Transition transferases prime bacterial capsule polymerization.

Capsules are long-chain carbohydrate polymers that envelop the surfaces of many bacteria, protecting them from host immune responses. Capsule biosynthesis enzymes are potential drug targets and valuable biotechnological tools for generating vaccine antigens. Despite their importance, it remains unknown how structurally variable capsule polymers of Gram-negative pathogens are linked to the conserved glycolipid anchoring these virulence factors to the bacterial membrane. Using Actinobacillus pleuropneumoniae as an example, we demonstrate that CpsA and CpsC generate a poly(glycerol-3-phosphate) linker to connect the glycolipid with capsules containing poly(galactosylglycerol-phosphate) backbones. We reconstruct the entire capsule biosynthesis pathway in A. pleuropneumoniae serotypes 3 and 7, solve the X-ray crystal structure of the capsule polymerase CpsD, identify its tetratricopeptide repeat domain as essential for elongating poly(glycerol-3-phosphate) and show that CpsA and CpsC stimulate CpsD to produce longer polymers. We identify the CpsA and CpsC product as a wall teichoic acid homolog, demonstrating similarity between the biosynthesis of Gram-positive wall teichoic acid and Gram-negative capsules.

A multi-enzyme machine polymerizes the Haemophilus influenzae type b capsule.

Bacterial capsules have critical roles in host-pathogen interactions. They provide a protective envelope against host recognition, leading to immune evasion and bacterial survival. Here we define the capsule biosynthesis pathway of Haemophilus influenzae serotype b (Hib), a Gram-negative bacterium that causes severe infections in infants and children. Reconstitution of this pathway enabled the fermentation-free production of Hib vaccine antigens starting from widely available precursors and detailed characterization of the enzymatic machinery. The X-ray crystal structure of the capsule polymerase Bcs3 reveals a multi-enzyme machine adopting a basket-like shape that creates a protected environment for the synthesis of the complex Hib polymer. This architecture is commonly exploited for surface glycan synthesis by both Gram-negative and Gram-positive pathogens. Supported by biochemical studies and comprehensive 2D nuclear magnetic resonance, our data explain how the ribofuranosyltransferase CriT, the phosphatase CrpP, the ribitol-phosphate transferase CroT and a polymer-binding domain function as a unique multi-enzyme assembly.

Efficient solid-phase synthesis of meningococcal capsular oligosaccharides enables simple and fast chemoenzymatic vaccine production.

Neisseria meningitidis serogroups A and X are among the leading causes of bacterial meningitis in the African meningitis belt. Glycoconjugate vaccines, consisting of an antigenic carrier protein coupled to the capsular polysaccharide of the bacterial pathogen, are the most effective strategy for prevention of meningococcal disease. However, the distribution of effective glycoconjugate vaccines in this region is limited by the high cost of cultivating pathogens and purification of their capsular polysaccharides. Moreover, chemical approaches to synthesize oligosaccharide antigens have proven challenging. In the current study, we present a chemoenzymatic approach for generating tailored oligosaccharide fractions ready for activation and coupling to the carrier protein. In a first step, the elongation modes of recombinant capsular polymerases from Neisseria meningitidis serogroups A (CsaB) and X (CsxA) were characterized. We observed that CsaB is a distributive enzyme, and CsxA is a processive enzyme. Sequence comparison of these two stealth family proteins revealed a C-terminal extension in CsxA, which conferred processivity because of the existence of a second product-binding site. Deletion of the C-terminal domain converted CsxA into a distributive enzyme, allowing facile control of product length by adjusting the ratio of donor to acceptor sugars. Solid-phase fixation of the engineered capsular polymerases enabled rapid production of capsular polysaccharides with high yield and purity. In summary, the tools developed here provide critical steps toward reducing the cost of conjugate vaccine production, which will increase access in regions with the greatest need. Our work also facilitates efforts to study the relationship between oligosaccharide size and antigenicity.

Time- and frequency-domain parameters of heart rate variability and sympathetic skin response in Parkinson's disease.

