Lucilia bufonivora, Not Lucilia silvarum (Diptera: Calliphoridae), Causes Myiasis in Anurans in North America With Notes About Lucilia elongata and Lucilia thatuna.

In North America, until recently, all cases of anuran myiasis were attributed to Lucilia silvarum (Meigen) or Lucilia elongata Shannon. The latter species is exceedingly rare and its life history is unknown, but L. silvarum is common and was thought to be capable of being either parasitic or saprophytic in North America. Until recently, the anuran parasite Lucilia bufonivora Moniez was thought to be strictly Palearctic, but a study in 2014 has determined this species is established throughout southern Canada. In 2019, a study demonstrated, with molecular and morphological evidence, that two adult flies formerly identified as L. silvarum and reared from amphibian myiasis cases from Canada, are actually L. bufonivora. Although the mentioned study detected relatively high genetic distances with European L. bufonivora, the lack of evident morphological differentiation suggest that they are the same species. The current study examined 12 adult males and eleven adult females morphologically from three additional North American studies. Specimens were examined which had been identified as L. silvarum or L. elongata, and they all proved to be L. bufonivora. We now suspect L. silvarum is strictly saprophagous in North America like they are in the Palearctic Region. We also provide evidence that the pattern of myiasis differs between European and North American specimens.

Ecological and geographical speciation in Lucilia bufonivora: The evolution of amphibian obligate parasitism.

Lucilia (Diptera: Calliphoridae) is a genus of blowflies comprised largely of saprophagous and facultative parasites of livestock. Lucilia bufonivora, however, exhibits a unique form of obligate parasitism of amphibians, typically affecting wild hosts. The evolutionary route by which amphibian myiasis arose, however, is not well understood due to the low phylogenetic resolution in existing nuclear DNA phylogenies. Furthermore, the timing of when specificity for amphibian hosts arose in L. bufonivora is also unknown. In addition, this species was recently reported for the first time in North America (Canada) and, to date, no molecular studies have analysed the evolutionary relationships between individuals from Eastern and Western hemispheres. To provide broader insights into the evolution of the amphibian parasitic life history trait and to estimate when the trait first arose, a time-scaled phylogeny was inferred from a concatenated data set comprising mtDNA, nDNA and non-coding rDNA (COX1, per and ITS2 respectively). Specimens from Canada, the UK, Poland, Switzerland, the Netherlands and Germany were analysed, as well as individuals from its sister taxa, the saprophage Lucilia silvarum and a Nearctic species also implicated in amphibian myiasis, Lucilia elongata. Obligate amphibian parasitism appears to have arisen ~4 mya, likely as a result of niche displacement of a saprophagous/facultative parasite ancestor. Consistent paraphyly of L. bufonivora with respect to L. elongata across single-gene phylogenies and high mtDNA genetic distances between Nearctic and Palearctic individuals suggest on-going cryptic speciation facilitated by geographical isolation. These findings suggest that recent reports of L. bufonivora in the Nearctic do not constitute a recent introduction, but instead suggest that it remained unrecorded due to taxonomic confusion and low abundance. This is the first study to confirm the involvement of L. bufonivora in amphibian myiasis in Canada using DNA-based identification methods.

DNA barcoding cannot reliably identify species of the blowfly genus Protocalliphora (Diptera: Calliphoridae).

In DNA barcoding, a short standardized DNA sequence is used to assign unknown individuals to species and aid in the discovery of new species. A fragment of the mitochondrial gene cytochrome c oxidase subunit 1 is emerging as the standard barcode region for animals. However, patterns of mitochondrial variability can be confounded by the spread of maternally transmitted bacteria that cosegregate with mitochondria. Here, we investigated the performance of barcoding in a sample comprising 12 species of the blow fly genus Protocalliphora, known to be infected with the endosymbiotic bacteria Wolbachia. We found that the barcoding approach showed very limited success: assignment of unknown individuals to species is impossible for 60% of the species, while using the technique to identify new species would underestimate the species number in the genus by 75%. This very low success of the barcoding approach is due to the non-monophyly of many of the species at the mitochondrial level. We even observed individuals from four different species with identical barcodes, which is, to our knowledge, the most extensive case of mtDNA haplotype sharing yet described. The pattern of Wolbachia infection strongly suggests that the lack of within-species monophyly results from introgressive hybridization associated with Wolbachia infection. Given that Wolbachia is known to infect between 15 and 75% of insect species, we conclude that identification at the species level based on mitochondrial sequence might not be possible for many insects. However, given that Wolbachia-associated mtDNA introgression is probably limited to very closely related species, identification at the genus level should remain possible.

