Rapid, efficient, and economical synthesis of PET tracers in a droplet microreactor: application to O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET).

BackgroundConventional scale production of small batches of PET tracers (e.g. for preclinical imaging) is an inefficient use of resources. Using O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET), we demonstrate that simple microvolume radiosynthesis techniques can improve the efficiency of production by consuming tiny amounts of precursor, and maintaining high molar activity of the tracers even with low starting activity.ProceduresThe synthesis was carried out in microvolume droplets manipulated on a disposable patterned silicon "chip" affixed to a heater. A droplet of [18F]fluoride containing TBAHCO3 was first deposited onto a chip and dried at 100 °C. Subsequently, a droplet containing 60 nmol of precursor was added to the chip and the fluorination reaction was performed at 90 °C for 5 min. Removal of protecting groups was accomplished with a droplet of HCl heated at 90 °C for 3 min. Finally, the crude product was collected in a methanol-water mixture, purified via analytical-scale radio-HPLC and formulated in saline. As a demonstration, using [18F]FET produced on the chip, we prepared aliquots with different molar activities to explore the impact on preclinical PET imaging of tumor-bearing mice.ResultsThe microdroplet synthesis exhibited an overall decay-corrected radiochemical yield of 55 ± 7% (n = 4) after purification and formulation. When automated, the synthesis could be completed in 35 min. Starting with < 370 MBq of activity, ~ 150 MBq of [18F]FET could be produced, sufficient for multiple in vivo experiments, with high molar activities (48-119 GBq/μmol). The demonstration imaging study revealed the uptake of [18F]FET in subcutaneous tumors, but no significant differences in tumor uptake as a result of molar activity differences (ranging 0.37-48 GBq/μmol) were observed.ConclusionsA microdroplet synthesis of [18F]FET was developed demonstrating low reagent consumption, high yield, and high molar activity. The approach can be expanded to tracers other than [18F]FET, and adapted to produce higher quantities of the tracer sufficient for clinical PET imaging

Розширення асортименту плавлених сирів з використанням соняшнико-вого ізоляту

The paper presents an analysis of the literature on the expansion of the range of processed cheeses using sunflower isolate. Four samples of processed cheese with different percentage of sunflower isolate were selected for the study: control, 1st – 3 %, 2nd – 5 % and 3rd – 7 %. Analyzing the results of organoleptic evaluation, it should be noted that the organoleptic characteristics of the control and the first samples were evaluated in the same number of points. Samples two and three as a result of tasting evaluation for taste lost to the first two by 0.2 and 0.4 points, respectively. The odor of the third sample with a percentage of sunflower isolate of 5 % was not inferior to the first sample and was estimated at 0.1 and 0.2 points higher than the control and third samples, respectively. The increase in the percentage of sunflower isolate led to a deterioration in the taste of the developed samples. Therefore, the introduction into the recipe of processed cheese sunflower isolate in the amount of 3 % allowed to obtain, according to the results of organoleptic evaluation, higher results compared to other samples. The results of studies of structural and mechanical parameters of the developed samples of processed cheese with sunflower isolate suggest that the introduction of a herbal additive in the recipe increased the structural and mechanical properties. An increase in the penetration force indicates a thickening of the consistency. Compared to the control sample, this figure increased by 1.8 kN/m2. The elastic index, which characterizes the elasticity of the cheese mass of the control sample, was lower than the first by 0.6 kN/m2. The obtained results allow us to conclude that the introduction of sunflower isolate into the formulation leads to minor structural changes within the regulatory documentation. The study found that the control sample contained 11.36 % protein, and sample 1 – 11.55 %. It should be noted that the protein content has not changed significantly. Taking into account the results of the described studies, we note that the developed processed cheese with 3 % sunflower isolate meets the requirements of regulatory documentation for organoleptic and structural-mechanical parameters. The structural and mechanical characteristics of the first sample indicate that the introduction of a herbal additive into the formulation has a positive effect on penetration efforts and elasticity. However, the introduction of three percent sunflower isolate did not affect the protein content of the product.У роботі представлено аналіз літературних джерел щодо питання розширення асортименту плавлених сирів з використанням соняшникового ізоляту. Для дослідження обрано чотири зразки плавленого сиру з різним відсотком соняшникового ізоляту: контроль, 1-й – 3 %, 2-й – 5 % і 3-й – 7 %. Аналізуючи отримані результати органолептичної оцінки слід зазначити, що органолептичні показники контрольного та першого зразків були оцінені в однакову кількість балів. Зразки два та три в результаті дегустаційної оцінки за смаком поступилися першим двом на 0,2 та 0,4 бали відповідно. Запах третього зразка із відсотком соняшникового ізоляту 5 % не поступався першому зразку та був оцінений на 0,1 та 0,2 бали вище ніж у контрольного й третього зразків відповідно. Збільшення відсотку соняшникового ізоляту призвело до погіршення смаку розроблених зразків. Отже, введення у рецептуру плавленого сиру соняшникового ізоляту у кількості 3 % дозволило отримати, за результатами органолептичної оцінки, більш високі результати порівняно з іншими зразками. Результати досліджень структурно-механічних показників розроблених зразків плавленого сиру з соняшниковим ізолятом дозволяють стверджувати, що введення у рецептуру рослинної добавки підвищили структурно-механічні властивості. Збільшення показника зусилля пенетрації свідчить про ущільнення консистенції. Відносно контрольного зразка цей показник збільшився на 1,8 kN/m2. Показник пружності, який характеризує еластичність сирної маси контрольного зразка, був нижчий ніж першого на 0,6 kN/m2. Отримані результати дозволяють зробити висновок, що введення у рецептуру соняшникового ізоляту веде до незначних структурних змін в межах нормативної документації. В ході дослідження встановили, що контрольний зразок містить 11,36 % білку, а зразок 1 – 11,55 %. Слід зазначити, що суттєво вміст білку не змінився. Враховуючи результати описаних досліджень зауважимо, що розроблений плавлений сир з 3 % соняшникового ізоляту відповідає вимогам нормативної документації за органолептичними та структурно-механічними показниками. Структурно-механічні харатеритики першого зразку вказують на те, що введення у рецептуру рослинної добавки позитивно впливає на зусилля пенетрації та пружність. Однак, введення трьох відсотків соняшникового ізоляту не вплинули на вміст у продукті білку

