Single cell RNA-sequencing and RNA-tomography of the avian embryo extending body axis.

Introduction: Vertebrate body axis formation initiates during gastrulation and continues within the tail bud at the posterior end of the embryo. Major structures in the trunk are paired somites, which generate the musculoskeletal system, the spinal cord-forming part of the central nervous system, and the notochord, with important patterning functions. The specification of these different cell lineages by key signalling pathways and transcription factors is essential, however, a global map of cell types and expressed genes in the avian trunk is missing. Methods: Here we use high-throughput sequencing approaches to generate a molecular map of the emerging trunk and tailbud in the chick embryo. Results and Discussion: Single cell RNA-sequencing (scRNA-seq) identifies discrete cell lineages including somites, neural tube, neural crest, lateral plate mesoderm, ectoderm, endothelial and blood progenitors. In addition, RNA-seq of sequential tissue sections (RNA-tomography) provides a spatially resolved, genome-wide expression dataset for the avian tailbud and emerging body, comparable to other model systems. Combining the single cell and RNA-tomography datasets, we identify spatially restricted genes, focusing on somites and early myoblasts. Thus, this high-resolution transcriptome map incorporating cell types in the embryonic trunk can expose molecular pathways involved in body axis development.

Managing outbreaks of invasive species--a new method to prioritize preemptive quarantine efforts across large geographic regions.

In pest risk assessment it is frequently necessary to make time-critical decisions regarding management of expanding pest populations. When an invasive pest outbreak is expanding rapidly, preemptive quarantine of areas that are under imminent threat of infestation is one of only a few available management tools that can be implemented quickly to help control the expansion. The preemptive quarantine of locations that surround an infested area also acts as a safeguard to counteract the risk of failed detections of the pest in field surveys. In this paper, we present a method that assesses the suitability of preemptive quarantine measures at the level of small geographical subdivisions (U.S. counties). The cost of a preemptive quarantine in a given county is weighed against the protective benefit of delaying the spread of an outbreak to other neighboring counties. We demonstrate the approach with a decision support model that estimates the suitability of preemptive quarantine across multiple counties that surround areas infested with the emerald ash borer (Agrilus planipennis Fairmaire (EAB), Coleoptera: Buprestidae), an emerging major threat to ash tree species (Fraxinus spp.) in North America. The model identifies the U.S. counties where the installation of preemptive quarantine would most effectively slow the spread of EAB populations and reduce risk to high-value areas.

Tissue engineered bone using select growth factors: A comprehensive review of animal studies and clinical translation studies in man.

There is a growing socio-economic need for effective strategies to repair damaged bone resulting from disease, trauma and surgical intervention. Bone tissue engineering has received substantial investment over the last few decades as a result. A multitude of studies have sought to examine the efficacy of multiple growth factors, delivery systems and biomaterials within in vivo animal models for the repair of critical-sized bone defects. Defect repair requires recapitulation of in vivo signalling cascades, including osteogenesis, chondrogenesis and angiogenesis, in an orchestrated spatiotemporal manner. Strategies to drive parallel, synergistic and consecutive signalling of factors including BMP-2, BMP-7/OP-1, FGF, PDGF, PTH, PTHrP, TGF-ß3, VEGF and Wnts have demonstrated improved bone healing within animal models. Enhanced bone repair has also been demonstrated in the clinic following European Medicines Agency and Food and Drug Administration approval of BMP-2, BMP-7/OP-1, PDGF, PTH and PTHrP. The current review assesses the in vivo and clinical data surrounding the application of growth factors for bone regeneration. This review has examined data published between 1965 and 2013. All bone tissue engineering studies investigating in vivo response of the growth factors listed above, or combinations thereof, utilising animal models or human trials were included. All studies were compiled from PubMed-NCBI using search terms including 'growth factor name', 'in vivo', 'model/animal', 'human', and 'bone tissue engineering'. Focus is drawn to the in vivo success of osteoinductive growth factors incorporated within material implants both in animals and humans, and identifies the unmet challenges within the skeletal regenerative area.

Evaluation of skeletal tissue repair, part 2: enhancement of skeletal tissue repair through dual-growth-factor-releasing hydrogels within an ex vivo chick femur defect model.