The autonomic nervous system (ANS) is regularly affected in Parkinson's disease (PD). Information on autonomic dysfunction can be derived from e.g. altered heart rate variability (HRV) and sympathetic skin response (SSR). Such parameters can be quantified easily and measured repeatedly which might be helpful for evaluating disease progression and therapeutic outcome. In this 2-center study, HRV and SSR of 45 PD patients and 26 controls were recorded. HRV was measured during supine metronomic breathing and analyzed in time- and frequency-domains. SSR was evoked by repetitive auditory stimulation. Various ANS parameters were compared (1) between patients and healthy controls, (2) to clinical scales (Unified Parkinson's disease rating scale, Mini-Mental State Examination, Becks Depression Inventory), and (3) to disease duration. Root mean square of successive differences (RMSSD) and low frequency/high frequency (LF/HF) ratio differed significantly between PD and controls. Both, HRV and SSR parameters showed low or no association with clinical scores. Time-domain parameters tended to be affected already at early PD stages but did not consistently change with longer disease duration. In contrast, frequency-domain parameters were not altered in early PD phases but tended to be lower (LF, LF/HF ratio), respectively higher (HF) with increasing disease duration. This report confirms previous results of altered ANS parameters in PD. In addition, it suggests that (1) these ANS parameters are not relevantly associated with motor, behavioral, and cognitive changes in PD, (2) time-domain parameters are useful for the assessment of early PD, and (3) frequency-domain parameters are more closely associated with disease duration.

Functional expression of the capsule polymerase of Neisseria meningitidis serogroup X: a new perspective for vaccine development.

Neisseria meningitidis (Nm) is a leading cause of bacterial meningitis and sepsis. A key feature in pathogenicity is the capsular polysaccharide (CPS) that prevents complement activation and thus supports bacterial survival in the host. Twelve serogroups characterized by immunologically and structurally different CPSs have been identified. Meningococcal CPSs elicit bactericidal antibodies and consequently are used for the development of vaccines. Vaccination against the epidemiologically most relevant serogroups was initially carried out with purified CPS and later followed by conjugate vaccines which consist of CPS covalently linked to a carrier protein. Of increasing importance in the African meningitis belt is NmX for which no vaccine is currently available. Here, we describe the molecular cloning, recombinant expression and purification of the capsule polymerase (CP) of NmX called CsxA. The protein expressed with N- and/or C-terminal epitope tags was soluble and could be purified to near homogeneity. With short oligosaccharide primers derived from the NmX capsular polysaccharide (CPSX), recombinant CsxA produced long polymer chains in vitro that in immunoblots were detected with NmX-specific antibodies. Moreover, the chemical identity of in vitro produced NmX polysaccharides was confirmed by NMR. Besides the demonstration that the previously identified gene csxA encodes the NmX CP CsxA, the data presented in this study pave the way for the use of the recombinant CP as a safe and economic way to generate the CPSX in vaccine developmental programs.

Evaluation of progression markers in the premotor phase of Parkinson's disease: the progression markers in the premotor phase study.

BACKGROUND: The clinical diagnosis of Parkinson's disease (PD) is proposed to be too late for the application of beneficial neuroprotective treatment. Thus, it is important to identify and follow individuals at risk for PD in order to gain knowledge about the prodromal course of the disease. Substantia nigra hyperechogenicity (SN+) has been confirmed as a risk factor for PD and appears promising as a predictor of PD, particularly in combination with other putative PD markers. We present the design and initial data of a 2-year longitudinal investigation of subjects proposed to be at high risk of developing PD (HRPD), compared to early PD patients and control subjects. The aim of the presented study is to monitor progression of the neurodegenerative process to motor PD. METHODS: In total, 40 HRPD, 16 PD and 41 control individuals were recruited. The HRPD subjects had SN+ and additionally either 1 cardinal PD motor sign or 2 further risk (e.g. positive family history) or prodromal markers (e.g. hyposmia). In this cohort, motor function, olfaction, mood and blood markers will be evaluated every 6 months, complemented by a comprehensive clinical, imaging and electrophysiological assessment. RESULTS: PD, HRPD and control subjects did not differ significantly regarding age, but the HRPD group consisted mainly of males (72.5% of HRPD subjects vs. 43.9% of controls; p = 0.013). Mean disease duration in PD patients was 31 months (range 15-56). HRPD subjects were predominantly recruited according to the occurrence of slight motor signs (HRPD 77.5%, PD 100%, p = 0.05; controls 0%, HRPD vs. controls, p < 0.017). The Unified Parkinson's Disease Rating Scale motor score (mean, range) indicated that the HRPD group (4, 0-12) had values between those of controls (0, 0-2; p < 0.017) and PD subjects (26, 9-55; p < 0.017). Among nonmotor symptoms, hyposmia was more common in both HRPD (47.5%) and PD subjects (75%) than in controls (5.1%; p < 0.017 for both comparisons). CONCLUSIONS: Here, we describe the recruitment of a highly enriched-risk cohort and a promising study design to assess progression to motor PD. Whether the HRPD group indeed suffers from early, PD-specific neurodegeneration remains to be verified in the ongoing follow-up examinations.

