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1.  Behaviour-Related Scalar Habitat Use by Cape Buffalo (Syncerus caffer caffer) 
PLoS ONE  2015;10(12):e0145145.
Studies of habitat use by animals must consider behavioural resource requirements at different scales, which could influence the functional value of different sites. Using Cape buffalo (Syncerus caffer caffer) in the Okavango Delta, Botswana, we tested the hypotheses that behaviour affected use between and within habitats, hereafter referred to as macro- and microhabitats, respectively. We fitted GPS-enabled collars to fifteen buffalo and used the distances and turning angles between consecutive fixes to cluster the resulting data into resting, grazing, walking and relocating behaviours. Distance to water and six vegetation characteristic variables were recorded in sites used for each behaviour, except for relocating, which occurred too infrequently. We used multilevel binomial and multinomial logistic regressions to identify variables that characterised seasonally-preferred macrohabitats and microhabitats used for different behaviours. Our results showed that macrohabitat use was linked to behaviour, although this was least apparent during the rainy season, when resources were most abundant. Behaviour-related microhabitat use was less significant, but variation in forage characteristics could predict some behaviour within all macrohabitats. The variables predicting behaviour were not consistent, but resting and grazing sites were more readily identifiable than walking sites. These results highlight the significance of resting, as well as foraging, site availability in buffalo spatial processes. Our results emphasise the importance of considering several behaviours and scales in studies of habitat use to understand the links between environmental resources and animal behavioural and spatial ecology.
PMCID: PMC4682657  PMID: 26673623
2.  A Pyrosequencing Investigation of Differences in the Feline Subgingival Microbiota in Health, Gingivitis and Mild Periodontitis 
PLoS ONE  2015;10(11):e0136986.
Periodontitis is the most frequently diagnosed health problem in cats yet little is known about the bacterial species important for the disease. The objective of this study was to identify bacterial species associated with health, gingivitis or mild periodontitis (<25% attachment loss) in feline plaque. Knowledge of these species is a first step in understanding the potential for improving oral health of cats via dietary interventions that alter the proportions of influential species. Subgingival plaque samples were collected from 92 cats with healthy gingiva, gingivitis or mild periodontitis. Pyrosequencing of the V1-V3 region of the 16S rDNA from these plaque samples generated more than one million reads and identified a total of 267 operational taxonomic units after bioinformatic and statistical analysis. Porphyromonas was the most abundant genus in all gingival health categories, particularly in health along with Moraxella and Fusobacteria. The Peptostreptococcaceae were the most abundant family in gingivitis and mild periodontitis. Logistic regression analysis identified species from various genera that were significantly associated with health, gingivitis or mild periodontitis. The species identified were very similar to those observed in canine plaque in the corresponding health and disease states. Such similarities were not observed between cat and human at the bacterial species level but with disease progression similarities did emerge at the phylum level. This suggests that interventions targeted at human pathogenic species will not be effective for use in cats but there is more potential for commonalities in interventions for cats and dogs.
PMCID: PMC4659563  PMID: 26605793
3.  The evolution of an integrated ultrasound curriculum (iUSC) for medical students: 9-year experience 
Interest in ultrasound education in medical schools has increased dramatically in recent years as reflected in a marked increase in publications on the topic and growing attendance at international meetings on ultrasound education. In 2006, the University of South Carolina School of Medicine introduced an integrated ultrasound curriculum (iUSC) across all years of medical school. That curriculum has evolved significantly over the 9 years. A review of the curriculum is presented, including curricular content, methods of delivery of the content, student assessment, and program assessment. Lessons learned in implementing and expanding an integrated ultrasound curriculum are also presented as are thoughts on future directions of undergraduate ultrasound education. Ultrasound has proven to be a valuable active learning tool that can serve as a platform for integrating the medical student curriculum across many disciplines and clinical settings. It is also well-suited for a competency-based model of medical education. Students learn ultrasound well and have embraced it as an important component of their education and future practice of medicine. An international consensus conference on ultrasound education is recommended to help define the essential elements of ultrasound education globally to ensure ultrasound is taught and ultimately practiced to its full potential. Ultrasound has the potential to fundamentally change how we teach and practice medicine to the benefit of learners and patients across the globe.
Electronic supplementary material
The online version of this article (doi:10.1186/s13089-015-0035-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4654731  PMID: 26589313
Ultrasound; Education; Medical students; Integrated curriculum
4.  Comparative Genomics of the Genus Porphyromonas Identifies Adaptations for Heme Synthesis within the Prevalent Canine Oral Species Porphyromonas cangingivalis 
Genome Biology and Evolution  2015;7(12):3397-3413.