There is an unmet need for improved, effective tissue engineering strategies to replace or repair bone damaged through disease or injury. Recent research has focused on developing biomaterial scaffolds capable of spatially and temporally releasing combinations of bioactive growth factors, rather than individual molecules, to recapitulate repair pathways present in vivo. We have developed an ex vivo embryonic chick femur critical size defect model and applied the model in the study of novel extracellular matrix (ECM) hydrogel scaffolds containing spatio-temporal combinatorial growth factor-releasing microparticles and skeletal stem cells for bone regeneration. Alginate/bovine bone ECM (bECM) hydrogels combined with poly(d,l-lactic-co-glycolic acid) (PDLLGA)/triblock copolymer (10-30% PDLLGA-PEG-PLDLGA) microparticles releasing dual combinations of vascular endothelial growth factor (VEGF), chondrogenic transforming growth factor beta 3 (TGF-ß3) and the bone morphogenetic protein BMP2, with human adult Stro-1+bone marrow stromal cells (HBMSCs), were placed into 2mm central segmental defects in embryonic day 11 chick femurs and organotypically cultured. Hydrogels loaded with VEGF combinations induced host cell migration and type I collagen deposition. Combinations of TGF-ß3/BMP2, particularly with Stro-1+HBMSCs, induced significant formation of structured bone matrix, evidenced by increased Sirius red-stained matrix together with collagen expression demonstrating birefringent alignment within hydrogels. This study demonstrates the successful use of the chick femur organotypic culture system as a high-throughput test model for scaffold/cell/growth factor therapies in regenerative medicine. Temporal release of dual growth factors, combined with enriched Stro-1+HBMSCs, improved the formation of a highly structured bone matrix compared to single release modalities. These studies highlight the potential of a unique alginate/bECM hydrogel dual growth factor release platform for bone repair.

Evaluation of skeletal tissue repair, part 1: assessment of novel growth-factor-releasing hydrogels in an ex vivo chick femur defect model.

Current clinical treatments for skeletal conditions resulting in large-scale bone loss include autograft or allograft, both of which have limited effectiveness. In seeking to address bone regeneration, several tissue engineering strategies have come to the fore, including the development of growth factor releasing technologies and appropriate animal models to evaluate repair. Ex vivo models represent a promising alternative to simple in vitro systems or complex, ethically challenging in vivo models. We have developed an ex vivo culture system of whole embryonic chick femora, adapted in this study as a critical size defect model to investigate the effects of novel bone extracellular matrix (bECM) hydrogel scaffolds containing spatio-temporal growth factor-releasing microparticles and skeletal stem cells on bone regeneration, to develop a viable alternative treatment for skeletal degeneration. Alginate/bECM hydrogels combined with poly (d,l-lactic-co-glycolic acid) (PDLLGA)/triblock copolymer (10-30% PDLLGA-PEG-PDLLGA) microparticles releasing VEGF, TGF-ß3 or BMP-2 were placed, with human adult Stro-1+ bone marrow stromal cells, into 2mm central segmental defects in embryonic chick femurs. Alginate/bECM hydrogels loaded with HSA/VEGF or HSA/TGF-ß3 demonstrated a cartilage-like phenotype, with minimal collagen I deposition, comparable to HSA-only control hydrogels. The addition of BMP-2 releasing microparticles resulted in enhanced structured bone matrix formation, evidenced by increased Sirius red-stained matrix and collagen expression within hydrogels. This study demonstrates delivery of bioactive growth factors from a novel alginate/bECM hydrogel to augment skeletal tissue formation and the use of an organotypic chick femur defect culture system as a high-throughput test model for scaffold/cell/growth factor therapies for regenerative medicine.

Visual regulation of refractive development: insights from animal studies.

Investigations employing animal models have demonstrated that ocular growth and refractive development are regulated by visual feedback. In particular, lens compensation experiments in which treatment lenses are used to manipulate the eye's effective refractive state have shown that emmetropization is actively regulated by signals produced by optical defocus. These observations in animals are significant because they indicate that it should be possible to use optical treatment strategies to influence refractive development in children, specifically to slow the rate of myopia progression. This review highlights some of the optical performance properties of the vision-dependent mechanisms that regulate refractive error development, especially those that are likely to influence the efficacy of optical treatment strategies for myopia. In this respect, the results from animal studies have been very consistent across species; however, to facilitate extrapolation to clinical settings, results are presented primarily for nonhuman primates. In agreement with preliminary clinical trials, the experimental data show that imposed myopic defocus can slow ocular growth and that treatment strategies that influence visual signals over a large area of the retina are likely to be most effective.

