Imaging joint infections using D-methyl-11C-methionine PET/MRI: initial experience in humans.

PURPOSE: Non-invasive imaging is a key clinical tool for detection and treatment monitoring of infections. Existing clinical imaging techniques are frequently unable to distinguish infection from tumors or sterile inflammation. This challenge is well-illustrated by prosthetic joint infections that often complicate joint replacements. D-methyl-11C-methionine (D-11C-Met) is a new bacteria-specific PET radiotracer, based on an amino acid D-enantiomer, that is rapidly incorporated into the bacterial cell wall. In this manuscript, we describe the biodistribution, radiation dosimetry, and initial human experience using D-11C-Met in patients with suspected prosthetic joint infections. METHODS: 614.5 ± 100.2 MBq of D-11C-Met was synthesized using an automated in-loop radiosynthesis method and administered to six healthy volunteers and five patients with suspected prosthetic joint infection, who were studied by PET/MRI. Time-activity curves were used to calculate residence times for each source organ. Absorbed doses to each organ and body effective doses were calculated using OLINDA/EXM 1.1 with both ICRP 60 and ICRP 103 tissue weighting factors. SUVmax and SUVpeak were calculated for volumes of interest (VOIs) in joints with suspected infection, the unaffected contralateral joint, blood pool, and soft tissue background. A two-tissue compartment model was used for kinetic modeling. RESULTS: D-11C-Met was well tolerated in all subjects. The tracer showed clearance from both urinary (rapid) and hepatobiliary (slow) pathways as well as low effective doses. Moreover, minimal background was observed in both organs with resident micro-flora and target organs, such as the spine and musculoskeletal system. Additionally, D-11C-Met showed increased focal uptake in areas of suspected infection, demonstrated by a significantly higher SUVmax and SUVpeak calculated from VOIs of joints with suspected infections compared to the contralateral joints, blood pool, and background (P < 0.01). Furthermore, higher distribution volume and binding potential were observed in suspected infections compared to the unaffected joints. CONCLUSION: D-11C-Met has a favorable radiation profile, minimal background uptake, and fast urinary extraction. Furthermore, D-11C-Met showed increased uptake in areas of suspected infection, making this a promising approach. Validation in larger clinical trials with a rigorous gold standard is still required.

Drosophila melanogaster Stress Odorant (dSO) Displays the Characteristics of an Interspecific Alarm Cue.

Organisms depend on visual, auditory, and olfactory cues to signal the presence of danger that could impact survival and reproduction. Drosophila melanogaster emits an olfactory alarm signal, termed the Drosophila stress odorant (dSO), in response to mechanical agitation or electric shock. While it has been shown that conspecifics avoid areas previously occupied by stressed individuals, the contextual underpinnings of the emission of, and response to dSO, have received little attention. Using a binary choice assay, we determined that neither age and sex of emitters, nor the time of the day, affected the emission or avoidance of dSO. However, both sex and mating status affected the response to dSO. We also demonstrated that while D. melanogaster, D. simulans, and D. suzukii, have different dSO profiles, its avoidance was not species-specific. Thus, dSO should not be considered a pheromone but a general alarm signal for Drosophila. However, the response levels to both intra- and inter-specific cues differed between Drosophila species and possible reasons for these differences are discussed.

Arabinofuranose-derived positron-emission tomography radiotracers for detection of pathogenic microorganisms.

