Markus Tschopp

Crystal structure of the anti-His tag antibody 3D5 single-chain fragment complexed to its antigen.

The crystal structure of a mutant form of the single-chain fragment (scFv), derived from the monoclonal anti-His tag antibody 3D5, in complex with a hexahistidine peptide has been determined at 2.7 A resolution. The peptide binds to a deep pocket formed at the interface of the variable domains of the light and the heavy chain, mainly through hydrophobic interaction to aromatic residues and hydrogen bonds to acidic residues. The antibody recognizes the C-terminal carboxylate group of the peptide as well as the main chain of the last four residues and the last three imidazole side-chains. The crystals have a solvent content of 77% (v/v) and form 70 A-wide channels that would allow the diffusion of peptides or even small proteins. The anti-His scFv crystals could thus act as a framework for the crystallization of His-tagged target proteins. Designed mutations in framework regions of the scFv lead to high-level expression of soluble protein in the periplasm of Escherichia coli. The recombinant anti-His scFv is a convenient detection tool when fused to alkaline phosphatase. When immobilized on a matrix, the antibody can be used for affinity purification of recombinant proteins carrying a very short tag of just three histidine residues, suitable for crystallization. The experimental structure is now the basis for the design of antibodies with even higher stability and affinity.

Impact of corneal cross-linking on drug penetration in an ex vivo porcine eye model

Purpose: To analyze the influence of corneal cross-linking (CXL) using ultraviolet-A and riboflavin on corneal drug penetration of topically applied drugs. Methods: In an ex vivo porcine eye model, eyes were randomly assigned to CXL or control treatment. Central corneal thickness and anterior chamber depth were measured with a Pentacam device. In the CXL group, eyes were treated with CXL using ultraviolet-A (370 nm) and riboflavin, whereas in the control group only riboflavin was applied without irradiation. Subsequently, 0.3% ofloxacin (n = 40 eyes) or 1% voriconazole (n = 40 eyes) eye drops were applied to the cornea every 5 minutes for 30 minutes. Aqueous humour samples were obtained performing an anterior chamber tap. The concentrations of ofloxacin and voriconazole were determined with high-pressure liquid chromatography. Groups were compared performing a Mann–Whitney test. Results: In the CXL group, the mean concentration of ofloxacin (13.33 ± 4.67 μg/mL) and voriconazole (52.70 ± 8.76 μg/mL) was significantly lower than in the untreated control group (ofloxacin: 18.51 ± 6.08 μg/mL, P = 0.005; voriconazole: 62.43 ± 13.5 μg/mL, P = 0.01). This corresponds to a reduction in permeability of 27.98% for ofloxacin and 15.59% for voriconazole. Central corneal thickness and anterior chamber depth were comparable in the CXL and control groups (P > 0.05, each). Conclusions: CXL reduces the corneal permeability of ofloxacin and voriconazole. This may be of clinical significance, for example, in keratitis treatment.

Characteristics of rod regeneration in a novel zebrafish retinal degeneration model using N-methyl-N-nitrosourea (MNU)

Primary loss of photoreceptors caused by diseases such as retinitis pigmentosa is one of the main causes of blindness worldwide. To study such diseases, rodent models of N-methyl-N-nitrosourea (MNU)-induced retinal degeneration are widely used. As zebrafish (Danio rerio) are a popular model system for visual research that offers persistent retinal neurogenesis throughout the lifetime and retinal regeneration after severe damage, we have established a novel MNU-induced model in this species. Histology with staining for apoptosis (TUNEL), proliferation (PCNA), activated Müller glial cells (GFAP), rods (rhodopsin) and cones (zpr-1) were performed. A characteristic sequence of retinal changes was found. First, apoptosis of rod photoreceptors occurred 3 days after MNU treatment and resulted in a loss of rod cells. Consequently, proliferation started in the inner nuclear layer (INL) with a maximum at day 8, whereas in the outer nuclear layer (ONL) a maximum was observed at day 15. The proliferation in the ONL persisted to the end of the follow-up (3 months), interestingly, without ongoing rod cell death. We demonstrate that rod degeneration is a sufficient trigger for the induction of Müller glial cell activation, even if only a minimal number of rod cells undergo cell death. In conclusion, the use of MNU is a simple and feasible model for rod photoreceptor degeneration in the zebrafish that offers new insights into rod regeneration.

