Functional characterisation of a nicotinic acetylcholine receptor α subunit from the brown dog tick, Rhipicephalus sanguineus

Ticks and tick-borne diseases have a major impact on human and animal health worldwide. Current control strategies rely heavily on the use of chemical acaricides, most of which target the CNS and with increasing resistance, new drugs are urgently needed. Nicotinic acetylcholine receptors (nAChRs) are targets of highly successful insecticides. We isolated a full-length nAChR α subunit from a normalised cDNA library from the synganglion (brain) of the brown dog tick, Rhipicephalus sanguineus. Phylogenetic analysis has shown this R. sanguineus nAChR to be most similar to the insect α1 nAChR group and has been named Rsanα1. Rsanα1 is distributed in multiple tick tissues and is present across all life-stages. When expressed in Xenopus laevis oocytes Rsanα1 failed to function as a homomer, with and without the addition of either Caenorhabditis elegans resistance-to-cholinesterase (RIC)-3 or X. laevis RIC-3. When co-expressed with chicken β2 nAChR, Rsanα1 evoked concentration-dependent, inward currents in response to acetylcholine (ACh) and showed sensitivity to nicotine (100 μM) and choline (100 μM). Rsanα1/β2 was insensitive to both imidacloprid (100 μM) and spinosad (100 μM). The unreliable expression of Rsanα1 in vitro suggests that additional subunits or chaperone proteins may be required for more robust expression. This study enhances our understanding of nAChRs in arachnids and may provide a basis for further studies on the interaction of compounds with the tick nAChR as part of a discovery process for novel acaricides.     

Detection of molecular alterations in methamphetamine‐activated Fos‐expressing neurons from a single rat dorsal striatum using fluorescence‐activated cell sorting (FACS)

Methamphetamine and other drugs activate a small proportion of all neurons in the brain. We previously developed a fluorescence‐activated cell sorting (FACS)‐based method to characterize molecular alterations induced selectively in activated neurons that express the neural activity marker Fos. However, this method requires pooling samples from many rats. We now describe a modified FACS‐based method to characterize molecular alterations in Fos‐expressing dorsal striatal neurons from a single rat using a multiplex pre‐amplification strategy. Fos and NeuN (a neuronal marker) immunohistochemistry indicate that 5–6% of dorsal striatum neurons were activated 90 min after acute methamphetamine injections (5 mg/kg, i.p.) while less than 0.5% of neurons were activated by saline injections. We used FACS to separate NeuN‐labeled neurons into Fos‐positive and Fos‐negative neurons and assessed mRNA expression using RT‐qPCR from as little as five Fos‐positive neurons. Methamphetamine induced 3–20‐fold increases of immediate early genes arc, homer‐2, c‐fos, fosB, and its isoforms (ΔfosB and a novel isoform ΔfosB‐2) in Fos‐positive but not Fos‐negative neurons. Immediate early gene mRNA induction was 10‐fold lower or absent when assessed in unsorted samples from single dorsal striatum homogenates. Our modified method makes it feasible to study unique molecular alterations in neurons activated by drugs or drug‐associated cues in complex addiction models.

     

Integrating molecular tools into freshwater ecology: developments and opportunities

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The equine CD1 gene family is the largest and most diverse yet identified

The CD1 family is a group of non-polymorphic MHC class I-like molecules that present lipid-based antigens to T cells. Previous work in our laboratory demonstrated that cytotoxic T lymphocytes from immune adult horses recognize lipids from the cell wall of an important equine pathogen, Rhodococcus equi. These findings suggest an important role for the equine CD1 antigen presentation system in protective immune responses to microbial pathogens in the horse. In this study, we characterized and mapped the equine CD1 gene cluster. The equine genome was found to contain 13 complete CD1 genes; seven genes were classified as homologues of human CD1a, two CD1b, one CD1c, one CD1d, and two CD1e, making it the largest CD1 family to date. All but one of the eqCD1 molecules were expressed in all antigen-presenting cells investigated. The major amino acid differences between equine CD1 isoforms are located in the predicted antigen binding site, suggesting that a variety of lipid antigens can be presented. R. equi survives and replicates within professional phagocytes by arresting phagosome maturation between the early endosome and late phagosome. Based on the absence of a tyrosine sorting motif in all eqCD1a, CD1a molecules are predicted to co-localize with R. equi in the early endosome. Here, they could acquire lipid antigen and present it to T lymphocytes. The extraordinarily large number of CD1 molecules in the horse may reflect their crucial role in immunity to R. equi.     