Tamoxifen stimulates cancellous bone formation in long bones of female mice.

Selective estrogen receptor modulators (SERMs) have been developed as a means of targeting estrogen's protective effect on the skeleton in the treatment of postmenopausal osteoporosis. Although it is well established that SERMs such as tamoxifen inhibit bone resorption in a similar manner to estrogen, whether this agent shares estrogen's stimulatory action on bone formation is currently unclear. To address this question, we compared the effect of treatment for 28 d with 17beta-estradiol (E2; 0.1, 1.0 mg/kg x d) and tamoxifen (0.1, 1.0, or 10 mg/kg x d) on cancellous bone formation at the proximal tibial metaphysis of intact female mice. E2 stimulated the formation of new cancellous bone throughout the metaphysis. A similar response was observed after administration of tamoxifen, the magnitude of which was approximately 50% of that seen after E2. As expected, E2 was found to suppress longitudinal bone growth, but in contrast, this parameter was stimulated by tamoxifen. We conclude that tamoxifen acts as an agonist with respect to estrogen's stimulatory action on bone formation but as an antagonist in terms of estrogen's inhibition of longitudinal growth, suggesting that the protective effect of SERMs on the skeleton is partly mediated by stimulation of osteoblast activity.

Wolbachia and genetic variability in the birdnest blowfly Protocalliphora sialia.

Wolbachia are widespread cytoplasmically inherited bacteria that induce various reproductive alterations in host arthropods, including cytoplasmic incompatibility (CI), an incompatibility between sperm and egg that typically results in embryonic death. CI has been invoked as a possible mechanism for reproductive isolation and speciation in arthropods, by restricting gene flow and promoting maintenance (and evolution) of genetic divergence between populations. Here we investigate patterns of Wolbachia infection and nuclear and mitochondrial differentiation in geographical populations of the birdnest blowfly Protocalliphora sialia. Blowflies in western North America are infected with two A-group Wolbachia, with some individuals singly and others doubly infected. Individuals in eastern North America mostly show single infections with a B-group Wolbachia. Populations in the Midwest are polymorphic for infections and show A- or B-group infection. There is a low level of mitochondrial divergence and perfect concordance of mitochondrial haplotype with infection type, suggesting that two Wolbachia-associated selective sweeps of the mitochondrion have occurred in this species. Amplified fragment length polymorphism analysis of nuclear genetic variation shows genetic differentiation between the eastern-Midwestern and western populations. Both Midwestern and eastern flies infected with A-Wolbachia show eastern nuclear genetic profiles. Current results therefore suggest that Wolbachia has not acted as a major barrier to gene flow between western and eastern-Midwestern populations, although some genetic differentiation between A-Wolbachia infected and B-Wolbachia infected individuals in eastern-Midwestern populations cannot be ruled out.

Upregulation of gamma-aminobutyric acid transporter expression: role of alkylated gamma-aminobutyric acid derivatives.

Pregabalin [(S)-(+)-3-isobutylgaba] and gabapentin [1-(aminomethyl)cyclohexane acetic acid] are gamma-aminobutyric acid (GABA) derivatives that are effective in the treatment of behavioural disorders, convulsions, epilepsy and hyperalgesia. The mechanisms underlying the diverse actions of these compounds in the brain have not been well elucidated. To test the hypothesis that these compounds exert some of their effects on GABAergic systems in the brain, we examined their role in regulating the rat brain GABA transporter GAT1, a plasma membrane protein involved in regulating synaptic transmitter levels. Prolonged incubation of hippocampal cultures, which endogenously express GAT1, with gabapentin and pregabalin caused a 2-fold increase in subsequent GABA uptake, which was concentration- and time-dependent. This increase in uptake was correlated with a redistribution of GAT1 protein from intracellular locations to the plasma membrane. Further experiments also suggested that the signal transduction cascade that modulates pregabalin-mediated GAT1 redistribution may involve pathways activated by specific GAT1 substrates and antagonists but does not involve protein kinase C and tyrosine kinases, two other pathways known to regulate GAT1 redistribution. These data suggest that pregabalin and gabapentin may exert some of their actions in the brain by altering GABAergic signalling.

Substrate-induced regulation of gamma-aminobutyric acid transporter trafficking requires tyrosine phosphorylation.