PURPOSE: Detection of bacteria-specific metabolism via positron emission tomography (PET) is an emerging strategy to image human pathogens, with dramatic implications for clinical practice. In silico and in vitro screening tools have recently been applied to this problem, with several monosaccharides including l-arabinose showing rapid accumulation in Escherichia coli and other organisms. Our goal for this study was to evaluate several synthetically viable arabinofuranose-derived 18 F analogs for their incorporation into pathogenic bacteria. PROCEDURES: We synthesized four radiolabeled arabinofuranose-derived sugars: 2-deoxy-2-[18 F]fluoro-arabinofuranoses (d-2-18 F-AF and l-2-18 F-AF) and 5-deoxy-5-[18 F]fluoro-arabinofuranoses (d-5-18 F-AF and l-5-18 F-AF). The arabinofuranoses were synthesized from 18 F- via triflated, peracetylated precursors analogous to the most common radiosynthesis of 2-deoxy-2-[18 F]fluoro-d-glucose ([18 F]FDG). These radiotracers were screened for their uptake into E. coli and Staphylococcus aureus. Subsequently, the sensitivity of d-2-18 F-AF and l-2-18 F-AF to key human pathogens was investigated in vitro. RESULTS: All 18 F radiotracer targets were synthesized in high radiochemical purity. In the screening study, d-2-18 F-AF and l-2-18 F-AF showed greater accumulation in E. coli than in S. aureus. When evaluated in a panel of pathologic microorganisms, both d-2-18 F-AF and l-2-18 F-AF demonstrated sensitivity to most gram-positive and gram-negative bacteria. CONCLUSIONS: Arabinofuranose-derived 18 F PET radiotracers can be synthesized with high radiochemical purity. Our study showed absence of bacterial accumulation for 5-substitued analogs, a finding that may have mechanistic implications for related tracers. Both d-2-18 F-AF and l-2-18 F-AF showed sensitivity to most gram-negative and gram-positive organisms. Future in vivo studies will evaluate the diagnostic accuracy of these radiotracers in animal models of infection.

Mirvetuximab after anaphylaxis to Paclitaxel: A case report.

Introduction: Patients with platinum resistant epithelial ovarian cancer have limited treatment options which are further limited by hypersensitivity reactions to first line medications such as paclitaxel. Paclitaxel is a taxane that inhibits microtubules and has a high incidence of hypersensitivity reactions. Mirvetuximab soravtansine-gynx (MIRV) is a folate receptor alpha (FRα) directed antibody and microtubule inhibitor that is approved for patients with FRα positive platinum resistant recurrent epithelial ovarian cancer. Both medications are microtubule-targeting agents with similar binding sites, therefore a theoretical risk of cross reactivity between paclitaxel and MIRV may exist. Additionally, phase II clinical trial, SORAYA, did not include data on patients with prior hypersensitivity to paclitaxel. Case: This is the case of a 33-year-old female with recurrent stage IIIC epithelial ovarian cancer with a history of severe anaphylaxis to paclitaxel. She was deemed eligible for MIRV after progression on multiple regimens, but MIRV was given with caution given her severe reaction history. With proper pre-treatment and monitoring, she was treated with MIRV without a reaction. Discussion: It is suspected that most paclitaxel reactions are due to the cremophor solvent rather than paclitaxel itself; however, cross reactivity with docetaxel which is suspended in a polysorbate solution can also occur. Therefore, there is no clear way to determine the risk of cross reactivity between paclitaxel and similar medications. MIRV is also suspended in polysorbate and has a similar mechanism to taxanes, therefore it was unknown if a patient with a prior grade 5 reaction to paclitaxel would also have a reaction to MIRV. Though this is one case, patients with a history of severe hypersensitivity to paclitaxel and meet the criteria for MIRV could be treated with MIRV with careful monitoring.

The gene "degrees of kevin bacon" (dokb) regulates a social network behaviour in Drosophila melanogaster.

Social networks are a mathematical representation of interactions among individuals which are prevalent across various animal species. Studies of human populations have shown the breadth of what can spread throughout a social network: obesity, smoking cessation, happiness, drug use and divorce. 'Betweenness centrality' is a key property of social networks that indicates an individual's importance in facilitating communication and cohesion within the network. Heritability of betweenness centrality has been suggested in several species, however the genetic regulation of this property remains enigmatic. Here, we demonstrate that the gene CG14109, referred to as degrees of kevin bacon (dokb), influences betweenness centrality in Drosophila melanogaster. We identify strain-specific alleles of dokb with distinct amino acid sequences and when the dokb allele is exchanged between strains, flies exhibit the betweenness centrality pattern dictated by the donor allele. By inserting a GAL4 reporter into the dokb locus, we confirm that dokb is expressed in the central nervous system. These findings define a novel genetic entry point to study social network structure and thereby establish gene-to-social structure relationships. While dokb sequence homology is exclusive to Diptera, we anticipate that dokb-associated molecular pathways could unveil convergent neural mechanisms of social behaviour that apply in diverse animal species.