Information needs of cancer patients and survivors regarding diet, exercise and weight management: a qualitative study.

While advanced cancer is often associated with weight loss, curative cancer treatment is often associated with weight gain. Weight gain during treatment may be associated with greater risk of cancer recurrence and development of lifestyle diseases. Currently, limited resources are available to cancer patients focussed on weight control. This study assessed the information needs of patients undergoing curative chemotherapy regarding diet, exercise and weight management for the purpose of developing weight management resources. Focus groups were held with oncology practitioners, patients and survivors to determine current information provision and needs. Focus groups highlighted a perception that information provision regarding diet, exercise and weight management is insufficient and no routine assessment of weight occurs during chemotherapy. Barriers to information provision described included lack of resources and time, and practitioners' uncertainty regarding appropriate messages to provide. Patients wanted more information regarding diet, exercise and weight during treatment time. The findings of this study suggest an increase in provision of diet, exercise and weight management information is needed. This information should be evidence-based and delivered at an appropriate time by the preferred health care professional. It would also be beneficial to implement protocols regarding assessment of weight during treatment.

A novel ex vivo culture model for inflammatory bone destruction.

Pathological alterations in the balance of bone metabolism are central to the progression of inflammatory bone diseases such as periodontal disease. We have developed and characterized a novel ex vivo murine mandible model of inflammatory bone destruction. Slices of mandible were cultured for 14 days in the presence or absence of P. gingivalis lipopolysaccharide (LPS) or pro-inflammatory cytokines. Following culture, cell viability and tissue histomorphometry were assessed with quantification of matrix proteins, resident osteoclasts, ligament cells, monocytes, macrophages, and neutrophils. In the absence of inflammatory factors, culture viability, osteoclasts, and matrix components were maintained. LPS or TNFα stimulation demonstrated an increase in cellular proliferation, monocyte cells, osteoclast differentiation, and matrix degradation. Pathophysiological bone metabolism can be induced via exposure to LPS and direct influence of TNFα within the model despite the absence of systemic circulation, providing a model for inflammatory bone destruction and investigation of the effects of novel therapeutics.

A humanised mouse model of cytokine release: comparison of CD3-specific antibody fragments.

CD3-specific antibodies have shown clinical efficacy in both transplantation and autoimmunity. However, targeting CD3 in this way can lead to T-cell activation and a serious cytokine release syndrome mediated by Fcγ receptor binding. An in vivo mouse model has been developed using severe combined immunodeficient (SCID) mice to detect human T-cell depletion and cytokine release into the circulation after administration of OKT3. This system has been used to evaluate OKT3 antibody fragments lacking the entire Fc region alongside whole antibody constructs. These data clearly show that cytokine release is detected with all OKT3 antibody constructs and fragments tested and these can be ranked from highest to lowest as follows: mIgG2a>hIgG1 (Ala-Ala)>hIgG1 diFab' maleimide (DFM)>hIgG1 F(ab')2>mIgG2a F(ab')2>hIgG1 Fab'. Furthermore, the monovalent hIgG1 Fab' fragment gives the least cytokine release but it does not deplete human T-cells in this assay format. This suggests that T-cell activation may be playing a role in the mechanism of action of anti-CD3 antibodies and consequently the unwanted cytokine release is potentially unavoidable for this class of molecules. This model system provides a useful tool to aid in understanding and reducing the potential risks of cytokine release following antibody therapy.

Local sensitivity to stimulus orientation and spatial frequency within the receptive fields of neurons in visual area 2 of macaque monkeys.