Porphyromonads play an important role in human periodontal disease and recently have been shown to be highly prevalent in canine mouths. Porphyromonas cangingivalis is the most prevalent canine oral bacterial species in both plaque from healthy gingiva and plaque from dogs with early periodontitis. The ability of P. cangingivalis to flourish in the different environmental conditions characterized by these two states suggests a degree of metabolic flexibility. To characterize the genes responsible for this, the genomes of 32 isolates (including 18 newly sequenced and assembled) from 18 Porphyromonad species from dogs, humans, and other mammals were compared. Phylogenetic trees inferred using core genes largely matched previous findings; however, comparative genomic analysis identified several genes and pathways relating to heme synthesis that were present in P. cangingivalis but not in other Porphyromonads. Porphyromonas cangingivalis has a complete protoporphyrin IX synthesis pathway potentially allowing it to synthesize its own heme unlike pathogenic Porphyromonads such as Porphyromonas gingivalis that acquire heme predominantly from blood. Other pathway differences such as the ability to synthesize siroheme and vitamin B12 point to enhanced metabolic flexibility for P. cangingivalis, which may underlie its prevalence in the canine oral cavity.
PMCID: PMC4700951  PMID: 26568374
periodontal disease; comparative genomics; Porphyromonas; canine; plaque
5.  Demonstrating frequency-dependent transmission of sarcoptic mange in red foxes 
Biology Letters  2014;10(10):20140524.
Understanding the relationship between disease transmission and host density is essential for predicting disease spread and control. Using long-term data on sarcoptic mange in a red fox Vulpes vulpes population, we tested long-held assumptions of density- and frequency-dependent direct disease transmission. We also assessed the role of indirect transmission. Contrary to assumptions typical of epidemiological models, mange dynamics are better explained by frequency-dependent disease transmission than by density-dependent transmission in this canid. We found no support for indirect transmission. We present the first estimates of R0 and age-specific transmission coefficients for mange in foxes. These parameters are important for managing this poorly understood but highly contagious and economically damaging disease.
PMCID: PMC4272203  PMID: 25296930
age-specific infection; basic reproductive number; frequency-dependent transmission; indirect transmission; susceptible–exposed–infected (SEI) model
6.  Reactive oxygen species derived from Nox4 mediate BMP2 gene transcription and osteoblast differentiation 
The Biochemical journal  2011;433(2):393-402.
BMP-2 (bone morphogenetic protein-2) promotes differentiation of osteoblast precursor cells to mature osteoblasts that form healthy bone. In the present study, we demonstrate a novel mechanism of BMP-2-induced osteoblast differentiation. The antioxidant NAC (N-acetyl-L-cysteine) and the flavoprotein enzyme NAD(P)H oxidase inhibitor DPI (diphenyleneiodonium) preventedBMP-2-stimulated alkaline phosphatase expression and mineralized bone nodule formation inmouse 2T3 pre-osteoblasts. BMP-2 elicited a rapid generation of ROS (reactive oxygen species) concomitant with increased activation of NAD(P)H oxidase. NAC and DPI inhibited BMP-2-induced ROS production and NAD(P)H oxidase activity respectively. NAD(P)H oxidases display structurally similar catalytic subunits (Nox1–5) with differential expression in various cells. We demonstrate that 2T3 pre-osteoblasts predominantly express the Nox4 isotype of NAD(P)H oxidase. To extend this finding, we tested the functional effects of Nox4. Adenovirus-mediated expression of dominant-negative Nox4 inhibited BMP-2-induced alkaline phosphatase expression. BMP-2 promotes expression of BMP-2 for maintenance of the osteoblast phenotype. NAC and DPI significantly blocked BMP-2-stimulated expression of BMP2 mRNA and protein due to a decrease in BMP2 gene transcription. Dominant-negative Nox4 also mimicked this effect of NAC and DPI. Our results provide the first evidence for a new signalling pathway linking BMP-2-stimulated Nox4-derived physiological ROS to BMP-2 expression and osteoblast differentiation.
PMCID: PMC4539275  PMID: 21029048
bone morphogenetic protein-2 (BMP-2); bone morphogenetic protein-2 gene autoregulation; bone morphogenetic protein-2 signalling; osteoblast differentiation; reactive oxygen species
7.  Light-emitting diode street lights reduce last-ditch evasive manoeuvres by moths to bat echolocation calls 
Royal Society Open Science  2015;2(8):150291.
The light-emitting diode (LED) street light market is expanding globally, and it is important to understand how LED lights affect wildlife populations. We compared evasive flight responses of moths to bat echolocation calls experimentally under LED-lit and -unlit conditions. Significantly, fewer moths performed ‘powerdive’ flight manoeuvres in response to bat calls (feeding buzz sequences from Nyctalus spp.) under an LED street light than in the dark. LED street lights reduce the anti-predator behaviour of moths, shifting the balance in favour of their predators, aerial hawking bats.
PMCID: PMC4555863  PMID: 26361558
artificial lighting; light-emitting diode; street lights; bats; moth predation
8.  Managing Conflict between Bats and Humans: The Response of Soprano Pipistrelles (Pipistrellus pygmaeus) to Exclusion from Roosts in Houses 
PLoS ONE  2015;10(8):e0131825.