(-)-[(18) F]Flubatine: evaluation in rhesus monkeys and a report of the first fully automated radiosynthesis validated for clinical use.

(-)-[(18) F]Flubatine was selected for clinical imaging of α4 ß2 nicotinic acetylcholine receptors because of its high affinity and appropriate kinetic profile. A fully automated synthesis of (-)-[(18) F]flubatine as a sterile isotonic solution suitable for clinical use is reported, as well as the first evaluation in nonhuman primates (rhesus macaques). (-)-[(18) F]Flubatine was prepared by fluorination of the Boc-protected trimethylammonium iodide precursor with [(18) F]fluoride in an automated synthesis module. Subsequent deprotection of the Boc group with 1-M HCl yielded (-)-[(18) F]flubatine, which was purified by semi-preparative HPLC. (-)-[(18) F]Flubatine was prepared in 25% radiochemical yield (formulated for clinical use at end of synthesis, n = 3), >95% radiochemical purity, and specific activity = 4647 Ci/mmol (171.9 GBq/µmol). Doses met all quality control criteria confirming their suitability for clinical use. Evaluation of (-)-[(18) F]flubatine in rhesus macaques was performed with a Concorde MicroPET P4 scanner (Concorde MicroSystems, Knoxville, TN). The brain was imaged for 90 min, and data were reconstructed using the 3-D maximum a posteriori algorithm. Image analysis revealed higher uptake and slower washout in the thalamus than those in other areas of the brain and peak uptake at 45 min. Injection of 2.5 µg/kg of nifene at 60 min initiated a slow washout of [(18) F]flubatine, with about 25% clearance from the thalamus by the end of imaging at 90 min.

An atypical basement membrane forms a midline barrier in left-right asymmetric gut development.

Correct intestinal morphogenesis depends on the early embryonic process of gut rotation, an evolutionarily conserved program in which a straight gut tube elongates and forms into its first loops. However, the gut tube requires guidance to loop in a reproducible manner. The dorsal mesentery (DM) connects the gut tube to the body and directs the lengthening gut into stereotypical loops via left-right (LR) asymmetric cellular and extracellular behavior. The LR asymmetry of the DM also governs blood and lymphatic vessel formation for the digestive tract, which is essential for prenatal organ development and postnatal vital functions including nutrient absorption. Although the genetic LR asymmetry of the DM has been extensively studied, a divider between the left and right DM has yet to be identified. Setting up LR asymmetry for the entire body requires a Lefty1+ midline barrier to separate the two sides of the embryo-without it, embryos have lethal or congenital LR patterning defects. Individual organs including the brain, heart, and gut also have LR asymmetry, and while the consequences of left and right signals mixing are severe or even lethal, organ-specific mechanisms for separating these signals are not well understood. Here, we uncover a midline structure composed of a transient double basement membrane, which separates the left and right halves of the embryonic chick DM during the establishment of intestinal and vascular asymmetries. Unlike other basement membranes of the DM, the midline is resistant to disruption by intercalation of Netrin4 (Ntn4). We propose that this atypical midline forms the boundary between left and right sides and functions as a barrier necessary to establish and protect organ asymmetry.

The Clinician's Guide to Baseball Pitching Biomechanics.