Extending the enzymatic toolbox for heparosan polymerization, depolymerization, and detection.

Heparosan is an acidic polysaccharide expressed as a capsule polymer by pathogenic and commensal bacteria, e.g. by E. coli K5. As a precursor in the biosynthesis of heparan sulfate and heparin, heparosan has a high biocompatibility and is thus of interest for pharmaceutical applications. However, due to its low immunogenicity, developing antibodies against heparosan and detecting the polymer in biological samples has been challenging. In this study, we exploited the enzyme repertoire of E. coli K5 and the E. coli K5-specific bacteriophage ΦK5B for the controlled synthesis and depolymerization of heparosan. A fluorescently labeled heparosan nonamer was used as a priming acceptor to study the elongation mechanism of the E. coli K5 heparosan polymerases KfiA and KfiC. We could demonstrate that the enzymes act in a distributive manner, producing labeled heparosan of low dispersity. The enzymatically synthesized heparosan was a useful tool to identify the tailspike protein KflB of ΦK5B as heparosan lyase and to characterize its endolytic depolymerization mechanism. Most importantly, using site-directed mutagenesis and rational construct design, we generated an inactive version of KflB for the detection of heparosan in ELISA-based assays, on blots, and on bacterial and mammalian cells.

A high-throughput screen for polysialyltransferase activity.

Polysialic acid is common to humans and a few bacterial pathogens and it holds great potential for the development of new therapeutic reagents. Currently, the bacterial polysialyltransferases (polySTs) are the only source of polysialic acid for research and biotechnological purposes either directly, by enzymatic polysialylation of therapeutic proteins, or indirectly, by harvest of polysialic acid from bacterial fermentation. Further engineering and optimization of these enzymes is hindered by the lack of high-throughput screening methodologies for polysialyltransferase activity. Here we report the development of an efficient in vivo activity screen for bacterial polySTs. The screen exploits complementation of a dormant capsule export complex in the expression strain, Escherichia coli BL21-Gold(DE3). This strain was metabolically engineered to synthesize CMP-Neu5Ac, the donor sugar for the polysialylation reaction. Using the new strain, a colony blotting procedure that enables the routine testing of more than 10(4) polyST genes was developed. To test the usefulness of the methodology, we screened a library of N-terminally truncated polySTs derived from the Neisseria meningitidis serogroup B (NmB)-polyST. We identified truncations that remove a putative membrane interaction domain, resulting in soluble and active enzymes.

Exploitation of Capsule Polymerases for Enzymatic Synthesis of Polysaccharide Antigens Used in Glycoconjugate Vaccines.

The exploitation of recombinant enzymes for the synthesis of complex carbohydrates is getting increasing attention. Unfortunately, the analysis of the resulting products often requires advanced methods like nuclear magnetic resonance spectroscopy and mass spectrometry. Here, we use the capsule polymerases Cps4B and Cps11D from Actinobacillus pleuropneumoniae serotypes 4 and 11, respectively, as examples for the in vitro synthesis of capsule polymers similar to those used in glycoconjugate vaccine formulations. We demonstrate how substrate turnover in an enzymatic reaction can be analyzed by HPLC-based anion exchange chromatography and provide the protocol for separation and detection of UV-active polymer. Moreover, we describe how UV-inactive polymer can be separated and visualized using polyacrylamide gel electrophoresis followed by combined alcian blue-silver staining.

Mix-and-Match System for the Enzymatic Synthesis of Enantiopure Glycerol-3-Phosphate-Containing Capsule Polymer Backbones from Actinobacillus pleuropneumoniae, Neisseria meningitidis, and Bibersteinia trehalosi.