Conflict can arise when bats roost in human dwellings and householders are affected adversely by their presence. In the United Kingdom, the exclusion of bats from roosts can be licensed under exceptional circumstances to alleviate conflict, but the fate of excluded bats and the impact on their survival and reproduction is not well understood. Using radio-tracking, we investigated the effects of exclusion on the soprano pipistrelle Pipistrellus pygmaeus, a species that commonly roosts in buildings in Europe. Exclusions were performed under licence at five roosts in England in spring, when females were in the early stages of pregnancy. Following exclusion, all bats found alternative roosts and colonies congregated in nearby known roosts that had been used by radio-tagged bats prior to exclusion. We found no difference in roosting behaviour before and after exclusion. Both the frequency of roost switching and the type of roosts used by bats remained unchanged. We also found no change in foraging behaviour. Bats foraged in the same areas, travelled similar distances to reach foraging areas and showed similar patterns of habitat selection before and after exclusion. Population modelling suggested that any reduction in survival following exclusion could have a negative impact on population growth, whereas a reduction in productivity would have less effect. While the number of soprano pipistrelle exclusions currently licensed each year is likely to have little effect on local populations, the cumulative impacts of licensing the destruction of large numbers of roosts may be of concern.
PMCID: PMC4526527  PMID: 26244667
9.  Bmp2 Deletion Causes an Amelogenesis Imperfecta Phenotype Via Regulating Enamel Gene Expression 
Journal of cellular physiology  2015;230(8):1871-1882.
Although Bmp2 is essential for tooth formation, the role of Bmp2 during enamel formation remains unknown in vivo. In this study, the role of Bmp2 in regulation of enamel formation was investigated by the Bmp2 conditional knock out (Bmp2 cKO) mice. Teeth of Bmp2 cKO mice displayed severe and profound phenotypes with asymmetric and misshaped incisors as well as abrasion of incisors and molars. Scanning electron microscopy analysis showed that the enamel layer was hypoplastic and enamel lacked a typical prismatic pattern. Teeth from null mice were much more brittle as tested by shear and compressive moduli. Expression of enamel matrix protein genes, amelogenin, enamelin, and enamel-processing proteases, Mmp-20 and Klk4 was reduced in the Bmp2 cKO teeth as reflected in a reduced enamel formation. Exogenous Bmp2 up-regulated those gene expressions in mouse enamel organ epithelial cells. This result for the first time indicates Bmp2 signaling is essential for proper enamel development and mineralization in vivo.
PMCID: PMC4469356  PMID: 25545831
10.  Hyperglycemia and xerostomia are key determinants of tooth decay in type 1 diabetic mice 
Insulin-dependent type 1 diabetes mellitus (DM) and oral diseases are closely interrelated. Poor metabolic control in diabetics is associated with a high risk of gingivitis, periodontitis and tooth loss. Salivary flow declines in diabetics and patients suffer from xerostomia. Reduced saliva predisposes to enamel hypomineralization and caries formation; however, the mechanisms that initiate and lead to progression of tooth decay and periodontitis in type 1 DM have not been explored. To address this issue, we analyzed tooth morphology in Akita −/− mice that harbor a point mutation in the Ins2 insulin gene, which leads to progressive hyperglycemia. Mandibles from Akita −/− and wild-type littermates were analyzed by microCT, scanning EM and histology; teeth were examined for amelogenin (Amel) and ameloblastin (Ambn) expression. Mice were injected with pilocarpine to assess saliva production. As hyperglycemia may alter pulp repair, the effect of high glucose levels on the proliferation/differentiation of cultured MD10-F2 pulp cells was also analyzed. Results showed that Akita −/− mice at 6 weeks of age showed chalky white incisors that correlated with marked hyperglycemia and impaired saliva production. MicroCT of Akita −/− teeth revealed excessive enamel wearing and hypomineralization; immunostaining for Amel and Ambn was decreased. A striking feature was invasion of dentinal tubules with Streptococcus mitis and microabcesses that originated in the coronal pulp and progressed to pulp necrosis and periapical periodontitis. High levels of glucose also inhibited MD10-F2 cell proliferation and differentiation. Our findings provide the first evidence that hyperglycemia in combination with reduced saliva in a model of type1 DM leads to decreased enamel mineralization/matrix proteins and predisposes to excessive wearing and decay. Importantly, hyperglycemia adversely affects enamel matrix proteins and pulp repair. Early detection and treatment of hyperglycemia and hyposalivation may provide a useful strategy for preventing the dental complications of diabetes and promoting oral health in this population.
PMCID: PMC4513945  PMID: 22449801
diabetes; enamel; hyperglycemia; pulpitis; saliva
11.  Parathyroid Hormone Induces Bone Cell Motility and Loss of Mature Osteocyte Phenotype through L-Calcium Channel Dependent and Independent Mechanisms 
PLoS ONE  2015;10(5):e0125731.