CONTEXT: Improper baseball pitching biomechanics are associated with increased stresses on the throwing elbow and shoulder as well as an increased risk of injury. EVIDENCE ACQUISITION: Previous studies quantifying pitching kinematics and kinetics were reviewed. STUDY DESIGN: Clinical review. LEVEL OF EVIDENCE: Level 5. RESULTS: At the instant of lead foot contact, the elbow should be flexed approximately 90° with the shoulder at about 90° abduction, 20° horizontal abduction, and 45° external rotation. The stride length should be about 85% of the pitcher's height with the lead foot in a slightly closed position. The pelvis should be rotated slightly open toward home plate with the upper torso in line with the pitching direction. Improper shoulder external rotation at foot contact is associated with increased elbow and shoulder torques and forces and may be corrected by changing the stride length and/or arm path. From foot contact to maximum shoulder external rotation to ball release, the pitcher should demonstrate a kinematic chain of lead knee extension, pelvis rotation, upper trunk rotation, elbow extension, and shoulder internal rotation. The lead knee should be flexed about 45° at foot contact and 30° at ball release. Corrective strategies for insufficient knee extension may involve technical issues (stride length, lead foot position, lead foot orientation) and/or strength and conditioning of the lower body. Improper pelvis and upper trunk rotation often indicate the need for core strength and flexibility. Maximum shoulder external rotation should be about 170°. Insufficient external rotation leads to low shoulder internal rotation velocity and low ball velocity. Deviation from 90° abduction decreases the ability to achieve maximum external rotation, increases elbow torque, and decreases the dynamic stability in the glenohumeral joint. CONCLUSION: Improved pitching biomechanics can increase performance and reduce risk of injury. SORT: Level C.

Dermatopathology Laboratory Green Initiatives.

OBJECTIVES: Climate change and the accumulation of greenhouse gases pose challenges for humanity. The laboratory can reduce the environmental impact of routine operations. METHODS: Our laboratory implemented several recycling initiatives in 2021, including shredding plastic and recycling 10% formalin, xylene, and reagent alcohols. Additional insulation has reduced electricity costs, and we have plans to derive 100% of our electricity from solar in 2022. RESULTS: Recycling leads to decreases in reagent purchase by several hundred gallons. Our lab reduced its carbon footprint by a minimum of 68.52 tons, with an anticipated $43,000 savings in purchases and waste disposal during 2022. CONCLUSIONS: Regardless of an individual's environmental consciousness, green initiatives have several tangible benefits. Recycling helps mitigate climate change and decrease the laboratory's carbon footprint. These measures also insulate the laboratory from supply shortages and lead to significant, tangible monetary savings.

Biomechanical effects of foot placement during pitching.

Baseball coaches often focus on the landing position of a pitcher's front foot as a key aspect of mechanics. Furthermore, controversy persists regarding positioning the rear foot on the first base or third base end of the rubber. The purpose of this study was to determine the effect of rear and front foot placement on pitching biomechanics. Our hypotheses were that there would be significant kinematic and kinetic differences associated with foot placement. This was a retrospective review including 144 healthy right-handed adult baseball pitchers divided into groups based on their rear and front foot placements: first base open (1B-Open), first base closed (1B-Closed), third base open (3B-Open), and third base closed (3B-Closed). Two-way ANOVAs detected no statistically significant main effects for kinetic variables but several for kinematic variables. Open pitchers had less shoulder abduction at the time of ball release and greater maximum shoulder internal rotation velocity in comparison with closed pitchers. They also had less forearm pronation at the time of ball release and greater maximum elbow extension velocity. Additional statistically significant results were found; however, low effect sizes may lessen the clinical significance of many of the results.

High Enantiomeric Excess In-Loop Synthesis of d-[methyl-11C]Methionine for Use as a Diagnostic Positron Emission Tomography Radiotracer in Bacterial Infection.

