-secreting B cells combined with a rise in FoxP3(+) regulatory T cells at the local site of infection.

The development of autoimmune features in aging mice is closely associated with alterations of the peripheral CD4⁺ T-cell compartment.

Some signs of potential autoimmunity, such as the appearance of antinuclear antibodies (ANAs) become prevalent with age. In most cases, elderly people with ANAs remain healthy. Here, we investigated whether the same holds true for inbred strains of mice. Indeed, we show that most mice of the C57BL/6 (B6) strain spontaneously produced IgG ANA at 8-12 months of age, showed IgM deposition in kidneys and lymphocyte infiltrates in submandibular salivary glands. Despite all of this, the mice remained healthy. ANA production is likely CD4(+) T-cell dependent, since old (40-50 weeks of age) B6 mice deficient for MHC class II do not produce IgG ANAs. BM chimeras showed that ANA production was not determined by age-related changes in radiosensitive, hematopoietic progenitor cells, and that the CD4(+) T cells that promote ANA production were radioresistant. Thymectomy of B6 mice at 5 weeks of age led to premature alterations in T-cell homeostasis and ANA production, by 15 weeks of age, similar to that in old mice. Our findings suggest that a disturbed T-cell homeostasis may drive the onset of some autoimmune features.

The amount of self-antigen determines the effector function of murine T cells escaping negative selection.

Autoimmune diseases develop when self-specific T cells that escaped negative selection initiate a harmful immune response against self. However, factors, which influence the initiation and progression of an autoimmune response remain incompletely understood. By establishing a double-transgenic BALB/c mouse system in which different amounts of a cell-surface neo-self-antigen are expressed under the CD11c promoter, we demonstrate that antigen dose dramatically influences T-cell tolerance mechanisms. Moderate antigen expression in both hematopoietic and nonhematopoietic cells favors the development of antigen-specific Treg cells and the establishment of a tolerogenic environment. In marked contrast, a high dose of antigen expression results in very stringent negative selection, in poor development of antigen-specific Treg cells and in the early onset of anemia and splenomegaly and the late development of arthritis and high titers of IgG auto Abs. Disease is initiated by autoreactive T cells, which escape negative selection by expressing a second TCR with a different specificity or an altered affinity. Transfer of Ag-specific Treg cells ameliorates the early onset signs of disease but does not prevent the development of long-term chronic pathologies. Altogether, our results suggest that Ag dose directly affects Treg-cell generation and thus, the set-up of T-cell tolerance.

Activation, exhaustion, and persistent decline of the antimicrobial MR1-restricted MAIT-cell population in chronic HIV-1 infection.

Mucosal-associated invariant T (MAIT) cells are an evolutionarily conserved antimicrobial MR1-restricted T-cell subset. MAIT cells are CD161(+), express a V7.2 TCR, are primarily CD8(+) and numerous in blood and mucosal tissues. However, their role in HIV-1 infection is unknown. In this study, we found levels of MAIT cells to be severely reduced in circulation in patients with chronic HIV-1 infection. Residual MAIT cells were highly activated and functionally exhausted. Their decline was associated with time since diagnosis, activation levels, and the concomitant expansion of a subset of functionally impaired CD161(+) V7.2(+) T cells. Such cells were generated in vitro by exposure of MAIT cells to Escherichia coli. Notably, whereas the function of residual MAIT cells was at least partly restored by effective antiretroviral therapy, levels of MAIT cells in peripheral blood were not restored. Interestingly, MAIT cells in rectal mucosa were relatively preserved, although some of the changes seen in blood were recapitulated in the mucosa. These findings are consistent with a model in which the MAIT-cell compartment, possibly as a result of persistent exposure to microbial material, is engaged, activated, exhausted, and progressively and persistently depleted during chronic HIV-1 infection.

Progression of clinical tuberculosis is associated with a Th2 immune response signature in combination with elevated levels of SOCS3.