Parathyroid Hormone (PTH) can exert both anabolic and catabolic effects on the skeleton, potentially through expression of the PTH type1 receptor (PTH1R), which is highly expressed in osteocytes. To determine the cellular and molecular mechanisms responsible, we examined the effects of PTH on osteoblast to osteocyte differentiation using primary osteocytes and the IDG-SW3 murine cell line, which differentiate from osteoblast to osteocyte-like cells in vitro and express GFP under control of the dentin matrix 1 (Dmp1) promoter. PTH treatment resulted in an increase in some osteoblast and early osteocyte markers and a decrease in mature osteocyte marker expression. The gene expression profile of PTH-treated Day 28 IDG-SW3 cells was similar to PTH treated primary osteocytes. PTH treatment induced striking changes in the morphology of the Dmp1-GFP positive cells in IDG-SW3 cultures and primary cells from Dmp1-GFP transgenic mice. The cells changed from a more dendritic to an elongated morphology and showed increased cell motility. E11/gp38 has been shown to be important for cell migration, however, deletion of the E11/gp38/podoplanin gene had no effect on PTH-induced motility. The effects of PTH on motility were reproduced using cAMP, but not with protein kinase A (PKA), exchange proteins activated by cAMP (Epac), protein kinase C (PKC) or phosphatidylinositol-4,5-bisphosphonate 3-kinase (Pi3K) agonists nor were they blocked by their antagonists. However, the effects of PTH were mediated through calcium signaling, specifically through L-type channels normally expressed in osteoblasts but decreased in osteocytes. PTH was shown to increase expression of this channel, but decrease the T-type channel that is normally more highly expressed in osteocytes. Inhibition of L-type calcium channel activity attenuated the effects of PTH on cell morphology and motility but did not prevent the downregulation of mature osteocyte marker expression. Taken together, these results show that PTH induces loss of the mature osteocyte phenotype and promotes the motility of these cells. These two effects are mediated through different mechanisms. The loss of phenotype effect is independent and the cell motility effect is dependent on calcium signaling.
PMCID: PMC4420268  PMID: 25942444
12.  Draft Genome Sequences of 26 Porphyromonas Strains Isolated from the Canine Oral Microbiome 
Genome Announcements  2015;3(2):e00187-15.
We present the draft genome sequences for 26 strains of Porphyromonas (P. canoris, P. gulae, P. cangingavalis, P. macacae, and 7 unidentified) and an unidentified member of the Porphyromonadaceae family. All of these strains were isolated from the canine oral cavity, from dogs with and without early periodontal disease.
PMCID: PMC4392144  PMID: 25858832
13.  A review of the clinical and biologic aspects of HPV-positive HNC 
Human papillomavirus (HPV), a known etiology of a subset of head and neck squamous cell carcinomas (HNCs), causes numerous alterations in normal cellular functions. This article reviews the biology, detection and treatment of HPV-positive HNC. The role of HPV oncoproteins in tumor development, the natural history of HPV infection, and risk factors for and prevention of transmission of oral HPV are considered. Commonly used methods for detecting HPV infection including limitations of these methods are discussed to aid the practicing clinician in utilizing these tests in their clinical practice. Clinical characteristics of HPV-positive HNC including potential explanations for the improved outcomes seen in patients with HPV-positive HNC are assessed. Ongoing clinical trials specific for patients with HPV-positive HNC are described and areas in need of additional research are summarized. Until the results of ongoing trials are known, treatment of HPV-positive HNC should not differ in clinical practice from treatment of similar non-HPV related cancers.
PMCID: PMC3990872  PMID: 24606845
Head and neck cancer; human papillomavirus; radiation therapy
14.  Early Canine Plaque Biofilms: Characterization of Key Bacterial Interactions Involved in Initial Colonization of Enamel 
PLoS ONE  2014;9(12):e113744.
Periodontal disease (PD) is a significant problem in dogs affecting between 44% and 63.6% of the population. The main etiological agent for PD is plaque, a microbial biofilm that colonizes teeth and causes inflammation of the gingiva. Understanding how this biofilm initiates on the tooth surface is of central importance in developing interventions against PD. Although the stages of plaque development on human teeth have been well characterized little is known about how canine plaque develops. Recent studies of the canine oral microbiome have revealed distinct differences between the canine and human oral environments and the bacterial communities they support, particularly with respect to healthy plaque. These differences mean knowledge about the nature of plaque formation in humans may not be directly translatable to dogs. The aim of this study was to identify the bacterial species important in the early stages of canine plaque formation in vivo and then use isolates of these species in a laboratory biofilm model to develop an understanding of the sequential processes which take place during the initial colonization of enamel. Supra-gingival plaque samples were collected from 12 dogs at 24 and 48 hour time points following a full mouth descale and polish. Pyrosequencing of the 16S rDNA identified 134 operational taxonomic units after statistical analysis. The species with the highest relative abundance were Bergeyella zoohelcum, Neisseria shayeganii and a Moraxella species. Streptococcal species, which tend to dominate early human plaque biofilms, had very low relative abundance. In vitro testing of biofilm formation identified five primary colonizer species, three of which belonged to the genus Neisseria. Using these pioneer bacteria as a starting point, viable two and three species communities were developed. Combining in vivo and in vitro data has led us to construct novel models of how the early canine plaque biofilm develops.
