Spatiotemporal analysis of gene flow in Chesapeake Bay Diamondback Terrapins (Malaclemys terrapin).

There is widespread concern regarding the impacts of anthropogenic activities on connectivity among populations of plants and animals, and understanding how contemporary and historical processes shape metapopulation dynamics is crucial for setting appropriate conservation targets. We used genetic data to identify population clusters and quantify gene flow over historical and contemporary time frames in the Diamondback Terrapin (Malaclemys terrapin). This species has a long and complicated history with humans, including commercial overharvesting and subsequent translocation events during the early twentieth century. Today, terrapins face threats from habitat loss and mortality in fisheries bycatch. To evaluate population structure and gene flow among Diamondback Terrapin populations in the Chesapeake Bay region, we sampled 617 individuals from 15 localities and screened individuals at 12 polymorphic microsatellite loci. Our goals were to demarcate metapopulation structure, quantify genetic diversity, estimate effective population sizes, and document temporal changes in gene flow. We found that terrapins in the Chesapeake Bay region harbour high levels of genetic diversity and form four populations. Effective population sizes were variable. Among most population comparisons, estimates of historical and contemporary terrapin gene flow were generally low (m ≈ 0.01). However, we detected a substantial increase in contemporary gene flow into Chesapeake Bay from populations outside the bay, as well as between two populations within Chesapeake Bay, possibly as a consequence of translocations during the early twentieth century. Our study shows that inferences across multiple time scales are needed to evaluate population connectivity, especially as recent changes may identify threats to population persistence.

Genome edited sheep and cattle.

Genome editing tools enable efficient and accurate genome manipulation. An enhanced ability to modify the genomes of livestock species could be utilized to improve disease resistance, productivity or breeding capability as well as the generation of new biomedical models. To date, with respect to the direct injection of genome editor mRNA into livestock zygotes, this technology has been limited to the generation of pigs with edited genomes. To capture the far-reaching applications of gene-editing, from disease modelling to agricultural improvement, the technology must be easily applied to a number of species using a variety of approaches. In this study, we demonstrate zygote injection of TALEN mRNA can also produce gene-edited cattle and sheep. In both species we have targeted the myostatin (MSTN) gene. In addition, we report a critical innovation for application of gene-editing to the cattle industry whereby gene-edited calves can be produced with specified genetics by ovum pickup, in vitro fertilization and zygote microinjection (OPU-IVF-ZM). This provides a practical alternative to somatic cell nuclear transfer for gene knockout or introgression of desirable alleles into a target breed/genetic line.

A mobility enabled inpatient monitoring system using a ZigBee medical sensor network.

This paper presents a ZigBee In-Patient Monitoring system embedded with a new ZigBee mobility management solution. The system enables ZigBee device mobility in a fixed ZigBee network. The usage, the architecture and the mobility framework are discussed in details in the paper. The evaluation shows that the new algorithm offers a good efficiency, resulting in a low management cost. In addition, the system can save lives by providing a panic button and can be used as a location tracking service. A case study focused on the Princes of Wales Hospital in Hong Kong is presented and findings are given. This investigation reveals that the developed mobile solutions offer promising value-added services for many potential ZigBee applications.

Genetic and morphological characterization of the freshwater mussel clubshell species complex (Pleurobema clava and Pleurobema oviforme) to inform conservation planning.