Illusionary Self-Motion Perception in Zebrafish

Zebrafish mutant belladonna (bel) carries a mutation in the lhx2 gene (encoding a Lim domain homeobox transcription factor) that results in a defect in retinotectal axon pathfinding, which can lead to uncrossed optic nerves failing to form an optic chiasm. Here, we report on a novel swimming behavior of the bel mutants, best described as looping. Together with two previously reported oculomotor instabilities that have been related to achiasmatic bel mutants, reversed optokinetic response (OKR) and congenital nystagmus (CN, involuntary conjugate oscillations of both eyes), looping opens a door to study the influence of visual input and eye movements on postural balance. Our result shows that looping correlates perfectly with reversed OKR and CN and is vision-dependent and contrast sensitive. CN precedes looping and the direction of the CN slow phase is predictive of the looping direction, but is absent during looping. Therefore, looping may be triggered by CN in bel. Moreover, looping in wild-type fish can also be evoked by whole-field motion, suggesting that looping in a bel mutant larvae is a result of self-motion perception. In contrary to previous hypotheses, our findings indicate that postural control in vertebrates relies on both direct visual input (afference signal) and eye-movement-related signals (efference copy or reafference signal).

Structure of ACC synthase inactivated by the mechanism-based inhibitor l-vinylglycine

l‐Vinylglycine (l‐VG) is both a substrate for and a mechanism‐based inhibitor of 1‐aminocyclopropane‐1‐carboxylate (ACC) synthase. The ratio of the rate constants for catalytic conversion to α‐ketobutyrate and ammonia to inactivation is 500/1. The crystal structure of the covalent adduct of the inactivated enzyme was determined at 2.25 Å resolution. The active site contains an external aldimine of the adduct of l‐VG with the pyridoxal 5′‐phosphate cofactor. The side chain γ‐carbon of l‐VG is covalently bound to the ε‐amino group of Lys273. This species corresponds to one of the two alternatives proposed by Feng and Kirsch [Feng, L. and Kirsch, J.F. (2000) l‐Vinylglycine is an alternative substrate as well as a mechanism‐based inhibitor of 1‐aminocyclopropane‐1‐carboxylate synthase. Biochemistry 39, 2436–2444] and presumably results from Michael addition to a vinylglycine ketimine intermediate.

Funduscopy in Adult Zebrafish and Its Application to Isolate Mutant Strains with Ocular Defects

Funduscopy is one of the most commonly used diagnostic tools in the ophthalmic practice, allowing for a ready assessment of pathological changes in the retinal vasculature and the outer retina. This non-invasive technique has so far been rarely used in animal model for ophthalmic diseases, albeit its potential as a screening assay in genetic screens. The zebrafish (Danio rerio) is well suited for such genetic screens for ocular alterations. Therefore we developed funduscopy in adult zebrafish and employed it as a screening tool to find alterations in the anterior segment and the fundus of the eye of genetically modified adult animals. A stereomicroscope with coaxial reflected light illumination was used to obtain fundus color images of the zebrafish. In order to find lens and retinal alterations, a pilot screen of 299 families of the F3 generation of ENU-treated adult zebrafish was carried out. Images of the fundus of the eye and the anterior segment can be rapidly obtained and be used to identify alterations in genetically modified animals. A number of putative mutants with cataracts, defects in the cornea, eye pigmentation, ocular vessels and retina were identified. This easily implemented method can also be used to obtain fundus images from rodent retinas. In summary, we present funduscopy as a valuable tool to analyse ocular abnormalities in adult zebrafish and other small animal models. A proof of principle screen identified a number of putative mutants, making funduscopy based screens in zebrafish feasible.

Impact of corneal cross-linking on topical drug penetration in humans

To analyse the influence of corneal cross‐linking (CXL) with ultraviolet‐A (UV‐A) and riboflavin on drug permeability in human subjects.

