Louise van der Weerd

Evolution of an Arsenal Structural and Functional Diversification of the Venom System in the Advanced Snakes (Caenophidia)

Venom is a key innovation underlying the evolution of advanced snakes (Caenophidia). Despite this, very little is known about venom system structural diversification, toxin recruitment event timings, or toxin molecular evolution. A multidisciplinary approach was used to examine the diversification of the venom system and associated toxins across the full range of the ∼100 million-year-old advanced snake clade with a particular emphasis upon families that have not secondarily evolved a front-fanged venom system (∼80% of the 2500 species). Analysis of cDNA libraries revealed complex venom transcriptomes containing multiple toxin types including three finger toxins, cobra venom factor, cysteine-rich secretory protein, hyaluronidase, kallikrein, kunitz, lectin, matrix metalloprotease, phospholipase A2, snake venom metalloprotease/a disintegrin and metalloprotease, and waprin. High levels of sequence diversity were observed, including mutations in structural and functional residues, changes in cysteine spacing, and major deletions/truncations. Morphological analysis comprising gross dissection, histology, and magnetic resonance imaging also demonstrated extensive modification of the venom system architecture in non-front-fanged snakes in contrast to the conserved structure of the venom system within the independently evolved front-fanged elapid or viperid snakes. Further, a reduction in the size and complexity of the venom system was observed in species in which constriction has been secondarily evolved as the preferred method of prey capture or dietary preference has switched from live prey to eggs or to slugs/snails. Investigation of the timing of toxin recruitment events across the entire advanced snake radiation indicates that the evolution of advanced venom systems in three front-fanged lineages is associated with recruitment of new toxin types or explosive diversification of existing toxin types. These results support the role of venom as a key evolutionary innovation in the diversification of advanced snakes and identify a potential role for non-front-fanged venom toxins as a rich source for lead compounds for drug design and development.

Cognitive deficits in Tsc1+/- mice in the absence of cerebral lesions and seizures.

Tuberous sclerosis complex (TSC) is characterized by brain lesions, epilepsy, increased incidence of mental retardation and autism. The causal link between lesion load and epilepsy on cognitive disabilities has been debated, and these factors explain only part of the intelligence quotient variability. A Tsc2 rat model of the disease provided evidence that the TSC genes are directly involved in neuronal function. However, these lesion‐ and epilepsy‐free animals did not show learning deficits, leaving open the possibility that the presence of brain lesions or epilepsy is a prerequisite for the cognitive deficits to fully develop. Here, we reinvestigated the relation among cerebral lesions, epilepsy, and cognitive function using Tsc1+/−mice.

The chronic vascular and haemodynamic response after permanent bilateral common carotid occlusion in newborn and adult rats

Vascular growth and redistribution of flow can compensate for arterial occlusion and possibly reduce the effects of hypoperfusion. As yet there is limited information on the age-dependent nature of vasculature remodelling. In this study, we have monitored the vascular and morphologic changes using magnetic resonance imaging and histology in a chronic bilateral common carotid artery occlusion (BCCAO) model in both newborn and adult rats. Acutely, cerebral blood flow (CBF) decreased immediately after BCCAO, producing a state of oligaemic hypoperfusion. At 6 months after BCCAO in both adult and neonatal rats, the CBF had normalised at control values. To investigate the underlying mechanism for the return of CBF to control values, intra- and extracerebral magnetic resonance angiograms (MRAs) were acquired. As expected, signal from the common carotid arteries was present in the sham-operated rats, but was absent in the BCCAO animals. India ink angiograms demonstrated more tortuous basilar arteries in the adult rats post-BCCAO and MRAs demonstrated more extracerebral midline collaterals in the neonatal rats post-BCCAO, indicating different modes of vascular adaptation dependent on the age at onset of the insult. Both groups had collateral vessels arising from the vertebral arteries, and BCCAO was also associated with increased diameter of basilar, posterior cerebral, posterior communicating, internal carotid, middle cerebral and anterior cerebral arteries. Our study suggests that the developing and mature animals exhibit different patterns of vascular remodelling and that the BCCAO hypoperfusion model will be useful for investigating age-dependent vascular events in response to vasoocclusive disease.

A central role for venom in predation by Varanus komodoensis (Komodo Dragon) and the extinct giant Varanus (Megalania) priscus

The predatory ecology of Varanus komodoensis (Komodo Dragon) has been a subject of long-standing interest and considerable conjecture. Here, we investigate the roles and potential interplay between cranial mechanics, toxic bacteria, and venom. Our analyses point to the presence of a sophisticated combined-arsenal killing apparatus. We find that the lightweight skull is relatively poorly adapted to generate high bite forces but better adapted to resist high pulling loads. We reject the popular notion regarding toxic bacteria utilization. Instead, we demonstrate that the effects of deep wounds inflicted are potentiated through venom with toxic activities including anticoagulation and shock induction. Anatomical comparisons of V. komodoensis with V. (Megalania) priscus fossils suggest that the closely related extinct giant was the largest venomous animal to have ever lived.

Evolution and diversification of the Toxicofera reptile venom system.