The shell morphologies of the freshwater mussel species Pleurobema clava (federally endangered) and Pleurobema oviforme (species of concern) are similar, causing considerable taxonomic confusion between the two species over the last 100 years. While P. clava was historically widespread throughout the Ohio River basin and tributaries to the lower Laurentian Great Lakes, P. oviforme was confined to the Tennessee and the upper Cumberland River basins. We used two mitochondrial DNA (mtDNA) genes, 13 novel nuclear DNA microsatellite markers, and shell morphometrics to help resolve this taxonomic confusion. Evidence for a single species was apparent in phylogenetic analyses of each mtDNA gene, revealing monophyletic relationships with minimal differentiation and shared haplotypes. Analyses of microsatellites showed significant genetic structuring, with four main genetic clusters detected, respectively, in the upper Ohio River basin, the lower Ohio River and Great Lakes, and upper Tennessee River basin, and a fourth genetic cluster, which included geographically intermediate populations in the Ohio and Tennessee river basins. While principal components analysis (PCA) of morphometric variables (i.e., length, height, width, and weight) showed significant differences in shell shape, only 3% of the variance in shell shape was explained by nominal species. Using Linear Discriminant and Random Forest (RF) analyses, correct classification rates for the two species' shell forms were 65.5% and 83.2%, respectively. Random Forest classification rates for some populations were higher; for example, for North Fork Holston (HOLS), it was >90%. While nuclear DNA and shell morphology indicate that the HOLS population is strongly differentiated, perhaps indicative of cryptic biodiversity, we consider the presence of a single widespread species the most likely biological scenario for many of the investigated populations based on our mtDNA dataset. However, additional sampling of P. oviforme populations at nuclear loci is needed to corroborate this finding.

Population dynamics of American horseshoe crabs--historic climatic events and recent anthropogenic pressures.

Populations of the American horseshoe crab, Limulus polyphemus, have declined, but neither the causes nor the magnitude are fully understood. In order to evaluate historic demography, variation at 12 microsatellite DNA loci surveyed in 1218 L. polyphemus sampled from 28 localities was analysed with Bayesian coalescent-based methods. The analysis showed strong declines in population sizes throughout the species' distribution except in the geographically isolated southern-most population in Mexico, where a strong increase in population size was inferred. Analyses suggested that demographic changes in the core of the distribution occurred in association with the recolonization after the Ice Age and also by anthropogenic effects, such as the past overharvest of the species for fertilizer or the current use of the animals as bait for American eel (Anguilla rostrata) and whelk (Busycon spp.) fisheries. This study highlights the importance of considering both climatic changes and anthropogenic effects in efforts to understand population dynamics--a topic which is highly relevant in the ongoing assessments of the effects of climate change and overharvest.

Development of an invasively monitored porcine model of acetaminophen-induced acute liver failure.

BACKGROUND: The development of effective therapies for acute liver failure (ALF) is limited by our knowledge of the pathophysiology of this condition, and the lack of suitable large animal models of acetaminophen toxicity. Our aim was to develop a reproducible invasively-monitored porcine model of acetaminophen-induced ALF. METHOD: 35kg pigs were maintained under general anaesthesia and invasively monitored. Control pigs received a saline infusion, whereas ALF pigs received acetaminophen intravenously for 12 hours to maintain blood concentrations between 200-300 mg/l. Animals surviving 28 hours were euthanased. RESULTS: Cytochrome p450 levels in phenobarbital pre-treated animals were significantly higher than non pre-treated animals (300 vs 100 pmol/mg protein). Control pigs (n = 4) survived 28-hour anaesthesia without incident. Of nine pigs that received acetaminophen, four survived 20 hours and two survived 28 hours. Injured animals developed hypotension (mean arterial pressure; 40.8 +/- 5.9 vs 59 +/- 2.0 mmHg), increased cardiac output (7.26 +/- 1.86 vs 3.30 +/- 0.40 l/min) and decreased systemic vascular resistance (8.48 +/- 2.75 vs 16.2 +/- 1.76 mPa/s/m3). Dyspnoea developed as liver injury progressed and the increased pulmonary vascular resistance (636 +/- 95 vs 301 +/- 26.9 mPa/s/m3) observed may reflect the development of respiratory distress syndrome.Liver damage was confirmed by deterioration in pH (7.23 +/- 0.05 vs 7.45 +/- 0.02) and prothrombin time (36 +/- 2 vs 8.9 +/- 0.3 seconds) compared with controls. Factor V and VII levels were reduced to 9.3 and 15.5% of starting values in injured animals. A marked increase in serum AST (471.5 +/- 210 vs 42 +/- 8.14) coincided with a marked reduction in serum albumin (11.5 +/- 1.71 vs 25 +/- 1 g/dL) in injured animals. Animals displayed evidence of renal impairment; mean creatinine levels 280.2 +/- 36.5 vs 131.6 +/- 9.33 mumol/l. Liver histology revealed evidence of severe centrilobular necrosis with coagulative necrosis. Marked renal tubular necrosis was also seen. Methaemoglobin levels did not rise >5%. Intracranial hypertension was not seen (ICP monitoring), but there was biochemical evidence of encephalopathy by the reduction of Fischer's ratio from 5.6 +/- 1.1 to 0.45 +/- 0.06. CONCLUSION: We have developed a reproducible large animal model of acetaminophen-induced liver failure, which allows in-depth investigation of the pathophysiological basis of this condition. Furthermore, this represents an important large animal model for testing artificial liver support systems.

