Coevolution with hosts underpins speciation in brood-parasitic cuckoos.

Coevolution between interacting species is thought to increase biodiversity, but evidence linking microevolutionary processes to macroevolutionary patterns is scarce. We leveraged two decades of behavioral research coupled with historical DNA analysis to reveal that coevolution with hosts underpins speciation in brood-parasitic bronze-cuckoos. At a macroevolutionary scale, we show that highly virulent brood-parasitic taxa have higher speciation rates and are more likely to speciate in sympatry than less-virulent and nonparasitic relatives. We reveal the microevolutionary process underlying speciation: Hosts reject cuckoo nestlings, which selects for mimetic cuckoo nestling morphology. Where cuckoos exploit multiple hosts, selection for mimicry drives genetic and phenotypic divergence corresponding to host preference, even in sympatry. Our work elucidates perhaps the most common, but poorly characterized, evolutionary process driving biological diversification.

Chalkbrood disease in honey bees.

Chalkbrood is a fungal disease of honey bee brood caused by Ascosphaera apis. This disease is now found throughout the world, and there are indications that chalkbrood incidence may be on the rise. In this review we consolidate both historic knowledge and recent scientific findings. We document the worldwide spread of the fungus, which is aided by increased global travel and the migratory nature of many beekeeping operations. We discuss the current taxonomic classification in light of the recent complete reworking of fungal systematics brought on by application of molecular methods. In addition, we discuss epidemiology and pathogenesis of the disease, as well as pathogen biology, morphology and reproduction. New attempts at disease control methods and management tactics are reviewed. We report on research tools developed for identification and monitoring, and also include recent findings on genomic and molecular studies not covered by previous reviews, including sequencing of the A. apis genome and identification of the mating type locus.

High mobility group (HMG-box) genes in the honeybee fungal pathogen Ascosphaera apis.

The genome of the honeybee fungal pathogen Ascosphaera apis (Maassen) encodes three putative high mobility group (HMG-box) transcription factors. The predicted proteins (MAT1-2, STE11 and HTF), each of which contain a single strongly conserved HMG-box, exhibit high similarity to mating type proteins and STE11-like transcription factors previously identified in other ascomycete fungi, some of them important plant and human pathogens. In this study we characterized the A. apis HMG-box containing genes and analyzed the structure of the mating type locus (MAT1-2) and its flanking regions. The MAT1-2 locus contains a single gene encoding a protein with an HMG-box. We also have determined the transcriptional patterns of all three HMG-box containing genes in both mating type idiomorphs and discuss a potential role of these transcription factors in A. apis development and reproduction. A multiplex PCR method with primers amplifying mat1-2-1 and Ste11 gene fragments is described. This new method allows for identification of a single mating type idiomorph and might become an essential tool for applied and basic research of chalkbrood disease in honeybees.

Genome sequences of the honey bee pathogens Paenibacillus larvae and Ascosphaera apis.

Genome sequences offer a broad view of host-pathogen interactions at the systems biology level. With the completion of the sequence of the honey bee, interest in the relevant pathogens is heightened. Here we report the genome sequences of two of the major pathogens of honey bees, the bacterium Paenibacillus larvae (causative agent for American foulbrood disease) and the fungus Ascosphaera apis. (causative agent for chalkbrood disease). Ongoing efforts to characterize the genomes of these species can be used to understand and mitigate the effects of two important pathogens, and will provide a contrast with pathogenic, benign and freeliving relatives.

N-methyl-D-aspartate receptor dependent transcriptional regulation of two calcium/calmodulin-dependent protein kinase type II isoforms in rodent cerebral cortex.