Sperm distribution and fertilization after unilateral and bilateral laparoscopic artificial insemination with frozen-thawed goat semen

Generally, laparoscopic artificial insemination (LAI) provides a higher success rate than of cervical insemination in goats. However, the sperm distribution after LAI in goats remains unknown, particularly when frozen-thawed semen is used. This study evaluated the distribution of frozen-thawed goat spermatozoa after LAI and compared the effects of sperm numbers and deposition sites (unilateral and bilateral sites) on pregnancy rate. In experiment 1, the frozen-thawed spermatozoa were stained either with CellTracker Green CMFDA (CT-Green) or CellTracker Red CMPTX (CT-Red), and in vitro evaluations of viability and motility were performed. In experiment 2, the labeled spermatozoa were deposited via LAI into the left (CT-Green) and right (CT-Red) uterine horns (n = 4). After ovariohysterectomy (6 hours after insemination), the distributions of green- and red-colored spermatozoa were assessed via tissue section, flushing, and the oviductal contents were also collected. Experiment 3 was designed to test the pregnancy rates in a group of 120 does after LAI using different numbers of spermatozoa (60 and 120 × 10⁶ sperm per LAI) and different deposition sites. The results demonstrated that the fluorochromes used in this study did not impair sperm motility or viability. Frozen-thawed goat spermatozoa can migrate transuterinally after LAI, as evidenced by the observations of both CT-Green– and CT-Red–labeled spermatozoa in both uterine horns. Lower numbers of spermatozoa (60 × 10⁶) that are inseminated unilaterally (either ipsilateral or contralateral to the site of ovulation) can efficiently be used for LAI in goats (with a 56.67% pregnancy rate).     

A Strategy for Using Weight-of-Evidence Methods in Ecological Risk Assessments

Our review of existing approaches and regulatory uses of weight-of-evidence (WOE) methods suggested the need for a practical strategy for deploying WOE within a predictive ecological risk assessment (ERA). WOE is the process of considering strengths and weaknesses of various pieces of information in order to inform a decision being made among competing alternatives. A predictive ERA uses existing information relating cause and effect to estimate the probability that today's action X will lead to tomorrow's adverse outcome Y. There appears to be no practical guidance for use of WOE in predictive assessments. We therefore propose a strategy for using a WOE approach, within an ERA framework, to weigh and integrate outcomes from various lines of evidence to estimate the probability of an adverse outcome in an assessment endpoint. An ERA framework is necessary to connect the results of an assessment to the management goals of concern to decision-makers and stakeholders. Within that framework, a WOE approach provides a consistent and transparent means of interpreting the myriad types of data and information gathered during a complex ecological assessment. Impediments to application of WOE are discussed, including limited regulatory guidance, limited prior regulatory use, and persistent reliance on threshold-based decision-making.     