Currently, there exists no accurate, noninvasive clinical imaging method to detect living bacteria in vivo. Our goal is to provide a positron emission tomography (PET) method to image infection by targeting bacteria-specific metabolism. Standard of care methodologies detect morphologic changes, image immunologic response to infection, or employ invasive tissue sampling with associated patient morbidity. These strategies, however, are not specific for living bacteria and are often inadequate to detect bacterial infection during fever workup. As such, there is an unmet clinical need to identify and validate new imaging tools suitable for noninvasive, in vivo (PET) imaging of living bacteria. We have shown that d-[methyl-11C]methionine (d-[11C]Met) can distinguish active bacterial infection from sterile inflammation in a murine infection model and is sensitive to both Gram-positive and Gram-negative bacteria. Here, we report an automated and >99% enantiomeric excess (ee) synthesis of d-[11C]Met from a linear d-homocysteine precursor, a significant improvement over the previously reported synthesis utilizing a d-homocysteine thiolactone hydrochloride precursor with approximately 75-85% ee. Furthermore, we took additional steps toward applying d-[11C]Met to infected patients. d-[11C]Met was subject to a panel of clinically relevant bacterial strains and demonstrated promising sensitivity to these pathogens. Finally, we performed radiation dosimetry in a normal murine cohort to set the stage for translation to humans in the near future.

Sensing Living Bacteria in Vivo Using d-Alanine-Derived 11C Radiotracers.

Incorporation of d-amino acids into peptidoglycan is a unique metabolic feature of bacteria. Since d-amino acids are not metabolic substrates in most mammalian tissues, this difference can be exploited to detect living bacteria in vivo. Given the prevalence of d-alanine in peptidoglycan muropeptides, as well as its role in several antibiotic mechanisms, we targeted this amino acid for positron emission tomography (PET) radiotracer development. d-[3-11C]Alanine and the dipeptide d-[3-11C]alanyl-d-alanine were synthesized via asymmetric alkylation of glycine-derived Schiff-base precursors with [11C]methyl iodide in the presence of a cinchonidinium phase-transfer catalyst. In cell experiments, both tracers showed accumulation by a wide variety of both Gram-positive and Gram-negative pathogens including Staphylococcus aureus and Pseudomonas aeruginosa. In a mouse model of acute bacterial myositis, d-[3-11C]alanine was accumulated by living microorganisms but was not taken up in areas of sterile inflammation. When compared to existing clinical nuclear imaging tools, specifically 2-deoxy-2-[18F]fluoro-d-glucose and a gallium citrate radiotracer, d-alanine showed more bacteria-specific uptake. Decreased d-[3-11C]alanine uptake was also observed in antibiotic-sensitive microbes after antimicrobial therapy, when compared to that in resistant organisms. Finally, prominent uptake of d-[3-11C]alanine uptake was seen in rodent models of discitis-osteomyelitis and P. aeruginosa pneumonia. These data provide strong justification for clinical translation of d-[3-11C]alanine to address a number of important human infections.

Synthesis and pre-clinical evaluation of a potential radiotracer for PET imaging of the dopamine D3 receptor.

There is considerable interest in using positron emission tomography (PET) imaging to understand the function of dopamine D3 receptors. Due to high sequence homology with D2 receptors, development of D3-selective PET radiotracers has been challenging. In an effort to overcome this issue, we report the radiosynthesis of a new selective D3 ligand with carbon-11 ([11C]1 ), and its initial preclincial evaluation as a potential PET radiotracer for in vivo imaging of D3 receptors. [11C]1 was prepared via [11C]CO2 fixation in 0.1% non-corrected radiochemical yield, good radiochemical purity (>95%) and high specific activity (>2000 Ci mmol-1). [11C]1 exhibited specific binding to D3 receptors using ex vivo autoradiography experiments with rat brain, but only 14-fold selectivity over D2 receptors which is lower than the 1400-fold value reported previously for cell studies. Rodent PET imaging revealed reasonable uptake of the radiotracer in areas of the brain known to be rich in D3 receptors.

Fluorine-18 patents (2009-2015). Part 1: novel radiotracers.

The most commonly utilized PET radionuclide is fluorine-18 ((18)F) because of its convenient half-life and excellent imaging properties. In this review, we present the first analysis of patents issued for radiotracers labeled with fluorine-18 (between 2009 and 2015), and provide perspective on current trends and future directions in PET radiotracer development.