Neurotransmitter transporters regulate synaptic transmitter levels and are themselves functionally regulated by a number of different signal transduction cascades. A common theme in transporter regulation is redistribution of transporter protein between intracellular stores and the plasma membrane. The triggers and mechanisms underlying this regulation are important in the control of extracellular transmitter concentrations and hence synaptic signaling. Previously, we demonstrated that the gamma-aminobutyric acid transporter GAT1 is regulated by direct tyrosine phosphorylation, resulting in an up-regulation of transporter expression on the plasma membrane. In the present report, we show that two tyrosine residues on GAT1 contribute to the phosphorylation and transporter redistribution. Tyrosine phosphorylation is concomitant with a decrease in the rate of transporter internalization from the plasma membrane. A decrease in GAT internalization rates also occurs in the presence of GAT1 substrates, suggesting the hypothesis that tyrosine phosphorylation is required for the substrate-induced up-regulation of GAT1 surface expression. In support of this hypothesis, incubation of GAT1-expressing cells with transporter ligands alters the amount of GAT1 tyrosine phosphorylation, and substrate-induced surface expression is unchanged in a GAT1 mutant lacking tyrosine phosphorylation sites. These data suggest a model in which substrates permit the phosphorylation of GAT1 on tyrosine residues and that the phosphorylated state of the transporter is refractory for internalization.

Recombinant GABA(C) receptors expressed in rat hippocampal neurons after infection with an adenovirus containing the human rho1 subunit.

1. A recombinant adenovirus was generated with the human rho1 GABA(C) receptor subunit (adeno-rho). Patch-clamp and antibody staining were employed to confirm functional expression of recombinant rho1 receptors after infection of human embryonic kidney cells (HEK293 cell line), human embryonic retinal cells (911 cell line), dissociated rat hippocampal neurons and cultured rat hippocampal slices. 2. Standard whole-cell recording and Western blot analysis using rho1 GABA(C) receptor antibodies revealed that recombinant rho1 receptors were expressed in HEK293 and 911 cells after adeno-rho infection and exhibited properties similar to those of rho1 receptors after standard transfection. 3. Cultured rat hippocampal neurons (postnatal day (P)3-P5) did not show a native GABA(C)-like current. After adeno-rho infection, however, a GABA(C)-like current appeared in 70-90 % of the neurons. 4. Five days after infection, expression of GABA(C) receptors in hippocampal neurons significantly decreased native GABA(A) receptor currents from 1200 +/- 300 to 150 +/- 70 pA (n = 10). The native glutamate-activated current was unchanged. 5. Hippocampal slices (P8) did not show a native GABA(C)-like current, although recombinant rho1 receptors could be expressed in cultured hippocampal slices after adeno-rho infection. 6. These data indicate that an adenovirus can be used to express recombinant GABA(C) receptors in hippocampal neurons. This finding could represent an important step towards the gene therapy of CNS receptor-related diseases.

Upregulation of surface alpha4beta2 nicotinic receptors is initiated by receptor desensitization after chronic exposure to nicotine.

It is hypothesized that desensitization of neuronal nicotinic acetylcholine receptors (nAChRs) induced by chronic exposure to nicotine initiates upregulation of nAChR number. To test this hypothesis directly, oocytes expressing alpha4beta2 receptors were chronically incubated (24-48 hr) in nicotine, and the resulting changes in specific [3H]nicotine binding to surface receptors on intact oocytes were compared with functional receptor desensitization. Four lines of evidence strongly support the hypothesis. (1) The half-maximal nicotine concentration necessary to produce desensitization (9.7 nM) was the same as that needed to induce upregulation (9.9 nM). (2) The concentration of [3H]nicotine for half-maximal binding to surface nAChRs on intact oocytes was also similar (11.1 nM), as predicted from cyclical desensitization models. (3) Functional desensitization of alpha3beta4 receptors required 10-fold higher nicotine concentrations, and this was mirrored by a 10-fold shift in concentrations necessary for upregulation. (4) Mutant alpha4beta2 receptors that do not recover fully from desensitization, but not wild-type channels, were upregulated after acute (1 hr) applications of nicotine. Interestingly, the nicotine concentration required for half-maximal binding of alpha4beta2 receptors in total cell membrane homogenates was 20-fold lower than that measured for surface nAChRs in intact oocytes. These data suggest that cell homogenate binding assays may not accurately reflect the in vivo desensitization affinity of surface nAChRs and may account for some of the previously reported differences in the efficacy of nicotine for inducing nAChR desensitization and upregulation.

Regulation of alpha4beta2 nicotinic receptor desensitization by calcium and protein kinase C.