Transgenic sheep designed for transplantation studies.

Lentiviral vectors have recently emerged as an efficient method of transgene delivery to the germline of animals. We now demonstrate that combining this efficiency with embryo splitting procedures enables the production of monozygotic twins, one of which is transgenic. We propose that this approach can be used to generate animals in which cell or tissue transplantation can be achieved without the use of immunosuppressive regimes.

Anti-colchicine Fab fragments prevent lethal colchicine toxicity in a porcine model: a pharmacokinetic and clinical study.

BACKGROUND: Colchicine poisoning is commonly lethal. Colchicine-specific Fab fragments increase rat urinary colchicine clearance and have been associated with a good outcome in one patient. We aimed to develop a porcine model of colchicine toxicity to study the pharmacokinetics and efficacy of ovine Fab. METHODS: A Göttingen minipig critical care model was established and serial blood samples taken for colchicine and Fab pharmacokinetics, clinical chemistry, and haematology. Animals were euthanised when the mean arterial pressure fell below 45 mmHg without response to vasopressor, or at study completion. RESULTS: Initial studies indicated that oral dosing produced variable pharmacokinetics and time-to-euthanasia. By contrast, intravenous infusion of 0.25 mg/kg colchicine over 1 h produced reproducible pharmacokinetics (AUC0-20 343 [SD = 21] µg/L/h), acute multi-organ injury, and cardiotoxicity requiring euthanasia a mean of 22.5 (SD = 3.2) h after dosing. A full-neutralising equimolar Fab dose given 6 h after the infusion (50% first hour, 50% next 6 h [to reduce renal-loss of unbound Fab]) produced a 7.35-fold increase in plasma colchicine (AUC0-20 2,522 [SD = 14] µg/L/h), and removed all free plasma colchicine, but did not prevent toxicity (euthanasia at 29.1 [SD = 3.4] h). Earlier administration over 1 h of the full-neutralising dose, 1 or 3 h after the colchicine, produced a 12.9-fold (AUC0-20 4,433 [SD = 607] µg/L/h) and 6.0-fold (AUC0-20 2,047 [SD = 51] µg/L/h) increase in plasma colchicine, respectively, absence of free plasma colchicine until 20 h, and survival to study end without marked cardiotoxicity. CONCLUSIONS: Colchicine-specific Fab given early, in equimolar dose, bound colchicine, eliciting its movement into the blood, and preventing severe toxicity. Clinical studies are now needed to determine how soon this antidote must be given to work in human poisoning.

The female and male mitochondrial genomes of Unio delphinus and the phylogeny of freshwater mussels (Bivalvia: Unionida).

We have sequenced the female and male mtDNA of Unio delphinus and inferred the Unionidae phylogeny using 41 complete mtDNA sequences. Additionally, we compared the concatenated mtDNA trees with those using single or combination of two mtDNA genes to identify the best genes to use in the absence of complete mitogenomes. The gender-specific mtDNAs of U. delphinus contain all Unionida mtDNA specific features. The mtDNA phylogeny supports the reciprocal monophyly of the gender-specific clades but it was inconclusive regarding Unionidae subfamilies relationships. The gene trees topologies using ND5 or 16S-rRNA with ND1 were the closest trees to the mtDNA trees.

Epitope-specific immunotherapy targeting CD4-positive T cells in coeliac disease: two randomised, double-blind, placebo-controlled phase 1 studies.