We used dynamic dense noise stimuli and local spectral reverse correlation methods to reveal the local sensitivities of neurons in visual area 2 (V2) of macaque monkeys to orientation and spatial frequency within their receptive fields. This minimized the potentially confounding assumptions that are inherent in stimulus selections. The majority of neurons exhibited a relatively high degree of homogeneity for the preferred orientations and spatial frequencies in the spatial matrix of facilitatory subfields. However, about 20% of all neurons showed maximum orientation differences between neighboring subfields that were greater than 25 deg. The neurons preferring horizontal or vertical orientations showed less inhomogeneity in space than the neurons preferring oblique orientations. Over 50% of all units also exhibited suppressive profiles, and those were more heterogeneous than facilitatory profiles. The preferred orientation and spatial frequency of suppressive profiles differed substantially from those of facilitatory profiles, and the neurons with suppressive subfields had greater orientation selectivity than those without suppressive subfields. The peak suppression occurred with longer delays than the peak facilitation. These results suggest that the receptive field profiles of the majority of V2 neurons reflect the orderly convergence of V1 inputs over space, but that a subset of V2 neurons exhibit more complex response profiles having both suppressive and facilitatory subfields. These V2 neurons with heterogeneous subfield profiles could play an important role in the initial processing of complex stimulus features.

Examination of nitrate concentration, loading and isotope dynamics in subsurface drainage under standard agricultural cropping in Atlantic Canada.

Intensive agricultural farming practices have the potential to cause high levels of nitrate-nitrogen (NO(3)(-)-N) to be released from tile drainage systems. A better understanding of the temporal dynamics of NO(3)(-)-N loading, δ(15)N and δ(18)O from standard drainage systems is needed, in order to improve our understanding of NO(3)(-)-N transport and transformation processes; particularly, with regards to the imperfectly drained agricultural soils found within Atlantic Canada. Three conventional subsurface drainage plots (48 × 48 m) placed at a 0.80 m soil depth were monitored over a seven month period on sandy loam soil in Onslow, Nova Scotia. Each plot received similar applications of both organic and inorganic fertilizer. Water samples were obtained and analyzed for NO(3)(-)-N concentrations and isotopic signatures of δ(15)N and δ(18)O for NO(3)(-)-N. Maximum NO(3)(-)-N loads were observed in the winter and fall, when both discharge and concentration of the NO(3)(-)-N were highest. Mean isotope values in NO(3)(-) ranged from 3.1 to 8.5‰ for δ(15)N and -3.2 to 17.7‰ for δ(18)O. Results suggest that NO(3)(-)-N from the drainage water was derived from organic sources (i.e. manure and soil organic matter) and that loss via denitrification does not impart an identifiable signature upon the NO(3)(-)-N pool. The dual isotope approach examined here provides insight into N source and transformation processes which may be contributing to the NO(3)(-)-N found within the drainage water.

Nitrate loading and isotopic signatures in subsurface agricultural drainage systems.

Artificially draining soils using subsurface tiles is a common practice on many agricultural fields. High levels of nitrate-nitrogen (NO-N) are often released from these systems; therefore, knowledge on the sources and processes controlling NO-N in drainage systems is needed. A dual isotope study (δN and δO) was used to investigate three subsurface drainage systems (shallow, conventional, and controlled) in Onslow, Nova Scotia, Canada. The objectives of this study were (i) to identify which drainage system more effectively reduced the NO-N loading, (ii) to examine differences in isotopic signatures under identical nutrient and cropping regimes for a fixed soil type, and (iii) to identify the utility of different drainage systems in controlling nutrient flows. Nitrate concentrations measured ranged from 0.92 to 11.8, from 2.3 to 17.3, and from 2.1 to 19.8 mg L for the shallow, conventional, and controlled drains, respectively. Total NO-N loading from shallow and controlled drains were 20 and 5.6 kg ha, respectively, lower than conventional (39.1 kg ha). The isotopic composition of NO-N for all drainage types appeared to be a mixture of two organic sources (manure and soil organic matter) via the process of nitrification. There was no evidence that denitrification played a significant role in removing NO-N during transport. Overall, shallow drainage reduced NO-N loading but offered no water conservation benefits. Combining the benefits of decreased NO-N loading from shallow systems with water control capability may offer the best solution to reducing nutrient loadings into water systems, achieving optimal crop yield, and decreasing drainage installation costs.

TGF-beta1 exposure from bone surfaces by chemical treatment modalities.