The diversification of the reptile venom system has been an area of major research but of great controversy. In this review we examine the historical and modern-day efforts of all aspects of the venom system including dentition, glands and secreted toxins and highlight areas of future research opportunities. We use multidisciplinary techniques, including magnetic resonance imaging of venom glands through to molecular phylogenetic reconstruction of toxin evolutionary history, to illustrate the diversity within this integrated weapons system and map the timing of toxin recruitment events over the toxicoferan organismal evolutionary tree.

Neuroprotective effects of HSP70 overexpression after cerebral ischaemia--an MRI study.

Heat shock proteins (HSPs) have been reported to increase cell survival in response to a wide range of cellular challenges. However, the role of HSP70 overexpression is still a matter of debate, with some reports showing protection and others not. In order to resolve these discrepancies and further investigate the action of these proteins in vivo, transgenic mice overexpressing HSP70 have been compared to wild-type mice in a middle cerebral artery occlusion model of permanent cerebral ischaemia. Previously, the effect of HSP70 was assessed histologically postmortem. In this report, magnetic resonance imaging (MRI) was used to assess the mice in vivo after the onset of stroke. The lesion volume, as measured at 24 h using T(2)-weighted MRI, was significantly smaller in HSP70 transgenic mice compared with wild-type mice. The smaller lesion size in HSP70 transgenic mice could not be attributed to differences in vascular anatomy or in cerebral blood flow during occlusion. Additionally, the apparent diffusion coefficient showed different spatial and temporal patterns between the groups, suggesting that the damage within the lesion may be less severe for HSP70 transgenic mice. Thus, we conclude that overexpression of HSP70 reduces the overall lesion size and may also limit the tissue damage within the lesion.

Neuroprotective effects of virally delivered HSPs in experimental stroke.

Heat shock proteins (HSPs) are molecular chaperones with essential roles in modulating the proteolytic machinery and accelerating cell repair. Heat shock protein overexpression has been observed in vivo and in vitro under stresses including heat, nutrient deprivation and ischemia. Experiments in in vivo models of stroke indicate that transgenically overexpressed or virally delivered HSPs can enhance cell survival, but cannot always reduce lesion size. This study aims to assess the effects of virally delivered HSPs in a rat middle cerebral artery occlusion model of reversible focal cerebral ischemia using noninvasive magnetic resonance imaging. Attenuated herpes simplex virus carrying HSP27, HSP70, or a LacZ control was microinjected into the striatum 3 days before ischemia. Multislice T2-weighted images at 24 h after ischemia indicated that lesion volume was reduced by 44% in HSP27-treated animals compared with controls (P 0.019). No significant differences were found between HSP70-treated and control animals (P 0.88). Immunohistochemistry and Western blots revealed that HSP27 and HSP70 expression levels were equally high in injected hemispheres, but only the former had an effect on lesion size. This is the first evidence of the efficacy of gene therapy with any viral vector expressing HSP27 in an experimental model of stroke.

Changes in GABAA receptor properties in amygdala kindled animals: in vivo studies using [11C]flumazenil and positron emission tomography.

Purpose:u2002 The purpose of the present investigation was to quantify alterations in GABAA receptor density in vivo in rats subjected to amygdala kindling.

Regional variation of cerebral blood flow and arterial transit time in the normal and hypoperfused rat brain measured using continuous arterial spin labeling MRI.

Continuous arterial spin labeling (CASL) is a noninvasive magnetic resonance (MR) method for measuring cerebral perfusion. In its most widely used form, CASL incorporates a postlabeling delay to minimize the sensitivity of the technique to transit time effects, which otherwise corrupt cerebral blood flow (CBF) quantification. For this delay to work effectively, it must be longer than the longest transit time present in the system. In this work, CASL measurements were made in four coronal slices in the rat brain using a range of postlabeling delays. By doing this, direct estimation of both CBF and arterial transit time (δa) was possible. These measurements were performed in the normal brain and during hypoperfusion induced by occlusion of the common carotid arteries. It was found that, in the normal rat brain, significant regional variation exists for both CBF and δa. Mean values of CBF and δa in the selected gray matter regions of interest were 233 mL/100 g min and 266 ms, respectively, with the latter ranging from 100 to 500 ms. Therefore, use of a 500-ms postlabeling delay is suitable for any location in the normal rat brain. After common carotid artery occlusion, CBF decreased and δa increased by regionally dependent amounts. In the sensory cortex, δa increased to a mean value of 740 ms, significantly greater than 500 ms. These results highlight the importance of either (a) determining δa as part of the CASL measurement or (b) knowing the approximate range of values δa is likely to take for a given application, so that the parameters of the CASL sequence can be chosen appropriately.

Evaluation of algorithms for analysis of NMR relaxation decay curves

Quantitative processing of NMR relaxation images depends on the characteristics of the used fitting algorithm. Therefore several common fitting algorithms are compared for decay curves with low signal-to-noise ratios. The use of magnitude data yields a non-zero base line, and is shown to result in an overestimation of the decay time. A simple base line correction is no solution since this yields an equally large underestimation due to overcorrection of the first part of the curve. The use of squared data does yield reliable results, but only in the case of monoexponential decays. The best fitting algorithm under all experimentally occurring conditions turns out to be using real data after phase correction. A phase correction scheme is proposed, which applies to all imaging experiments for which the phase of the pixels is constant over the echo train. This scheme is validated for a phantom and for a tulip bulb.