An X-ray micro-fluorescence study to investigate the distribution of Al, Si, P and Ca ions in the surrounding soft tissue after implantation of a calcium phosphate-mullite ceramic composite in a rabbit animal model

Synthetic calcium phosphates, despite their bioactivity, are brittle. Calcium phosphate-mullite composites have been suggested as potential dental and bone replacement materials which exhibit increased toughness. Aluminium, present in mullite, has however been linked to bone demineralisation and neurotoxicity: it is therefore important to characterise the materials fully in order to understand their in vivo behaviour. The present work reports the compositional mapping of the interfacial region of a calcium phosphate-20 wt% mullite biocomposite/soft tissue interface, obtained from the samples implanted into the long bones of healthy rabbits according to standard protocols (ISO-10993) for up to 12 weeks. X-ray micro-fluorescence was used to map simultaneously the distribution of Al, P, Si and Ca across the ceramic-soft tissue interface. A well defined and sharp interface region was present between the ceramic and the surrounding soft tissue for each time period examined. The concentration of Al in the surrounding tissue was found to fall by two orders of magnitude, to the background level, within similar to 35 mu m of the implanted ceramic.

How checking breeds doubt:reduced performance in a simple working memory task

A paradox of memory research is that repeated checking results in a decrease in memory certainty, memory vividness and confidence [van den Hout, M. A., & Kindt, M. (2003a). Phenomenological validity of an OCD-memory model and the remember/know distinction. Behaviour Research and Therapy, 41, 369–378; van den Hout, M. A., & Kindt, M. (2003b). Repeated checking causes memory distrust. Behaviour Research and Therapy, 41, 301–316]. Although these findings have been mainly attributed to changes in episodic long-term memory, it has been suggested [Shimamura, A. P. (2000). Toward a cognitive neuroscience of metacognition. Consciousness and Cognition, 9, 313–323] that representations in working memory could already suffer from detrimental checking. In two experiments we set out to test this hypothesis by employing a delayed-match-to-sample working memory task. Letters had to be remembered in their correct locations, a task that was designed to engage the episodic short-term buffer of working memory [Baddeley, A. D. (2000). The episodic buffer: a new component in working memory? Trends in Cognitive Sciences, 4, 417–423]. Of most importance, we introduced an intermediate distractor question that was prone to induce frustrating and unnecessary checking on trials where no correct answer was possible. Reaction times and confidence ratings on the actual memory test of these trials confirmed the success of this manipulation. Most importantly, high checkers [cf. VOCI; Thordarson, D. S., Radomsky, A. S., Rachman, S., Shafran, R, Sawchuk, C. N., & Hakstian, A. R. (2004). The Vancouver obsessional compulsive inventory (VOCI). Behaviour Research and Therapy, 42(11), 1289–1314] were less accurate than low checkers when frustrating checking was induced, especially if the experimental context actually emphasized the irrelevance of the misleading question. The clinical relevance of this result was substantiated by means of an extreme groups comparison across the two studies. The findings are discussed in the context of detrimental checking and lack of distractor inhibition as a way of weakening fragile bindings within the episodic short-term buffer of Baddeley's (2000) model. Clinical implications, limitations and future research are considered.

Recombinant human endostatin reduces hypertrophic scar formation in rabbit ear model through down- regulation of VEGF and TIMP-1.

Background: Recombinant human endostatin (Endostar) has been widely used to suppress angiogenesis in carcinoma patients. Hypertrophic scar (HS) tissue, much like a carcinoma, is often associated with angiogenesis. However, there have been few studies conducted on the effects of Endostar on HS or its mechanism. Objective: This paper investigated the effects Endostar on the HS of rabbit ears and studied the effects of Endostar on VEGF and TIMP-1 expression. Methods: Sixteen New Zealand white rabbits were used to establish HS models. Then, rabbit ears containing HS were randomly assigned to either the Endostar group or the control group. The changes of appearance and histology were evaluated using the naked eye, hematoxylin eosin staining, and a scar elevation index. The VEGF and TIMP-1 expressions were detected by immunohistochemical staining, RT-PCR, and western blot. Results: The thickness of the connective tissue in the Endostar group were thinner, the numbers of micro vessels and fibroblasts were fewer, and the collagen fibers were smoother. Moreover, the mRNA and protein expressions of VEGF and TIMP-1 in the Endostar group were significantly lower than those in the control group. Conclusion: The results suggested that Endostar reduced the formation of HS by down-regulation of VEGF and TIMP-1 expressions.