PMCID: PMC4252054  PMID: 25463050
15.  A longitudinal assessment of periodontal disease in 52 miniature schnauzers 
BMC Veterinary Research  2014;10:166.
Periodontal disease (PD) is the most widespread oral disease in dogs and has been associated with serious systemic diseases. The disease is more prevalent in small breeds compared to large breeds and incidence increases with advancing age. In prevalence studies 84% of beagles over the age of 3 and 100% of poodles over the age of 4 were diagnosed with PD. Current knowledge of the rate of progression of PD is limited. The objective of this study was to determine the rate of PD progression in miniature schnauzers, an at risk small breed of dog.
Dogs (n = 52, age 1.3-6.9 years) who had received a regular oral care regime prior to this study were assessed for levels of gingivitis and periodontitis around the whole gingival margin in every tooth under general anaesthetic. Assessments were conducted approximately every six weeks for up to 60 weeks following the cessation of the oral care regime.
All of the 2155 teeth assessed entered the study with some level of gingivitis. 23 teeth entered the study with periodontitis, observed across 12 dogs aged between 1.3 and 6.9 years. 35 dogs had at least 12 teeth progress to periodontitis within 60 weeks. Of the teeth that progressed to periodontitis, 54% were incisors. The lingual aspect of the incisors was significantly more likely to be affected (p < 0.001). The severity of gingivitis in periodontitis-affected teeth was variable with 24% of the aspects affected having very mild gingivitis, 36% mild gingivitis and 40% moderate gingivitis. Periodontitis progression rate was significantly faster in older dogs. Only one dog (age 3.5) did not have any teeth progress to periodontitis after 60 weeks.
This is the first study to have assessed the progression rate of periodontitis in miniature schnauzers and highlights that with no oral care regime, the early stages of periodontitis develop rapidly in this breed. An oral care regime and twice yearly veterinary dental health checks should be provided from an early age for this breed and other breeds with similar periodontitis incidence rates.
PMCID: PMC4236762  PMID: 25179569
Dog; Gingivitis; Periodontitis; Periodontal disease; Miniature schnauzer
16.  The Unculturables: targeted isolation of bacterial species associated with canine periodontal health or disease from dental plaque 
BMC Microbiology  2014;14:196.
The current inability to culture the entirety of observed bacteria is well known and with the advent of ever more powerful molecular tools, that can survey bacterial communities at previously unattainable depth, the gap in our capacity to culture and define all of these species increases exponentially. This gap has essentially become the rate limiting step in determining how the knowledge of which species are present in a sample can be applied to understand the role of these species in an ecosystem or disease process. A case in point is periodontal disease, which is the most widespread oral disease in dogs. If untreated the disease results in significant pain, eventual loss of the dentition and potentially an increased risk of systemic diseases. Previous molecular based studies have identified the bacterial species associated with periodontal disease in dogs; however without cultured strains from many of these species it has not been possible to study whether they play a role in the disease process.
Using a quantitative polymerase chain reaction (qPCR) directed approach a range of microbiological media were screened and optimized to enrich for previously uncultivated target species. A systematic screening methodology was then employed to isolate the species of interest. In cases where the target species were not cultivable in isolation, helper strains grown underneath a nitrocellulose membrane were used to provide the necessary growth factors. This guided media optimization approach enabled the purification of 14 species, 8 of which we had previously been unable to cultivate in isolation. It is also applicable to the targeted isolation of isolates from species that have previously been cultured (for example to study intra-species variation) as demonstrated by the successful isolation of 6 targeted isolates of already cultured species.
To our knowledge this is the first time this combination of qPCR guided media optimization, strategic screening and helper strain support has been used successfully to isolate previously uncultured bacteria. This approach can be applied to any uncultured bacterial species where knowledge of their nutritional requirements or low relative abundance impedes their isolation.
PMCID: PMC4236526  PMID: 25085267
Periodontal disease; Periodontitis; Uncultivable; Quantitative PCR; QPCR; Bacterial isolation; Helper strain; Unculturable
17.  Habitat Selection by African Buffalo (Syncerus caffer) in Response to Landscape-Level Fluctuations in Water Availability on Two Temporal Scales 
PLoS ONE  2014;9(7):e101346.
Seasonal fluctuations in water availability cause predictable changes in the profitability of habitats in tropical ecosystems, and animals evolve adaptive behavioural and spatial responses to these fluctuations. However, stochastic changes in the distribution and abundance of surface water between years can alter resource availability at a landscape scale, causing shifts in animal behaviour. In the Okavango Delta, Botswana, a flood-pulsed ecosystem, the volume of water entering the system doubled between 2008 and 2009, creating a sudden change in the landscape. We used African buffalo (Syncerus caffer) to test the hypotheses that seasonal habitat selection would be related to water availability, that increased floodwater levels would decrease forage abundance and affect habitat selection, and that this would decrease buffalo resting time, reduce reproductive success and decrease body condition. Buffalo selected contrasting seasonal habitats, using habitats far from permanent water during the rainy season and seasonally-flooded habitats close to permanent water during the early and late flood seasons. The 2009 water increase reduced forage availability in seasonally-flooded habitats, removing a resource buffer used by the buffalo during the late flood season, when resources were most limited. In response, buffalo used drier habitats in 2009, although there was no significant change in the time spent moving or resting, or daily distance moved. While their reproductive success decreased in 2009, body condition increased. A protracted period of high water levels could prove detrimental to herbivores, especially to smaller-bodied species that require high quality forage. Stochastic annual fluctuations in water levels, predicted to increase as a result of anthropogenically-induced climate change, are likely to have substantial impacts on the functioning of water-driven tropical ecosystems, affecting environmental conditions within protected areas. Buffer zones around critical seasonal resources are essential to allow animals to engage in compensatory behavioural and spatial mechanisms in response to changing environmental conditions.