Capsule polymers are crucial virulence factors of pathogenic bacteria and are used as antigens in glycoconjugate vaccine formulations. Some Gram-negative pathogens express poly(glycosylglycerol phosphate) capsule polymers that resemble Gram-positive wall teichoic acids and are synthesized by TagF-like capsule polymerases. So far, the biotechnological use of these enzymes for vaccine developmental studies was restricted by the unavailability of enantiopure CDP-glycerol, one of the donor substrates required for polymer assembly. Here, we use CTP:glycerol-phosphate cytidylyltransferases (GCTs) and TagF-like polymerases to synthesize the poly(glycosylglycerol phosphate) capsule polymer backbones of the porcine pathogen Actinobacillus pleuropneumoniae, serotypes 3 and 7 (App3 and App7). GCT activity was confirmed by high-performance liquid chromatography, and polymers were analyzed using comprehensive nuclear magnetic resonance studies. Solid-phase synthesis protocols were established to allow potential scale-up of polymer production. In addition, one-pot reactions exploiting glycerol-kinase allowed us to start the reaction from inexpensive, widely available substrates. Finally, this study highlights that multidomain TagF-like polymerases can be transformed by mutagenesis of active site residues into single-action transferases, which in turn can act in trans to build-up structurally new polymers. Overall, our protocols provide enantiopure, nature-identical capsule polymer backbones from App2, App3, App7, App9, and App11, Neisseria meningitidis serogroup H, and Bibersteinia trehalosi serotypes T3 and T15. IMPORTANCE Economic synthesis platforms for the production of animal vaccines could help reduce the overuse and misuse of antibiotics in animal husbandry, which contributes greatly to the increase of antibiotic resistance. Here, we describe a highly versatile, easy-to-use mix-and-match toolbox for the generation of glycerol-phosphate-containing capsule polymers that can serve as antigens in glycoconjugate vaccines against Actinobacillus pleuropneumoniae and Bibersteinia trehalosi, two pathogens causing considerable economic loss in the swine, sheep, and cattle industries. We have established scalable protocols for the exploitation of a versatile enzymatic cascade with modular architecture, starting with the preparative-scale production of enantiopure CDP-glycerol, a precursor for a multitude of bacterial surface structures. Thereby, our approach not only allows the synthesis of capsule polymers but might also be exploitable for the (chemo)enzymatic synthesis of other glycerol-phosphate-containing structures such as Gram-positive wall teichoic acids or lipoteichoic acids.

Time course of eye and head deviation in spatial neglect.

Spatial neglect is characterized by a deviation of the eyes and the head during active search, as well as at rest. Here the authors investigate the hitherto unknown relationship between these striking behaviors in the course of recovery. Gaze, eye-in-head, and head-on-trunk positions were recorded separately under two experimental conditions: (i) at rest (i.e., without any specific requirements, doing nothing) and (ii) during active exploratory search in a large visual array of 240 degrees x 80 degrees over a 10-month period. The authors observed a parallel decrease of eye and head (= gaze) deviation in both conditions, accompanied by a comparable decline in neglect severity. The results strengthen the view that the marked gaze deviation toward the ipsilesional side in patients with spatial neglect is due to a very elementary disturbance of human spatial information processing.

A New Family of Capsule Polymerases Generates Teichoic Acid-Like Capsule Polymers in Gram-Negative Pathogens.

Group 2 capsule polymers represent crucial virulence factors of Gram-negative pathogenic bacteria. They are synthesized by enzymes called capsule polymerases. In this report, we describe a new family of polymerases that combine glycosyltransferase and hexose- and polyol-phosphate transferase activity to generate complex poly(oligosaccharide phosphate) and poly(glycosylpolyol phosphate) polymers, the latter of which display similarity to wall teichoic acid (WTA), a cell wall component of Gram-positive bacteria. Using modeling and multiple-sequence alignment, we showed homology between the predicted polymerase domains and WTA type I biosynthesis enzymes, creating a link between Gram-negative and Gram-positive cell wall biosynthesis processes. The polymerases of the new family are highly abundant and found in a variety of capsule-expressing pathogens such as Neisseria meningitidis, Actinobacillus pleuropneumoniae, Haemophilus influenzae, Bibersteinia trehalosi, and Escherichia coli with both human and animal hosts. Five representative candidates were purified, their activities were confirmed using nuclear magnetic resonance (NMR) spectroscopy, and their predicted folds were validated by site-directed mutagenesis.IMPORTANCE Bacterial capsules play an important role in the interaction between a pathogen and the immune system of its host. During the last decade, capsule polymerases have become attractive tools for the production of capsule polymers applied as antigens in glycoconjugate vaccine formulations. Conventional production of glycoconjugate vaccines requires the cultivation of the pathogen and thus the highest biosafety standards, leading to tremendous costs. With regard to animal husbandry, where vaccines could avoid the extensive use of antibiotics, conventional production is not sufficiently cost-effective. In contrast, enzymatic synthesis of capsule polymers is pathogen-free and fast, offers high stereo- and regioselectivity, and works with high efficacy. The new capsule polymerase family described here vastly increases the toolbox of enzymes available for biotechnology purposes. Representatives are abundantly found in human pathogens but also in animal pathogens, paving the way for the exploitation of polymerases for the development of a new generation of vaccines for animal husbandry.