Green approaches to late-stage fluorination: radiosyntheses of (18)F-labelled radiopharmaceuticals in ethanol and water.

Green strategies for late-stage fluorination with (18)F, in which ethanol and water are the only solvents used throughout the entire radiolabeling process (azeotropic drying, nucleophilic fluorination, purification and formulation), have been developed and applied to the radiosyntheses of a range of radiopharmaceuticals commonly employed in clinical PET imaging.

Comparative Study of Eis-like Enzymes from Pathogenic and Nonpathogenic Bacteria.

Antibiotic resistance is a growing problem worldwide. Of particular importance is the resistance of Mycobacterium tuberculosis (Mtb) to currently available antibiotics used in the treatment of infected patients. Up-regulation of an aminoglycoside (AG) acetyltransferase, the enhanced intracellular survival (Eis) protein of Mtb (Eis_Mtb), is responsible for resistance to the second-line injectable drug kanamycin A in a number of Mtb clinical isolates. This acetyltransferase is known to modify AGs, not at a single position, as usual for this type of enzyme, but at multiple amine sites. We identified, using in silico techniques, 22 homologues from a wide variety of bacteria, that we then cloned, purified, and biochemically studied. From the selected Eis homologues, 7 showed the ability to modify AGs to various degrees and displayed both similarities and differences when compared to Eis_Mtb. In addition, an inhibitor proved to be active against all homologues tested. Our findings show that this family of acetyltransferase enzymes exists in both mycobacteria and non-mycobacteria and in both pathogenic and nonpathogenic species. The bacterial strains described herein should be monitored for rising resistance rates to AGs.

Radiosyntheses using fluorine-18: the art and science of late stage fluorination.

Positron (ß(+)) emission tomography (PET) is a powerful, noninvasive tool for the in vivo, three-dimensional imaging of physiological structures and biochemical pathways. The continued growth of PET imaging relies on a corresponding increase in access to radiopharmaceuticals (biologically active molecules labeled with short-lived radionuclides such as fluorine-18). This unique need to incorporate the short-lived fluorine-18 atom (t1/2 = 109.77 min) as late in the synthetic pathway as possible has made development of methodologies that enable rapid and efficient late stage fluorination an area of research within its own right. In this review we describe strategies for radiolabeling with fluorine-18, including classical fluorine-18 radiochemistry and emerging techniques for late stage fluorination reactions, as well as labeling technologies such as microfluidics and solid-phase radiochemistry. The utility of fluorine-18 labeled radiopharmaceuticals is showcased through recent applications of PET imaging in the healthcare, personalized medicine and drug discovery settings.

Establishing genomic tools and resources for Guizotia abyssinica (L.f.) Cass.-the development of a library of expressed sequence tags, microsatellite loci, and the sequencing of its chloroplast genome.

We present an EST library, chloroplast genome sequence, and nuclear microsatellite markers that were developed for the semi-domesticated oilseed crop noug (Guizotia abyssinica) from Ethiopia. The EST library consists of 25 711 Sanger reads, assembled into 17 538 contigs and singletons, of which 4781 were functionally annotated using the Arabidopsis Information Resource (TAIR). The age distribution of duplicated genes in the EST library shows evidence of two paleopolyploidizations-a pattern that noug shares with several other species in the Heliantheae tribe (Compositae family). From the EST library, we selected 43 microsatellites and then designed and tested primers for their amplification. The number of microsatellite alleles varied between 2 and 10 (average 4.67), and the average observed and expected heterozygosities were 0.49 and 0.54, respectively. The chloroplast genome was sequenced de novo using Illumina's sequencing technology and completed with traditional Sanger sequencing. No large re-arrangements were found between the noug and sunflower chloroplast genomes, but 1.4% of sites have indels and 1.8% show sequence divergence between the two species. We identified 34 tRNAs, 4 rRNA sequences, and 80 coding sequences, including one region (trnH-psbA) with 15% sequence divergence between noug and sunflower that may be particularly useful for phylogeographic studies in noug and its wild relatives.