BACKGROUND: A gluten-free diet is the only means to manage coeliac disease, a permanent immune intolerance to gluten. We developed a therapeutic vaccine, Nexvax2, designed to treat coeliac disease. Nexvax2 is an adjuvant-free mix of three peptides that include immunodominant epitopes for gluten-specific CD4-positive T cells. The vaccine is intended to engage and render gluten-specific CD4-positive T cells unresponsive to further antigenic stimulation. We assessed the safety and pharmacodynamics of the vaccine in patients with coeliac disease on a gluten-free diet. METHODS: We did two randomised, double-blind, placebo-controlled, phase 1 studies at 12 community sites in Australia, New Zealand, and the USA, in HLA-DQ2·5-positive patients aged 18-70 years who had coeliac disease and were on a gluten-free diet. In the screening period for ascending dose cohorts, participants were randomly assigned (1:1) by central randomisation with a simple block method to a double-blind crossover, placebo-controlled oral gluten challenge. Participants with a negative interferon γ release assay to Nexvax2 peptides after the screening oral gluten challenge were discontinued before dosing. For the biopsy cohorts, the screening period included an endoscopy, and participants with duodenal histology who had a Marsh score of greater than 1 were discontinued before dosing. Participants were subsequently randomly assigned to either Nexvax2 or placebo in ascending dose cohorts (2:1) and in biopsy cohorts (1:1) by central randomisation with a simple block method. In the three-dose study, participants received either Nexvax2 60 µg, 90 µg, or 150 µg weekly, or placebo over 15 days; in a fourth biopsy cohort, patients received either Nexvax2 at the maximum tolerated dose (MTD) or placebo. In the 16-dose study, participants received Nexvax2 150 µg or 300 µg or placebo twice weekly over 53 days; in a third biopsy cohort, patients also received either Nexvax2 at the MTD or placebo. In the 4-week post-treatment period, ascending dose cohorts underwent a further double-blind crossover, placebo-controlled oral gluten challenge, which had a fixed sequence, and biopsy cohorts had a gastroscopy with duodenal biopsies and quantitative histology within 2 weeks without oral gluten challenge. Participants, investigators, and study staff were masked to the treatment assignment, except for the study pharmacist. The primary endpoint was the number and percentage of adverse events in the treatment period in an intention-to-treat analysis. Both trials were completed and closed before data analysis. Trials were registered with the Australian New Zealand Clinical Trials Registry, numbers ACTRN12612000355875 and ACTRN12613001331729. FINDINGS: Participants were enrolled from Nov 28, 2012, to Aug 14, 2014, in the three-dose study, and from Aug 3, 2012, to Sept 10, 2013, in the 16-dose study. Overall, 62 (57%) of 108 participants were randomly assigned after oral gluten challenge and 20 (71%) of 28 participants were randomly assigned after endoscopy. In the three-dose study, nine participants were randomly allocated to Nexvax2 60 µg and three to placebo (first cohort), nine were allocated to Nexvax2 90 µg and four to placebo (second cohort), eight were allocated to Nexvax2 150 µg and four to placebo (third cohort), and three were allocated to Nexvax2 150 µg and three to placebo (biopsy cohort). In the 16-dose study, eight participants were randomly allocated to Nexvax2 150 µg and four to placebo (first cohort), ten were allocated to Nexvax2 300 µg and three to placebo (second cohort), and seven were allocated to Nexvax2 150 µg and seven to placebo (biopsy cohort). The MTD for Nexvax2 was 150 µg because of transient, acute gastrointestinal adverse events with onset 2-5 h after initial doses of the vaccine, similar to those caused by gluten ingestion. In the ascending dose cohorts in the three-dose study, six (55%) of 11 placebo recipients, five (56%) of nine who received Nexvax2 60 µg, seven (78%) of nine who received Nexvax2 90 µg, and five (63%) of eight who received Nexvax2 150 µg had at least one treatment-emergent adverse event, as did all three (100%) placebo recipients and one (33%) of three Nexvax2 150 µg recipients in the biopsy cohort. In the ascending dose cohorts of the 16-dose study, five (71%) of seven placebo-treated participants, six (75%) of eight who received Nexvax2 150 µg, and all ten (100%) who received Nexvax2 300 µg had at least one treatment-emergent adverse event, as did six (86%) of seven placebo recipients and five (71%) of seven Nexvax2 150 µg recipients in the biopsy cohort. Vomiting, nausea, and headache were the only treatment-emergent adverse events that occurred in at least 5% of participants in either study. Among participants given the MTD, eight gastrointestinal treatment-emergent adverse events occurred in four (50%) of eight participants in the third cohort and none (0%) of three participants in the biopsy cohort in the three-dose study, and five events occurred in five (63%) of eight participants in the first cohort and three events in two (29%) of seven participants in the biopsy cohort of the 16-dose study. Median villous height to crypt depth ratio in distal duodenal biopsies was not significantly different between those who received the vaccine at the MTD on either schedule and those who received placebo. Of the participants who completed the post-treatment oral gluten challenge per protocol, interferon γ release assay to Nexvax2 peptides was negative (responders to treatment) in two (22%) of nine placebo-treated participants in the three-dose study versus two (33%) of six who received Nexvax2 60 µg, five (63%) of eight who received Nexvax2 90 µg, and six (100%) of six who received Nexvax2 150 µg (p=0·007); in the 16-dose study, none (0%) of five placebo-treated participants had a negative assay versus six (75%) of eight who received Nexvax2 150 µg (p=0·021). INTERPRETATION: The MTD of Nexvax2 was 150 µg for twice weekly intradermal administration over 8 weeks, which modified immune responsiveness to Nexvax2 peptides without deterioration in duodenal histology. The gastrointestinal symptoms that followed the first intradermal administration of the vaccine resembled those associated with oral gluten challenge. These findings support continued clinical development of this potential therapeutic vaccine for coeliac disease. FUNDING: ImmusanT.