The Use of Mucoadhesive Polymers to Enhance the Hypotensive Effect of a Melatonin Analogue, 5-MCA-NAT, in Rabbit Eyes

Purpose.: 5-Methoxy-carbonylamino-N-acetyltryptamine (5-MCA-NAT, a melatonin receptor agonist) produces a clear intraocular pressure (IOP) reduction in New Zealand White rabbits and glaucomatous monkeys. The goal of this study was to evaluate whether the hypotensive effect of 5-MCA-NAT was enhanced by the presence of cellulose derivatives, some of them with bioadhesive properties, as well as to determine whether these formulations were well tolerated by the ocular surface. Methods.: Formulations were prepared with propylene glycol (0.275%), carboxymethyl cellulose (CMC, 0.5% and 1.0%) of low and medium viscosity and hydroxypropylmethyl cellulose (0.3%). Quantification of 5-MCA-NAT (100 μM) was assessed by HPLC. In vitro tolerance was evaluated by the MTT method in human corneal-limbal epithelial cells and normal human conjunctival cells. In vivo tolerance was analyzed by biomicroscopy and specular microscopy in rabbit eyes. The ocular hypotensive effect was evaluated measuring IOP for 8 hours in rabbit eyes. Results.: All the formulations demonstrated good in vitro and in vivo tolerance. 5-MCA-NAT in CMC medium viscosity 0.5% was the most effective at reducing IOP (maximum IOP reduction, 30.27%), and its effect lasted approximately 7 hours. Conclusions.: The hypotensive effect of 5-MCA-NAT was increased by using bioadhesive polymers in formulations that are suitable for the ocular surface and also protective of the eye in long-term therapies. The use of 5-MCA-NAT combined with bioadhesive polymers is a good strategy in the treatment of ocular hypertension and glaucoma.

Affinity purification of recombinant human plasminogen activator from transgenic rabbit milk using a novel polyolresponsive monoclonal antibody

Purpose: To develop processes for effective isolation and purification of recombinant human plasminogen activator (rhPA) from transgenic rabbit milk. Methods: Immunoaffinity chromatography was selected and improved by a special polyol-responsive monoclonal antibody (PR-mAb). Alteplase was used as immunogen because of its similarity to rhPA in terms of structure. The PR-mAb was prepared by hybridoma technology and screened by ELISA-elution assay. Screening antibody was performed using rhPA milk in an ELISA-elution assay. The antibody clone C4-PR-mAb was selected for immunoaffinity chromatography. The rhPA was effectively bound to immobilized C4-PR-mAb on the column and was eluted with Tris buffer comprising 0.75 mol/L ammonium sulfate and 40n% propanediol (pH7.9). The rhPA was further purified by passing through Chromdex75 gel filtration column. Results: There were 12 hybridoma strains selected into the polyol-responsive mAbs screen step and three hybridoma strains were superior for producing PR-mAbs (C1, C4, C8). The rhPA can be purified from transgenic rabbit milk and maintained a higher thrombolytic activity in vitro by FAPA. Conclusion: The results demonstrate the suitability of the alternative approach used in this study. Using immunoaffinity chromatography and gel filtration column is feasible and convenient for extracting rhPA from milk, and should be useful for purifying other tPA mutants or other novel recombinant milkderived proteins.

Congenital thoracic vertebral malformations in brachycephalic “screw-tailed” dog breeds: Validation of a human classification scheme and a method of Cobb angle measurement in the assessment of vertebral column deformity

Congenital vertebral malformations are common in brachycephalic “screw-tailed” dog breeds such as French bulldogs, English bulldogs, Boston terriers, and Pugs. Those vertebral malformations disrupt the normal vertebral column anatomy and biomechanics, potentially leading to deformity of the vertebral column and subsequent neurological dysfunction. The initial aim of this work was to study and determine whether the congenital vertebral malformations identified in those breeds could be translated in a radiographic classification scheme used in humans to give an improved classification, with clear and well-defined terminology, with the expectation that this would facilitate future study and clinical management in the veterinary field. Therefore, two observers who were blinded to the neurologic status of the dogs classified each vertebral malformation based on the human classification scheme of McMaster and were able to translate them successfully into a new classification scheme for veterinary use. The following aim was to assess the nature and the impact of vertebral column deformity engendered by those congenital vertebral malformations in the target breeds. As no gold standard exists in veterinary medicine for the calculation of the degree of deformity, it was elected to adapt the human equivalent, termed the Cobb angle, as a potential standard reference tool for use in veterinary practice. For the validation of the Cobb angle measurement method, a computerised semi-automatic technique was used and assessed by multiple independent observers. They observed not only that Kyphosis was the most common vertebral column deformity but also that patients with such deformity were found to be more likely to suffer from neurological deficits, more especially if their Cobb angle was above 35 degrees.

