Construction of Species-Specific PCR Primers for Detection of Coccidia Parasites in Captive-Reared Northern Bobwhites

Captive rearing and subsequent release of game birds, including northern bobwhites (Colinus virginianus), has become common in certain areas. In this practice, bobwhites are often raised in confinement to ‘flight ready’ and subsequently released for hunting. It is estimated that 30–40 million bobwhites are raised in captivity annually and some farms in the USA produce upwards of 1 million birds annually for this market. Raising game birds in these densities greatly facilitates the transmission of pathogenic organisms. Coccidiosis has been previously identified as an important disease in captive bobwhites and infection can lead to weight loss, diarrhea, poor feather growth, dehydration and, in severe cases, death. Eimeria lettyae, E. colini, and E. dispersa are the three described coccidia species from bobwhites. We investigated the prevalence and distribution of species of coccidia in captive bobwhite facilities throughout the United States. We collected litter or intestinal samples from 31captive bobwhite facilities originating from 13 states. Species-specific PCR primers were constructed against the internal transcribed spacer region 1 (ITS-1) of the ribosomal RNA gene of the various Eimeria spp. to aid in parasite detection and distinction. Primers were used to detect the specific Eimeria spp. in the collected samples. All 31 samples were positive for coccidia. Results of the primer survey disclosed E. lettyae, E. dispersa, and an unidentified Eimeria sp. in 20 (64.5%), 22 (72%), and 29 (93.5%) of the samples, respectively. Thirteen (41.9%) samples had 3 Eimeria spp. detected, 14 (45.2%) samples had 2 spp. detected, and 4 (12.9%) samples had 1 sp. detected. Flock age or geographical location was not associated with the presence of any particular Eimeria spp. To our knowledge, this is the first study of coccidia in captive bobwhites. Previous studies of Eimeria spp. in wild northern bobwhite are rare and disclosed variable prevalence rates ranging from 0 to 36%; no efforts were made to distinguish the coccidia species in these studies It would be helpful to use the species-specific primers constructed in this study to examine the prevalence and distribution of the Eimeria spp. in wild bobwhites from throughout their range to investigate the potential for captive-raised bobwhites to be a source of coccidiosis for wild bobwhites

Synthesis of 2,2-difluoro-1,3-diketone and 2,2-difluoro-1,3-ketoester derivatives using fluorine gas

Solutions of 1,3-diketones and 1,3-ketoester derivatives react with fluorine to give the corresponding 2,2-difluoro-1,3-dicarbonyl derivatives in the presence of quinuclidine. Quinuclidine reacts with fluorine in situ to generate a fluoride ion that facilitates limiting enolization processes, and an electrophilic N-F fluorinating agent that is reactive towards neutral enol species

Characterisation of an atherosclerotic micro-calcification model using ApoE-/- mice and PET/CT

Intraplaque calcification is a prominent feature of advanced atherosclerotic plaque development. Current clinical evidence suggests that the size of calcium deposit may confer different effects on plaque stability [1], [2], [3]. Macro-calcified deposits (CT detected) are thought to confer plaque stability whereas micro-calcification ([18F]NaF PET detected) are thought to be a feature of high-risk ‘vulnerable’ plaques which are prone to rupture. Following on from the emerging role of micro-calcification in high risk plaques within the clinic [4], there is now an urgent need for preclinical atherosclerotic models with this feature to gain mechanistic insights and assess the impact of calcification-targeted therapies. Using a combination of invasive and ex vivo methods, ApoE−/− mice placed on an atherogenic diet have been shown to develop intraplaque calcification [5]. Additionally, [18F]NaF PET/CT has been used to assess the impact of exercise on calcification in ApoE−/− mice on a western diet [6]. In this study, we set out to determine if [18F]NaF PET/CT could be used to non-invasively detect and quantify micro-calficiation in the ApoE−/− high cholesterol diet (HCD) mouse model, and examine the temporal nature of this process

Standardization of Preclinical PET/CT Imaging to Improve Quantitative Accuracy, Precision, and Reproducibility: A Multicenter Study