Neuronal nicotinic acetylcholine receptor (nAChR) desensitization is hypothesized to be a trigger for long-term changes in receptor number and function observed after chronic administration of nicotine at levels similar to those found in persons who use tobacco. Factors that regulate desensitization could potentially influence the outcome of long-lasting exposure to nicotine. The roles of Ca2+ and protein kinase C (PKC) on desensitization of alpha4beta2 nAChRs expressed in Xenopus laevis oocytes were investigated. Nicotine-induced (300 nM; 30 min) desensitization of alpha4beta2 receptors in the presence of Ca2+ developed in a biphasic manner with fast and slow exponential time constants of tauf = 1.4 min (65% relative amplitude) and taus = 17 min, respectively. Recovery from desensitization was reasonably well described by a single exponential with taurec = 43 min. Recovery was largely eliminated after replacement of external Ca2+ with Ba2+ and slowed by calphostin C (taurec = 48 min), an inhibitor of PKC. Conversely, the rate of recovery was enhanced by phorbol-12-myristate-13-acetate (taurec = 14 min), a PKC activator, or by cyclosporin A (with taurec = 8 min), a phosphatase inhibitor. alpha4beta2 receptors containing a mutant alpha4 subunit that lacks a consensus PKC phosphorylation site exhibited little recovery from desensitization. Based on a two-desensitized-state cyclical model, it is proposed that after prolonged nicotine treatment, alpha4beta2 nAChRs accumulate in a "deep" desensitized state, from which recovery is very slow. We suggest that PKC-dependent phosphorylation of alpha4 subunits changes the rates governing the transitions from "deep" to "shallow" desensitized conformations and effectively increases the overall rate of recovery from desensitization. Long-lasting dephosphorylation may underlie the "permanent" inactivation of alpha4beta2 receptors observed after chronic nicotine treatment.

Clinical ladder to professional advancement program. An evolutionary process.

Since the early 1970s, clinical ladder programs have been a method of defining, recognizing, and rewarding nursing practice. As clinical practice in an institution grows and evolves, so must the program that supports the development of the practitioner. An in-depth evaluation of one clinical ladder program was conducted to determine if it was reflective of current practice. The authors discuss the method of evaluation, findings, and the revised program.

Proteinases from pollen and pests.

An examination of the proteinases present in two very different systems is described, in order to illustrate the diversity in function of this class of enzymes. In the first case we have noted the importance of gut proteinases from the fire ant Solenopsis invicta in relation to the nutritional requirements of the entire colony. In the second we have investigated the properties of endoproteases from both ragweed and mesquite pollen, relative to their role in the development of allergies and asthma. If the function of each type of enzyme(s) is correct, then it is clear that addition of exogenous inhibitors might be useful in a) controlling the infestation associated with the fire ant, and b) reducing the deleterious effects associated with the development of asthma.

Implementing a clinically focused advancement system. One institution's experience.

Clinical ladders were designed to recognize, reward, and retain professional nurses who chose to remain in direct clinical practice. One institution implemented a clinically focused advancement system that addresses many of the pitfalls that have historically caused clinical ladders to fail. The authors describe unique features of the advancement system, along with the process for advancement, budgetary considerations, and transition to the new advancement system.

Sequence of the feline cardiac sarcoplasmic reticulum Ca(2+)-ATPase.

The complementary DNA for the feline cardiac sarcoplasmic reticulum (SR) Ca(2+)-ATPase has been cloned and sequenced. The deduced amino acid sequence consists of 997 amino acid residues which shows greater than 98% identity with the pig, rabbit and human SR Ca(2+)-ATPase. The 5' and 3' untranslated regions are also strikingly similar to the published rabbit sequence.

Five month extrauterine survival in a female triploid (69,XXX) child.

The case of a (69,XXX) triploid infant who survived 160 days is reported. Previously, the longest survival of a non-mosaic triploid had been 9 days. Possible reasons for this extensive survival are discussed.

The matching law in and within groups of rats.

In each of the two experiments, a group of five rats lived in a complex maze containing four small single-lever operant chambers. In two of these chambers, food was available on variable-interval schedules of reinforcement. In Experiment I, nine combinations of variable intervals were used, and the aggregate lever-pressing rates (by the five rats together) were studied. The log ratio of the rates in the two chambers was linearly related to the log ratio of the reinforcement rates in them; this is an instance of Herrnstein's matching law, as generalized by Baum. Summing over the two food chambers, food consumption decreased, and response output increased, as the time required to earn each pellet increased. In Experiment II, the behavior of individual rats was observed by time-sampling on selected days, while different variable-interval schedules were arranged in the two chambers where food was available. Individual lever-pressing rates for the rats were obtained, and their median bore the same "matching" relationship to the reinforcement rates as the group aggregate in Experiment I. There were differences between the rats in their distribution of time and responses between the two food chambers; these differences were correlated with differences in the proportions of reinforcements the rats obtained from each chamber.