Alpha Calcium/calmodulin-dependent protein kinase type II (CaMKII-alpha) expression is regulated in an activity-dependent manner, but it is not known whether other CaMKII isoforms (beta, delta, and gamma) are similarly regulated. We examined the activity-dependent regulation of these CaMKII isoforms in vivo, using a model of generalized seizures caused by i.p. injection of kainic acid. Following seizure induction, CaMKII-alpha expression was downregulated and CaMKII-delta expression upregulated while CaMKII-beta and CaMKII-gamma expression was unaffected. A transient downregulation in CaMKII-alpha and a transient increase in CaMKII-delta occurred throughout neocortex in the same temporal order. Although CaMKII-alpha mRNA was decreased by seizure activity, the less abundant, alternatively spliced, CaMKII-alpha33 mRNA was unaffected. Organotypic cortical slice cultures treated with bicuculline and 4-aminopyridine to induce seizure activity also showed a downregulation of CaMKII-alpha mRNA and an upregulation of CaMKII-delta mRNA. Prior exposure to tetrodotoxin prevented the changes in CaMKII-alpha and CaMKII-delta mRNA regulation and this was mimicked by D-L-2-amino-5-phosphonovaleric acid, but not by 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline, suggesting that CaMKII-alpha and CaMKII-delta mRNA expression is regulated in an N-methyl-D-aspartate receptor-dependent manner. Regulation was also transcription dependent. Blocking transcription with actinomycin-D prevented activity-dependent changes in CaMKII-alpha and CaMKII-delta mRNA, but produced opposite effects on basal transcription, resulting in more stabilized CaMKII-alpha mRNA and less stabilized CaMKII-delta mRNA. These results reveal unique patterns of seizure-induced alterations in CaMKII mRNAs. Activity-dependent changes in subunit composition could, therefore, differentially influence the functional attributes of the CaMKII holoenzyme.

Characterisation of LMD virus-like nanoparticles self-assembled from cationic liposomes, adenovirus core peptide mu and plasmid DNA.

Liposome:mu:DNA (LMD) is a ternary nucleic acid delivery system built around the mu peptide associated with the condensed core complex of the adenovirus. LMD is prepared by precondensing plasmid DNA (D) with mu peptide (M) in a 1:0.6 (w/w) ratio and then combining these mu:DNA (MD) complexes with extruded cationic liposomes (L) resulting in a final lipid:mu:DNA ratio of 12:0.6:1 (w/w/w). Correct buffer conditions, reagent concentrations and rates of mixing are all crucial to success. However, once optimal conditions are established, homogeneous LMD particles (120 +/- 30 nm) will result that each appear to comprise an MD particle encapsulated within a cationic bilammellar liposome. LMD particles can be formulated reproducibly, they are amenable to long-term storage (>1 month) at -80 degrees C and are stable to aggregation at a plasmid DNA concentration up to 5 mg/ml (15 mM nucleotide concentration). Furthermore, LMD transfections are significantly more time and dose efficient in vitro than cationic liposome-plasmid DNA (LD) transfections. Transfection times as short as 10 min and plasmid DNA doses as low as 0.001 microg/well result in significant gene expression. LMD transfections will also take place in the presence of biological fluids (eg up to 100% serum) giving 15-25% the level of gene expression observed in the absence of serum. Results from confocal microscopy experiments using fluorescent-labelled LMD particles suggest that endocytosis is not a significant barrier to LMD transfection, although the nuclear membrane still is. We also confirm that topical lung transfection in vivo by LMD is at least equal in absolute terms with transfection mediated by GL-67:DOPE:DMPE-PEG(5000) (1:2:0.05 m/m/m), an accepted 'gold-standard' non-viral vector system for topical lung transfection, and is in fact at least six-fold more dose efficient. All these features make LMD an important new non-viral vector platform system from which to derive tailor-made non-viral delivery systems by a process of systematic modular upgrading.

Inhibition of polyglutamine aggregation in R6/2 HD brain slices-complex dose-response profiles.

Huntington's disease (HD) is a late onset neurodegenerative disorder caused by a CAG/polyglutamine (polyQ) repeat expansion. PolyQ aggregates can be detected in the nuclei and processes of neurons in HD patients and mouse models prior to the onset of symptoms. The misfolding and aggregation pathway is an important therapeutic target. To better test the efficacy of aggregation inhibitors, we have developed an organotypic slice culture system. We show here that the formation of polyQ aggregates in hippocampal slices established from the R6/2 mouse follows the same prescribed sequence as occurs in vivo. Using this assay, we show that Congo red and chrysamine G can modulate aggregate formation, but show complex dose-response curves. Oral administration of creatine has been shown to delay the onset of all aspects of the phenotype and neuropathology in R6/2 mice. We show here that creatine can similarly inhibit aggregate formation in the slice culture assay.