Maintenance of pregnancy in pigs with limited viable embryos.

Interest is increasing in assisted reproductive technologies in the pig involving embryo cryopreservation, cloning, and genetic modification. Although inherently inefficient and variable in their outcome, the successful application of these techniques in this species is confounded by unique mechanisms for pregnancy recognition and maintenance that require a minimum number of viable embryos during a critical window of time early in gestation. These mechanisms require both local and systemic interactions between conceptuses and the maternal reproductive axis. Here, we describe a method wherein cotransfer of parthenogenetic embryos with the capacity for limited development can be used to sustain the pregnancy of fewer than four viable conceptuses to term.

Hiding in Plain Sight: A Case for Cryptic Metapopulations in Brook Trout (Salvelinus fontinalis).

A fundamental issue in the management and conservation of biodiversity is how to define a population. Spatially contiguous fish occupying a stream network have often been considered to represent a single, homogenous population. However, they may also represent multiple discrete populations, a single population with genetic isolation-by-distance, or a metapopulation. We used microsatellite DNA and a large-scale mark-recapture study to assess population structure in a spatially contiguous sample of Brook Trout (Salvelinus fontinalis), a species of conservation concern. We found evidence for limited genetic exchange across small spatial scales and in the absence of barriers to physical movement. Mark-recapture and stationary passive integrated transponder antenna records demonstrated that fish from two tributaries very seldom moved into the opposite tributary, but movements between the tributaries and mainstem were more common. Using Bayesian genetic clustering, we identified two genetic groups that exhibited significantly different growth rates over three years of study, yet survival rates were very similar. Our study highlights the importance of considering the possibility of multiple genetically distinct populations occurring within spatially contiguous habitats, and suggests the existence of a cryptic metapopulation: a spatially continuous distribution of organisms exhibiting metapopulation-like behaviors.

Mammalian interspecies substitution of immune modulatory alleles by genome editing.

We describe a fundamentally novel feat of animal genetic engineering: the precise and efficient substitution of an agronomic haplotype into a domesticated species. Zinc finger nuclease in-embryo editing of the RELA locus generated live born domestic pigs with the warthog RELA orthologue, associated with resilience to African Swine Fever. The ability to efficiently achieve interspecies allele introgression in one generation opens unprecedented opportunities for agriculture and basic research.

Live lambs born from zona-pellucida denuded embryos.