In this study, we explored the local cytokine/chemokine profiles in patients with active pulmonary or pleural tuberculosis (TB) using multiplex protein analysis of bronchoalveolar lavage and pleural fluid samples. Despite increased pro-inflammation compared to the uninfected controls; there was no up-regulation of IFN-γ or the T cell chemoattractant CCL5 in the lung of patients with pulmonary TB. Instead, elevated levels of IL-4 and CCL4 were associated with high mycobacteria-specific IgG titres as well as SOCS3 (suppressors of cytokine signaling) mRNA and progression of moderate-to-severe disease. Contrary, IL-4, CCL4 and SOCS3 remained low in patients with extrapulmonary pleural TB, while IFN-γ, CCL5 and SOCS1 were up-regulated. Both SOCS molecules were induced in human macrophages infected with Mycobacterium tuberculosis in vitro. The Th2 immune response signature found in patients with progressive pulmonary TB could result from inappropriate cytokine/chemokine responses and excessive SOCS3 expression that may represent potential targets for clinical TB management.

Gene transfer and diversification of microbial eukaryotes.

The importance of lateral gene transfer in genome evolution of microbial eukaryotes is slowly being appreciated. Acquisitions of genes have led to metabolic adaptation in diverse eukaryotic lineages. In most cases the metabolic genes have originated from prokaryotes, often followed by sequential transfers between eukaryotes. However, the knowledge of gene transfer in eukaryotes is still mainly based on anecdotal evidence. Some of the observed patterns may be biases in experimental approaches and sequence databases rather than evolutionary trends. Rigorous systematic studies of gene acquisitions that allow for the possibility of exchanges of all categories of genes from all sources are needed to get a more objective view of gene transfer in eukaryote evolution. It may be that the role of gene transfer in the diversification process of microbial eukaryotes currently is underestimated.

Ionic liquids as stationary phases in gas chromatography--an LSER investigation of six commercial phases and some applications.

The separation properties of six novel stationary phases for gas chromatography, commercially available from Sigma-Aldrich (Supelco) and based on ionic liquids (ILs), were investigated. The linear solvation energy relationship model (LSER) was used to describe the molecular interactions between these stationary phases and 30 solutes. The solutes belong to different groups of compounds, like haloalkanes, alcohols, ketones, aromatics, aliphatics, and others. A good description of different interactions, as described by the LSER model, could be achieved. The calculated values of system constants for the ionic liquid phases were compared with constants of commonly used standard phases like a 5 % phenyl/95 % dimethyl siloxane and a polyethylene glycol phase. The solute descriptors are in good agreement with those found by previous authors who have used the LSER model for 44 different ionic liquids as stationary phase. The experiments were carried out at two temperatures to evaluate the influence on the phase parameters and separation characteristics. The interactions of different functional groups with the IL phases are discussed. These novel IL phases are a promising replacement of or an addition to common polar phases. Based on the evaluated phase properties, several possibilities for applications of these novel phases are shown.

64Cu production via the 68Zn(p,nα)64Cu nuclear reaction: An untapped, cost-effective and high energy production route.