Favourable areas for expansion and reintroduction of Iberian lynx accounting for distribution trends and genetic diversity of the European rabbit

Although on a local scale Iberian lynx distribution is determined by the availability of prey rabbits, recent modelling analyses have uncovered broad-scale disagreements between these two species’ distribution trends. These analyses showed also that the lynx had become restricted to only a fraction of the rabbit’s genetic diversity, and that this could be jeopardising its survival in the face of environmental hazards and uncertainty. In the present paper, a follow-up was carried out through the building of lynx and rabbit distribution models based on the most recent Spanish mammal atlas. Environmental favourability values for lynx and rabbit were positively correlated within the lynx’s current distribution area, but they were negatively correlated within the total Spanish area where lynx used to occur in the 1980’s. Environmental favourability for rabbits was significantly higher where lynx maintains reproductive populations than where it recently disappeared, indicating that rabbit favourability plays an important role and can be a good predictor of lynx persistence. The lynx and rabbit models were extrapolated to predict favourable areas for both species in Spain as well as in Portugal, on the original scale of the distribution data (10x10 km) and on a 100 times finer spatial resolution (1x1 km). The lynx and rabbit models were also combined through fuzzy logic to forecast the potential for lynx occurrence incorporating information on favourable areas for its main prey. Several areas are proposed as favourable for lynx expansion or re-introduction,

Conservation biogeography of ecologically interacting species: The case of the Iberian lynx and the European rabbit

Aim To relate the recent Iberian lynx decline to changes in the distribution of the European rabbit after the haemorrhagic disease outbreak of 1989. As Iberian rabbits evolved in two geographically separated lineages, being the recent lynx range practically restricted to the southwestern lineage, we also test if differential range dynamics exists for these lineages, with the consequent implications for lynx conservation and reintroduction planning.

Distribution modelling of wild rabbit hunting yields in its original area (S Iberian Peninsula)

In this work we used the information of the Annual Hunting Reports (AHRs) to obtain a high-resolution model of the potential favourableness for wild rabbit harvesting in Andalusia (southern Spain), using environmental and land-use variables as predictors. We analysed 32,134 AHRs from the period 1993/2001 reported by 6049 game estates to estimate the average hunting yields of wild rabbit in each Andalusian municipality (n5771). We modelled the favourableness for obtaining good hunting yields using stepwise logistic regression on a set of climatic, orographical, land use, and vegetation variables. The favourability equation was used to create a downscaled image representing the favourableness of obtaining good hunting yields for the wild rabbit in 161 km squares in Andalusia, using the Idrisi Image Calculator. The variables that affected hunting yields of wild rabbit were altitude, dry wood crops (mainly olive groves, almond groves, and vineyards), temperature, pasture, slope, and annual number of frost days. The 161 km squares with high favourableness values are scattered throughout the territory, which seems to be caused mainly by the effect of vegetation. Finally, we obtained quality categories for the territory by combining the probability values given by logistic regression with those of the environmental favourability function.

A real time Taqman RT-PCR for the detection of rabbit hemorrhagic disease virus 2 (RHDV2)

As key prey, the wild rabbit downsize constitutes a major drawback on the endangered Iberian lynx (Lynx pardinus) re-introduction in the Iberia. Several captive breeding units mostly located in Alentejo, endeavour the wild rabbit repopulation of depleted areas assigned for the lynx re-introduction. Here we report an RHDV2 outbreak that occurred in early 2016 in a wild rabbit captive breeding unit located in Barrancos municipality. The estimated mortality rate between March and April 2016 was approximately 8.67%. Anatomopathologic examination was carried out for 13 victimized rabbits. Molecular characterization was based on the complete vp60 capsid gene. The 13 rabbit carcasses investigated showed typical macroscopic RHD lesions testing positive to RHDV2- RNA. Comparison of the vp60 nucleotide sequences obtained from two specimens with others publically available disclosed similarities below 98.22% with RHDV2 strains originated in the Iberia and Azores and revealed that the two identical strains from Barrancos-2016 contain six unique single synonymous nucleotide polymorphisms. In the phylogenetic analysis performed, the Barrancos-2016 strains clustered apart from other known strains, meaning they may represent new evolutionary RHDV2 lineages. No clear epidemiological link could be traced for this outbreak where the mortalities were lower compared with previous years. Yet, network analysis suggested a possible connection between the missing intermediates from which the strains from Barrancos 2013, 2014 and 2016 have derived. It is therefore possible that RHDV2 has circulated endemically in the region since 2012, with periodic epizootic occurrences. Still, six years after its emergence in wild rabbits, RHDV2 continues to pose difficulties to the establishment of natural wild rabbit populations that are crucial for the self-sustainability of the local ecosystems.