Progress with techniques using zona-pellucida denuded embryos has resulted in the birth of live cattle, pigs, and mice. The application of zona-free methods in sheep has been restricted to in vitro studies. In this report, we demonstrate that live lambs can be produced from zona-free IVF embryos. We are pursuing this method as a prerequisite to developing viral vector co-culture delivery strategies.

Efficient generation of transgenic pigs using equine infectious anaemia virus (EIAV) derived vector.

Traditional methods of transgene delivery in livestock are inefficient. Recently, human immunodeficiency virus (HIV-1) based lentiviral vectors have been shown to offer an efficient transgene delivery system. We now extend this method by demonstrating efficient generation of transgenic pigs using an equine infectious anaemia virus derived vector. We used this vector to deliver a green fluorescent protein expressing transgene; 31% of injected/transferred eggs resulted in a transgenic founder animal and 95% of founder animals displayed green fluorescence. This compares favourably with results using HIV-1 based vectors, and is substantially more efficient than the standard pronuclear microinjection method, indicating that lentiviral transgene delivery may be a general tool with which to efficiently generate transgenic mammals.

Immunohistochemical characterization of cloned lamb nephropathy.

Kidneys from lambs derived by nuclear transfer are frequently abnormal and are characterized by an enlarged pelvis and narrow medulla, consistent with lower urinary tract obstruction and development of variable hydronephrosis. The precise pathogenesis of this entity is unknown. Immunohistochemical staining for intermediate filaments was used to further characterize the lesions seen in this condition and was compared with age-matched control tissue. Major findings were upregulation of cytokeratin on damaged tubules, desmin and vimentin in undifferentiated mesenchyme, and smooth muscle actin in mesenchyme and on smooth muscle "collars" around dilated tubules. In addition, some cases showed reexpression of vimentin and desmin on proximal tubular epithelial cells. Taken together, these findings provide a valuable database for tracking the expression of intermediate filaments throughout renal development in sheep and have further characterized the nature of the response to injury by the developing kidney, a response that is characterized by proliferation of mesenchyme and both reexpression and upregulation of intermediate filaments within renal cells. In addition, the study has confirmed that the changes in cloned lamb nephropathy are established by day 85 of development.

Somatic cell nuclear transfer: recent progress and challenges.

Somatic cell nuclear transfer (NT) offers new and exciting opportunities in many areas of research and biotechnology. However, the field as a whole is still in its infancy, with continuing inefficiencies in the process proving many early expectations premature. The technical steps of NT are complex, and success is highly susceptible to minor variations. Furthermore, the biological process of reprogramming is not fully understood, making it difficult to optimize the protocols for providing ideal recipient oocytes and donor cells. In this paper, we describe recent advances and novel approaches, which resulted in progress during the last year, including the birth of cloned piglets and farm animals with precise genetic changes. Key problems hindering further progress are addressed.

In vitro and in vivo developmental competence of ovulated and in vitro matured porcine oocytes activated by electrical activation.

The objective of this study was to evaluate the in vitro and in vivo developmental competence of parthenogenetic (parthenote) pig embryos derived from ovulated and in vitro matured (IVM) oocytes. A total of four experiments were carried out. These demonstrated that the mean blastocyst rates from stimulated ovulated and IVM pig oocytes were not significantly different (61% vs. 46%, p > 0.05) following in vitro culture. Both ovulated and IVM pig parthenotes were able to develop in vivo for 30 days. Parthenote fetuses collected 21 and 30 days post estrus were morphologically normal but significantly smaller and lighter than fertilized controls (p < 0.01). IVM pig parthenotes stopped development around 31 days post estrus.

Application of reproductive biotechnology in animals: implications and potentials. Applications of reproductive cloning.

The development of new methods of nuclear transfer in mammals is creating many new opportunities in research, medicine and agriculture. The method of cloning is repeatable and has been established in many laboratories worldwide. However, the present procedure is inefficient with fewer than 4% of embryos becoming viable offspring. A considerable improvement in efficiency is required before wide scale use for livestock improvement. The opportunity to introduce precise genetic changes to livestock is available for the first time through the use of gene targeting procedures in cultured cells that are used as nuclear donors. This has potential application in the production of organs for transplantation to humans, studies of human genetic disease and basic research in to the control of gene expression and function.