Combined Chemical Synthesis and Tailored Enzymatic Elongation Provide Fully Synthetic and Conjugation-Ready Neisseria meningitidis Serogroup X Vaccine Antigens.

Studies on the polymerization mode of Neisseria meningitidis serogroup X capsular polymerase CsxA recently identified a truncated construct that can be immobilized and used for length controlled on-column production of oligosaccharides. Here, we combined the use of a synthetic acceptor bearing an appendix for carrier protein conjugation and the on-column process to a novel chemo-enzymatic strategy. After protein coupling of the size optimized oligosaccharide produced by the one-pot elongation procedure, we obtained a more homogeneous glycoconjugate compared to the one previously described starting from the natural polysaccharide. Mice immunized with the conjugated fully synthetic oligomer elicited functional antibodies comparable to controls immunized with the current benchmark MenX glycoconjugates prepared from the natural capsule polymer or from fragments of it enzymatically elongated. This pathogen-free technology allows the fast total in vitro construction of predefined bacterial polysaccharide fragments. Compared to conventional synthetic protocols, the procedure is more expeditious and drastically reduces the number of purification steps to achieve the oligomers. Furthermore, the presence of a linker for conjugation in the synthetic acceptor minimizes manipulations on the enzymatically produced glycan prior to protein conjugation. This approach enriches the methods for fast construction of complex bacterial carbohydrates.

Restricted ocular exploration does not seem to explain simultanagnosia.

One major function of parietal cortex is to direct our attention towards salient stimuli. The present data suggest that it also plays an important role in visual gestalt perception. Patients with simultanagnosia following lesions in this area are not able to extract the meaning of a visual scene whereas being perfectly able to recognise individual objects of this scene. We tested two patients with simultanagnosia with hierarchical Navon figures combined with eye movements recordings. The patients' performance allowed us to compare directly the scan paths in trials in which the global letter shape was recognised with trials in which the global letter shape was not recognised. We did not find any obvious differences in the eye movement pattern related to the two perceptual situations. The two patients did not show a significant problem in shifting their eyes (and thus possibly also their attentional focus) to all aspects of the complex visual stimulus when attempting to bind together the different elements of spatially distributed information. The results demonstrate that restricted ocular exploration cannot be the reason for the patients' inability to recognise the global shape of stimuli. Our data rather suggest a role of parietal cortex in visual gestalt perception that is beyond its role of directing attention towards relevant objects.

The role of the right superior temporal gyrus in visual search-insights from intraoperative electrical stimulation.

Intraoperative electrical stimulation in awake patients is a seminal technique during brain surgery allowing one to infer the function of brain areas by temporary inactivation. Using this technique, we found that inactivation of the middle portion of the superior temporal gyrus (STG) leads to disturbed serial exploratory visual search. The data supplement recent findings by Ellison et al. [Ellison, A., Schindler, I., Pattison, L. L., & Milner, A. D. (2004). An exploration of the role of the superior temporal gyrus in visual search and spatial perception using TMS. Brain, 127, 2307-2315] using repetitive transcranial magnetic stimulation over the STG in healthy subjects. Our data demonstrate that the STG is integral to human exploration behaviour and challenge the traditional view that only the right posterior parietal cortex is involved in the mediation of visual search processes.

Disturbed line bisection is associated with posterior brain lesions.

Neglect patients classically fail to orient and respond to stimuli appearing on their contralesional side. Traditionally, the neglect syndrome has been associated with damage to the right inferior parietal lobule (IPL) and the right temporo-parietal junction (TPJ). Neglect is popularly assessed by two different tasks: line bisection and cancellation. In a previous study (S. Ferber, H.-O. Karnath, How to assess spatial neglect-line bisection or cancellation tasks. J. Clin. Exp. Neuropsychol. 23 (2001) 599-607), we observed that performance on the cancellation task correlates well with the characteristic behavioral disorders used to clinically diagnose spatial neglect, while line bisection was a poor predictor. This might indicate that the disability to correctly bisect lines is a distinct disorder separable from spatial neglect. Here, we assess the anatomy of the patients investigated in that study, and reveal that damage to the temporo-occipital junction correlates with poor performance in the line bisection task. This work extends previous work by Binder et al. (J. Binder, R. Marshall, R. Lazar, J. Benjamin, J.P. Mohr, Distinct syndromes of hemineglect. Arch. Neurol. 49 (1992) 1187-1194) suggesting that line bisection and cancellation identify distinct syndromes. The data suggest that these two tasks dissociate both in terms of behavior and anatomy. This anatomical distinction may help reconcile our recent finding that spatial neglect is associated with damage to the superior temporal cortex and insula, while others have identified the IPL and TPJ. Specifically, we note that our previous anatomical studies did not use the line bisection task to select neglect patients, while many others used this task. We suggest that anatomical studies that combine patients from both of these two distinct groups may result in misleading findings.