Enhanced cationic liposome-mediated transfection using the DNA-binding peptide mu (mu) from the adenovirus core.

Promising advances in nonviral gene transfer have been made as a result of the production of cationic liposomes formulated with synthetic cationic lipids (cytofectins) that are able to transfect cells. However few cationic liposome systems have been examined for their ability to transfect CNS cells. Building upon our earlier use of cationic liposomes formulated from 3beta-[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol (DC-Chol) and dioleoyl-L-alpha-phosphatidyl-ethanolamine (DOPE), we describe studies using two cationic viral peptides, mu (mu) and Vp1, as potential enhancers for cationic liposome-mediated transfection. Mu is derived from the condensed core of the adenovirus and was selected to be a powerful nucleic acid charge neutralising and condensing agent. Vp1 derives from the polyomavirus and harbours a classical nuclear localisation signal (NLS). Vp1 proved disappointing but lipopolyplex mixtures formulated from pCMVbeta plasmid, mu peptide and DC-Chol/DOPE cationic liposomes were able to transfect an undifferentiated neuronal ND7 cell line with beta-galactosidase reporter gene five-fold more effectively than lipoplex mixtures prepared from pCMVbeta plasmid and DC-Chol/DOPE cationic liposomes. Mu was found to give an identical enhancement to cationic liposome-mediated transfection of ND7 cells as poly-L-lysine (pLL) or protamine sulfate (PA). The enhancing effects of mu were found to be even greater (six- to 10-fold) when differentiated ND7 cells were transfected with mu-containing lipopolyplex mixtures. Differentiated ND7 cells represent a simple ex vivo-like post-mitotic CNS cell system. Successful transfection of these cells bodes well for transfection of primary neurons and CNS cells in vivo. These findings have implications for experimental and therapeutic uses of cationic liposome-mediated delivery of nucleic acids to CNS cells.

Ultrasound study of the motion of the residual femur within a trans-femoral socket during daily living activities other than gait.

This study analyses the residual femur motion of a single amputee within a transfemoral socket during a series of daily living activities. Two simultaneously transmitting, socket mounted transducers were connected to two ultrasound scanners. Displacement measurements of the ultrasound image of the femur were video recorded and measured on "paused" playback. Abduction/adduction and flexion/extension of the residual femur within the socket at any instant during these activities were estimated, knowing the relative positions of the two transducers and the position of the residual femur on the ultrasound image. Consistent motion patterns of the residual femur within the trans-femoral socket were noted throughout each monitored daily living activity of the single amputee studied. Convery and Murray (2000) reported that during level walking, relative to the socket, the residual femur extends 6 degrees and abducts 9 degrees by mid-stance while flexing 6 degrees and adducting 2 degrees by toe-off. Uphill/downhill, turning to the right and stepping up/down altered this reported pattern of femoral motion by approximately 1 degree. During the standing activity from a seated position the femur initially flexed 4 degrees before moving to 7 degrees extension, while simultaneously adducting 6 degrees. During the sitting activity from a standing position the femur moved from 7 degrees extension and 6 degrees adduction to 3 degrees flexion and 1 degree abduction. The activity of single prosthetic support to double support introduced only minor femoral motion whereas during the activity of prosthetic suspension the femur flexed 8 degrees while simultaneously adducting 9 degrees. Additional studies of more amputees are required to validate the motion patterns presented in this investigation.

The calibration of ultrasound transducers used to monitor motion of the residual femur within a trans-femoral socket during gait.

During gait the motion of the residual femur within a trans-femoral socket may be estimated using video recorded data from two ultrasound transducers attached to the socket wall. This paper reviews possible measurement errors and identifies the magnitude of the inaccuracies. Inaccuracies due to equipment limitations and those due to human error are identified and quantified. Ranges of flexion/extension and abduction/adduction of the residual femur within the socket during gait have been estimated with a cumulative level of inaccuracy of <1 degree.