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology

In Vitro and In Vivo Studies Identify Important Features of Dengue Virus pr-E Protein Interactions

Flaviviruses bud into the endoplasmic reticulum and are transported through the secretory pathway, where the mildly acidic environment triggers particle rearrangement and allows furin processing of the prM protein to pr and M. The peripheral pr peptide remains bound to virus at low pH and inhibits virus-membrane interaction. Upon exocytosis, the release of pr at neutral pH completes virus maturation to an infectious particle. Together this evidence suggests that pr may shield the flavivirus fusion protein E from the low pH environment of the exocytic pathway. Here we developed an in vitro system to reconstitute the interaction of dengue virus (DENV) pr with soluble truncated E proteins. At low pH recombinant pr bound to both monomeric and dimeric forms of E and blocked their membrane insertion. Exogenous pr interacted with mature infectious DENV and specifically inhibited virus fusion and infection. Alanine substitution of E H244, a highly conserved histidine residue in the pr-E interface, blocked pr-E interaction and reduced release of DENV virus-like particles. Folding, membrane insertion and trimerization of the H244A mutant E protein were preserved, and particle release could be partially rescued by neutralization of the low pH of the secretory pathway. Thus, pr acts to silence flavivirus fusion activity during virus secretion, and this function can be separated from the chaperone activity of prM. The sequence conservation of key residues involved in the flavivirus pr-E interaction suggests that this protein-protein interface may be a useful target for broad-spectrum inhibitors

Humoral Immune Responses of Dengue Fever Patients Using Epitope-Specific Serotype-2 Virus-Like Particle Antigens

Dengue virus (DENV) is a serious mosquito-borne pathogen causing significant global disease burden, either as classic dengue fever (DF) or in its most severe manifestation dengue hemorrhagic fever (DHF). Nearly half of the world's population is at risk of dengue disease and there are estimated to be millions of infections annually; a situation which will continue to worsen with increasing expansion of the mosquito vectors and epidemic DF/DHF. Currently there are no available licensed vaccines or antivirals for dengue, although significant effort has been directed toward the development of safe and efficacious dengue vaccines for over 30 years. Promising vaccine candidates are in development and testing phases, but a better understanding of immune responses to DENV infection and vaccination is needed. Humoral immune responses to DENV infection are complex and may exacerbate pathogenicity, yet are essential for immune protection. In this report, we develop DENV-2 envelope (E) protein epitope-specific antigens and measure immunoglobulin responses to three distinct epitopes in DENV-2 infected human serum samples. Immunoglobulin responses to DENV-2 infection exhibited significant levels of individual variation. Antibody populations targeting broadly cross-reactive epitopes centered on the fusion peptide in structural domain II were large, highly variable, and greater in primary than in secondary DENV-2 infected sera. E protein domain III cross-reactive immunoglobulin populations were similarly variable and much larger in IgM than in IgG. DENV-2 specific domain III IgG formed a very small proportion of the antibody response yet was significantly correlated with DENV-2 neutralization, suggesting that the highly protective IgG recognizing this epitope in murine studies plays a role in humans as well. This report begins to tease apart complex humoral immune responses to DENV infection and is thus important for improving our understanding of dengue disease and immunological correlates of protection, relevant to DENV vaccine development and testing

Segmented flow generator for serial crystallography at the European X-ray free electron laser

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported

Analysis of Epitopes on Dengue Virus Envelope Protein Recognized by Monoclonal Antibodies and Polyclonal Human Sera by a High Throughput Assay