Growth factors are known to be sequestered to the mineralised matrix of bone. The aim of this study was to investigate the ability of citric acid, EDTA, calcium hydroxide and sodium hydroxide to release active growth factors from bone surfaces, able to promote osteoblast differentiation. All chemical treatments increased surface levels of TGF-beta1 (used as a biomarker of growth factor release), compared to control bone surfaces treated with PBS. Differences were observed in the kinetics of TGF-beta1 exposure at the surface and its subsequent release into the aqueous environment for the different chemical treatments. Surface levels of growth factor following chemical treatment were low, but of sufficient concentration to stimulate cell expansion and osteoblast differentiation of bone marrow stromal cells grown on EDTA and calcium hydroxide treated surfaces compared to PBS treated surfaces. The increased osteogenic potential on these surfaces may relate to an increase in growth factor availability and changes to the surface chemistry and topography.

Neuronal responses in visual area V2 (V2) of macaque monkeys with strabismic amblyopia.

Amblyopia, a developmental disorder of spatial vision, is thought to result from a cascade of cortical deficits over several processing stages beginning at the primary visual cortex (V1). However, beyond V1, little is known about how cortical development limits the visual performance of amblyopic primates. We quantitatively analyzed the monocular and binocular responses of V1 and V2 neurons in a group of strabismic monkeys exhibiting varying depths of amblyopia. Unlike in V1, the relative effectiveness of the affected eye to drive V2 neurons was drastically reduced in the amblyopic monkeys. The spatial resolution and the orientation bias of V2, but not V1, neurons were subnormal for the affected eyes. Binocular suppression was robust in both cortical areas, and the magnitude of suppression in individual monkeys was correlated with the depth of their amblyopia. These results suggest that the reduced functional connections beyond V1 and the subnormal spatial filter properties of V2 neurons might have substantially limited the sensitivity of the amblyopic eyes and that interocular suppression was likely to have played a key role in the observed alterations of V2 responses and the emergence of amblyopia.

A pilot study of the effects of chronic paroxetine administration on hippocampal N-acetylaspartate in generalized anxiety disorder.

The neural basis of generalized anxiety disorder (GAD) is poorly characterized. The effect of chronic administration (12 weeks) of paroxetine, a selective serotonin reuptake inhibitor, on N-acetylaspartate (NAA), a marker of neuronal viability, was evaluated in adults with GAD using proton magnetic resonance spectroscopic imaging ((1)H MRSI) at 1.5 T. We hypothesized that, pretreatment abnormalities in hippocampal NAA/creatine (NAA/Cr) would normalize with symptomatic improvement. Nine GAD patients (mean age = 41.7 year; 4 females) received 12 weeks of open-label paroxetine treatment, flexibly dosed up to 60 mg/day. Clinical outcome was assessed with the Hamilton Anxiety Rating Scale (HAM-A). Multislice ( 1)H MRSI scans were performed at unmedicated baseline and following 6 and 12 weeks of treatment. Ten untreated healthy volunteers (HVs) (mean age = 37.1 year; 4 females) received scans at the same intervals. All patients achieved remission (HAM-A

Postnatal development of disparity sensitivity in visual area 2 (v2) of macaque monkeys.

Macaque monkeys do not reliably discriminate binocular depth cues until about 8 wk of age. The neural factors that limit the development of fine depth perception in primates are not known. In adults, binocular depth perception critically depends on detection of relative binocular disparities and the earliest site in the primate visual brain where a substantial proportion of neurons are capable of discriminating relative disparity is visual area 2 (V2). We examined the disparity sensitivity of V2 neurons during the first 8 wk of life in infant monkeys and compared the responses of V2 neurons to those of V1 neurons. We found that the magnitude of response modulation in V2 and V1 neurons as a function of interocular spatial phase disparity was adult-like as early as 2 wk of age. However, the optimal spatial frequency and binocular response rate of these disparity sensitive neurons were more than an octave lower in 2- and 4-wk-old infants than in adults. Consequently, despite the lower variability of neuronal firing in V2 and V1 neurons of infant monkeys, the ability of these neurons to discriminate fine disparity differences was significantly reduced compared with adults. This reduction in disparity sensitivity of V2 and V1 neurons is likely to limit binocular depth perception during the first several weeks of a monkey's life.