PMCID: PMC4077810  PMID: 24983377
18.  High Risks of Losing Genetic Diversity in an Endemic Mauritian Gecko: Implications for Conservation 
PLoS ONE  2014;9(6):e93387.
Genetic structure can be a consequence of recent population fragmentation and isolation, or a remnant of historical localised adaptation. This poses a challenge for conservationists since misinterpreting patterns of genetic structure may lead to inappropriate management. Of 17 species of reptile originally found in Mauritius, only five survive on the main island. One of these, Phelsuma guimbeaui (lowland forest day gecko), is now restricted to 30 small isolated subpopulations following severe forest fragmentation and isolation due to human colonisation. We used 20 microsatellites in ten subpopulations and two mitochondrial DNA (mtDNA) markers in 13 subpopulations to: (i) assess genetic diversity, population structure and genetic differentiation of subpopulations; (ii) estimate effective population sizes and migration rates of subpopulations; and (iii) examine the phylogenetic relationships of haplotypes found in different subpopulations. Microsatellite data revealed significant population structure with high levels of genetic diversity and isolation by distance, substantial genetic differentiation and no migration between most subpopulations. MtDNA, however, showed no evidence of population structure, indicating that there was once a genetically panmictic population. Effective population sizes of ten subpopulations, based on microsatellite markers, were small, ranging from 44 to 167. Simulations suggested that the chance of survival and allelic diversity of some subpopulations will decrease dramatically over the next 50 years if no migration occurs. Our DNA-based evidence reveals an urgent need for a management plan for the conservation of P. guimbeaui. We identified 18 threatened and 12 viable subpopulations and discuss a range of management options that include translocation of threatened subpopulations to retain maximum allelic diversity, and habitat restoration and assisted migration to decrease genetic erosion and inbreeding for the viable subpopulations.
PMCID: PMC4070904  PMID: 24963708
19.  In vitro and in vivo approaches to study osteocyte biology 
Bone  2012;54(2):296-306.
Osteocytes, the most abundant cell population of the bone lineage, have been a major focus in the bone research field in recent years. This population of cells that resides within mineralized matrix is now thought to be the mechanosensory cell in bone and plays major roles in regulation of bone formation and resorption. Studies of osteocytes had been impaired by their location, resulting in numerous attempts to isolate primary osteocytes and to generate cell lines representative of the osteocytic phenotype. Progress has been achieved in recent years by utilizing in vivo genetic technology and generation of osteocyte directed transgenic and gene deficiency mouse models.
We will provide an overview of the current in vitro and in vivo models utilized to study osteocyte biology. We discuss generation of osteocyte-like cell lines and isolation of primary osteocytes and summarize studies that have utilized these cellular models to understand the functional role of osteocytes. Approaches that attempt to selectively identify and isolate osteocytes using fluorescent protein reporters driven by regulatory elements of genes that are highly expressed in osteocytes will be discussed.
In addition, recent in vivo studies utilizing overexpression or conditional deletion of various genes using dentin matrix protein (Dmp1) directed Cre recombinase are outlined. In conclusion, evaluation of the benefits and deficiencies of currently used cell lines/genetic models in understanding osteocyte biology underlines the current progress in this field. The future efforts will be directed towards developing novel in vitro and in vivo models that would additionally facilitate understanding the multiple roles of osteocytes.
PMCID: PMC3566324  PMID: 23072918
osteocyte; cell lines; transgenic mice; Dmp1; Sost; Cre-recombinase; GFP
20.  Ecological Effects of the Invasive Giant Madagascar Day Gecko on Endemic Mauritian Geckos: Applications of Binomial-Mixture and Species Distribution Models 
PLoS ONE  2014;9(4):e88798.