Use of 18F-Fluorodeoxyglucose Positron Emission Tomography–Computed Tomography to Aid in Diagnosing Intestinal Adenocarcinoma in 2 Rhesus Macaques (Macaca mulatta)

Two aged female rhesus macaques (Macaca mulatta) presented with weight loss and intermittent inappetence. The signalment and constellation of clinical signs led clinicians to suspect the presence of intestinal adenocarcinoma. Because of each animal''s advanced age and inconclusive radiographic findings, a noninvasive diagnostic tool was preferred over exploratory laparotomy to assist in determining a diagnosis. Consequently, 2-[¹⁸F]fluoro-2-deoxy-d-glucose (FDG) positron emission tomography–CT (FDG-PET–CT) was chosen to aid in confirming a suspicion of gastrointestinal adenocarcinoma in both animals. FDG is a glucose analogue labeled with fluorine-18 and is taken up by highly metabolically active cells, as observed in many cancers. Tomography revealed an annular constriction of the small intestine with focal FDG uptake in one animal, and an FDG avid transmural mass in the ascending colon of the second animal. Necropsy later confirmed both sites to be adenocarcinomas. This report supports the use of FDG-PET–CT as an adjunct to conventional radiography in the diagnosis of intestinal adenocarcinoma in nonhuman primates.Abbreviations: FDG, 2-[¹⁸F]fluoro-2-deoxy-d-glucose; PET, positron emission tomographyThe noninvasive imaging modality 2-[¹⁸F]fluoro-2-deoxy-d-glucose positron emission tomography-computed tomography (FDG-PET–CT) is used primarily for the detection and staging of cancer and for the assessment of therapeutic response.²⁸ This modality also is useful for the identification of some infectious and inflammatory diseases, as demonstrated in the recent report of intense FDG uptake in a case of pneumonia in a miniature pig.¹⁴ Furthermore, FDG-PET–CT has been helpful in the detection and monitoring of inflammatory bowel disease and complicating infections of the gastrointestinal tract.²⁸PET detects the three-dimensional distribution of radioactivity based on the annihilation photons emitted by radiotracers labeled with positron-emitting radionuclides, such as ¹⁸F-labeled FDG. In this manner, PET enables noninvasive assessment of biochemical processes. In contrast, CT uses X-rays to generate high-resolution images, allowing clear visualization of anatomic structures. In PET–CT, both the multidetector CT apparatus and the PET detectors are mounted in the same scanner, one behind the other. The PET data are superimposed on the CT data (coregistration), enabling precise anatomic localization of FDG activity.²⁹ Combining PET imaging with CT allows the fusion of functional and anatomic information acquired almost simultaneously, facilitating the visualization and localization of metabolic information noninvasively.Intestinal adenocarcinoma is a substantial cause of morbidity and mortality in aged rhesus macaques.^25,26,30 Rhesus macaques often are maintained on long term-studies, resulting in a population of animals exhibiting morbidities associated with advanced age.^20,25,26 As the age of the animals in our care advances, so too must our diagnostic capabilities advance. Here we report the use of a noninvasive method, FDG-PET–CT, to support the diagnosis of intestinal adenocarcinoma in 2 aged rhesus macaques. We describe the physics of PET, biochemical rationale for the use of FDG, and pitfalls of interpretation.     

No Evidence of Harms of Probiotic Lactobacillus rhamnosus GG ATCC 53103 in Healthy Elderly—A Phase I Open Label Study to Assess Safety,Tolerability and Cytokine Responses

Background

Although Lactobacillus rhamnosus GG ATCC 53103 (LGG) has been consumed by 2 to 5 million people daily since the mid 1990s, there are few clinical trials describing potential harms of LGG, particularly in the elderly.

Objectives

The primary objective of this open label clinical trial is to assess the safety and tolerability of 1×10¹⁰ colony forming units (CFU) of LGG administered orally twice daily to elderly volunteers for 28 days. The secondary objectives were to evaluate the effects of LGG on the gastrointestinal microbiome, host immune response and plasma cytokines.

Methods

Fifteen elderly volunteers, aged 66–80 years received LGG capsules containing 1×10¹⁰ CFU, twice daily for 28 days and were followed through day 56. Volunteers completed a daily diary, a telephone call on study days 3, 7 and 14 and study visits in the Clinical Research Center at baseline, day 28 and day 56 to determine whether adverse events had occurred. Assessments included prompted and open-ended questions.