Preclinical PET/CT is a well-established noninvasive imaging tool for studying disease development/progression and the development of novel radiotracers and pharmaceuticals for clinical applications. Despite this pivotal role, standardization of preclinical PET/CT protocols, including CT absorbed dose guidelines, is essentially nonexistent. This study (1) quantitatively assesses the variability of current preclinical PET/CT acquisition and reconstruction protocols routinely used across multiple centers and scanners; and (2) proposes acquisition and reconstruction PET/CT protocols for standardization of multicenter data, optimized for routine scanning in the preclinical PET/CT laboratory. Methods: Five different commercial preclinical PET/CT scanners in Europe and the United States were enrolled. Seven different PET/CT phantoms were used for evaluating biases on default/general scanner protocols, followed by developing standardized protocols. PET, CT, and absorbed dose biases were assessed. Results: Site default CT protocols were the following: greatest extracted Hounsfield units (HU) were 133 HU for water and −967 HU for air; significant differences in all tissue equivalent material (TEM) groups were measured. The average CT absorbed doses for mouse and rat were 72 mGy and 40 mGy, respectively. Standardized CT protocol were the following: greatest extracted HU were −77 HU for water and −990 HU for air; TEM precision improved with a reduction in variability for each tissue group. The average CT absorbed dose for mouse and rat decreased to 37 mGy and 24 mGy, respectively. Site default PET protocols were the following: uniformity was substandard in one scanner, recovery coefficients (RCs) were either over- or underestimated (maximum of 43%), standard uptake values (SUVs) were biased by a maximum of 44%. Standardized PET protocols were the following: scanner with substandard uniformity improved by 36%, RC variability decreased by 13% points, and SUV accuracy improved to 10%. Conclusion: Data revealed important quantitative biases in preclinical PET/CT and absorbed doses with default protocols. Standardized protocols showed improvements in measured PET/CT accuracy and precision with reduced CT absorbed dose across sites. Adhering to standardized protocols generates reproducible and consistent preclinical imaging datasets, thus augmenting translation of research findings to the clinic

Cupriphication of gold to sensitize d10–d10 metal–metal bonds and near-unity phosphorescence quantum yields

Outer-shell s0/p0 orbital mixing with d10 orbitals and symmetry reductionuponcupriphicationofcyclic trinucleartrigonal-planargold(I) complexes are found to sensitize ground-state Cu(I)–Au(I) covalent bonds and near-unity phosphorescence quantum yields. Heterobimetallic Au4Cu2 {[Au4(μ-C2,N3-EtIm)4Cu2(μ-3,5-(CF3)2Pz)2], (4a)}, Au2Cu {[Au2(μ-C2,N3-BzIm)2Cu(μ-3,5-(CF3)2Pz)], (1) and [Au2(μ-C2, N3-MeIm)2Cu(μ-3,5-(CF3)2Pz)], (3a)}, AuCu2 {[Au(μ-C2,N3-MeIm)Cu2(μ3,5-(CF3)2Pz)2], (3b) and [Au(μ-C2,N3-EtIm)Cu2(μ-3,5-(CF3)2Pz)2], (4b)} and stacked Au3/Cu3 {[Au(μ-C2,N3-BzIm)]3[Cu(μ-3,5-(CF3)2Pz)]3, (2)} formuponreactingAu3 {[Au(μ-C2,N3-(N-R)Im)]3 ((N-R)Im = imidazolate; R =benzyl/methyl/ethyl =BzIm/MeIm/EtIm)} with Cu3 {[Cu(μ-3,5(CF3)2Pz)]3 (3,5-(CF3)2Pz = 3,5-bis(trifluoromethyl)pyrazolate)}. The crystal structures of 1 and 3a reveal stair-step infinite chains whereby adjacent dimer-of-trimer units are noncovalently packed via twoAu(I)⋯Cu(I)metallophilicinteractions,whereas 4a exhibitsa hexanuclear cluster structure wherein two monomer-of-trimer units are linked by a genuine d10–d10 polar-covalent bond with ligandunassisted Cu(I)–Au(I) distances of 2.8750(8) Å each—the shortest such an intermolecular distance ever reported between any two d10 centers so as to deem it a “metal–metal bond” vis-à-vis “metallophilic interaction.” Density-functional calculations estimate 35– 43kcal/molbindingenergy,akintotypicalM–Msingle-bondenergies. Congruently, FTIR spectra of4a showmultiple far-IR bands within 65– 200 cm−1, assignable to vCu-Au as validated by both the Harvey–Gray method of crystallographic-distance-to-force-constant correlation and dispersive density functional theory computations. Notably, the heterobimetallic complexes herein exhibit photophysical properties that are favorable to those for their homometallic congeners, due to threefold-to-twofold symmetry reduction, resulting in cuprophilicsensitizationinextinctioncoefficientandsolid-state photoluminescence quantum yields approaching unity (ΦPL = 0.90–0.97 vs. 0–0.83 for Au3 and Cu3 precursors), which bodes well for potential future utilization in inorganic and/or organic LED applications