Altered mRNA expression for brain-derived neurotrophic factor and type II calcium/calmodulin-dependent protein kinase in the hippocampus of patients with intractable temporal lobe epilepsy.

The expression of brain-derived neurotrophic factor and the alpha subunit of calcium/calmodulin-dependent protein kinase II mRNA in hippocampi obtained during surgical resections for intractable temporal lobe epilepsy were examined. Both calcium/calmodulin-dependent protein kinase II and brain-derived neurotrophic factor are localized heavily within the hippocampus and have been implicated in regulating hippocampal activity (Kang and Schuman [1995] Science 267:1658-1662; Suzuki [1994] Intl J Biochem 26:735-744). Also, the autocrine and paracrine actions of brain-derived neurotrophic factor within the central nervous system make it a likely candidate for mediating morphologic changes typically seen in the epileptic hippocampus. Quantitative assessments of mRNA levels in epileptic hippocampi relative to autopsy controls were made by using normalized densitometric analysis of in situ hybridization. In addition, correlations between clinical data and mRNA levels were studied. Relative to autopsy control tissue, decreased hybridization to mRNA of the alpha subunit of calcium/calmodulin-dependent protein kinase II and increased hybridization to brain-derived neurotrophic factor mRNA were found throughout the granule cells of the epileptic hippocampus. There also was a significant negative correlation between the duration of epilepsy and the expression of mRNA for brain-derived neurotrophic factor. These results are similar qualitatively to those found in animal models of epilepsy and suggest that chronic seizure activity in humans leads to persistent alterations in gene expression. Furthermore, these alterations in gene expression may play a role in the etiology of the epileptic condition.

Ultrasound study of the motion of the residual femur within a trans-femoral socket during gait.

This study analyses the motion of the residual femur within a trans-femoral socket during gait using ultrasound data from two simultaneously transmitting transducers connected to two ultrasound scanners. Calibration tests accurately established the orientation of the two transducers mounted on the lateral wall of the socket. Relative positions of the ultrasound image of the femur were measured on video playback. Motion of the residual femur, relative to the lateral wall of the socket, at any instant during gait may be estimated, if the relative positions of the two transducers and the motion of the ultrasound image are known. A consistent pattern of femoral motion during 10 gait cycles is displayed graphically. The femoral motion in this paper is expressed as abduction/adduction or flexion/extension relative to the socket. However, without a full gait analysis study, the orientation of the socket relative to the ground or relative to the pelvis cannot be determined. Only one ultrasound scanner may be available for clinical use. Hence data collection may be restricted to only one transducer during gait. In order to simulate the single transducer mode, the ultrasound data recorded during the 10 previous gait cycles, was averaged at any instant of the gait cycle. The angular orientation of the femur was calculated based on the averaged data. Similar patterns of femoral motion were obtained irrespective of the technique adopted.

Procainamide-induced postoperative pyrexia.

Procainamide is an effective antiarrhythmic that is often used to convert atrial fibrillation to normal sinus rhythm. A side effect of procainamide, rarely reported in the surgical literature, is pyrexia. The pyrexia is a manifestation of an allergic response to this medication. If unrecognized, procainamide-induced pyrexia can lead to unnecessary testing, hospitalization, and treatment. We present a case of a post-coronary artery bypass surgery patient who repeatedly displayed pyrexia when reexposed to procainamide indicating an allergic response to this drug.

Cationic liposome-mediated DNA transfection in organotypic explant cultures of the ventral mesencephalon.