The invasion of the giant Madagascar day gecko Phelsuma grandis has increased the threats to the four endemic Mauritian day geckos (Phelsuma spp.) that have survived on mainland Mauritius. We had two main aims: (i) to predict the spatial distribution and overlap of P. grandis and the endemic geckos at a landscape level; and (ii) to investigate the effects of P. grandis on the abundance and risks of extinction of the endemic geckos at a local scale. An ensemble forecasting approach was used to predict the spatial distribution and overlap of P. grandis and the endemic geckos. We used hierarchical binomial mixture models and repeated visual estimate surveys to calculate the abundance of the endemic geckos in sites with and without P. grandis. The predicted range of each species varied from 85 km2 to 376 km2. Sixty percent of the predicted range of P. grandis overlapped with the combined predicted ranges of the four endemic geckos; 15% of the combined predicted ranges of the four endemic geckos overlapped with P. grandis. Levin's niche breadth varied from 0.140 to 0.652 between P. grandis and the four endemic geckos. The abundance of endemic geckos was 89% lower in sites with P. grandis compared to sites without P. grandis, and the endemic geckos had been extirpated at four of ten sites we surveyed with P. grandis. Species Distribution Modelling, together with the breadth metrics, predicted that P. grandis can partly share the equivalent niche with endemic species and survive in a range of environmental conditions. We provide strong evidence that smaller endemic geckos are unlikely to survive in sympatry with P. grandis. This is a cause of concern in both Mauritius and other countries with endemic species of Phelsuma.
PMCID: PMC4005729  PMID: 24785293
21.  Abuse Potential of Intravenous Oxycodone/Naloxone Solution in Nondependent Recreational Drug Users 
Clinical Drug Investigation  2014;34(6):421-429.
Background and Objective
Abuse of opioid analgesics has become a public health issue. Some opioid abusers use intravenous administration to increase the magnitude of positive reinforcing effects. Intravenous co-administration of oxycodone with naloxone, an opioid antagonist, may reduce these rewarding effects and discourage abuse. A 2:1 oxycodone:naloxone (OXN) tablet formulation has been studied in the USA for the management of moderate-to-severe chronic pain. Intravenous administration of a 2:1 oxycodone:naloxone solution (sOXN) reflects the oxycodone:naloxone ratio found in laboratory studies of OXN following tampering for intravenous administration. The objective of this study was to characterize abuse-deterrent properties of sOXN.
This single-center, double-blind, randomized, placebo-controlled, active-controlled, crossover study enrolled nondependent recreational opioid users with experience using multiple (two or more) routes of administration. Following demonstration that subjects could discern between placebo and oxycodone, 24 eligible male and female subjects were randomized to receive intravenous injections of 0.07 mg/kg oxycodone (OXY), 0.07 mg/kg oxycodone and 0.035 mg/kg naloxone solution (sOXN), or matching placebo over three visits. Pharmacokinetics, pharmacodynamics, safety, and tolerability were assessed at scheduled times up to 8 h post-dose. Parameters were computed and statistically compared among treatments.
Pharmacokinetics were similar between OXY and sOXN. Subjects reported significantly fewer rewarding effects with sOXN compared with OXY; differences between sOXN and placebo were generally not significant. sOXN was well tolerated.
Significant reductions in drug liking and other subjective effects following administration of sOXN compared with OXY indicate that naloxone concentrations were sufficient to antagonize the effects of oxycodone when abused by the intravenous route of administration in opioid-experienced drug users.
PMCID: PMC4026623  PMID: 24756361
22.  Breeding systems, hybridization and continuing evolution in Avon Gorge Sorbus 
Annals of Botany  2013;111(4):563-575.
Background and Aims
Interspecific hybridization and polyploidy are key processes in plant evolution and are responsible for ongoing genetic diversification in the genus Sorbus (Rosaceae). The Avon Gorge, Bristol, UK, is a world ‘hotspot’ for Sorbus diversity and home to diploid sexual species and polyploid apomictic species. This research investigated how mating system variation, hybridization and polyploidy interact to generate this biological diversity.
Mating systems of diploid, triploid and tetraploid Sorbus taxa were analysed using pollen tube growth and seed set assays from controlled pollinations, and parent–offspring genotyping of progeny from open and manual pollinations.
Key Results
Diploid Sorbus are outcrossing and self-incompatible (SI). Triploid taxa are pseudogamous apomicts and genetically invariable, but because they also display self-incompatibility, apomictic seed set requires pollen from other Sorbus taxa – a phenomenon which offers direct opportunities for hybridization. In contrast tetraploid taxa are pseudogamous but self-compatible, so do not have the same obligate requirement for intertaxon pollination.
The mating inter-relationships among Avon Gorge Sorbus taxa are complex and are the driving force for hybridization and ongoing genetic diversification. In particular, the presence of self-incompatibility in triploid pseudogamous apomicts imposes a requirement for interspecific cross-pollination, thereby facilitating continuing diversification and evolution through rare sexual hybridization events. This is the first report of naturally occurring pseudogamous apomictic SI plant populations, and we suggest that interspecific pollination, in combination with a relaxed endosperm balance requirement, is the most likely route to the persistence of these populations. We propose that Avon Gorge Sorbus represents a model system for studying the establishment and persistence of SI apomicts in natural populations.
PMCID: PMC3605949  PMID: 23408832
Hybridization; evolution; polyploidy; apomixis; pseudogamy; self-incompatibility; Sorbus
23.  Genome Wide Analysis Indicates Genes for Basement Membrane and Cartilage Matrix Proteins as Candidates for Hip Dysplasia in Labrador Retrievers 
PLoS ONE  2014;9(1):e87735.