Pseudomonas aeruginosa isolates co-incubated with Acanthamoeba castellanii exhibit phenotypes similar to chronic cystic fibrosis isolates

The opportunistic pathogen, Pseudomonas aeruginosa , is ubiquitous in the environment, and in humans is capable of causing acute and chronic infections. P. aeruginosa , when co-incubated with the bacterivorous amoeba, Acanthamoeba castellanii , for extended periods, produced genetic and phenotypic variants. Sequencing of late-stage amoeba-adapted P. aeruginosa isolates demonstrated single nucleotide polymorphisms within genes that encode known virulence factors, and this correlated with a reduction in expression of virulence traits. Virulence towards the nematode, Caenorhabditis elegans , was attenuated in late-stage amoeba-adapted P. aeruginosa compared to early stage amoeba-adapted and non-adapted counterparts. Late-stage amoeba-adapted P. aeruginosa lost competitive fitness compared to non-adapted counterparts when grown in nutrient rich media. However, non-adapted P. aeruginosa were rapidly cleared by amoeba predation, whereas late-stage amoeba-adapted isolates remained in higher numbers 24 h after ingestion by amoeba. In addition, there was reduced uptake by macrophage of amoeba-adapted isolates and reduced uptake by human neutrophils as well as increased survival in the presence of neutrophils. Our findings indicate that the selection imposed by amoeba on P. aeruginosa resulted in reduced virulence over time. Importantly, the genetic and phenotypic traits possessed by late-stage amoeba-adapted P. aeruginosa are similar to what is observed for isolates obtained from chronic cystic fibrosis infections. This notable overlap in adaptation to different host types suggests similar selection pressures among host cell types. Author Summary Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute infections in plants and animals, including humans and also causes chronic infections in immune compromised and cystic fibrosis patients. This bacterium is commonly found in soils and water where bacteria are constantly under threat of being consumed by the bacterial predators, protozoa. To escape being killed, bacteria have evolved a suite of mechanisms that protect them from being consumed or digested. Here we examined the effect of long-term predation on the genotype and phenotypes expressed by P. aeruginosa. We show that long-term co-incubation with protozoa resulted in mutations in the bacteria that made them less pathogenic. This is particularly interesting as we see similar mutations arise in bacteria associated with chronic infections. Thus, predation by protozoa and long term colonization of the human host may represent similar environments that select for similar losses in gene functions

Community survey results show that standardisation of preclinical imaging techniques remains a challenge

PURPOSE: To support acquisition of accurate, reproducible and high-quality preclinical imaging data, various standardisation resources have been developed over the years. However, it is unclear the impact of those efforts in current preclinical imaging practices. To better understand the status quo in the field of preclinical imaging standardisation, the STANDARD group of the European Society of Molecular Imaging (ESMI) put together a community survey and a forum for discussion at the European Molecular Imaging Meeting (EMIM) 2022. This paper reports on the results from the STANDARD survey and the forum discussions that took place at EMIM2022. PROCEDURES: The survey was delivered to the community by the ESMI office and was promoted through the Society channels, email lists and webpages. The survey contained seven sections organised as generic questions and imaging modality-specific questions. The generic questions focused on issues regarding data acquisition, data processing, data storage, publishing and community awareness of international guidelines for animal research. Specific questions on practices in optical imaging, PET, CT, SPECT, MRI and ultrasound were further included. RESULTS: Data from the STANDARD survey showed that 47% of survey participants do not have or do not know if they have QC/QA guidelines at their institutes. Additionally, a large variability exists in the ways data are acquired, processed and reported regarding general aspects as well as modality-specific aspects. Moreover, there is limited awareness of the existence of international guidelines on preclinical (imaging) research practices. CONCLUSIONS: Standardisation of preclinical imaging techniques remains a challenge and hinders the transformative potential of preclinical imaging to augment biomedical research pipelines by serving as an easy vehicle for translation of research findings to the clinic. Data collected in this project show that there is a need to promote and disseminate already available tools to standardise preclinical imaging practices

T.A. Ward

respectively). A combination of one habitat assessment and Proper Functioning Condition should be utilized to conduct a comprehensive assessment of riparian/stream health. Site characteristics, which were significantly associated with assessment outcomes included entrenchment ratio, substrate size, channel width to depth and slope. This presents a problem in that comparison of assessment outcomes across different streams and stream reaches are confounded by factors such as slope and substrate type, which may not always be indicative of riparian/stream health. The Rosgen Stream Morphology Classification system was used to successfully control for the effect of these site-specific effects on assessment outcome, allowing for comparison of riparian/stream health assessments across streams