We have examined the potential of cationic liposomes as a tool for approaches to gene therapy in the CNS. Our previous work has shown that cationic liposomes formulated from 3 beta-[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol (DC-Chol) and dioleoyl-L-alpha-phosphatidylethanolamine (DOPE) could achieve high transfection levels in a neuronal cell line (McQuillin et al. Neuroreport 1997; 8: 1481-1484). We therefore wished to assess transfection efficiencies in organotypic cultures from the brain with a reporter plasmid expressing E. coli beta-galactosidase in order to mimic an in vivo model. Explant cultures were generated according to the method of Stoppini et al (J Neurosci Meth 1991; 37: 173-182) with slight modifications. Brain slices were maintained on transparent porous membranes and were observed to maintain their intrinsic connectivity and cytoarchitecture to a large degree over periods of up to 6 weeks in culture. CNS tissue was obtained from rats at birth or 5 days after birth. After transfection beta-galactosidase expression was detected in cells of both neuronal and non-neuronal morphology. Control cultures were exposed to liposome alone and a plasmid that had the beta-galactosidase gene insert removed. Only low levels of endogenous beta-galactosidase reactivity were seen in these control cultures. DC-Chol/DOPE-mediated transfection was confirmed using a RT-PCR protocol capable of differentiating between untranscribed plasmid DNA and RNA generated from the transfected vector. These results suggest that cationic liposomes, particularly DC-Chol/DOPE liposomes, will be useful as delivery agents for gene transfer to CNS cells in vitro and possibly in vivo.

Intraoperative lymphatic mapping for non-small cell lung cancer: the sentinel node technique.

BACKGROUND: The purpose of the study was to determine the accuracy and role of the sentinel node technique in patients with non-small cell lung cancer. METHODS: This study was carried out on 36 consecutive patients undergoing lung resection. Peritumoral tissue was infiltrated with isosulfan blue dye and the first lymph node to stain was identified as a sentinel node. Sensitivity and specificity of the sentinel node in predicting the status of other lymph node stations were determined. RESULTS: Seventeen patients had sentinel lymph nodes. In 9 of these 17 cases neither the sentinel node nor any other lymph node contained metastatic carcinoma. In 5 cases the sentinel node was in the mediastinum and documented unexpected N2 disease. In 19 patients no sentinel node was found. Final lymph node statuses were N0 in 13 patients, N1 in 5, and N2 in 1. CONCLUSIONS: The use of isosulfan blue for intraoperative lymphatic mapping is feasible. The specificity in our experience was good; 9 of 9 patients with negative sentinel nodes were found to be N0 on the final pathology report. Unexpected N2 disease was found in 5 patients. The accumulation of further experience will determine the role of the sentinel node technique in patients with non-small cell lung cancer.

Attenuation of the seizure-induced expression of BDNF mRNA in adult rat brain by an inhibitor of calcium/calmodulin-dependent protein kinases.

We have examined the potential involvement of calcium/calmodulin-dependent protein kinases in the regulation of brain-derived neurotrophic factor mRNA in vivo following kainic acid (kainate)-induced seizure activity by in situ hybridization. KN-62, a specific inhibitor of calcium/calmodulin-dependent protein kinase type II and IV, blocked the characteristic induction of brain-derived neurotrophic factor mRNA seen following seizure activity. This blockade was specific to calcium/calmodulin-dependent protein kinase type II and IV as inhibitors of both protein kinase C and cAMP-dependent protein kinase had no effect. Inhibition of brain-derived neurotrophic factor mRNA increases varied between brain regions; an almost complete inhibition was seen throughout cortical regions, whereas only partial inhibitory effects were noted within hippocampus. A similar inhibition of increased c-fos mRNA was observed throughout cortical, hippocampal and diencephalic regions. The two predominant brain-derived neurotrophic factor transcripts induced by kainate, containing exons I or III, were differentially affected by KN-62. The cortical induction of exon I was blocked by KN-62, whereas exon III was not, providing additional evidence for the differential regulation of individual brain-derived neurotrophic factor transcripts and demonstrating that inhibition of brain-derived neurotrophic factor induction was not due to general blockade of seizure activity throughout the neocortex. These data implicate calcium/calmodulin-dependent protein kinase type II or IV in the regulation of brain-derived neurotrophic factor mRNA in vivo and suggest regionally specific mechanisms occur throughout the brain.

The messenger RNA encoding VGF, a neuronal peptide precursor, is rapidly regulated in the rat central nervous system by neuronal activity, seizure and lesion.