Results

There were no serious adverse events. The 15 volunteers had a total of 47 events (range 1–7 per volunteer), 39 (83%) of which were rated as mild and 40% of which were considered related to consuming LGG. Thirty-one (70%) of the events were expected, prompted symptoms while 16 were unexpected events. The most common adverse events were gastrointestinal (bloating, gas, and nausea), 27 rated as mild and 3 rated as moderate. In the exploratory analysis, the pro-inflammatory cytokine interleukin 8 decreased during LGG consumption, returning towards baseline one month after discontinuing LGG (p = 0.038) while there was no difference in other pro- or anti-inflammatory plasma cytokines.

Conclusions

Lactobacillus rhamnosus GG ATCC 53103 is safe and well tolerated in healthy adults aged 65 years and older.

Trial Registration

ClinicalTrials.gov NCT 01274598     

Flame Experiments at the Advanced Light Source: New Insights into Soot Formation Processes

The following experimental protocols and the accompanying video are concerned with the flame experiments that are performed at the Chemical Dynamics Beamline of the Advanced Light Source (ALS) of the Lawrence Berkeley National Laboratory^1-4. This video demonstrates how the complex chemical structures of laboratory-based model flames are analyzed using flame-sampling mass spectrometry with tunable synchrotron-generated vacuum-ultraviolet (VUV) radiation. This experimental approach combines isomer-resolving capabilities with high sensitivity and a large dynamic range^5,6. The first part of the video describes experiments involving burner-stabilized, reduced-pressure (20-80 mbar) laminar premixed flames. A small hydrocarbon fuel was used for the selected flame to demonstrate the general experimental approach. It is shown how species’ profiles are acquired as a function of distance from the burner surface and how the tunability of the VUV photon energy is used advantageously to identify many combustion intermediates based on their ionization energies. For example, this technique has been used to study gas-phase aspects of the soot-formation processes, and the video shows how the resonance-stabilized radicals, such as C₃H₃, C₃H₅, and i-C₄H₅, are identified as important intermediates⁷. The work has been focused on soot formation processes, and, from the chemical point of view, this process is very intriguing because chemical structures containing millions of carbon atoms are assembled from a fuel molecule possessing only a few carbon atoms in just milliseconds. The second part of the video highlights a new experiment, in which an opposed-flow diffusion flame and synchrotron-based aerosol mass spectrometry are used to study the chemical composition of the combustion-generated soot particles⁴. The experimental results indicate that the widely accepted H-abstraction-C₂H₂-addition (HACA) mechanism is not the sole molecular growth process responsible for the formation of the observed large polycyclic aromatic hydrocarbons (PAHs).     

What are the benefits of parental care? The importance of parental effects on developmental rate

The evolution of parental care is beneficial if it facilitates offspring performance traits that are ultimately tied to offspring fitness. While this may seem self‐evident, the benefits of parental care have received relatively little theoretical exploration. Here, we develop a theoretical model that elucidates how parental care can affect offspring performance and which aspects of offspring performance (e.g., survival, development) are likely to be influenced by care. We begin by summarizing four general types of parental care benefits. Care can be beneficial if parents (1) increase offspring survival during the stage in which parents and offspring are associated, (2) improve offspring quality in a way that leads to increased offspring survival and/or reproduction in the future when parents are no longer associated with offspring, and/or (3) directly increase offspring reproductive success when parents and offspring remain associated into adulthood. We additionally suggest that parental control over offspring developmental rate might represent a substantial, yet underappreciated, benefit of care. We hypothesize that parents adjust the amount of time offspring spend in life‐history stages in response to expected offspring mortality, which in turn might increase overall offspring survival, and ultimately, fitness of parents and offspring. Using a theoretical evolutionary framework, we show that parental control over offspring developmental rate can represent a significant, or even the sole, benefit of care. Considering this benefit influences our general understanding of the evolution of care, as parental control over offspring developmental rate can increase the range of life‐history conditions (e.g., egg and juvenile mortalities) under which care can evolve.