Dengue virus is the leading cause of arboviral diseases worldwide. The envelope protein is the major target of neutralizing antibodies and vaccine development. While previous studies have reported several epitopes on envelope protein, the possibility of interdomain epitopes and the relationship of epitopes to neutralizing potency remain unexplored. We developed a high throughput dot blot assay by using 67 alanine mutants of surface-exposed envelope residues as a systematic approach to identify epitopes recognized by mouse monoclonal antibodies and polyclonal human sera. Our results suggested the presence of interdomain epitopes more frequent than previously appreciated. Compared with monoclonal antibodies generated by traditional protocol, the potent neutralizing monoclonal antibodies generated by a new protocol showed several unique features of their epitopes. Moreover, the predominant epitopes of antibodies against envelope protein in polyclonal sera can be identified by this assay. These findings have implications for future development of epitope-specific diagnostics and epitope-based dengue vaccine, and add to our understanding of humoral immune responses to dengue virus at the epitope level

The Development of Therapeutic Antibodies That Neutralize Homologous and Heterologous Genotypes of Dengue Virus Type 1

Antibody protection against flaviviruses is associated with the development of neutralizing antibodies against the viral envelope (E) protein. Prior studies with West Nile virus (WNV) identified therapeutic mouse and human monoclonal antibodies (MAbs) that recognized epitopes on domain III (DIII) of the E protein. To identify an analogous panel of neutralizing antibodies against DENV type-1 (DENV-1), we immunized mice with a genotype 2 strain of DENV-1 virus and generated 79 new MAbs, 16 of which strongly inhibited infection by the homologous virus and localized to DIII. Surprisingly, only two MAbs, DENV1-E105 and DENV1-E106, retained strong binding and neutralizing activity against all five DENV-1 genotypes. In an immunocompromised mouse model of infection, DENV1-E105 and DENV1-E106 exhibited therapeutic activity even when administered as a single dose four days after inoculation with a heterologous genotype 4 strain of DENV-1. Using epitope mapping and X-ray crystallographic analyses, we localized the neutralizing determinants for the strongly inhibitory MAbs to distinct regions on DIII. Interestingly, sequence variation in DIII alone failed to explain disparities in neutralizing potential of MAbs among different genotypes. Overall, our experiments define a complex structural epitope on DIII of DENV-1 that can be recognized by protective antibodies with therapeutic potential

Green and efficient synthesis of the radiopharmaceutical [ 18 F]FDOPA using a microdroplet reactor

From an efficiency standpoint, microdroplet reactors enable significant improvements in the preparation of radiopharmaceuticals due to the vastly reduced reaction volume. To demonstrate these advantages, we adapt the conventional (macroscale) synthesis of the clinically-important positron-emission tomography tracer [18F]FDOPA, following the nucleophilic diaryliodonium salt approach, to a newly-developed ultra-compact microdroplet reaction platform. In this first microfluidic implementation of [18F]FDOPA synthesis, optimized via a high-throughput multi-reaction platform, the radiochemical yield (non-decay-corrected) was found to be comparable to macroscale reports, but the synthesis consumed significantly less precursor and organic solvents, and the synthesis process was much faster. In this initial report, we demonstrate the production of [18F]FDOPA in 15 MBq [400 μCi] amounts, sufficient for imaging of multiple mice, at high molar activity

A simple and efficient automated microvolume radiosynthesis of [18F]Florbetaben.

BackgroundCurrent automated radiosynthesizers are generally optimized for producing large batches of PET tracers. Preclinical imaging studies, however, often require only a small portion of a regular batch, which cannot be economically produced on a conventional synthesizer. Alternative approaches are desired to produce small to moderate batches to reduce cost and the amount of reagents and radioisotope needed to produce PET tracers with high molar activity. In this work we describe the first reported microvolume method for production of [18F]Florbetaben for use in imaging of Alzheimer's disease.ProceduresThe microscale synthesis of [18F]Florbetaben was adapted from conventional-scale synthesis methods. Aqueous [18F]fluoride was azeotropically dried with K2CO3/K222 (275/383 nmol) complex prior to radiofluorination of the Boc-protected precursor (80 nmol) in 10 μL DMSO at 130 °C for 5 min. The resulting intermediate was deprotected with HCl at 90 °C for 3 min and recovered from the chip in aqueous acetonitrile solution. The crude product was purified via analytical scale HPLC and the collected fraction reformulated via solid-phase extraction using a miniature C18 cartridge.ResultsStarting with 270 ± 100 MBq (n = 3) of [18F]Fluoride, the method affords formulated product with 49 ± 3% (decay-corrected) yield,> 98% radiochemical purity and a molar activity of 338 ± 55 GBq/μmol. The miniature C18 cartridge enables efficient elution with only 150 μL of ethanol which is diluted to a final volume of 1.0 mL, thus providing a sufficient concentration for in vivo imaging. The whole procedure can be completed in 55 min.ConclusionsThis work describes an efficient and reliable procedure to produce [18F]Florbetaben in quantities sufficient for large-scale preclinical applications. This method provides very high yields and molar activities compared to reported literature methods. This method can be applied to higher starting activities with special consideration given to automation and radiolysis prevention