Hip dysplasia, an abnormal laxity of the hip joint, is seen in humans as well as dogs and is one of the most common skeletal disorders in dogs. Canine hip dysplasia is considered multifactorial and polygenic, and a variety of chromosomal regions have been associated with the disorder. We performed a genome-wide association study in Dutch Labrador Retrievers, comparing data of nearly 18,000 single nucleotide polymorphisms (SNPs) in 48 cases and 30 controls using two different statistical methods. An individual SNP analysis based on comparison of allele frequencies with a χ2 statistic was used, as well as a simultaneous SNP analysis based on Bayesian variable selection. Significant association with canine hip dysplasia was observed on chromosome 8, as well as suggestive association on chromosomes 1, 5, 15, 20, 25 and 32. Next-generation DNA sequencing of the exons of genes of seven regions identified multiple associated alleles on chromosome 1, 5, 8, 20, 25 and 32 (p<0.001). Candidate genes located in the associated regions on chromosomes 1, 8 and 25 included LAMA2, LRR1 and COL6A3, respectively. The associated region on CFA20 contained candidate genes GDF15, COMP and CILP2. In conclusion, our study identified candidate genes that might affect susceptibility to canine hip dysplasia. These genes are involved in hypertrophic differentiation of chondrocytes and extracellular matrix integrity of basement membrane and cartilage. The functions of the genes are in agreement with the notion that disruptions in endochondral bone formation in combination with soft tissue defects are involved in the etiology of hip dysplasia.
PMCID: PMC3907504  PMID: 24498183
24.  A Cross-Sectional Survey of Bacterial Species in Plaque from Client Owned Dogs with Healthy Gingiva, Gingivitis or Mild Periodontitis 
PLoS ONE  2013;8(12):e83158.
Periodontal disease is the most widespread oral disease in dogs which if left untreated results in significant pain to the pet and loss of dentition. The objective of this study was to identify bacterial species in canine plaque that are significantly associated with health, gingivitis and mild periodontitis (<25% attachment loss). In this survey subgingival plaque samples were collected from 223 dogs with healthy gingiva, gingivitis and mild periodontitis with 72 to 77 samples per health status. DNA was extracted from the plaque samples and subjected to PCR amplification of the V1-V3 region of the 16S rDNA. Pyrosequencing of the PCR amplicons identified a total of 274 operational taxonomic units after bioinformatic and statistical analysis. Porphyromonas was the most abundant genus in all disease stages, particularly in health along with Moraxella and Bergeyella. Peptostreptococcus, Actinomyces, and Peptostreptococcaceae were the most abundant genera in mild periodontitis. Logistic regression analysis identified species from each of these genera that were significantly associated with health, gingivitis or mild periodontitis. Principal component analysis showed distinct community profiles in health and disease. The species identified show some similarities with health and periodontal disease in humans but also major differences. In contrast to human, healthy canine plaque was found to be dominated by Gram negative bacterial species whereas Gram positive anaerobic species predominate in disease. The scale of this study surpasses previously published research and enhances our understanding of the bacterial species present in canine subgingival plaque and their associations with health and early periodontal disease.
PMCID: PMC3862762  PMID: 24349448
25.  Do Formal Inspections Ensure that British Zoos Meet and Improve on Minimum Animal Welfare Standards? 
Simple Summary
Key aims of the formal inspections of British zoos are to assess compliance with minimum standards of animal welfare and promote improvements in animal care and husbandry. We compared reports from two consecutive inspections of 136 British zoos to see whether these goals were being achieved. Most zoos did not meet all the minimum animal welfare standards and there was no clear evidence of improving levels of compliance with standards associated with the Zoo Licensing Act 1981. The current system of licensing and inspection does not ensure that British zoos meet and maintain, let alone exceed, the minimum animal welfare standards.
We analysed two consecutive inspection reports for each of 136 British zoos made by government-appointed inspectors between 2005 and 2011 to assess how well British zoos were complying with minimum animal welfare standards; median interval between inspections was 1,107 days. There was no conclusive evidence for overall improvements in the levels of compliance by British zoos. Having the same zoo inspector at both inspections affected the outcome of an inspection; animal welfare criteria were more likely to be assessed as unchanged if the same inspector was present on both inspections. This, and erratic decisions as to whether a criterion applied to a particular zoo, suggest inconsistency in assessments between inspectors. Zoos that were members of a professional association (BIAZA) did not differ significantly from non-members in the overall number of criteria assessed as substandard at the second inspection but were more likely to meet the standards on both inspections and less likely to have criteria remaining substandard. Lack of consistency between inspectors, and the high proportion of zoos failing to meet minimum animal welfare standards nearly thirty years after the Zoo Licensing Act came into force, suggest that the current system of licensing and inspection is not meeting key objectives and requires revision.
PMCID: PMC4494370  PMID: 26479752
animal welfare; captive wild animals; enforcement; government inspections; improving standards; legislative oversight; local authority; Zoo Licensing Act

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