Normalized perfusion MRI to identify common areas of dysfunction: patients with basal ganglia neglect.

Perfusion-weighted imaging (PWI) is used to identify brain regions that are receiving enough blood supply to remain structurally intact, but not enough to function normally. Previous observations suggest that spatial neglect due to subcortical stroke can be explained by dysfunction of cortical areas rather than through the neuronal loss in the subcortical structures itself. The present study aimed to identify the dysfunctional cortical regions induced by basal ganglia stroke in patients with spatial neglect. In a patient group with stroke lesions centring on the basal ganglia, we examined the common area(s) of structurally intact but dysfunctional cortical tissue by using spatial normalization of PWI maps as well as symmetric voxel-wise inter-hemispheric comparisons. These new techniques allow comparison of the structurally intact but abnormally perfused areas of different individuals in the same stereotaxic space, and at the same time avoid problems due to regional perfusion differences and to possible observer-dependent biases. We found that strokes centring on the right basal ganglia which provoke spatial neglect induce abnormal perfusion in a circumscribed area of intact cortex that typically involves those three regions that have previously been described to provoke spatial neglect when damaged directly by cortical infarction: the superior temporal gyrus, the inferior parietal lobule and the inferior frontal gyrus. The data suggest that spatial neglect following a right basal ganglia lesion typically is caused by the dysfunction of (part of) these specific cortical areas.

An efficient cell free enzyme-based total synthesis of a meningococcal vaccine candidate.

Invasive meningococcal disease (IMD) is a global health problem and vaccination has proven the most effective way of disease control. Neisseria meningitidis serogroup X (NmX) is an emerging threat in the African sub-Saharan meningitis belt, but no vaccine is available today. Leading vaccines against Nm are glycoconjugates, in which capsular polysaccharides isolated from large-scale pathogen cultures are conjugated to adjuvant proteins. Though safe and efficacious even in infants, high costs and biohazard associated with the production limit abundant application of glycoconjugate vaccines particularly in the most afflicted nations. An existing NmX vaccine candidate (CPSXn-CRM197) produced by established protocols from NmX capsule polysaccharide (CPSX) has been shown to elicit high bactericidal immunoglobulin G titres in mice. Here we describe the scalable in vitro synthesis of CPSXiv from chemically pure precursors by the use of recombinant NmX capsule polymerase. Application of the described coupling chemistry gives CPSXiv-CRM197, which in mouse vaccination experiments behaves identical to the benchmark CPSXn-CRM197. Excluding any biohazards, this novel process represents a paradigm shift in vaccine production and a premise towards vaccine manufacturing in emerging economies.

Spontaneous eye and head position in patients with spatial neglect.

The most prominent deficit in patients with spatial neglect is a bias of their active behaviour, i. e. a deviation of exploratory movements towards the right. When searching for targets, copying, or reading, the patients direct their eye and hand movements towards the ipsilesional side, leading to neglect of the contralesional side. The present study investigated whether spatial neglect is predominantly linked with such active behaviour or if it is obvious also without any explicit requirements, namely in the patients' spontaneous eye and head position. To address this issue we investigated the patients' spontaneous resting position while "doing nothing", i. e. just sitting and waiting for an experiment to start. Using magnetic search coil technique, we recorded spontaneous eye-in-head and head-on-trunk orientation in that waiting period in 24 patients with and without spatial neglect. In contrast to controls, neglect patients showed a marked deviation of spontaneous eye and head orientation of about 30 degrees (= gaze position) towards the right. The findings strengthen the view that one component of the behaviour in neglect patients is due to a very elementary disturbance of spatial information processing. The deviation of eye and head may be understood as a pathological adjustment of the subject's normal resting position to a more rightward position. While the position in healthy subjects is in line with trunk orientation, this "default position" is shifted to a new origin in patients with spatial neglect.