The VGF gene encodes a neuronal secretory-peptide precursor that is rapidly induced by neurotrophic growth factors and by depolarization in vitro. VGF expression in the animal peaks during critical periods in the developing peripheral and central nervous systems. To gain insight into the possible functions and regulation of VGF in vivo, we have used in situ hybridization to examine the regulation of VGF messenger RNA by experimental manipulations, and have found it to be regulated in the CNS by paradigms that affect electrical activity and by lesion. Inhibition of retinal electrical activity during the critical period of visual development rapidly repressed VGF messenger RNA in the dorsal lateral geniculate nucleus of the thalamus. In the adult, kainate-induced seizures transiently induced VGF messenger RNA in neurons of the dentate gyrus, hippocampus, and cerebral cortex within hours. Cortical lesion strongly induced VGF messenger RNA in ipsilateral cortex within hours, and strongly repressed expression in ipsilateral striatum. Ten days postlesion there was a delayed induction of VGF messenger RNA in a portion of deafferented striatum where compensatory cortical sprouting has been detected. Expression of the neuronal secretory-peptide precursor VGF is therefore modulated in vivo by monocular deprivation, seizure, and cortical lesion, paradigms which lead to neurotrophin induction, synaptic remodeling and axonal sprouting.

Cell-specific expression of type II calcium/calmodulin-dependent protein kinase isoforms and glutamate receptors in normal and visually deprived lateral geniculate nucleus of monkeys.

In situ hybridization histochemistry and immunocytochemistry were used to map distributions of cells expressing mRNAs encoding alpha, beta, gamma, and delta isoforms of type II calcium/calmodulin-dependent protein kinase (CaMKII), alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA)/ kainate receptor subunits, (GluR1-7), and N-methyl-D-aspartate (NMDA) receptor subunits, NR1 and NR2A-D, or stained by subunit-specific immunocytochemistry in the dorsal lateral geniculate nuclei of macaque monkeys. Relationships of specific isoforms with particular glutamate receptor types may be important elements in neural plasticity. CaMKII-alpha is expressed only by neurons in the S laminae and interlaminar plexuses of the dorsal lateral geniculate nucleus, but may form part of a more widely distributed matrix of similar cells extending from the geniculate into adjacent nuclei. CaMKII-beta, -gamma, and -delta isoforms are expressed by all neurons in principal and S laminae and interlaminar plexuses. In principal laminae, they are down-regulated by monocular deprivation lasting 8-21 days. All glutamate receptor subunits are expressed by neurons in principal and S laminae and interlaminar plexuses. The AMPA/kainate subunits, GluR1, 2, 5, and 7, are expressed at low levels, although GluR1 immunostaining appears selectively to stain interneurons. GluR3 is expressed at weak, GluR 6 at moderate and GluR 4 at high levels. NMDA subunits, NR1 and NR2A, B, and D, are expressed at moderate to low levels. GluR4, GluR6 and NMDA subunits are down-regulated by visual deprivation. CaMKII-alpha expression is unique in comparison with other CaMKII isoforms which may, therefore, have more generalized roles in cell function. The results demonstrate that all of the isoforms are associated with NMDA receptors and with AMPA receptors enriched with GluR4 subunits, which implies high calcium permeability and rapid gating.

The importance of pulsatile and nonpulsatile flow in the design of blood pumps.

The traditional approach of total artificial heart (TAH) and ventricular assist device (VAD) development has been the mimicking of the native heart. Nonpulsatile flow using cardiopulmonary bypass has provided evidence of short-term physiologic tolerances. The design of nonpulsatile TAHs and VADs has eliminated the need for valves, flexing diaphragms, and large ventricular volumes. However, these devices require high efficiency power sources and reliable bearing seals or electromagnetic bearings while simultaneously attempting to avoid thromboemboli. The physiologic response to nonpulsatile flow is complex and variable. When compared to a pulsatile device, a nonpulsatile TAH or VAD needs to produce increased flow and higher mean intravascular pressures to maintain normal organ function. Despite its maintaining normal organ function, nonpulsatile flow does cause alterations in biochemical functions and organ specific blood flow. The combination of bioengineering superiority and the maintenance of physiologic homeostasis has directed future TAH and VAD research towards nonpulsatile systems.