Identification of biomarkers for the antiangiogenic and antitumour activity of the superoxide dismutase 1 (SOD1) inhibitor tetrathiomolybdate (ATN-224).

Tetrathiomolybdate (choline salt; ATN-224), a specific, high-affinity copper binder, is currently being evaluated in several phase II cancer trials. ATN-224 inhibits CuZn superoxide dismutase 1 (SOD1) leading to antiangiogenic and antitumour effects. The pharmacodynamics of tetrathiomolybdate has been followed by tracking ceruloplasmin (Cp), a biomarker for systemic copper. However, at least in mice, the inhibition of angiogenesis occurs before a measurable decrease in systemic copper is observed. Thus, the identification and characterisation of other biomarkers to follow the activity of ATN-224 in the clinic is of great interest. Here, we present the preclinical evaluation of two potential biomarkers for the activity of ATN-224: (i) SOD activity measurements in blood cells in mice and (ii) levels of endothelial progenitor cells (EPCs) in bonnet macaques treated with ATN-224. The superoxide dismutase activity in blood cells in mice is rapidly inhibited by ATN-224 treatment at doses at which angiogenesis is maximally inhibited. Furthermore, ATN-224 dosing in bonnet macaques causes a profound and reversible decrease in EPCs without significant toxicity. Thus, both SOD activity measurements and levels of EPCs may be useful biomarkers of the antiangiogenic activity of ATN-224 to be used in its clinical development.

A 5.8-kDa component of manuka honey stimulates immune cells via TLR4.

Honey is used as a therapy to aid wound healing. Previous data indicate that honey can stimulate cytokine production from human monocytes. The present study further examines this phenomenon in manuka honey. As inflammatory cytokine production in innate immune cells is classically mediated by pattern recognition receptors in response to microorganisms, bacterial contamination of honey and the effect of blocking TLR2 and -4 on stimulatory activity were assessed. No vegetative bacteria were isolated from honey; however, bacterial spores were cultured from one-third of samples, and low levels of LPS were detected. Blocking TLR4 but not TLR2 inhibited honey-stimulated cytokine production significantly. Cytokine production did not correlate with LPS levels in honey and was not inhibited by polymyxin B. Further, the activity was reduced significantly following heat treatment, indicating that component(s) other than LPS are responsible for the stimulatory activity of manuka honey. To identify the component responsible for inducing cytokine production, honey was separated by molecular weight using microcon centrifugal filtration and fractions assessed for stimulatory activity. The active fraction was analyzed by MALDI-TOF mass spectroscopy, which demonstrated the presence of a number of components of varying molecular weights. Additional fractionation using miniaturized, reverse-phase solid-phase extraction resulted in the isolation of a 5.8-kDa component, which stimulated production of TNF-alpha via TLR4. These findings reveal mechanisms and components involved in honey stimulation of cytokine induction and could potentially lead to the development of novel therapeutics to improve wound healing for patients with acute and chronic wounds.

Development of temporal response properties and contrast sensitivity of V1 and V2 neurons in macaque monkeys.

The temporal contrast sensitivity of human infants is reduced compared to that of adults. It is not known which neural structures of our visual brain sets limits on the early maturation of temporal vision. In this study we investigated how individual neurons in the primary visual cortex (V1) and visual area 2 (V2) of infant monkeys respond to temporal modulation of spatially optimized grating stimuli and a range of stimulus contrasts. As early as 2 wk of age, V1 and V2 neurons exhibited band-pass temporal frequency tuning. However, the optimal temporal frequency and temporal resolution of V1 neurons were much lower in 2- and 4-wk-old infants than in 8-wk-old infants or adults. V2 neurons of 8-wk-old monkeys had significantly lower optimal temporal frequencies and resolutions than those of adults. Onset latency was longer in V1 at 2 and 4 wk of age and was slower in V2 even at 8 wk of age than in adults. Contrast threshold of V1 and V2 neurons was substantially higher in 2- and 4-wk-old infants but became adultlike by 8 wk of age. For the first 4 wk of life, responses to high-contrast stimuli saturated more readily in V2. The present results suggest that although the early development of temporal vision and contrast sensitivity may largely depend on the functional maturation of precortical structures, it is also likely to be limited by immaturities that are unique to V1 and V2.