Vestibular deficits do not underlie looping behavior in achiasmatic fish

Zebrafish belladonna (bel) mutants carry a mutation in the lhx2 gene that encodes a Lim domain homeobox transcription factor, leading to a defect in the retinotectal axon pathfinding. As a result, a large fraction of homozygous bel mutants is achiasmatic. Achiasmatic bel mutants display ocular motor instabilities, both reserved optokinetic response (OKR) and spontaneous eye oscillations, and an unstable swimming behavior, described as looping. All these unstable behaviors have been linked to the underlying optic nerve projection defect. Looping has been investigated under different visual stimuli and shown to be vision dependent and contrast sensitive. In addition, looping correlates perfectly with reversed OKR and the spontaneous oscillations of the eyes. Hence, it has been hypothesized that looping is a compensatory response to the perception of self-motion induced by the spontaneous eye oscillations. However, both ocular and postural instabilities could also be caused by a yet unidentified vestibular deficit. In this paper, we performed a preliminary test of the vestibular function in achiasmatic bel larval mutants in order to clarify the potential role of a vestibular deficit in looping. We found that the vestibular ocular reflex (VOR) is normally directed in both bel mutants and wild types and therefore exclude the possibility that nystagmus and looping in reverse to the rotating optokinetic drum can be attributed to an underlying vestibular deficit.

The Challenge of Evaluating the Intensity of Short Actions in Soccer: A New Methodological Approach Using Percentage Acceleration.

Purpose There are several approaches to quantifying physical load in team sports using positional data. Distances in different speed zones are most commonly used. Recent studies have used acceleration data in addition in order to take short intense actions into account. However, the fact that acceleration decreases with increasing initial running speed is ignored and therefore introduces a bias. The aim of our study was to develop a new methodological approach that removes this bias. For this purpose, percentage acceleration was calculated as the ratio of the maximal acceleration of the action (amax,action) and the maximal voluntary acceleration (amax) that can be achieved for a particular initial running speed (percentage acceleration [%] = amax,action / amax * 100). Methods To define amax, seventy-two highly trained junior male soccer players (17.1 ± 0.6 years) completed maximal sprints from standing and three different constant initial running speeds (vinit; trotting: ~6.0 km·h–1; jogging: ~10.8 km·h–1; running: ~15.0 km·h–1). Results The amax was 6.01 ± 0.55 from a standing start, 4.33 ± 0.40 from trotting, 3.20 ± 0.49 from jogging and 2.29 ± 0.34 m·s–2 from running. The amax correlated significantly with vinit (r = –0.98) and the linear regression equation of highly-trained junior soccer players was: amax = –0.23 * vinit + 5.99. Conclusion Using linear regression analysis, we propose to classify high-intensity actions as accelerations >75% of the amax, corresponding to acceleration values for our population of >4.51 initiated from standing, >3.25 from trotting, >2.40 from jogging, and >1.72 m·s–2 from running. The use of percentage acceleration avoids the bias of underestimating actions with high and overestimating actions with low initial running speed. Furthermore, percentage acceleration allows determining individual intensity thresholds that are specific for one population or one single player.

Inhibition of the TGFβ Pathway Enhances Retinal Regeneration in Adult Zebrafish

In contrast to the mammalian retina, the zebrafish retina exhibits the potential for lifelong retinal neurogenesis and regeneration even after severe damage. Previous studies have shown that the transforming growth factor beta (TGFβ) signaling pathway is activated during the regeneration of different tissues in the zebrafish and is needed for regeneration in the heart and the fin. In this study, we have investigated the role of the TGFβ pathway in the N-methyl-N-nitrosourea (MNU)-induced chemical model of rod photoreceptor de- and regeneration in adult zebrafish. Immunohistochemical staining for phosphorylated Smad3 was elevated during retinal regeneration, and phosphorylated Smad3 co-localized with proliferating cell nuclear antigen and glutamine synthetase, indicating TGFβ pathway activation in proliferating Müller glia. Inhibiting the TGFβ signaling pathway using a small molecule inhibitor (SB431542) resulted in accelerated recovery from retinal degeneration. Accordingly, we observed increased cell proliferation in the outer nuclear layer at days 3 to 8 after MNU treatment. In contrast to the observations in the heart and the fin, the inhibition of the TGFβ signaling pathway resulted in increased proliferation after the induction of retinal degeneration. A better understanding of the underlying pathways with the possibility to boost retinal regeneration in adult zebrafish may potentially help to stimulate such proliferation also in other species.