INTRODUCTION: Copper-64 (64Cu, t1/2 = 12.7 h) is a positron emitter well suited for theranostic applications with beta-emitting 67Cu for targeted molecular imaging and radionuclide therapy. The present work aims to evaluate the radionuclidic purity and radiochemistry of 64Cu produced via the 68Zn(p,nα)64Cu nuclear reaction. Macrocyclic chelators DOTA, NOTA, TETA, and prostate-specific membrane antigen ligand PSMA I&T were radiolabeled with purified 64Cu and tested for in vitro stability. [64Cu]Cu-PSMA I&T was used to demonstrate in vivo PET imaging using 64Cu synthesized via the 68Zn(p,nα)64Cu production route and its suitability as a theranostic imaging partner alongside 67Cu therapy. METHODS: 64Cu was produced on a 24 MeV TR-24 cyclotron at a beam energy of 23.5 MeV and currents up to 70 µA using 200 mg 68Zn encapsulated within an aluminum­indium-graphite sealed solid target assembly. 64Cu semi-automated purification was performed using a NEPTIS Mosaic-LC synthesis unit employing CU, TBP, and TK201 (TrisKem) resins. Radionuclidic purity was measured by HPGe gamma spectroscopy, while ICP-OES assessed elemental purity. Radiolabeling was performed with NOTA at room temperature and DOTA, TETA, and PSMA I&T at 95 °C. 64Cu incorporation was studied by radio-TLC. 64Cu in vitro stability of [64Cu]Cu-NOTA, [64Cu]Cu-DOTA, [64Cu]Cu-TETA, and [64Cu]Cu-PSMA I&T was assessed at 37 °C from 0 to 72 h in human blood serum. Preclinical PET imaging was performed at 1, 24, and 48 h post-injection with [64Cu]Cu-PSMA I&T in LNCaP tumor-bearing mice and compared with [68Ga]Ga-PSMA I&T. RESULTS: Maximum purified activity of 4.9 GBq [64Cu]CuCl2 was obtained in 5 mL of pH 2-3 solution, with 2.9 GBq 64Cu concentrated in 0.5 mL. HPGe gamma spectroscopy of purified 64Cu detected <0.3 % co-produced 67Cu at EOB with no other radionuclidic impurities. ICP-OES elemental analysis determined <1 ppm Al, Zn, In, Fe, and Cu in the [64Cu]CuCl2 product. NOTA, DOTA, TETA, and PSMA I&T were radiolabeled with 64Cu, resulting in maximum molar activities of 164 ± 6 GBq/µmol, 155 ± 31 GBq/µmol, 266 ± 34 GBq/µmol, and 117 ± 2 GBq/µmol, respectively. PET imaging in PSMA-expressing LNCaP xenografts resulted in high tumor uptake (SUVmean = 1.65 ± 0.1) using [64Cu]Cu-PSMA I&T, while [68Ga]Ga-PSMA I&T yielded an SUVmean of 0.76 ± 0.14 after 60 min post-injection. CONCLUSIONS: 64Cu was purified in a small volume amenable for radiolabeling, with yields suitable for preclinical and clinical application. The 64Cu production and purification process and the favourable PET imaging properties confirm the 68Zn(p,nα)64Cu nuclear reaction as a viable 64Cu production route for facilities with access to a higher energy proton cyclotron, compared to using expensive 64Ni target material and the 64Ni(p,n)64Cu nuclear reaction. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: Our 64Cu production technique provides an alternative production route with the potential to improve 64Cu availability for preclinical and clinical studies alongside 67Cu therapy.

Position paper to facilitate patient access to radiopharmaceuticals: considerations for a suitable pharmaceutical regulatory framework.

BACKGROUND: Nuclear medicine has made enormous progress in the past decades. However, there are still significant inequalities in patient access among different countries, which could be mitigated by improving access to and availability of radiopharmaceuticals. MAIN BODY: This paper summarises major considerations for a suitable pharmaceutical regulatory framework to facilitate patient access to radiopharmaceuticals. These include the distinct characteristics of radiopharmaceuticals which require dedicated regulations, considering the impact of the variable complexity of radiopharmaceutical preparation, personnel requirements, manufacturing practices and quality assurance, regulatory authority interfaces, communication and training, as well as marketing authorisation procedures to ensure availability of radiopharmaceuticals. Finally, domestic and regional supply to ensure patient access via alternative regulatory pathways, including in-house production of radiopharmaceuticals, is described, and an outlook on regulatory challenges faced by new developments, such as the use of alpha emitters, is provided. CONCLUSIONS: All these considerations are an outcome of a dedicated Technical Meeting organised by the IAEA in 2023 and represent the views and opinions of experts in the field, not those of any regulatory authorities.